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ENZYME ACTION IN THE MARINE ALGAE

A: R. DAVIS

Research Assistant to the Missouri Botanical Garden,
Formerly Rufus J. Lackland Fellow in the Henry Shaw School of Botany of
Washington University

In a previous contribution from this laboratory! attention
has been called to the difficulties experienced in demonstrating
enzyme action in Fucus vesiculosus. Because of the negative
results there obtained it was deemed worth while to extend
the study to certain representative forms of the three great
groups of marine algae, the ‘‘greens,’’ the ‘‘browns,’’ and
the ‘‘reds’’; first, to ascertain whether this apparent inac-
tivity were generally characteristic of the algae, and second,
because of the light such an investigation might shed upon
the general metabolism of the group.

HIsTorIcaL

Knowledge concerning enzyme activity and the distribution
of enzymes in the algae is extremely meagre. The few papers
that have found their way into the literature have been, for
the most part, by-products of other studies and as such have
dealt merely with isolated phases of the subject. From time
to time, previous to actual demonstration, the presence of
enzymes has been suggested by the work of various investi-
gators. Arber (’01), attacking the problem of carbon as-
similation in Ulva latissima,? found that the accumulation of
starch in the tissue disappeared very slowly when the plant
was subjected to darkness. This would suggest the presence
of a diastase acting slowly. Spargo (718) observed that
Chlamydomonas began growth more slowly when the medium
contained sucrose as a source of carbon than when dextrose
was supplied. She suggests that the sugar is probably assim-

1 Duggar, B. M. and Davis, A. R. Enzyme action in Fucus vesiculosus. Ann.
Mo. Bot. Gard. 1:419-426. 1914.

?The binomials used throughout the historical review are those employ2d
by the original investigators, no attempt being made to have them conform to
any different existing nomenclature.

ANN. Mo. Bort. GArD., VOL. 2, 1915 (771)

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IP ANNALS OF THE MISSOURI BOTANICAL GARDEN

ilated in the hexose form and that sucrose must be split by
invertase before becoming available. It is a well-known fact
that diverse fresh-water algae can be grown in pure culture
on media where asparagin and peptone are sources of
nitrogen. It is hardly conceivable that the large protein
molecule is assimilated directly and, a priori, this would argue
for the presence of both an ereptase and a desamidizing
enzyme.
ENZYMES FOUND IN THE MARINE ALGAE

Few workers have demonstrated enzymes present in either
the fresh- or salt-water algae. Fischer (’05), working on the
storage carbohydrates of Anabaena and Oscillatoria, found
that the specific carbohydrate involved, which he named ana-
baenin, disappeared when the algal tissue was autolysed at
40°C. Microchemical tests showed glycogen split off. The
action here, if it be due to ferments of the alga, is interest-
ing in that the action was inhibited by .1 per cent acetic acid,
by 1 per cent carbolic acid, and still more strangely, by con-
centrations of ethyl alcohol as low as 5 per cent. One per
cent carbolic acid is quite often used as an antiseptic in enzyme
experimentation, and the resistance of enzymes to even high
concentrations of aleohol is common knowledge. No attempt
was made to isolate the enzyme or to carry on experiments
outside the cell.

Teodoresco (712) found that Chlamydomonas in pure cul-
ture gave rise to an extracellular enzyme that decomposed
sodium nucleate with the liberation of phosphorus. Later,
(712°) he demonstrated nucleases present in certain ‘‘blue-
greens,’’ ‘‘browns,’’ and ‘‘reds.’’ Unfortunately, differences
in methods do not permit a true comparison of activity with
that of the nuclease isolated by Dox (’10) from Penicilliwm
camemberti, nor with that determined by Zaleski (’07) in the
growing tips of Vicia faba, yet even a crude comparison is in-
teresting. Dox added 2 grams of mold powder to 100 ce. of a
2 per cent solution of yeast nucleic acid, and maintaining his
flasks at a temperature of 35-37°C. for forty-five days, found
51 milligrams of phosphorus (calculated as phosphoric acid)
liberated. Teodoresco used a .5 per cent solution of sodium

1915]
DAVIS—ENZYME ACTION IN MARINE ALGAE 773

nucleate with an unstated amount of crushed seaweed. The
temperature during the incubation period varied from 21
to 26°C. for the different forms used. The following are his
results for 100 ec. of substrate:

TABLE I
Phosphorus as P205
Alga Days megms.
Cladophora frusta...........- 57 54.3
Cerammm rubrum............ 51 76.6
Griffithsia setacea............ 37 62.5
Phormidiwm sp.............-- 15 90.0

Zaleski crushed growing tips of Vicia faba, added water
and an antiseptic, and allowed this material to autolyse at
34°C. for 4 days. At the end of that time the control flask
showed a free phosphorus content of 13.6 milligrams and the
one containing the active enzyme 51.2 milligrams. We have
no means of knowing even the relative amount of enzyme
present in any of these experiments and yet it seems that the
algal nuclease compares very favorably with that isolated
from the fungi and the higher plants.

The classes in plant physiology at the Marine Biological
Laboratory, Woods Hole, for several years past have quali-
tatively determined diastase in Ulva lactuca. Bartholemew
(714), working on the question of starch in the Florideae,
conclusively demonstrated diastase present in such ‘‘reds”’
as Polysiphoma variegata, Dasya elegans, Agardiuella tenera,
and Ceramium sp. In order to isolate the enzyme, he used
the ordinary method of precipitation by alcohol from an
aqueous extract of crushed tissue. Starch as paste was
hydrolysed rather slowly to an undetermined reducing sugar,
presumably dextrose, 5 cc. of .25 per cent starch paste with
a relatively large amount of the enzyme material requiring
from 6 to 9 days for the completion of hydrolysis. Micro-
scopic observation of the attacked starch grain showed corro-
sion similar to that caused by the translocation diastase of
the barley. Torup (Krefting and Torup, ’09) had previously
isolated an enzyme from fresh Lamar that hydrolysed the
characteristic storage carbohydrate of that alga, laminarin,
to dextrose.

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774 ANNALS OF THE MISSOURI BOTANICAL GARDEN

Atkins (’14) investigated the oxidases and peroxidases of
twenty-nine diverse algae. Using guaiacum as a reagent,
oxidases were demonstrated in but one—Furcellaria fasti-
giata—while peroxidases were shown present in seven. Alpha
naphthol gave negative reactions for all the forms studied,
while with it peroxidases could be determined in but two—
Delesseria sanguinea and Furcellaria fastigiata. He calls at-
tention to the reducing power of the tissues of certain algae
and suggests that such agents may be responsible for the
failure to obtain positive tests in the other forms. Reed
(715, 715°), on the other hand, holds that many of these
algae may show a specific oxidative ability. Like Atkins, he
found that the ordinary reagents, such as gum guaiac, alpha
naphthol, and aloin, gave negative results in all but one or two
instances. When, however, alpha naphthol and para-pheny-
lenediamine, para-phenylenediamine alone, or the hydro-
chlorides of these two were used in the presence of peroxide,
positive tests were very generally obtained.

As earlier indicated, the results obtained by Duggar and
Davis (714) for Fucus vesiculosus were very generally nega-
tive. This was true even though a great variety of substrates
were used under varying conditions, and only vigorously
growing plants, fresh crushed, or dried and powdered, were
employed for enzyme action. The results are exceedingly
difficult to explain. It might well be that the enzymes were
present but in such small amounts as to escape detection by
the ordinary methods. Methods of enzyme isolation are still
crude and they undoubtedly involve some loss of the ferments.
Another factor suggested in the preliminary paper, was that
the death of the cell might liberate certain substances which
would then be free to unite with the enzyme complex, throw-
ing it out of the sphere of action.

SOME STORAGE PRODUCTS OF THE ALGAE

It is often assumed that the presence of storage products
in the plant is generally linked with the presence of specific
enzymes—stareh with diastase, inulin with inulase, fats with
lipase, hemicelluloses with cytase, ete. These enzymes may
be present at all times, as the diastase of the potato tuber and

1915]
DAVIS—ENZYME ACTION IN MARINE ALGAE 775

the diastase and maltase of the barley grain, or they may only
arise when there is food transformation and translocation, as
in germinating seeds. However, in the light of such possi-
bilities of association, it is worth while to call attention briefly
to some of the work that has been done on the chief storage
products of the algae.

The carbohydrates have been more worked over in this
respect than has any other chemical group, but much confusion
still exists regarding their exact status in assimilation. Much
of the study has been on the cleavage products, obtained by
acid hydrolysis, of undetermined carbohydrates. These, how-
ever, are not a true index of the distribution and more re-
stricted chemical nature of assimilable carbohydrates in the
living plant; one must look rather to the work of those who
have limited themselves to the isolation and determination of
unaltered carbohydrates.

CHLOROPHYCEAE

Polysaccharides.—Nageli (’63) reported ‘‘spharokristalle”’
in Acetabularia which Leitgeb (’87) later showed were inulin.
The former worker also demonstrated the presence of this
carbohydrate in various members of the Dasycladaceae.
Kiister (’99) has more recently found characteristic crystal
formations in Derbesia and Bryopsis which, from the many
reactions they gave, appear to have been inulin. Famintzin
(767) and Krause (’70) worked on the effect of light on
starch formation in Spirogyra, and within recent years, Tim-
berlake (’01) has contributed observations on the starch of
Hydrodictyon. Oltmanns (’05, p. 147) speaks of starch ac-
cumulation in the Conjugales, Volvocales, Ulotrichales,
Charales, Siphonocladiales, and some of the Siphonales. He
considers it the first visible product of assimilation, but
thinks that it may also function as a reserve. Starch in the
marine forms seems to be quite widely distributed. In the
work of Arber (’01), to which reference has already been
made, starch accumulation in the tissues of Ulva, Cladophora,
and Enteromorpha was easily demonstrated by means of
iodine. Swartz (’11) isolated starch from Ulva but was
unable to prove its presence in Enteromorpha, a closely re-

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776 ANNALS OF THE MISSOURI BOTANICAL GARDEN

lated genus. She concluded that the carbohydrates existed in
the form of hemicelluloses, probably as pentosans.

Glycogen, although frequently found in the ‘‘blue-greens,’’
where, as held by some authors (Fischer, ’05), it functions
as the chief reserve carbohydrate, has been demonstrated in
but one case, as far as is known, in the Chlorophyceae, and
that by Beyerinck (’04) in Chlorella variegata.

Simple sugars.—The nature of the simple sugars in the
group is indefinite. Klebs (’96) reported a substance in the
cells of certain Heterokonteae that reduced Fehling’s solu-
tion, but this means little since most algae contain non-
carbohydrate reducing substances made up chiefly of tannins
and tannoidal bodies. Tihomirov (’10) used the phenyl-
hydrazine method as modified by Senft (’04) for the detection
of osozone-forming sugars in algal tissues in this group,
chiefly those of Codiwm bursa and C. tomentosum. After a
period of thirty days, for these two forms, yellow amorphous
deposits appeared in the cells indicating a sugar reaction.
The definite sugars these osozones represented could not be
determined, but he suggests the possibility of dextrose and
d-galactose. It seems evident that they must be present in
very small quantities in the tissues investigated.

PHAEOPHYCEAE

Polysaccharides.—Starch is conspicuously absent from the
great group of ‘‘browns,’’ but there are, however, certain less
highly condensed polysaccharides present. Schmiedeberg
(85) speaks of a dextrin-like compound which he isolated
from Laminaria. He gave to it the name ‘‘laminarin’’ and
the general formula, 10(C601005)-9H20. There seems, how-
ever, to be some confusion regarding his method of arriving
at these figures. Torup (’09) was able to extract a dextrin
from Laminaria sp. with warm water, that gave dextrose on
hydrolysis. This could be isolated only during the winter
months. He called it ‘‘kreftin.’’ Kylin (’13), extracting
crushed Laminaria saccharina, Fucus vesiculosus, and Asco-
phyllum nodosum, obtained a dextrin-like compound similar
to that described by Schmiedeberg and he retained Schmiede-

1915]
DAVIS—ENZYME ACTION IN MARINE ALGAE Hay

berg’s name, ‘‘laminarin.’’ He showed also that Torup’s
‘“‘kreftin’’ was without doubt a modification of ‘‘laminarin.’’
Kylin ascribes to ‘‘laminarin’’ the same physiological func-
tion that starch performs in the higher plants, i. e., that of a
reserve product. In a more recent paper (715) he shows that
there is an accumulation of the ‘‘laminarin’’ in the tissues of
the algae during the summer months, while during the winter
and spring this reserve is drawn upon by the young fronds
until by the end of March very little of it is demonstrable.
Kylin was also able to clear up much of the confusion that
has attended observation of the light-refracting granules
present in the cells of many members of the group. They
had been variously considered as of fatty nature, protein-
aceous, tannin-like, and glucosidal. Reinke (’76) demon-
strated fat-like bodies in the cells of Fucus that he looked
upon as the first visible products of assimilation, a point of
view later supported by Hansen (’93). Schmitz (’83) claimed
two distinct bodies present, one of which, although it did not
react with iodine, he called ‘‘phaeophyceenstarke,’’ the other
giving the ordinary reactions for fats. Hansteen (’92) had
observed bodies in the same plant which he maintained were
of carbohydrate composition and to which he applied the
term, ‘‘fucosankorner.’’ Crato (’92, ’93), the same year, in-
vestigating the fat globules observed by Schmitz, suggested
that they were either phloroglucin or a derivative of it, since
they colored red with vanillin-hydrochloric acid. This con-
ception was held by Bruns (’94) as well. In a later paper,
Hansteen (’00) observed that the ‘‘fucosankorner’’ were
formed in the presence of light, and this to his mind indicated
that they function as the first assimilable products. Hunger’s
(702) work two years later pointed to Hansteen’s ‘‘fucosan-
korner’’ as being glucosidal in nature, the carbohydrate at-
tached being bound up with phloroglucin, or at times, with
tannic acid. Some of the larger ‘‘korner”’ gave fat reactions,
some protein. Kylin found three definite bodies in the cell,
the nature of which had been confused by earlier workers—
fat globules, proteinaceous particles, and tannin-like bodies—
these latter probably representing the ‘‘fucosankorner’’ of

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778 ANNALS OF THE MISSOURI BOTANICAL GARDEN

Hansteen. He holds that none of these are to be considered
the first visible products of assimilation, and suggests that
here, as in most phanerogams, carbohydrates function in that
role.

Simple sugars.—As far as is known, Tihomirov (’10) was
the first to definitely demonstrate simple sugars in these
plants. He used the same phenylhydrazine method employed
with the‘‘ greens,’’ but as was the case there, was unable to con-
nect the osozones with definite sugars. The osozones took con-
siderable periods of time to form, in some cases as long as five
months, evidence pointing to the low concentration of sugars
in the cell. It is a question, too, whether during this long
period of incubation some of the more highly condensed carbo-
hydrates in the cell were not hydrolysed far enough to give
the sugar tests. Using the same method, Kylin (713) was
unable to substantiate these results. However, by using 40
per cent alcohol as an extracting agent, precipitating the in-
organic material with lead acetate, and then purifying with
alcohol, he was able to obtain reducing sugars from several
of the Fucoideae, particularly Laminaria digitata, L. sac-
charina, Ascophyllum nodosum, and Fucus vesiculosus. In
all cases Seliwanoff’s test for fructose was positive, while
dextrose was demonstrated by its osozone. These sugars he
considers the first products of assimilation referred to above.

RHODOPHYCEAE

Polysaccharides —The so-called Florideae-starch has been
the source of many investigations, from the time of Nageli
(758) and Van Tieghem (’65) to the present day. Although
not identical perhaps, it is very similar to the starch of the
higher plants, and as very generally held, it undoubtedly funce-
tions in the same manner. Meyer (’95), Kolkwitz (’00),
and Bartholemew (’14) hold the opinion that it repre-
sents a combination between true starch and dextrin,
while Biitschli (’03) suggests the possibility of its being a
transitional stage between amyloporphyrin and amyloery-
thrin. Kylin (’13) considers it as standing midway between
starch and dextrin. This investigator succeeded in isolating

1915]
DAVIS—ENZYME ACTION IN MARINE ALGAE 779

Florideae-starch from Furcellaria fastigiata, that was readily
hydrolysed to dextrose by malt diastase, and it will be re-
membered that Bartholemew (714) isolated diastase from
several of the ‘‘reds’’ that split phanerogamic starch to re-
ducing sugars.

Simple sugars.—Very little work has been done on the di-
and monosaccharides of the ‘‘reds.’? Tihomirov (’10) suc-
ceeded in obtaining the same yellow amorphous osozone de-
posits in the tissues of Sphaerococcus crispus and Gigartina
mamillosa that he had in certain members of the ‘‘greens’’
and ‘‘browns,’’ but here, as in the other groups, the specific
osozone involved could not be determined.

FATS AS STORAGE PRODUCTS

Many observations have made it evident that fats in some
form or other are generally present in the algae, their
peculiar role, however, having been very little investigated.
In some of the siphonaceous forms, particularly Vaucheria,
they seem to replace carbohydrates. Whether fats are to be
regarded as the first visible products of assimilation in these
forms is disputed. Some workers hold them to be reserve
products, some by-products of metabolism. If they are
utilized as a reserve or storage product in any of the forms,
one might expect to find evidences of lipolytic action, yet none
has been reported so far.

As stated by Czapek (713, p. 761), Loew and Bokorny find
that Spirogyra and other filamentous forms contain 6 to 9
per cent of the dry weight as fat. This probably includes
lecithin. The same authority gives the following results as
obtained by Sestini, the figures being percentages of the dry

weight :
ViGUeneian PIS Sarco atneie evi. 2 ha ise le wae 6 2.94
UO GUGHUSSUNUD: eck ee Dae iete & 8, De dike odovere Al
A CWS: VCSTOUMLOSUSS ao orate iorate alteie ell, Se a.as 0) 0s 67
WQONAG GEOAGTO DUG. s.60 1s tos hese ee aes ess pelle)
Gracilaria confervoideS .......00eceecenes mlelt

Konig and Bettels (’05) made a large number of analyses of
the dry tissues of a variety of marine algae and found a fat

[VoL. 2
780 ANNALS OF THE MISSOURI BOTANICAL GARDEN

content ranging from .20 per cent in Enteromorpha to .98
per cent in Porphyra.

RELATION OF THE ALGAE TO NITROGEN

Some of the recent work on pure culture methods with
fresh-water algae, such as that of Beyerinck (’90), Charpen-
tier (’03, ’03*), Chick (’03), Artari (’13), Spargo (’13), and
Schramm (’14) have conclusively proved that these forms can
utilize organic nitrogen. Furthermore, the work of Letts and
Hawthorne (711), and Foster (’14) point to the fact that the
marine forms may have this capacity as well. Letts and Haw-
thorne and also Letts and Richards (’11) showed that Ulva
latissuma grew better in sewage-contaminated sea-water than
in water from the open sea. Foster placed strips of Ulva
lactuca in normal and artificial sea-water, containing in addi-
tion compounds of nitrogen in varying concentrations. When
urea or ammonium sulphate was added to either solution an
accelerated growth took place.

The current conception concerning the assimilation of
organic nitrogen by the animal organism is that the protein
and amino acid molecule must be completely desamidized be-
fore the building-up process can begin. In the absence of
definite information to the contrary, we can conceive of a par-
allel situation existing in the plant. The question at once
arises in regard to the algae, whether this be due to the agency
of amidases formed by the tissue, or to the activity of desamid-
izing bacteria, the presence of which Brandt (’99), Gran (’02),
Baur (’02), Reinke (’03), Benecke and Keutner (’03), and
others have shown to exist abundantly in harbor waters.
Neither Letts and Hawthorne nor Foster worked with pure
cultures, and these bacteria may have been the agency in their
experiments to render the amino-nitrogen assimilable,

CARBOHYDRATES AND CARBOHYDRATE CLEAVAGE PRODUCTS OF
ALGAL SLIME
Besides the carbohydrates that may be directly assimilable,
we find those whose function in metabolism is more or less
disputed. The so-called algal slime is made up chiefly of such
products.

1915]
DAVIS—ENZYME ACTION IN MARINE ALGAE 781

Chemical composition—Greenish (’81) found agar from
Fucus amylaceus to consist of 37.21 per cent gelose (probably
galactan since it passed to galactose on hydrolysis) and that
from Sphaerococcus crispus of 60 per cent of the same carbo-
hydrate. Konig and Bettels (’05) give the carbohydrate com-
position of agar-agar from Gelidiwm as 33 per cent galactans
and 3.1 per cent pentosans; by hydrolysis, d-galactose and
levulinic acid were split off. Giimther and Tollens (’90)
found fucosan in Fucus from which the methyl-pentose, fucose,
was split off. Galactose was also demonstrated. Sebor (’00)
obtained galactose, glucose, and fructose from the slime of
Chondrus crispus by acid hydrolysis. He held that the slime
is a very complex carbohydrate of high molecular weight,
made up chiefly of galactosan, glucosan, and fructosan.

The cleavage products of Porphyra laciniata, as investi-
gated by Oshima and Tollens (’01), were found to consist
chiefly of l-galactose and mannose, but glucose, fucose, and
other pentoses were also obtained. Miither and Tollens (’04)
found methyl-pentosans in several of the Fucaceae. Konig
and Bettels (’05), working on the carbohydrate hydrolytic
products of various species of Porphyra, Gelidium, Laminaria,
Cystophyllum, and Enteromorpha, found them to consist of
such hexoses as galactose, dextrose, and fructose, as well as
several pentoses, chiefly methyl-pentoses. Enteromorpha
yielded a pentose—rhamnose. The results of Swartz (’11)
agree with those above, namely, that for all forms studied,
representatives of the ‘‘greens,’’ ‘‘browns,’’ and ‘‘reds,’’
pentosans were always present, and galactans frequently so.
Kylin (713), by direct extraction with warm water of crushed
Ceramium, Furcellaria, and Dumotia, obtained substances
that gave the mucic acid test for galactose, as well as
the phloroglucin test for pentosans. Substances giving pen-
tosan reactions alone were isolated from the slime of Asco-
phyllum nodosum, Fucus vesiculosus, and Laminaria sp. He
was apparently unable to substantiate the finding of galactan
in Fucus by Giinther and Tollens, and this negative result also
conflicts with the statement of Swartz, who says that the
gelatinization in the algae is due to the galactan groups.

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782 ANNALS OF THE MISSOURI BOTANICAL GARDEN

Kylin (’14) and others have also demonstrated pectin-like
compounds forming the middle lamella in various members of
the F'ucaceae. These exist as the calcium salts of pectic-like
acids which Kylin designates ‘‘Fucinséure’’ and ‘‘ Algin-
saure.’’

PHYSIOLOGICAL SIGNIFICANCE OF ALGAL SLIME

It is seen that algal slime is made up chiefly of the an-
hydrides of hexoses and pentoses—carbohydrates that must
be broken down to simpler form before assimilation by the
plant would be possible. Two questions naturally arise: (1)
Do the algae concerned form enzymes that will hydrolyse these
highly condensed carbohydrates to assimilable form? (2)
Does the slime itself arise through the breaking down of the
hemicelluloses of the cell wall through enzymic or other
causes, or does it represent a final stage in the condensation
of those hemicelluloses?

Algal slime as a reserve product.—Galactanases and man-
nases have been demonstrated in the phanerogams and in the
fungi by Bourquelot and Hérissey (’99), Griiss (’02), and
Hérissey (’03). The last worker especially has clearly shown
the distinct role that galactans and mannans may play as re-
serve products in the tubers of the Orchidaceae and in many
of the Leguminoseae. It is significant that Gran (’02°) was
able to isolate a marine bacillus, B. gelaticus, that acted on
part of the constituents of agar-agar to give a reducing sugar.
From the standpoint of a possible symbiosis it would be in-
teresting to know if this organism has the ability to fix free
nitrogen. Saiki (’06) experimented with a number of algal
and lichen preparations containing a large proportion of
carbohydrates as galactans and pentosans, and concluded that
the latter could not be transformed into sugars readily by
carbohydrate digesting enzymes of animal origin and scarcely
more so by the vegetable enzymes, either of the higher plants
or of bacteria.

Still less is known of the digestion of pentosans by the
higher plants. Schone and Tollens (’92) found no decrease
in the amount of pentosans during germination and conclude

1915]
DAVIS—ENZYME ACTION IN MARINE ALGAE 783

that they cannot function as reserves. Cross, Bevan, and
Smith (’95) consider the pentosans as by-products of metab-
olism and once formed remain unalterable. Ravenna and
Cereser (’09), on the other hand, in some very interesting ex-
periments, found that when dextrose was supplied as the sole
nutrient to the leaves, pentosans increased greatly, especially
in the light. If, however, the function of chlorophyll is in-
hibited, a decrease in the amount of pentosans takes place.
These results form the basis for their conclusion that pento-
sans may sometimes function as reserves.

The origin of algal slime.—The question concerning the
origin of the slimy and gummy constituents of cells, whether
they arise through enzyme action or through other causes,
has provoked much discussion. There is considerable doubt
whether such gums can arise directly from true cellulose or
whether they are, at least in the case of the plant mucilages,
laid down as such.

One might roughly group the plant gums into those arising
as a result of some external excitant, such as, for example,
cherry gum, acacia gum, gums of citrus, ete., and those which
seem to be normal constituents of the plant, as the mucilages
found in the epidermis of many seeds and plant organs. The
former arise as a result of a pathological condition; the latter,
as far as we know, are normal physiological products and as
such are more nearly comparable to the algal slime.

Klebs (’84), investigating slime formation in some of the
lower algae, particularly some of the Desmidiaceae, held that
it was not a conversion product of cellulose. Hauptfleisch
(’88) substantiated the conclusion of Klebs, and going further,
states that it arises in this particular case through the activity
of the protoplasm, being excreted through pores. Oltmanns
(704, p. 76) illustrates very clearly the arrangement of these
pores. Tschirsch (’89) differentiates these slimes or muci-
lages into those giving a cellulose reaction and those not doing
so, the former having some relation perhaps to the cellulose,
but the latter being laid down on the cell wall as such by the
protoplasm. He holds the epidermal slime of Spirogyra to
be of this latter type, which he calls ‘‘echter Schleim.’’ In

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784 ANNALS OF THE MISSOURI BOTANICAL GARDEN

the same work the author concludes the slime of the Fucaceae
and of the Florideae to be of the ‘‘echter’’ type, occurring
here, however, not as a layer laid down on the inner cell wall,
but as an intercellular substance. Guignard (’93) held much
the same view, and in an excellent histological investigation,
clearly demonstrated the presence of slime or mucilage ducts
in the Laminariaceae.

Mucilages very similar in nature and origin to the algal
slimes occur in the higher plants, and much more work has
been done with them than with those occurring in the algae.
It is hardly necessary to go into the historical aspect of this
phase of the work. The current conception of its origin is
voiced by Walliczek (’93), who, investigating rather fully the
location of different types of normal mucilages by means of
suitable stains, found that in almost all cases they were laid
down as such. According to him, the slime forms secondary
layers on the cell wall which he designates ‘‘ Membranverdic-
kungsschichten’’—layers that in many instances almost com-
pletely fill the cell. Where the epidermal layer of seeds be-
comes gelatinous, as, for example, in those of flax, mistletoe,
various Cruciferae, ete., it is this inner cell wall which Wal-
liczek holds to be the seat of slime formation. Upon contact
with water the slime swells remarkably, filling the cell and
at times even bursting it. There may or may not be an actual
hydrolysis of the true cellulose, but if there is it seems rarely
to enter into mucilage formation.

EXPERIMENTAL

Forms used.—The algae to be used for enzyme investiga-
tion were collected in the vicinity of Woods Hole, Massa-
chusetts, during the summers of 1913-14, at which time the
plants were also dried for winter work at the Missouri
Botanical Garden. Work with the fresh tissue was carried
on at the Marine Biological Laboratory, Woods Hole, during
the latter summer. The selection of forms with which to work
was limited to those relatively abundant in the neighboring
waters, a further limiting factor in selection being relative
freedom from adhering marine organisms. Only those plants

1915]
DAVIS—ENZYME ACTION IN MARINE ALGAE 785

were selected that were ‘‘clean.’’ This was an important pre-
caution, since many adhering organisms have been found to
be quite active enzymatically, and the presence of even a few
might well lead to serious errors in the final results. The fol-
lowing forms lent themselves most readily to the work:!

Chlorophyceae
Ulva lactuca (l.) Le Jolis
Enteromorpha intestinalis (L.) Link

Phaeophyceae
Laminaria Agardhu Kjellm.
Ascophyllum nodosum (.) Le Jolis
Mesogloea dwaricata (Ag.) Kutz

Rhodophyceae
Ceramium rubrum (Huds.) Ag.
Agardiuella tenera (J. Ag.) Schmitz
Rhodymenia palmata (L.) Grev.
Chondrus crispus (l.) Stack.

Preparation of algal material.—In addition to the question
of cleanliness, great care was taken to select only plants that
were in a young, vigorously growing condition. These were
brought into the laboratory, placed in large aquarium jars
containing salt water, picked over, and all detectable foreign
matter removed. A thorough washing in running salt water
for two hours was then given, after which, with the exception
of one or two forms that rapidly gelatinized, the plants were
placed in running fresh water for 10 or 15 minutes. This
fresh water treatment was very efficacious in causing small
snails and other minute marine organisms to loosen their hold.

The plants so washed were either crushed and used at once
with the substrate for enzyme action, or they were dried for
future use. In either case, two general ways of using the ma-

*With the exception of Laminaria Agardhii and Agardhiella tenera, these
binomials conform to the nomenclature as given by Farlow (Marine algae of
New England, pp. 1-210. pl. 1-14. 1881); these two forms are as given by De
Toni (Sylloge Algarum 3: p. 349. 1895) and Engler and Prantl (Nat. Pflanzenfam.
17371. 1896), respectively.

[VoL 2
786 ANNALS OF THE MISSOURI BOTANICAL GARDEN

terial for such action were employed. The tissue was added
directly to the substrate, or it was extracted with water by
the method to be described later and a water-diffusion used of
the alcohol precipitate. If the fresh tissue were to be used
directly, it was ground in a meat chopper two or three
times, then pounded in a large mortar with an equal amount
of fine, clean, quartz sand. This treatment gave a very homo-
geneous pulp, one in which a large number of the cells were
broken down. If desired for future use, the plants were either
dried at room temperature or dehydrated by the following
modified Buchner ‘‘dauerhefe’’ process:

3 volumes 95 per cent alcohol for 15 minutes.

3 volumes acetone for 15 minutes.
3 volumes 95 per cent alcohol for 10 minutes.
3 volumes acetone for 5 minutes.
2 volumes absolute alcohol

or ether for 5 minutes.

After each treatment, the dehydrating liquid was pressed
out through two thicknesses of cheese cloth by making a
tourniquet. Upon the removal of the absolute alcohol or
ether, the tissue was spread out on adsorbent paper, either
filter paper or paper toweling, until all the dehydrating agent
had evaporated. A uniformly dry, brittle, easily crushed
material usually resulted that was roughly broken up and
stored in tightly stoppered bottles for future use. Those
plants that were dried at room temperature were simply
wrapped in paper or placed in paper bags until needed.

The crushing of the dry material was accomplished in the
same manner as was the fresh. Usually it was ground twice
or more in an ordinary meal mill, then pounded in a mortar
with an equal weight of quartz sand until a very fine powder
was obtained. The sand was dispensed with if the tissue were
easily crushed.

Methods of isolating the enzymes.—As indicated above,
there were two general methods of using the material for
enzyme action: first, adding the crushed tissue directly to
the substrate, either as fresh pulp or as ‘‘dauerhefe’’ powder ;

1915]
DAVIS—ENZYME ACTION IN MARINE ALGAE 787

second, by extracting the tissue with water and precipitating
the protein-enzyme complex with several volumes of 95 per
cent alcohol. Wherever possible the first method was used,
since it was thought that in this way the maximum enzymic
activity would be obtained. However, the fresh pulp and the
powdered material contained a substance, or substances
(probably tannoidal bodies), that reduced copper from Fehl-
ing’s solution, and so in all experiments where sugar deter-
minations were involved, it was found necessary to use the
extraction and precipitation method; by this means all the
unknown reducing substances were avoided. The method was
as follows:

To a known amount of the crushed, fresh algal material,
3-5 volumes by weight of distilled water were added; to the
powdered tissue, 8-10 volumes. The amounts varied owing
to the differences in viscosity produced by the different algae.
In some forms a relatively large amount of water was neces-
sary in order to overcome difficulties in handling due to this
high viscosity. Two per cent toluene was generally added as
an antiseptic, or in some cases, 1 per cent chloroform-thymol
mixture was used (5 per cent thymol dissolved in chloroform),
and the extraction allowed to go for 12 hours at room tempera-
ture, or for 4 hours at 35° C. The water extract, if at all
viscous, was then filtered off through two thicknesses of
cheese cloth and the algal tissue pressed out as completely
as possible by making a tourniquet of the cloth. Filtering
through cotton was tried at first, both with pressure and
without, but the method had the disadvantage of slowness and
also that of adsorption by the cotton. Neither did filter paper
lend itself efficiently to the filtration of such viscous liquids,
a drier residue being obtainable in a shorter time by the
cheese cloth-tourniquet method. A press would have been
desirable but none was at hand. If the medium were not
viscous, it was filtered with pressure through a thin layer of
cotton or a coarse filter paper in the bottom of a Buchner
funnel.

The protein-enzyme complex was precipitated with 3
volumes of 95 per cent alcohol. After a few moments the

[Vou. 2
788 ANNALS OF THE MISSOURI BOTANICAL GARDEN

coagulum either came to the top or settled to the bottom of
the vessel—if to the top, it was usually very much aggregated
and little difficulty was experienced in the filtering, if to the
bottom, it was generally in a very finely divided condition and
unless care was exercised in the decantation of the super-
natant liquid the pores of the filter soon became clogged, re-
sulting in extremely slow filtration. Time was therefore given
for a complete settling out (15 minutes to half an hour sufficed)
and all the clear fluid filtered off before the coagulum reached
the filter paper.

A homogeneous diffusion of the precipitate was made by
placing the filter paper with the attached coagulum in a known
volume of distilled water. The paper could soon be removed
without loss of material, and the weight of the original fresh
or dry tissue represented by an aliquot portion of the solu-
tion easily reckoned. If the precipitate were not required im-
mediately, it was dried on a filter paper at room temperature
and stored in stoppered jars. In none of the experiments
was the enzyme material purified further.

When dissolved in water, the precipitates behaved differ-
ently. Some, especially those where much slime had been
noticed in the extraction, gave an extremely viscous suspen-
sion, others a suspension of low viscosity. In Laminaria and
Chondrus, where the extract had been quite viscous and slimy,
the protein was caught up in the precipitated slime in such a
way as to make the freeing of it practically impossible. The
precipitate in these cases was very large and when diffused
in water gave a suspension difficult to handle. Rhodymenia,
Ceramuum, and Enteromorpha, on the other hand, gave a
finely divided precipitate that produced no viscosity.

Glassware, antiseptics, solutions, etc.—With few exceptions,
the various experiments were set up in 125 cc. Erlenmeyer
flasks. All glassware was thoroughly cleaned with strong
soap and then with chromic-sulphuric cleaning mixture, after
which it was rinsed several times with tap and distilled water.

Solutions were made up from either Merck’s or Kahlbaum’s
‘‘ouarantiert’’ chemicals.

1915]
DAVIS—ENZYME ACTION IN MARINE ALGAE 789

Three general antiseptics were used—toluene, alcohol to 20
per cent, and 5 per cent thymol in chloroform. Toluene was,
in general, the most satisfactory. Usually it was used to 2
per cent concentration, but where large surfaces were exposed,
as high as 4 per cent was found necessary. The chloroform-
thymol was also very efficacious, but in the carbohydrate ex-
periments chloroform could not be used because of its power
of reducing copper. In the lipase work the substrate was made
up to 20 per cent alcohol since the action seemed to proceed
best in the presence of this antiseptic. In all cases where the
experiments were maintained over a considerable period of
time, it was necessary to add additional antiseptic from time
to time.

Checks were set up in all experiments—on the substrate, on
the material used to demonstrate enzyme action, and on the
substrate plus such enzyme material boiled to destroy any
ferments that might be present.

CARBOHYDRASES OF THE ALGAE

In these experiments the alcohol precipitate from an
aqueous extract of crushed, fresh or dried, algal tissue was
employed as an enzyme source, this precipitate being diffused
in such a volume of distilled water that one gram of the
original material was represented by 5 cc. of the diffusion.
Thus one can more closely compare the amounts of enzyme
present in definite amounts of different algal tissue. The
number of cubic centimeters of diffusion will be noted in con-
nection with each set of experiments.

Substrates.—Starch, dextrin, inulin, sucrose, maltose, lac-
tose, glycogen, and in one or two cases, laminarin isolated
from Laminaria Agardhu, were used as substrates. These
were made up in 1 per cent concentrations with the ex-
ceptions of maltose and glycogen, where .25 per cent, and
laminarin, where .5 per cent concentrations were employed.

Of the many suggested methods for making up starch paste,
the following one used by Clark (’11) was found to give the
best satisfaction. Ten grams of potato starch were weighed
out and placed in a beaker with 250-300 ce. of distilled water.

[Vou. 2
790 ANNALS OF THE MISSOURI BOTANICAL GARDEN

This was brought to a boil with constant stirring, and when
an opalescent solution resulted the paste was transferred with
rinsing to a 2-liter flask containing about 500 ec. of boiling
water. The lot was boiled under a reflux condenser for two
hours, cooled, and made up to a liter. Although, as is stated
by Clark, this treatment is very effective in breaking down
the starch grain physically, no detectable hydrolysis: takes
place, and the additional advantage is gained in obtaining a
paste that will not settle out, even after long standing. Two
per cent toluene was employed as an antiseptic if the starch
were not to be used immediately.

Since all dextrin obtainable contained some reducing sugar,
it was found necessary to purify it by making a concentrated
solution in hot distilled water, and then precipitating out with
several volumes of 95 per cent alcohol. The dextrin was
caught on a filter paper and dried at a low constant tem-
perature.

Laminarin, a dextrin-like carbohydrate found in many of
the Fucaceae, was isolated from Laminaria Agardhu accord-
ing to the method employed by Kylin (713), with some few
slight modifications. Freshly collected Lamimaria was
crushed in the usual way and 1,680 grams of the pulp were
boiled with 7 liters of water for 24 hours, water being added
from time to time to replace that lost through evaporation.
The extract was then filtered off through a double thickness of
cheese cloth, and the residue pressed out with a tourniquet.
About 3,000 ec. of a dirty brown filtrate were obtained which
was divided into three lots of 1,000 ce. each. To the first of
these was added a concentrated Ba(OH)2 solution until the
precipitation of the inorganic matter was complete. The pre-
cipitate was caught on a cotton filter in a Buchner funnel,
the filtrate being a clear, golden-colored liquid. The inorganic
material in the other two lots was precipitated with basic
lead acetate, the liquid filtered off through cotton, and the
excess of lead removed with H28. The solutions were filtered
while hot through double filter paper to remove the lead
sulphide, and then the excess of HS was driven off with
heat. The three portions were first evaporated to about one-

1915]
DAVIS—ENZYME ACTION IN MARINE ALGAE 791

third their volume, when the scum that formed was filtered
off; this filtrate was then further evaporated to about one-
fifth the original volume on the water bath. At this point
the two lead acetate portions were placed together. Ninety-
five per cent alcohol was added to each of the lots to about
80 per cent concentration when a flocculent precipitate came
down rather slowly. With the Ba(OH)2 portion this was
copious, with the lead acetate, slight. After two hours the
precipitates were filtered off, washed with absolute alcohol,
redissolved in a small amount of distilled water, and then
reprecipitated with 4 volumes of absolute alcohol, the result-
ing precipitate being dried over CaCle. From the Ba(OH):.
portion, 4.2 grams of a creamy white powder were obtained
that gave a very slightly reddish tinge with iodine, did not
reduce Fehling’s, and was easily soluble in water, giving a
clear solution. Upon hydrolysis with weak H2SOu, a reducing
sugar was split off. The lead acetate portion gave but two
grams of the same material. This powder was taken to be the
laminarin described by Kylin.

The determination of reducing sugars.—The reduction of
copper, or in the case of maltose and lactose, the increase in
the reducing value of the substrate plus the enzyme over that
of the checks, was taken as the measure of carbohydrate
hydrolysis. In this determination the permanganate titra-
tion method, as modified and described by Shaffer (714), was
used, it being possible with it to determine amounts of sugars
as low as 2 milligrams! very accurately and quickly. Shaffer’s
description may not be generally available to plant workers
who may desire to use this really splendid method, and so
the various steps in the process as used here are set down in
some detail.

Ten cc. of the carbohydrate-enzyme substrate were placed
in a large test-tube containing 5 cc. of water, and just brought
to a boil. At this point a drop of 50 per cent acetic acid was
added. When the slight protein precipitate formed, 5 ce. of

1 Shaffer determines values below two milligrams, but as used here, con-

sistent results could not be obtained where less than that amount was involved.
Below this point the relative increase in the experimental error is large.

[Vou. 2
792 ANNALS OF THE MISSOURI BOTANICAL GARDEN

colloidal iron (Iron dialysed, Merck) were pipetted in and the
tube well shaken, the iron then being flocked out with .25 gram
of NazSO4. Upon the addition of this latter the mixture was
again thoroughly shaken and the iron precipitate thrown
down by centrifuging, the resulting clear, supernatant liquid
then being decanted off through a small filter. This filtrate
was entirely free of proteins or other substances which,
through oxidation later, would lead to errors in the perman-
ganate values. Ten ce. of this filtrate were placed in a
50 ec. lipped centrifuge tube, and standard Fehling’s solu-
tion added, the copper content of which was in excess of that
reducible by the sugar present.t The tube was then placed
in a boiling water bath for 10 minutes, at the end of which
time it was centrifuged at a moderate speed for 2 minutes,
the supernatant unreduced Fehling’s carefully decanted off,
a like volume of distilled water added, and the cuprous oxide
again thrown down by a 2-minute centrifuging. All but 1 or
2 ce. of this wash water was carefully decanted off, and the
copper dissolved in the smallest amount necessary of a mix-
ture of equal parts of 10 per cent ammonium ferric sulphate
and 50 per cent sulphuric acid. It was found that if the cop-
per were stirred up with a glass rod just before dissolving, it
went into solution more readily. The dissolved copper was
titrated directly in the centrifuge tube against N/50 KMn0Os.2

By calculation it is found that 1 ce. of N/50 KMn0Osz is
equivalent to 1.27 milligrams of copper, and for the conver-

sion of this into glucose use was made of the table prepared
by Shaffer.*

As stated by Shaffer, care must be observed on the three
following points: (1) to eliminate all oxidizable substances
other than sugar, (2) to titrate the cuprous oxide immedi-
ately after dissolving, (3) to use poor conductors of heat as
containers of the centrifuge tubes in the water bath, else many
broken tubes will result. As employed here, circular wire

1In the determinations made here this amount never exceeded 10 ee.
* It is necessary to titrate immediately after dissolving because of the danger

of oxidation of the cuprous oxide. If larger amounts of sugar are concerned,
N/10 KMnO, may be used.

1915]
DAVIS—ENZYME ACTION IN MARINE ALGAE 793

baskets having wooden bottoms and tops were used, the tops
containing holes large enough for the free insertion of the cen-
trifuge tubes, and the bottoms, slight depressions into which
the tubes might rest. It is always necessary to run blanks
with Fehling’s solution since some reduction always takes
place. The cuprous oxide solvent must be free from ferrous
iron, and this can be assured by the addition of a trace of per-
manganate.

Method of setting up expervments.—Fifty ec. of the sub-
strate to be used were placed in 125 cc. Erlenmeyer flasks with
2 per cent toluene as an antiseptic. If the series were main-
tained longer than six weeks, another 2 per cent toluene was
added. As previously noted, in these carbohydrate experi-
ments the material used for enzyme action was an alcohol
precipitate from a water extract of algal powder or pulp.
This was diffused in water so that 10 cc. of the diffusion
represented 2 grams of the original tissue. Usually this
amount was added to the substrate to be tested. Duplicates
and checks were set up in accordance with the following model
series for starch:

1. 50 ce. starch, 10 cc. enzyme diffusion.

2. 90 ee. starch, 10 cc. enzyme diffusion.

3. 50 ce. starch, 10 cc. boiled enzyme diffusion.
4. 50 ce. starch, 10 cc. boiled enzyme diffusion.
5. 90 ee. starch, 10 ce. distilled water.

6. 950 ce. starch, 10 ce. distilled water.

°To make this table more generally available, it is printed here in full.

Shaffer’s table of copper-glucose equivalents

mgms. mgms. mgms. mgms. mgms. mgms.

copper glucose copper glucose copper glucose
OFT 47 6.0 2.74 20.0 9.71
1.0 .62 7.0 Bie7Al 25-0 119) 245)
Nats) .88 8.0 3.68 30.0 14.80
2.0 i odlit 9.0 4.15 35.0 17.40
Zao iL By? 10.0 4.65 40.0 20.00
3.0 1.50 12.0 5.61 50.0 25.00
Ba 1.67 14.0 6.61 60.0 30.10
4.0 1.82 16.0 7.61 80.0 40.40
5.0 Dei 18.0 8.65 100.0 50.70

[Vou 2
794 ANNALS OF THE MISSOURI BOTANICAL GARDEN

In addition, at the end of a complete carbohydrate series there
were included for each alga the following checks:

1. 50 ce. distilled water, 10 ec. enzyme diffusion.

2. 90 ce. distilled water, 10 cc. enzyme diffusion.
50 ec. distilled water, 10 ec. boiled enzyme diffusion.
50 ec. distilled water, 10 ec. boiled enzyme diffusion.

fm co

Where the enzyme diffusion referred to above actually con-
tained carbohydrases, it was extremely difficult to render them
inactive by heating—10 minutes at the boiling point not being
sufficient in most cases to more than slow down the action.
This was probably due to the impurities contained, the rela-
tively large amounts of protein and slime present tending to
protect the enzymes. Those extracts relatively richer in such
constituents proved the more difficult to render inactive. The
expedient was finally adopted of placing the enzyme material
in the autoclave and bringing the pressure in the latter up to
15 pounds. This proved quite effective.

THE CARBOHYDRASES OF ULVA LACTUCA

The effect of an extract of Ulva lactuca on different starches.
—Since starches were to be used in many of the following

TABLE II
THE ACTION OF ULVA LACTUCA “DIFFUSION-EXTRACT'* UPON CERTAIN STARCHES

30 days

Starch Sugar as
50 ce. |
1 per cent BrCese Iodine test
P in 5 cc.
mgms.

Rotatos-oceseeer Blue, trace red 17.4 Complete hydrolysis
Arrowroot......... Blue, trace red 16.9 Complete hydrolysis
Wiheats.c eerie: Blue, trace red alfa at Complete hydrolysis
Gori Wiyssits scores 10.8 Reddish purple
Solublesseoc00oent 15.9 Traces dextrin

Reco troree Aes ‘Trace t¢|.e eee eee

* Wherever the term “diffusion-extract” is employed, it refers to a diffusion
in water of the alcohol precipitate from an aqueous extract of the alga under
discussion.

+ The sugar values in this and the following tables are net, i. e., sugar values
for all checks have been deducted.

{In all the following experiments an amount of sugar below 2 mgms. is
designated a “trace.”

1915]
DAVIS—ENZYME ACTION IN MARINE ALGAE 795

experiments, it was desired to know which, if any, were the
most favorable substrates for the diastases of the algae. The
action of diastase from Ulva lactuca was taken as an index.
Potato, arrowroot, wheat, corn, and soluble starch, as well as
inulin, were made up in 1 per cent concentrations in the man-
ner previously described. To 50 ce. of each of these sub-
strates were added 10 ce. of a diffusion of an alcohol precipi-
tate from a water extract of dehydrated Ulva lactuca. Two
per cent toluene was added as an antiseptic, and the flasks
maintained at a temperature of 35°C. for 30 days. The re-
sults of the experiments are given in table m1.

The data show but slight differences in the rate of digestion
of the starches with the exception of corn starch, and the
reason for this is not clear. One would expect it to be due to
some impurity in the starch rather than to an inherent dif-
ference in the granule. The action on inulin was so slight as
not to warrant the assumption of hydrolysis due to inulase.

The action of an extract of Ulwa lactuca upon various carbo-
hydrates.—A series was arranged using a ‘‘diffusion-
extract’’ from Ulva lactuca with the following substrates:
potato starch, dextrin, glycogen, sucrose, maltose, and lactose.
Ten ce. of the ‘‘diffusion-extract’’ were added to each flask
with 50 ce. of substrate, 2 per cent toluene used as an anti-
septic, and the flasks maintained at a temperature of 35°C.
for 30 days. The data are given in table m.

TABLE III
THE ACTION OF AN EXTRACT OF ULVA LACTUCA UPON VARIOUS CARBOHYDRATES

Sugar as glucose in 5 ce.

Substrate mss:
15 days 30 days
Starch Wctirdecukra seis 10.20 15.50
Dextrose sehen 6.30 9.95
GIN COEONs oonosodonedeous 2.25 3.50
SIUCKOSe Soe. ON Caer Trace
WACEOSO eereeeve cs tus cheese, 0) atcha! 2 WN © 8 taken
IWiaILOSOuyytocrercners crop uettsrdeilly » bl. fo ctarevehens Trace

The two polysaccharides, starch and dextrin, are very
readily attacked even though the action is slow. Glycogen,
which is hydrolysed by most diastatic enzymes with about the

[VoL. 2
796 ANNALS OF THE MISSOURI BOTANICAL GARDEN

same ease as starch, seems very slightly acted upon by the
carbohydrases of Ulva. The failure of action on sucrose and
lactose is not so surprising as is that on maltose, for one
would expect the action on polysaccharides to continue to
what is generally held to be directly assimilable sugars, i. e.,
the hexoses.
THE CARBOHYDRASES OF ENTEROMORPHA INTESTINALIS

This series (table 1v) was run under exactly the same con-
ditions as the one preceding. The ‘‘diffusion-extract’’ was
from dehydrated tissue about two months old. Ten ce. of this
were used with each 50 ce. of substrate, toluene added as an
antiseptic, and the flasks kept at a temperature of 35°C. for

30 days.
TABLE IV

THEACTION OF A “DIFFUSION-EXTRACT" FROM AIR-DRIED ENTEROMORPHA TISSUE
UPON CERTAIN CARBOHYDRATES

Sugar as glucose in 5 ce.

Substrate mgms.

15 days 30 days
Starch. sain ccitecean oe 9.7 13.1
DEXtrin'. . aoc coe 5. 9.8
Gly covenier. i act ance eerie 2.8 3.9
NUTTY SevSasos eee orci ere Trace Trace
SUCIOSG.. 5. 2me fee nis see arte LNereie Trace
hactose ha aireics mere circ
Maltose- ern esse ote

The results for this closely related form are consistent with
those obtained for Ulva, the action in the present case, how-
ever, being somewhat slower. The more common polysac-
charides are acted upon while the disaccharides are not
attacked.

THE CARBOHYDRASES OF LAMINARIA AGARDHII

The water extract from air-dried Laminaria tissue was
extremely viscous and upon addition of alcohol, a very heavy
precipitate was thrown down that contained a large amount
of algal slime. When water was added to this precipitate in
the usual ratio a very viscous diffusion was obtained. Ten
ce. of the ‘‘diffusion-extract’’ were used with 50 ce. of the sub-
strate and 2 per cent toluene added as an antiseptic. The
flasks were kept at a temperature of 20-22°C. for 100 days,

1915]
DAVIS—ENZYME ACTION IN MARINE ALGAE 797

portions being removed and sugar determinations made at
the definite intervals noted in table v.

The carbohydrases in this form appear to be limited to those
acting on starch and dextrin, and with these the hydrolysis
proceeds much more slowly than was true with either of the
preceding ‘‘greens.’’ The lower temperature at which the
hydrolysis occurred does not explain completely the lessened
action. Inhibiting substances or else an actually smaller con-
centration of the enzyme seem to be important factors.

TABLE V

THE ACTION OF A “DIFFUSION-EXTRACT” FROM AIR-DRIED LAMINARIA TISSUE
UPON CERTAIN CARBOHYDRATES

Sugar as glucose in 5 cc.
mgms.

Substrate

Another series (table vr) of flasks was set up with the same
form, using a ‘‘diffusion-extract’’ from the fresh tissue. Ten
ce. of this diffusion represented 6 grams of the Laminaria
pulp. In addition to the usual substrates, .5 per cent lamin-
arin was used. Toluene was added and the flasks maintained
for 60 days at room temperature (22-23°C.).

TABLE VI

THE ACTION OF A*DIFFUSION-EXTRACT” FROM FRESH LAMINARIA TISSUE UPON
CERTAIN CARBOHYDRATES

Sugar as glucose in 5 cc.

Substrate

[VoL. 2
798 ANNALS OF THE MISSOURI BOTANICAL GARDEN

The diastases of fresh Laminaria seem slightly more active
than those isolated from the dried tissue; however, no other
carbohydrases were evident than those shown in the previous
table.

THE CARBOHYDRASES OF ASCOPHYLLUM NODOSUM AND MESOGLOEA
DIVARICATA

The Mesogloea material was dehydrated as soon as brought
into the laboratory, the preliminary fresh-water washing
being omitted because of the rapid gelatinization of the tissue.
The crushed dried tissue, extracted in the usual way, gave a
very heavy, stringy precipitate with alcohol, consisting, as did
that from Laminaria, mostly of slime. This, when diffused
in the usual volume of water, gave a very viscous mixture.
Crushed fresh Ascophyllum was extracted directly. The
viscosity of the extract was high, but the alcohol precipitate
from it came down in a flocculent mass that gave only a
slightly viscous diffusion with water.

Experiments were set up with the various carbohydrates
heretofore employed, including laminarin, and in the different
series, amounts of the ‘‘diffusion-extract’’ were used varying
from 5-15 ec. As was true with the Fucus reported in the
previous study, in no case were there evidences of hydrolysis
even after 60 days at room temperature.

THE CARBOHYDRASES OF RHODYMENIA PALMATA

The air-dried Rhodymenia tissue proved to give rise to
one of the most viscous extracts encountered in the algae, 20
volumes of water being necessary to make handling possible.
With alcohol, a very rubbery, white precipitate came down
that was made up of a large proportion of algal slime. This
diffused very slowly, giving an extremely viscous mixture.
Ten cc. of the ‘‘diffusion-extract’’ were used with the sub-
strate to determine action, and toluene was added. The flasks
were kept at a temperature of 21-22°C. for 100 days, sugar
determinations being made from time to time, the results of
which are given in table vi.

The results here are quite comparable to those obtained
with Ulva and Enteromorpha, the same carbohydrates being

1915]
DAVIS—ENZYME ACTION IN MARINE ALGAE 799

acted upon, although perhaps a little more slowly. This action
is definitely progressive with starch, dextrin, and laminarin,
but with glycogen it takes a sudden jump during the 15—45-
day period, then remains practically stationary for the rest
of the time the series is being maintained. As was true of the
results shown in the previous tables, this carbohydrate was
less favorable as a substrate than any of the other polysac-

charides employed.
TABLE VII

THE ACTION OF A “DIFFUSION-EXTRACT”’ FROM AIR-DRIED RHODYMENIA
TISSUE UPON VARIOUS CARBOHYDRATES

Sugar as glucose in 5 cc.

Substrate mgms.

15 days 45 days 75 days 100 days
Starchey eerie eerie: 9.2 LZ 2, 14.8 18.2
Dextringea. se ny eas 8.25 9.7 10.3 ial oa
Glycogen seat. saci srariee: Trace 6.1 6.4 Onl
aminarine seer ee. 4.7 les 9.6 10.5
Manlineercn ee arene Sa eits Shon Trace Trace
SUCKOSC Hy. sac roti s Trace Trace Trace
ACTOS te Retiree ae Giese ; ab Trace
IMaltose sso sn aoe oer Trace Trace

THE CARBOHYDRASES OF AGARDHIELLA TENERA

The very succulent nature of the freshly collected material
compelled its partial dehydration immediately. Two 15-
minute treatments with 95 per cent alcohol were used, then
the tissue spread out on paper toweling to dry at room tem-
perature. After drying, it was very easily powdered without
the aid of quartz sand. The alcohol precipitate from a water
extract of this powder was quite fine and flocculent, differing
much from that of Rhodymema, both in amount and in nature.

TABLE VIII

THE ACTION OF A “DIFFUSION-EXTRACT” FROM DEHYDRATED AGARDHIELLA
TISSUE UPON CERTAIN CARBOHYDRATES

Sugar as glucose in 5 cc.

Substrate mgms.

15 days 45 days 75 days 100 days
Starchie mer ove cheese 6.35 9.4 14.5 20.9
DExtrinteee tee 7.00 10.5 15.95 19.7
@lycogenterscansn steer 2.35 6.15 6.65 6.85
Laminarin..... Arcee 5.8 8.3 11.6 Si,
IGRYCh Eas (tae eee oe ee renenes Le Trace Trace Trace
SIICKOSCEP ENE ern erase Trace Trace Trace Trace
LEA GLOSE MS iens eects aoe Trace

[Vou. 2
800 ANNALS OF THE MISSOURI BOTANICAL GARDEN

It diffused readily in water with no resulting viscosity. Ten
ec. of the ‘‘diffusion-extract’’ were used for enzyme action,
toluene added, and the flasks kept at a temperature of 21-23°C.
for 100 days. The data here obtained are given in table vim.

Dextrin here more nearly approaches starch as a favorable
substrate, differing from the action evidenced by the other
algae with the exception of Lammaria, where all action was
slow. There is also a slightly increased action over that evi-
denced by Rhodymenia, for all the carbohydrates hydrolysed.

THE CARBOHYDRASES OF CERAMIUM RUBRUM

As was the case with Rhodymenia, it was necessary here
to use 20 volumes of the water-extracting medium, not, how-
ever, because of the great viscosity, but on account of the
great adsorption of water by the tissue particles. The alcohol
precipitate was copious and finely flocculent. It diffused in
water rather slowly, giving a mixture that was only slightly
viscous. Ten ce. of the ‘‘diffusion-extract’’ were used for
action, the usual percentage of toluene added, and the flasks
maintained at a temperature of 21-23°C. for 100 days. The
data are given in table rx.

TABLE IX

THE ACTION OF A “DIFFUSION-EXTRACT” FROM FRESH CERAMIUM TISSUE UPON
CERTAIN CARBOHYDRATES

Sugar as glucose in 5 cc.

Substrate mgms.

15 days 45 days 75 days 100 days
Starchipeae sy teers tere 6.85 Sep nS) 16.9
Dextiritiaem erecta 1S 15.0 il7/ 55) 19.6
Glycogen jaccrncr steelers rs Trace 6.2 3 8.85
[Waminiariniecc ice dace tho?! 9.4 1251 112.2
JGayed boat. waren oadion oS eee oie Trace Trace
SUCKOSC scene die see ene Trace Trace Trace
WWaAGCEOSe Aarens ere re se Trace
IMaltoseaceceme eaeee Trace Trace Trace

Dextrin proved the most favorable substrate for the carbo-
hydrate enzymes of this alga, the hydrolysis being about the
same as that evidenced by Agardhiella. With the exception
of glycogen, the other carbohydrates showed a decreased
hydrolysis when compared with this latter form, and when

1915]
DAVIS—ENZYME ACTION IN MARINE ALGAE 801

compared with Ulva the difference is quite marked. As in
the other algae, Ceramiwm showed no ability to hydrolyse the
disaccharides used.

A COMPARISON OF THE DIASTATIC ACTIVITY OF ULVA LACTUCA
WITH THAT OF LEAF TISSUE FROM SOLANUM TUBEROSUM

One of the very evident facts brought out by the data in
the preceding tables was the relative slowness with which
hydrolysis was carried on. This point made it seem worth
while to compare, in a general way, the activity of such a form
as Ulva with the starch-forming leaf tissue of a higher plant,
one from which diastase could be isolated rather easily. The
potato (Solanum tuberosum) was chosen.

The Ulva tissue was from an air-dried lot that had been
tried out earlier and had been found quite active. Fresh
potato tops were brought into the laboratory, and both these
and the Ulva given the ‘‘dauerhefe’’ treatment. After de-
hydrating and drying at room temperature, both lots were
ground in a mill, then reduced to a fine powder in a mortar.
Exactly 18.5 grams of each were extracted with 250 cc. of
water for 12 hours at room temperature with toluene added as
an antiseptic, and then the protein-enzyme complex precipi-
tated with 2.5 volumes of 95 per cent alcohol. The Ulva pre-
cipitate was the characteristic heavy white mass to which
attention has been called before, while that of the potato was
finely divided and dark.

The entire amount of each precipitate was diffused in 60 ce.
of water. The Ulva precipitate gave a rather viscous diffu-
sion, due to the adsorption of water by the protein particles;
that from the potato did not all go into solution, making it
necessary to shake the flask so that a true sample might be
obtained. Five cc. of the ‘‘diffusion-extract’’ represented 1.84
grams of the original dehydrated tissue, and this volume was
used with 50 ce. of a starch and dextrin substrate. Toluene
was added as an antiseptic, and the flasks kept at a tempera-
ture of 31°C. for 42 days. Portions of the substrate were
removed from time to time and sugar determinations made,
the results of which are shown in table x.

[Vou. 2
802 ANNALS OF THE MISSOURI BOTANICAL GARDEN

The action of the potato extract upon starch was about two
and one-half times that of Ulva, and its action on dextrin
about twice in all of the determinations made. For some un-
known reason the hydrolysis of dextrin by the diastase from
Ulva ceased after the twenty-eighth day.

TABLE X

A COMPARISON OF THE DIASTATIC ACTIVITY OF ULVA WITH THAT OF POTATO
LEAF TISSUE

Sugar as glucose in 5 cc.

Substrate |= = EEE
50 ce. 14 days 21 days 28 days 35 days 42 days

starch. .....|/ 8.7)|) 18/1) | 976)" 2623) 1178) 2850) 11229) | S33E Sassi Solo
Dextrins 7. |/ 10553] 617-3 pds 250 Ae Sie ie On ede) | SO Sam | lide ne el

ACTION OF VARIOUS ALGAL EXTRACTS UPON THE CARBOHYDRATE
CONSTITUENTS OF AGAR-AGAR, AND OF VARIOUS GUMS,
AS WELL AS EXPERIMENTS UPON THE AUTOLYSIS
OF ALGAL SLIME

Because of the large amounts of carbohydrate-containing
slime formed by many algae, and because of the rdle this
might play as a reserve product, it was deemed advisable to
try out the various algae for enzymes capable of hydrolysing
such complex carbohydrates to assimilable sugars. It was
assumed on the basis of the work done by Konig and Bettels
(’05) and others, that such hydrolytic products would be re-
ducing sugars, in all probability galactoses and pentoses.

A series was set up with each of the several algae, using
50 ec. of .25 per cent agar as a substrate and varying amounts
of a ‘‘diffusion-extract’’ from fresh tissue. The agar sub-
strate was slightly viscous in the cold, but when kept at a
temperature of 40°C., the optimum temperature for diastase,
this was not noticeable. Toluene was used as an antiseptic.
The flasks were shaken at regular intervals during a 30-day
period and at the end of that time aliquot portions were re-
moved and tested for reducing sugars. There was no reduc-
tion in any case.

As a parallel series, thin strips of agar were placed in test-
tubes and 20 ce. of ‘‘diffusion-extract’’ added. Toluene was
used as an antiseptic and the tubes kept at a temperature of
40°C. for two months. At the end of that time no hydrolytic

1915]
DAVIS—ENZYME ACTION IN MARINE ALGAE 803

action was observable, either by reduction of Fehling’s or by
microscopical examination.

In the experiments on the hydrolysis of various poly- and
disaccharides, checks were set up in which the usual amount
of ‘‘diffusion-extract’’ was placed in distilled water. This
was to determine the reduction of copper, if any, due to the
‘‘diffusion-extract’’ itself. In no case was there more than
a very slight trace that might have been due to other
causes than enzymic. However, it was thought that a
self-digestion series would more definitely determine whether
the hydrolysis of the carbohydrates of the slime could be
brought about by specific algal enzymes. With this in mind,
a series was arranged in which the flasks contained 50 cc. of
a water extract from each of the forms investigated. Checks
were set up in which the ‘‘diffusion-extract’’ was inactivated
in the autoclave. Toluene was used as an antiseptic and the
flasks maintained at a temperature of 22-23°C. for two
months. Aliquot portions removed from time to time failed
to show the slightest trace of hydrolysis.

It will be remembered that Tihomirov (’10) had found
osozone-forming sugars in the conceptacles of Ascophyllum
and Fucus that he thought might be dextrose and d-galactose,
possibly also fucose and arabinose. ‘Thinking that these
might possibly have arisen from their corresponding an-
hydrides contained in the conceptacle slime, a self-digestion
series was set up with an extract from the abscised, crushed
conceptacles of those two forms. The Fucus was in a fruit-
ing state. The series were set up in duplicate, one kept at
room temperature and the other at 32-33°C. Fehling’s test
showed no hydrolysis after a month.

Pentosans alone were then used as substrates. Two series
of flasks for each of the algae investigated were set up, each
containing a .5 per cent solution of gum arabic.t To one
series was added 10 ec., to the other 20 ec. of ‘‘diffusion-
extract,’’ and the flasks placed at room temperature with
toluene as an antiseptic. No hydrolysis was apparent either

1The gum arabic was dissolved in water, then precipitated with several
volumes of 95 per cent alcohol to get rid of reducing sugars.

[Vou 2
804 ANNALS OF THE MISSOURI BOTANICAL GARDEN

by the phloroglucin test or by sugar determinations, even after

60 days.

THE ACTION OF ALGAL “DIFFUSION-EXTRACTS” UPON CELLULOSE
AND HEMICELLULOSE

Experiments were carried out to determine the presence
or absence of cellulose hydrolysing enzymes in the algae, and
to this end several methods were employed. First, strips of
filter paper were placed in test-tubes and entirely covered with
20 cc. of ‘‘diffusion-extract.’’ Checks were maintained with
distilled water and also with the ‘‘diffusion-extract’’ alone.
The series were set up in duplicate—one kept at room tem-
perature and the other at 35°C., both with toluene as an anti-
septic. After definite intervals during a 60-day period, the
contents of the tubes were tested for reduction. None was
observable in any case, and microscopic examination of the
filter paper failed to reveal any decomposition whatsoever.

A double series was then set up in a similar way, except
that 2 grams of fresh, crushed algal tissue were added to the
tubes instead of the ‘‘diffusion-extract,’’ together with 20 ce.
of distilled water. At the periods noted above, microscopic
examination revealed no attack. It was thought an inherent
difference between algal and filter paper cellulose might be
responsible for this absence of action. Accordingly, cellulose
was prepared from the tissue of Ascophyllum after the
method described by Fowler (711, p. 159) and used by Cooley
(714). Fifty grams of air-dried tissue were placed in a liter
flask, 500 ce. of distilled water added, and the lot placed in
the autoclave at 15 pounds for 15 minutes to destroy any
cellulase that might be present, and also to extract as much
as possible of the water-soluble substances. The water was
filtered from the tissue, fresh water added, and the flask
placed in an incubator at 35°C. It was kept at this tempera-
ture with daily changes of water for 10 days, at which time
the water-soluble constituents seemed to be almost entirely
removed. The treatment from here on was the same as that
described by Cooley. To the tissue was added a liter of
potassium-chlorate-nitric-acid solution made up in the pro-
portion of 30 grams of potassium chlorate to 520 ee. of nitric

1915]
DAVIS—ENZYME ACTION IN MARINE ALGAE 805

acid (sp. gr. 1.1). The flask was kept in the ice-box for two
weeks, when the oxidizing mixture was changed and the new
lot allowed to remain another fortnight. At the end of this
time a yellowish white tissue was obtained, representing fairly
pure algal cellulose. This was filtered off, washed well with
distilled water, and dried in the oven at 75-80°C. The final
product weighed 19.7 grams.

This cellulose was used in a way similar to the filter paper
in the first series. One gram was placed in each flask and well
shaken up with 50 cc. of distilled water. A concentrated
‘‘diffusion-extract’’ was prepared from Ascophyllum, Lamin-
aria, Ulva, and Chondrus, 10 ec. of which represented 5 grams
of the original dried tissue. This volume was added to the
flasks, and the series set away at 30°C. with toluene as an
antiseptic. At the end of two months no reduction of Fehl-
ing’s was observable and under the microscope there seemed
to be no decomposition of the cellulose particles.

Action on hemicelluloses—Hemicellulose was used from
two sources—from date seeds, and from the seeds of the wild
persimmon, Diospyros virginiana. In both cases the experi-
ments were essentially the same. The horny coats were
broken and the embryos removed. Small pieces of the hemi-
cellulose were then taken, placed in a flask with water, and
heated in the autoclave at 15 pounds for 15 minutes to kill
the cytase present. Upon removal from the autoclave the
pieces were washed several times in distilled water, being left
in the last wash water for several days with toluene as an
antiseptic—this to get rid of any reducing sugars present.
Two of these washed pieces were placed in test-tubes with
10 cc. of the concentrated ‘‘diffusion-extract’’ used in the ex-
periments with cellulose. Another lot was covered with 10 ce.
of distilled water and 2 grams of the dried algal powder
added. In a third series shavings of the hemicelluloses were
mounted in a Van Tieghem cell with a drop of enzyme solu-
tion. All the algae under investigation were tried out, but in
no case was there the slightest trace of decomposition, either
microscopically or by the reduction of copper.

[Vou. 2
806 ANNALS OF THE MISSOURI BOTANICAL GARDEN

Results —These negative results do not necessarily argue
against the production of slime through the agency of
enzymes. It is impossible to exactly reproduce the conditions
of the cell in vitro, and enzymes which might act upon cellu-
lose in the living tissue to produce slime might easily be in-
hibited from action on cellulose or hemicellulose under the
conditions of the experiments. Grtss (710) found that fresh
cherry gum contained cytase, but that none was demonstrable
in the older gum. He also found that malt diastase would
not act upon such gum until the tannins had been removed.
It is known that the algae do contain tannins or ‘‘tannoidal’’
bodies, the writer having demonstrated a ‘‘tannoid’’ content
in Ascophyllum of 1.1 per cent of the dry weight. These, or
other agents, could be involved in the partial or complete in-
hibition of cytolytic action. On the other hand, indirect evi-
dence, at least, points to the presence of the galactan and
pentosan groups as due to their being laid down as such, that
is, they do not arise as the direct result of hydrolytic enzyme
action, but probably represent the final step in the condensa-
tion of those particular hemicelluloses. Tschirsch (’89) and
his students have shown that the algal slime exists as an in-
tracellular substance, and they hold that in most instances,
at least, it does not arise from the cellulose. This seems to
be the logical view, and we in turn seem justified in looking
upon the galactan and pentosan groups in the algae as normal
products of the plant’s metabolism, present at all stages in
the plant’s growth, and capable of giving rise to gelatiniza-
tion at any time upon the adsorption of water. If one ex-
amines, for instance, such forms as Fucus, Mesogloea, and
Chondrus, the slime is hardly detectable when the plants are
growing under normal conditions, but when brought into the
laboratory and placed in fresh water, a rapid adsorption
begins at once. The dissolved salts in sea-water are un-
doubtedly the inhibiting factors in such adsorption under
normal conditions.

That this inhibition is not bound up with the living cell may
be shown by the simple experiment of killing two fronds of
Chondrus, for example, and placing one in fresh, the other

1915]
DAVIS—ENZYME ACTION IN MARINE ALGAE 807

in salt water, with toluene to keep down bacterial action.
Very slight, if indeed any, gelatinization is evident with the
frond placed in salt water, while that in fresh water begins
to gelatinize immediately. It is also a well-known fact that
in histological or cytological work with these forms, the kill-
ing fluids must be made up in sea-water or water containing
a high percentage of salts, else gelatinization interferes.
These facts, together with the apparent absence of cellulase
and cytase, tend to show that the galactan and pentosan
groups are always present as final condensation forms of their
particular ‘‘generic’’ carbohydrate line, and that sliming in
the marine algae, at least, is the result of the adsorption of
water by these already existing carbohydrate groups.

DISCUSSION OF RESULTS OF CARBOHYDRASE EXPERIMENTS

It is seen from the data presented in the foregoing tables
that carbohydrases in the algae, at least those that can be
isolated by standard methods, are very few. Furthermore, in
all cases where such carbohydrase action is evident, it is
limited to the polysaccharides—starch, dextrin, laminarin,
and glycogen. In no case were the disaccharides hydrolysed.
As groups, the ‘‘greens’’ are more active than the ‘‘reds,’’
while of the ‘‘browns,’’ Laminaria is the only form in which
carbohydrate action is demonstrable. Moreover, the action
here is extremely slow and is limited to starch, dextrin, and
laminarin. Mesogloea and Ascophyllum are similar to Fucus
in failing to show the presence of carbohydrases. Within the
groups there is little difference in the rate of carbohydrase
action. This is especially true in the ‘‘greens.’’ Of the
‘‘reds,’’? Agardhiella is a little more active than the other
forms investigated, while Ceramium is slightly the slowest.
Bartholemew (714), in the work already referred to, also
found that Ceramium was less active than the other ‘‘reds”’
with which he worked.

The various polysaccharides, with two exceptions, prove
favorable as substrates for the various algae in the same
order, viz., starch, dextrin, laminarin, and glycogen. The
carbohydrases of Ceramwm act more rapidly upon dextrin
than upon starch and this is also true of Laminaria, although

[Vou. 2
808 ANNALS OF THE MISSOURI BOTANICAL GARDEN

to a lesser extent. Glycogen, which is very generally
hydrolysed by diastase, is here decidedly less readily attacked
than the other polysaccharides. This would seem to indicate
that we are dealing with a distinct enzyme, one that might be
placed in the same category with dextrinases. These latter
always occur with the diastases but are held by many workers
to be distinct.

Some of the substrates tested for hydrolysis do not, as far
as we know, occur in the plants investigated. This is true of
sucrose, lactose, and inulin. However, although this might
reconcile us to the failure to find their specific enzymes, it does
not argue conclusively against such enzymes being formed. It
is well known that tissues do form ferments that have no de-
tectable substrates upon which to act—the rennen of the
bird’s stomach and the urease of the Soja bean being notable
examples. Inulin, as pointed out previously, does occur in
certain ‘‘greens,’’ as in Acetabularia and members of the
Dasycladaceae. Unfortunately, none of these forms were
available for investigation.

The absence of lactase and sucrase is not so significant as
is that of maltase. It is very generally considered that in the
plant, as well as in the animal organism, poly- and disac-
charides must be hydrolysed to simple sugars before assimila-
tion can take place. It is hardly possible that the algae are an
exception to this general rule and yet it is difficult to account
for this important negative result. It is known that inhibit-
ing agents do not affect all enzymes alike, and it may be here
that if such agents are liberated on the death of the cell, the
maltase might prove more sensitive to them than the other
carbohydrate enzymes. According to the findings of Kylin
(713), both dextrose and fructose have been demonstrated in
the tissues of Ascophyllum, Fucus, and Laminaria, but in ex-
tremely small quantities. These results would tend to con-
vince one that an enzyme giving rise to them is probably
present in the algal cell.

Such carbohydrates as galactans, pentosans, and mannans,
are very frequently met with in the algae and are potentially
capable of being split to assimilable sugars. That they are

1915]
DAVIS—ENZYME ACTION IN MARINE ALGAE 809

not so split, however, seems evident, at least not through the
activity of demonstrable algal enzymes, and in the face of
the negative evidence obtained, we would consider them as by-
products of metabolism rather than as playing the role of
reserves. As such, they would not be so comparable to the
reserve carbohydrates of the date as they would be perhaps to
the mucilaginous constituents of various seeds, as those of
flax, mistletoe, ete. These latter adsorb water readily with
gelatinization, and as far as is known, never function as re-
serves but act in a purely mechanical way (Czapek, ’13,
p. 705).
LIPASES IN THE ALGAE

The almost universal presence of fats in the marine algae
led to the question of their assimilation. Accordingly, experi-
ments were set up to determine the lipolytic activity upon
emulsions of neutral fats as well as upon certain esters of
the lower fatty acids. For the neutral fats olive oil was
chosen as a substrate, and two general methods were employed
in forming the emulsion.

The first, an olive oil-casein emulsion was made up after a
method described by Bloor (714). Four grams of casein were
placed in a warm mortar on a water bath and water added
until the whole formed a paste of medium viscosity. A drop
of phenylphthalein was added, then N/1 NaOH poured in and
stirred with the casein until the latter had been dissolved, this
point being indicated by a permanent pink tinge of the mix-
ture. Hight ce. of olive oil were stirred into the hot solution
and then ground with a pestle until all the oil globules had
disappeared. At this point the mortar was removed from the
bath and the emulsion cooled. During the cooling it was
found necessary to stir the mixture occasionally. The thick,
creamy mass resulting was diluted up to the required concen-
tration by the careful addition of water. If this dilution is
too great, the oil globules tend to rise to the surface.

The second method was also suggested by Doctor Bloor,
but, as far as is known, has not been described. Hight ce. of
olive oil were dissolved in the smallest amount of absolute
alcohol necessary. This solution was run through a hot fun-

[VoL 2
810 ANNALS OF THE MISSOURI BOTANICAL GARDEN

nel to which a drawn-out piece of glass tubing had been at-
tached, into about 100 ce. of cold distilled water, the water
being stirred constantly while the olive oil was being run in.
A milk-white emulsion made up of extremely small suspended
globules of oil resulted. In an emulsion carefully made, most
of these globules are small enough to show Brownian move-
ment. The alcohol was driven off finally by heating and the
emulsion made up to the desired concentration.

Both emulsions stand up well. In the latter, however, there
is a tendency toward flocking out by some of the smaller par-
ticles upon the addition of any salt-containing substance, such
as, for instance, algal powder; but, on the other hand, it has
the advantage of being more easily checked up because of its
simpler composition.

TABLE XI
LIPOLYTIC ACTION OF THE SEVERAL ALGAE UPON OLIVE OIL-CASEIN EMULSION

Number cc. of N/10 NaOH to neutralize 10 cc. of substrate

4 days 10 days 15 days

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In all the lipolytic experiments, algal powder or fresh algal
tissue crushed with fine quartz sand, was used as a source of
enzyme action. In some of the original series the olive oil-
casein emulsion was employed, but on account of the danger
arising from a possible hydrolysis of the casein with a re-
sulting increase in acidity, the alcohol emulsion was used in
the later work.

Lipolytic action of the several algae upon olive oil-casein
emulsion.—In this experimental series (table xt) flasks were
set up containing 50 ee. of olive oil-casein emulsion as a sub-

1915]
DAVIS—ENZYME ACTION IN MARINE ALGAE 811

strate, 5 grams of crushed algal tissue for enzyme action, and
10 ce. of 95 per cent alcohol as an antiseptic. Checks were
employed wherein the flasks in one case contained the emul-
sion alone, and in another case, the same weight of algal pulp
in distilled water. The flasks were maintained for 15 days
except for the forms especially noted. At intervals 10 cc. por-
tions were removed and titrated against N/10 NaOH with
phenylphthalein as an indicator.

Inpolytic action on alcohol-water-olive ow emulsion—Be-
cause of the possibility of the hydrolysis of the casein in the
emulsion used in the preceding experiments, a series (table
x11) employing the alcohol-water emulsion was set up as a
check. This emulsion alone was practically neutral but a

TABLE XII

LIPOLYTIC ACTION OF THE SEVERAL ALGAE UPON ALCOHOL-WATER-OLIVE-OIL
EMULSION

Number cc. N/10 NaOH to neutralize 10 cc.
substrate after 10 days

Alga
Emulsion | Emulsion Water+ |Water+] Net
+ tissue alone boiled tissue | tissue | acidity

Of Resch che ci trciy RRC eT eT 9 .00 .05 .05 85
EMLErOMOrPnG). 45. ee. + ice 8 .00 .025 sil a
WAH sea pean doce .65 .00 .05 .075 SOS
ASCOPNVILUMer a aniston cians or: aD .00 aS ailh .05
MUTT PETE soa caooncooessoe 1.15 .00 .02 ne .85
CHONALUS creas josie ecalede tine 1.45 .00 sll .35 1.10
GU TANS 66. 600b0 00 50M 25 .00 .05 .05 .20
COUNT oe .85 .00 zi .20 .65
IRHODVINENTG tense ee ele .525 .00 .05 als .375
CHOTPI on ee a 5125) .00 5 lS) LS .00

slight acidity was produced by the addition of the algal
powder. A negligible amount of the oil globules ran together
and collected at the surface of the liquid after some days, but
the bulk of the emulsion stood up well. As in the preceding
series, 5 grams of the fresh tissue were used as a source of
lipolytic activity, and the alcohol in which the olive oil had
been dissolved served as an antiseptic. Fifty cc. of the emul-
sion were used as a substrate, and the flasks maintained at a
temperature of 22-23°C. for 10 days.

Inpolytic action on triacetin—The lipolytic activity of dry
tissue powder of Ulva, Mesogloea, and Chondrus was tested,
using a .5 per cent solution of triacetin as a substrate. Two

[Vou. 2
812 ANNALS OF THE MISSOURI BOTANICAL GARDEN

grams of the tissue powder were used, otherwise the series
(table x11) was arranged exactly as the preceding and kept
at room temperature for 25 days.

Action on other esters——A series was set up with methyl
acetate, ethyl acetate, and ethyl butyrate in .25 per cent solu-
tion, using 2 grams of algal powder with 50 ce. of the sub-

TABLE XIII
THE ACTION OF POWDERED TISSUE FROM CERTAIN ALGAE UPON TRIACETIN

Number cc. of N/10 NaOH to neutralize
10 cc. substrate after

Alga 10 days 25 days
Tri Tri Tri
~ |Water+ . : Water+ t
eae A tissue nee acidity pcan tissue ae acidity
OLR coat Aoade ae) .025 sil 175 5 15 SS) 2
Mesogloea...... 529 .05 all ail 4 ae, aS .05
Chondrus...... aes .35 sit ail 8 4 aus) as)

strate in 20 per cent alcohol. Titrations were made from
time to time against N/10 NaOH with phenylphthalein as an
indicator. Even after 60 days at room temperature no in-
crease in acidity was observable over the checks.

General results for experiments with lipases.—The results
serve to show that, although slight, there is distinct lipolytic
activity in most of the forms investigated. The various
groups of algae are not so distinct regarding this activity as
was the case with the carbohydrases, nor does the activity of
the individual alga in this case relate itself particularly to the
activity shown by the form in its carbohydrase action. Agard-
hiella hydrolyses the polysaccharides more rapidly than any
other alga, yet its lipolytic activity is very low. Likewise,
Laminaria, so inactive in the previous group of enzymes, is
among the most active on fats. Fucus, on the other hand, was
found in previous work to have no action on either carbo-
hydrates or fats.

The action is especially evidenced by use of the olive oil-
casein emulsion. In general, the increases were less where the
aleohol-water emulsion was used—a difference probably ex-

1915]
DAVIS—ENZYME ACTION IN MARINE ALGAE 813

plainable on the ground that the casein gave rise to a slight
acidity.

Specificity of action might explain the failure to obtain
action on most of the esters. Euler (’12) differentiates the
lipases into true lipases and esterases, the former acting on
neutral fats particularly, the latter on the methyl and ethyl
esters of the lower fatty acids. Even in this latter restricted
field, great specificity may be shown. Reed (’12) found that
ethyl acetate was quite rapidly acted upon by an esterase
isolated from Glomerella rufomaculans, while ethyl butyrate
was only slightly hydrolysed.

THE PROTEINASES

The proteolytic activity of the various algae was tested on
albumin, casein, legumin, peptone, gelatin, and in certain
cases, on proteins isolated from the algal tissue—most of
these under acid, alkaline, and neutral conditions. The first
four were made up in 1 per cent concentrations. Albumin
and peptone went into solution quite readily; legumin and
casein, being insoluble in water, were either weighed out
directly, or dissolved in N/10 NaOH. ‘The albumin and
gelatin were also tested in the form of Mett’s tubes, and the
gelatin alone in test-tubes where it was held at a temperature
high enough to keep it in a liquid state while in contact with
the algal powder. In all cases, algal tissue was used directly,
either fresh crushed, or dry powdered—usually 2 grams of
the powder or 5 grams of the fresh tissue to each 50 cc. of
substrate.

Determination of hydrolysis——Proteolytic action was de-
termined in several ways, each acting as a check on the others.
The biuret test was used for the demonstration of tryptic
action, the proteins being precipitated by (NH4)eSO4 in
saturated solution and the test applied in the usual way. The
tryptophane test was employed for ereptic action and this
also furnished a check on the action of trypsin. In this, 1 ce.
of the protein solution was placed in a small evaporating dish,
a drop of glacial acetic acid added, and then a few drops of
strong chlorine water. The hydrolysis to the amino acid stage

[VoL. 2
814 ANNALS OF THE MISSOURI BOTANICAL GARDEN

was also demonstrated in two other ways—by the formal-
dehyde-titration method of Sorenson (’08), and the determina-
tion of the amino-nitrogen by the micro-Kjeldahl method of
Folin (713). The Soérenson method consisted in adding 2 ce.
of formalin, made alkaline to a faint pink tinge with N/20
NaOH, to 10 ce. of the filtered protein solution, made alkaline
to the same color. Upon mixing, the color disappeared and
the acidity resulting was titrated against N/50 NaOH, using
phenylphthalein as an indicator.

In the determination of the amino-nitrogen by the ‘‘micro’’
method of Folin, the protein in a 5 ee. filtered portion of the
solution was precipitated with 2 ec. of a 25 per cent solution
of phosphotungstic acid in 5 per cent H2SO4. The precipitate
was filtered off and a 2 cc. portion of the filtrate removed for
the determination of the nitrogen. Duplicate determinations
were made in all cases. These portions were placed in Jena
test-tubes, 20200 mm., 1 ee. concentrated H2SO4 added, then
1 gram of K2SOx4, and a drop of 5 per cent CuSO4. The
digestion was carried on over the flame from a micro burner,
the fumes being carried away by the fume adsorbers described
by Folin. Usually 20 minutes sufficed for the completion of
the digestion, although in a few instances 25 minutes were
required. After cooling slightly, 6 ec. of distilled water were
carefully added. The tubes were then transferred to the
distilling apparatus where concentrated NaOH was added to
alkalinity, and the tube contents distilled over for three
minutes, the NH; being collected in a known volume of N/10
HCl. The acid in the collection flask was titrated against N/10
NaOH with alizarin red (alizarin sulfonsaure Natrium,
Merck), and the amount of nitrogen represented by the
acid neutralized, determined.

In the method originally described by Folin, the NHs3 was
not distilled but was forced over from an alkaline solution by
a strong air current. However, students in his laboratory
have made use of a micro distilling apparatus, and the sug-
gestion for the ones employed here owes its origin to one of
Folin’s assistants. Distillation has the advantage of quick-
ness, and from the writer’s experience, of accuracy as well,

1915]
DAVIS—ENZYME ACTION IN MARINE ALGAE 815

at least where suction instead of compressed air is employed
in the air method. The results with the air current were very
often below the theoretical. The distilling tubes used were
made in the laboratory from glass tubing, the outer jacket
measuring 402 cem., and the inner being 5 mm. in diameter.
The lower end of tkis latter, where it dipped into the collec-
tion acid, was fitted with a larger tube 14 mm. in diameter—
this to prevent a back flow of the acid; to the upper end of
this inner tube was attached a safety bulb made from a 10 ce.
pipette, and this in turn fitted into the Jena tube containing
the distilling mixture, by means of a two-hole rubber stopper.
Through the second hole in this stopper was a small piece of
glass tubing closed at the upper end with a bit of rubber
tubing and a pinch clamp; it was through this that the alkali
was added after the apparatus was connected up for dis-
tillation.

Considerable trouble was experienced at first with bump-
ing, especially after the digestion mixture had become con-
centrated. Neither bits of glass nor pebbles would overcome
it. Finally the expedient was adopted of using short pieces
of glass tubing sealed at one end and this end placed upper-
most. These were of such a diameter that after digestion,
the digestion mixture drawn up into them by the cooling of
the contained air, would easily drain out when the boiling tube
was forced up on the side of the test-tube by a quick down-
ward motion.

The action of Enteromorpha, Mesogloea, and Chondrus
powder upon various proteins—Fifty cc. lots of casein,
legumin, albumin, and peptone were used as substrates in this
series—all in 1 per cent concentrations. The albumin and
peptone were dissolved directly in distilled water, the legumin
and casein in N/10 NaOH. Two grams of air-dried tissue
powder were used for proteolytic action, with the exception,
however, of Mesogloea, which, as before stated, was partially
dehydrated before being air-dried. The various substrates
were made neutral by the addition of N/10 alkali and then
acid or alkaline by further addition of 2.5 cc. of N/10 HCl
or NaOH. In the formaldehyde titrations 10 ec. of the sub-

[Vou. 2

ANNALS OF THE MISSOURI BOTANICAL GARDEN

816

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"HORN 20 1DH O1/N YUM oureyye Jo prow 00Z/N epeur asoM sazejsqns oy,

cL 50° Of" re Sr 7 ee DG
— 08° OF” = = 06'T OST aa = 0°72 O1'Z “aye
ar c3's 06°7 = = Slee co'T oI + Olas; Se “aye
am 09'T scot = ca Siar COmD ae == cot 09'T pre
= OL 7 OL'T = = $o'T OFT qP + 09°¢ 00°C pre
=a co'T cst <= = 00°C 06'T 2 ad co'T Os 'T yneu
= SiG O17 a =r OL'T Coal oF + S6'P O17? “nou
= So°? Orc => = 0772 0£°C a ae SVC Ole “aye
ar ST'OT | O€°S = =e SAG 09°7 + + Of CI | SOP “aye
= S6'7 08°C at = Sv 7 OF 7 = ae 01°C 09°C proe
Sa $9°7Z SIE a =a ce? OTe = po Ore SLEC pre
= SO56 0c 7 — = cS °C (OSA ame = OL OF 7 jnou
ate Shek ore — = cv 7 OfsG he aie O8 TT |SLT'€ jnou
ee 0z°1 cg" — feel ore | ort — [errr] oz 7 S6 aye
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See SO'T 06 By) peptone C1 — feel opr 06° arial
ee ee eee SO ZI S6'F aa O16; 6) @\60 16 GEC OL V4 a oR Ge alene Ole! CES *ynou
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= ¢9° OF" a ae OL” Os” ie ar OF o¢° “qnou
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sAep Q¢ |sAvp CT sAep (¢ |SAep CT sAep ¢ |sAep cy

eller geen Ts reenact w |—————_|# @
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+ 3
snapuoyy paopsosayy DYdaomosaqury

SNIGLOUd SNOIAVA NOdN AVOTV NIVLUGO WOMA YAACMOd ANSSIL AO NOILOV AHL
AIX ATEVL

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a7e1}sqns

1915]
DAVIS—ENZYME ACTION IN MARINE ALGAE 817

strate were titrated against N/50 NaOH. One per cent chloro-
form-thymol was used as an antiseptic, and the flasks were
kept at a temperature of 22—23°C. for 30 days.

The forms used in table x1v show a general ability to
hydrolyse proteins. All four proteins employed were acted
upon by one alga or another, but peptone and casein in
neutral and alkaline solution were the most readily attacked.
Enteromorpha split albumin and legumin but poorly; Chon-
drus acted upon legumin only in alkaline solution, and then
slightly; Mesogloea failed to hydrolyse casein and legumin,
and its action on albumin and peptone was very slow.

The action of Ulva, Laminaria, and Agardhiella powder
on peptone and casein in alkaline and neutral solution.—tIn-
dications in the preceding experiment seemed to point to the
fact that peptone and casein were more easily acted upon
than the other proteins—and these more especially in neutral
and alkaline solution. Accordingly, a series was set up with
these two substrates, similar in all respects to the preceding
one, except that the acid substrate was omitted and that Ulva,
Laminaria, and Agardhiella were used for proteolytic action.
Five grams of air-dried tissue were employed with 100 cc. of
substrate. One per cent chloroform-thymol served as an anti-
septic, and the flasks were kept at 35°C. for 30 days. Formal-
dehyde titrations were made after 15 and 30 days and trypto-
phane tests and amino-nitrogen determinations after 30 days.
In the titrations 10 ce. of substrate were titrated against N/50
NaOH, and the amino-nitrogen represents that in 2 ce. of the
filtrate from phosphotungstic precipitated protein.

The data in table xv tend to substantiate that of table xiv
concerning the hydrolysis of peptone and casein. The higher
temperature at which the flasks were maintained undoubtedly
had something to do with the larger amounts of amino acids
split off from these two proteins than was the case in the pre-
ceding series, yet if we can judge by the action on carbohy-
drates and fats, we are dealing here with the more active
members, enzymatically, of their respective groups.

On the whole, peptone and casein seem to be the most favor-
able substrates of those used for proteolytic activity, and

[VoL. 2

ANNALS OF THE MISSOURI BOTANICAL GARDEN

818

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6F° = Or'9 co's 6f = or'9 go's 8h = or'9 ccs SPOTS | |e Geneon ulose’)
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NIASVO GNV ANOLddd NOdN AVOTY NIVLUYGD WOU ANSSIL GHAAGMOd AO NOILOV AHL
AX HTAVL

1915]
DAVIS—ENZYME ACTION IN MARINE ALGAE 819

these in neutral and slightly alkaline solution. This was
shown in the formol titrations! and in the determination of
the nitrogen in the amino acids split off. Albumin was slowly
acted upon by Enteromorpha, Mesogloea, and Chondrus. The
first and last also hydrolysed the vegetable protein, legumin,
to a slight extent—an action that was not shared by Mesogloea.

The action of algal powder on the proteolysis of gelatin and
albumin in Mett’s tubes.—T wo lots of Mett’s tubes were made
up, one containing coagulated egg-white, and the other, 15 per
cent gelatin. In each of a series of flasks containing 50 cc.
of distilled water, N/200 NaOH and N/200 HCl respectively,
were placed one tube each of egg-white and gelatin. Two
grams of the powdered tissue from each of the several forms
under investigation were added for enzyme action and the
usual percentage of toluene used as an antiseptic. The sev-
eral series were kept for two months at room temperature.
At the end of that time the albumin tubes in the alkaline solu-
tion containing the algal powder of Ulva, Enteromorpha,
Chondrus, and Agardhiella showed a slight digestion. The
checks in the alkaline solution alone showed swelling. How-
ever, although this was indicative of action, it was not definite,
since the great length of time the protein was in contact with
the complex constituents of the tissue may have been a factor
in either causing a slight hydrolysis or a contraction of the
albumin. On the other hand, Laminaria, Ascophyllum, Meso-
gloea, and Ceramiwum caused no such action. The gelatin tubes
showed no evidences of action even after 60 days.

The effect of proteinases on the hardening of gelatin.—Dox
(710) describes a method for testing the hydrolysis of gelatin
which consists in keeping the protein in a liquid state during
contact with the material being tested for proteolytic activity,
then at the end of a stated period noting whether the gelatin
congeals when placed in cold water. This method was used
in the following way: Five cc. of 20 per cent gelatin were
placed in each of a series of test-tubes, and 5 ce. of the standard

1 The formaldehyde titrations, as used here, were satisfactory only in a general
way, i. e., to show relative rather than exact differences in the amounts of amino

acids split off. The differences brought out by the amino-nitrogen determinations
are much more exact.

[Vou. 2
820 ANNALS OF THE MISSOURI BOTANICAL GARDEN

““diffusion-extract,’’ described under ‘‘carbohydrases,’’ used
for action. The contents of the tubes were made neutral,
and acid and alkaline to N/200, as was done in the other pro-
teolytic experiments. Five drops of chloroform-thymol were
added as an antiseptic. Checks were set up containing the
gelatin together with 5 ce. of boiled ‘‘diffusion-extract.’’ The
tubes were placed in an incubator at 35°C. for a week, at which
time they were removed and cooled in running water. All
tubes hardened in a short time, showing that no hydrolysis
had taken place.

General results for experiments on proteolysis——The pro-
teolytic activity, although slow, as was the case with the
other enzymes investigated, is definite enough to warrant
the statement that proteinases and peptases are very gen-
erally present in the algae. When present, such enzymes
act best under neutral and alkaline conditions. This last
finding is interesting in the light of the existing differ-
ences of opinion regarding the relative value of acid and
alkaline substrates for vegetable proteinases. It will be re-
called that Vines (’97) found that acidity favored the pro-
teinase contained in the leaf pitchers of Nepenthes, and in a
later paper, he states that peptase (hydrolysing albumoses
and peptones to amino acids) always act best under faintly
acid conditions. Emmerling (’02), on the other hand, demon-
strated that the papain of Carica papaya acted more rapidly
when the substrate was alkaline. Euler (’12) states in a
general way that peptases require a neutral or faintly alka-
line substrate, and proteinases (tryptases) an acid one.

Of the proteins employed, solutions of casein and peptone
prove the most favorable substrates. Albumin in solution is
acted upon slowly, but when employed in the form of Mett’s
tubes, doubt exists regarding its digestion. Legumin appears
to be slowly hydrolysed by Enteromorpha and Chondrus, but
not by Mesogloea. Gelatin, either in the liquid state or in
the form of Mett’s tubes, is not attacked. As groups, the
‘‘reds’’? appear more active in proteolysis than do the
‘‘oreens,’’ while, as was true for carbohydrases, the ‘‘browns’’
show the least activity.

1915]
DAVIS—-ENZYME ACTION IN MARINE ALGAE 821

THE AMIDASES

The tissues from the several algae were tested for their
ability to split NHs from such amino and amido compounds
as urea, acetamid, asparagin, and methyl amine. These com-
pounds were used in 1 per cent concentrations. Series were
set up in which 50 ee. of the substrate to be tested were placed
in flasks together with 2 grams of the powdered tissue and
chloroform-thymol as an antiseptic. Checks were used with
the nitrogen compounds alone and with the algal tissue in
distilled water. The flasks of duplicate series were kept at
room temperature and at 35°C. respectively, for 30 days, at
the end of which time Folin’s method was employed for the
determination of any NH; that might have been split off. In
the collection of the NHs, Friedrich’s improved gas washing
bottles containing 250 cc. of N/50 HCl were used. Air was
bubbled through by means of a suction pump for two hours,
then 25 ce. portions of the collection acid were removed and
titrated against N/50 NaOH, with alizarin red as an indicator.
In no case was there any action over that evidenced by the
checks.

These results are extremely interesting, in the case of Ulva
especially. This form, as has been shown, thrives in waters
where the organic nitrogen content is high. The question
would at once arise whether this increased growth were due
to the ability of the Ulva to break down the protein molecule
and thus obtain an increased supply of nitrogen as NHs, or
whether it were due to the activities of the denitrifying bac-
teria rendering available a larger assimilable supply. That
such bacteria are relatively abundant in sewage-contaminated
water has been shown in the review of literature. We can
conceive of another factor entering in—that of selective for-
mation of enzymes. It might well be that with plenty of the
amino-nitrogen available through the activity of bacteria, no
amidases would be formed. The possibility of shedding some
light on this point led to the experiments following.

Experiments on amidase formation by Chlamydomonas.—
Chlamydomonas was grown in pure culture upon two differ-
ent media; one (with one or two modifications, that used by

[Vou, 2
822 ANNALS OF THE MISSOURI BOTANICAL GARDEN

Schramm (’14) ), containing (NH4)2SOx4 as a source of nitro-
gen, the other with nitrogen supplied as peptone and aspar-
agin. These media complete were as follows:

A. B:
UNC) CES OIC eI IERIE OCH LOFOONoramse Acar ererctsrrler st eee: 10.00 grams
(NE) SOR reece: .20-grams) ‘Peptone) <2). + teeter 4.00 grams
Me SO stip oO merit cters vO; grams Aspararingi erect 1.00 grams
KiSHiP Ornette sree: 210 grams) MegSO727 HeO) vie .10 grams
LS 0 Vin charmenmta cig bam AM OF trace GIBURO ooogob ene egD0 .10 grams
Glucosegss-raeeiaeeussrtrete 10/100) grams) HeSOw lace cielo arene trace
Distilled HsO <......... 500.00 ce. GlucoseWeretreeiarctacraeie 10.00 grams
Distilled HsOV ee 500.00 ce.

These media, designated ‘‘A’’ and ‘‘B,’’ were placed in
125 ec. Hrlenmeyers, about 25 ee. to each flask, and the flasks
placed horizontally until the agar hardened. A relatively
large surface was obtained in this way and the harvesting of
the alga later was facilitated. In inoculation, the alga was
smeared over the surface of the agar to give an even growth.
After a growth of thirty days, it was harvested by scraping
from the agar surface with a stiff, platinum needle. The cells
were then dehydrated with alcohol, acetone and ether, dried,
and ground with an equal weight of fine quartz sand. Flasks
were set up in duplicate in Wollf wash bottles, using 1 per
cent asparagin as a substrate, with an amount representing
.oo grams of sand-free algal powder. Checks were run on
both the asparagin and the algal powder alone. One-half the
series was taken down at the end of 7 days, the other half at
the end of 15 days, and the NHg split off determined by the
Folin method previously employed. The flasks were kept at
a temperature of 35°C. The results are given in table xvi.

TABLE XVI
THE ACTION OF DEHYDRATED CHLAMYDOMONAS CELLS UPON ASPARAGIN
Suhetrate Weight Nitrogen as NH; in 50 cc. substrate
50 ce. algal sae ee ee
1 per cent powder 7 days Net N 15 days Net N
.35 grams ‘‘A”’ 36 .16 45 mail
Asparagin .35 grams “B” sl) .96 1.84 1.60
BA Nr Cae Gols 18 A aes sahsinne 22 fies ote
Water .35 grams ‘‘A”’ OZ * alist 102i herent
.35 grams ‘‘B” £03! 9 ieee i 07: Ra ore ee icles

The amount of nitrogen in the checks is so small as to be
well within experimental error. The NHzg split off by powder

1915]
DAVIS—ENZYME ACTION IN MARINE ALGAE 823

‘‘B’’ would be almost negligible were the findings not so con-
sistent. There is a definiteness about the increase over the
checks that can hardly be ignored. In order to get further
evidence on this point, however, another series (table xvir)
was set up, using urea and asparagin in 1 per cent concentra-
tions as substrates. The flasks were maintained at a tempera-
ture of 35°C. for 30 days.

TABLE XVII
THE ACTION OF DEHYDRATED CHLAMYDOMONAS CELLS UPON ASPARAGIN
AND UREA

Weight Nitrogen as NH; split

Substrate algal off in 30 days Net nitrogen
powder mgms. mgms.
ONC AIMS yr Awe aS 220
Asparagin .5 grams ‘‘B”’ 3.10 2.20
Soe COR Cee 65 die he hoes eRe RTE
.5 grams ‘‘A”’ 1.45 5 lle)
Urea -o grams b: 3.70 2.47
Bp sais Car aheceeereth aide .98 Bee tare tors
Water 25 grams AY ESO wo MPMI eta cs ewes ayer
.5 grams “B”’ SL se TE CR ae eae ea re

In this, as in table xvi, the evidence goes to show that al-
though the desamidization is practically negligible where the
alga is grown with (NH3)2SOu, as a source of nitrogen, it is
definite where the nitrogen is supplied in the amino and amido
form. The actual splitting is small in any case.

On the basis of the above, we can simply reason by analogy,
and yet this analogy points to the fact that the probable
reason for the failure to demonstrate amidase in Ulva lies
in the failure to form that enzyme. This in turn would indi-
cate that the great growth of Ulva in sewage-contaminated
waters is probably due to the abundance of desamidizing bac-
teria which those waters maintain—bacteria which break
down the protein molecule with the ultimate setting free of
NH3. Nitrogen, as such, becomes directly available to the
plant.

NUCLEASES

The presence of nucleases in the algae has already been

reported by Teodoresco (712), but since he investigated only

[Vou 2
824 ANNALS OF THE MISSOURI BOTANICAL GARDEN

one of the forms falling within the scope of this study, experi-
ments were carried on to determine the presence or absence
of nucleases in one representative of each group, Ulva, Ce-
ramium, and Ascophyllum.

One-half per cent nuclein was dissolved in N/10 NaOH,
the complete solution of the compound being shown by a
drop of phenylphthalein. One hundred ce. of this neutral
solution were added to each flask together with 3 grams of
air-dried algal powder. Toluene was added as an antiseptic.
Checks were set up by adding autoclaved algal powder to the
nuclein and also by using nuclein solution alone. The flasks
were placed at 35-36°C. for 38 days, at the end of which time
the phosphoric acid split off was determined as P2Os by the
uranium-acetate method. Five ec. of a sodium acetate solu-
tion? were added to 25 ec. of the nuclein substrate, this brought
to a boil and titrated while hot. Potassium ferrocyanide was
used as an indicator—a drop of the titration mixture being
removed from time to time and brought into contact with a
drop of the indicator on a porcelain plate. The results ob-
tained are given in table xvii.

TABLE XVIII
THE ACTION OF POWDERED TISSUE FROM CERTAIN ALGAE UPON NUCLEIN

Substrate Free HsPOs as P2O5| Net amount
100 cc. Weight algal powder in 100 cc. in 38 days} POs in 100 cc.
1 per cent mgms. mgms.
3 gms. Ceramium 70.00 56.25
3 gms. Coromsuim boiled Ute = «Il “ Goaes
: 3 gms. Ulva 56.70 39.20
Nuclein 3 gms. Ulva boiled 1750.8 9 ben ees
3 gms. Ascophyllum d/25 Se)
3 gms. Ascophyllum bld. 16:90" - 5. oR eels

These findings substantiate those of Teodoresco (712) re-
garding the general presence of nucleases in the algae. The
values for Ceramium agree very well with those he obtained
for the same form, 1. e., 56.25 milligrams in 38 days at 35°C., as
compared with 76.6 milligrams in 51 days at 22-26°C. Ulva

1 The standard solution of the uranium acetate contained 8.8652 grams of the
salt in 250 ce. of water, and each cc. by calculation was equivalent to 5 milligrams
of P2Os5.

*The sodium acetate solution contained 25 grams of sodium acetate and 25
ec. of 30 per cent acetic acid in 250 ce.

1915]
DAVIS—ENZYME ACTION IN MARINE ALGAE 825

shows less nuclease activity than does Ceramium, while Asco-
phyllum, true to its reputation for inactivity, gives a value so
small as to be negligible.

An interesting point is brought out by the use of nuclein—
one that proves a check on some of the previous proteinase
experiments. Nuclein is composed of nucleic acid bound up
with some protein (according to Abderhalden, ’11, this is
albumin) which must be split off by a proteinase before the
nuclein residue is exposed to the attack of the nuclease. That
unmistakable nuclease activity was evident, only serves to
show again the presence of proteolytic enzymes.

OXIDASES AND CATALASES

Oxidases.—Direct and indirect tests for oxidase action, that
is, for the oxidases and the so-called peroxidases, were carried
out in all cases with fresh tissue. The general method de-
seribed by Clark (’10) was employed, using guaiacum, alpha
naphthol, and phenylphthalin as reagents. Five grams of the
fresh tissue, crushed with an equal weight of fine quartz sand,
were extracted for half an hour with 25 cc. of distilled water.
The extracting fluid was then filtered off, the tissue residue
pressed out, and the filtrate made up to 50 cc. Five cc.-
portions were placed in test-tubes, and for the direct test, ten
drops of the reagent were added; for the indirect test, this
amount plus 1 ce. of fresh 3 per cent hydrogen peroxide. In
only two cases was direct oxidization observable—with
Agardhiella and Ulva. With the former, direct action was
strong with all three reagents, and when peroxide was added
an immediate deepening of the color occurred, showing the
presence of peroxidases as well. With Ulva, however, both
direct and indirect tests were only weakly positive. Atkins
(714), it will be remembered, obtained direct tests with but
one of twenty-nine diverse algae investigated and indirect
tests with but seven. He thought that reducing substances
prevent the demonstration of oxidases in other forms. As
brought out in the review of literature, Reed (’15*) has since
demonstrated indirect oxidation of the alpha naphthol-para-
phenylenediamine group of compounds by many of these

[VoL. 2
826 ANNALS OF THE MISSOURI BOTANICAL GARDEN

forms. In the filamentous forms he showed the presence of
oxidases by the formation of colored granules within the cells
surrounded by these reagents. Reed concludes that oxidases
of specific oxidative ability are very generally present in the
algae, and where negative results are obtained, either the
necessary specific compound is not present or other factors
enter in, such as the destruction of the oxidase equilibrium of
the cell upon crushing.

Catalases—Both fresh and air-dried tissue were used for
catalase demonstration. In a preliminary series, the addition
of 5 ce. of 3 per cent hydrogen peroxide to about a gram of
fresh crushed algal tissue showed evolution of oxygen in all
cases except one, that of Mesogloea. Later, a series (table x1x)

TABLE XIX
CATALASE ACTIVITY OF CERTAIN ALGAE

Number cc. O: evolved at 21.5°C.
Alga

2 minutes 5 minutes 10 minutes
Ascophyllumt. 05 ose cn oe 3 9 a9)
LOMANGTI Aes ae ee 1.4 hee} D6)
Mesogloeay is « «insei- clei ioeteys 0.0 0.0 0.0
OUT a aicectoinin itoman ete oo a2, A a5
Algardhiellas v2: ace oce Sisk: 4.6 5.6
ChONGTUS pee. A Sere A 2.0 Deak)
Rhodymenta.......0..5.0- 9 1.4 2.0
Ceram eee Soll 5.9 8.2
Potato leaf tissue......... 22.6 ots s See

was set up in which 1 gram of powder was placed in 125 ce.
Erlenmeyer flasks, 10 cc. of 38 per cent hydrogen peroxide
added, and the oxygen evolved collected in a gas burette over
water. The flask in which the action was taking place was
shaken every 15 seconds, and the volume of oxygen evolved
read at the end of 2, 5, and 10 minutes. The temperature of
the room was practically constant during the experiments and
no especial precautions were taken to control the temperature
of the flask other than keeping the hands away from it dur-
ing the action. The results are not meant to be quantitatively
exact, but they do give the relative catalase activity of the
several forms. In addition, air-dried potato leaf tissue that
had been in the laboratory about the same length of time as
the algal tissue was tested for comparison.

1915]
DAVIS—ENZYME ACTION IN MARINE ALGAE 827

Catalase, so wide-spread in all plant tissues, is found here
in all the forms investigated except Mesogloea. The ‘‘reds”’
prove more active than the ‘‘browns,’’ and these latter slightly
more active than the ‘‘greens.’’ No alga is strikingly active,
however, when compared with potato leaf tissue. Strangely,
Ulva, most active in regard to the other enzyme groups, is
one of the least so here.

GENERAL DISCUSSION AND CONCLUSIONS

The data obtained in the foregoing investigation serve to
show that the number of enzymes in the algae that can be iso-
lated, by standard methods at least, is quite limited. This is
especially true of the ‘‘browns,’’ in two forms of which,
Ascophyllum, and the Fucus of the earlier study, such action
is limited to catalase alone. In this group the demonstrable
carbohydrases are restricted to very slowly acting diastases
in Laminaria; in neither Ascophyllum nor Mesogloea is there
the slightest trace of what might be termed carbohydrate
hydrolysis. Moreover, negative results are obtained in these
forms for most of the other enzymes sought. Lamaria
shows lipases and catalases (it was not tested for proteolytic
or nuclease activity), and action in Mesogloea is restricted to
lipases and proteinases, both tryptic and ereptic. On the
other hand, very general enzymic activity is demonstrable in
the ‘‘greens’’ and the ‘‘reds’’—diastases, dextrinases, lipases,
proteinases (tryptic and ereptic), nuclease, and catalase being
isolated from the crushed tissue. Oxidase is shown present
in one ‘‘red,’’ Agardiella, and in one ‘‘green,’’ Ulva. Such
action, as a whole, appears a little more rapid in the ‘‘reds’’
than in the ‘‘greens,’’ but no enzyme stands out as being
specific for either a group or an alga within a group.

The carbohydrases demonstrated are restricted in their
action to those hydrolysing starch, dextrin, glycogen, and
laminarin of the polysaccharides used as substrates, and in
Laminaria, such action was further limited by a failure to act
upon glycogen. Inno case, in any member of the three groups
was there evidence of disaccharides being attacked. While

[VoL. 2
828 ANNALS OF THE MISSOURI BOTANICAL GARDEN

this is not so surprising perhaps for sucrose and lactose, it is
difficult to understand the failure of enzymic hydrolysis of
maltose. The results obtained by Kylin (713) indicate that
both dextrose and fructose are found in algal tissues, and
reasoning from results found for plant and animal tissues in
general, it seems, as is true in those cases, that in the algae,
maltose must be broken down to glucose before assimilation
can take place. The failure to isolate this enzyme points to
the possible presence of some inhibiting factor, rather than
to the non-formation of the ferment.

Lipases, acting very slowly, appear wide-spread in algae,
being demonstrable in all the forms used in this study except-
ing Ascophyllum. Along with the fact that fats are very
generally found in the algae, these results are significant in
that they indicate the importance of the role these compounds
may play as assimilatory products. It is not thought, as was
advanced by Reinke (’76), Hansen (’93), and others, that these
fats function as the first products of assimilation, but rather,
that they act as storage products of more or less importance.

The algae, in general, show the presence of enzymes capable
of hydrolysing certain proteins. Casein and peptone in alka-
line and neutral solution prove the most favorable substrates
of those tested, although legumin and albumin are also slightly
attacked. The ‘‘greens’’ and the ‘‘reds’”’ are about equally
active in this way, the ‘‘browns,’’ as usual, acting more slowly.
The fact that both native proteins and peptones were
hydrolysed, points to the presence of both tryptic and ereptic
enzymes. Still further evidence of the presence of the first
of these was the splitting of the protein molecule from nuclein
preceding the action of nuclease.

Amidases seem not to be formed by any of these algae. The
results obtained with Chlamydomonas, from which the
amidases were isolated when the alga was grown on a medium
containing asparagin and peptone as a source of nitrogen but
not when the nitrogen was in the form of ammonium sulphate,
indicate that such amidase formation may depend upon the
nature of the supply of assimilable organic nitrogen. This has
a distinct bearing upon the reason for the increased growth of

1915]
DAVIS—ENZYME ACTION IN MARINE ALGAE 829

Ulwa in sewage-contaminated waters. In order to break down
the proteins present in the surrounding waters and even those
in close contact with the plant itself, it would be necessary for
the Ulva to secrete an extracellular enzyme, since the large
protein molecule is not diffusible into the cell. If so secreted,
the enzyme would be quickly dissipated in the large volume
of surrounding water. Desamidizing bacteria, on the other
hand, have been demonstrated in harbor and shore waters
where such algae abound. They can come into much more
intimate contact with the protein than can the plant, and un-
doubtedly play an important réle in rendering available at all
times an abundant supply of organic nitrogen.

The demonstration of nucleases acting upon the previously
split nuclein molecule, substantiates the findings of Teodoresco
for this enzyme. Both Ulva and Ceramium showed the
presence of the ferment, while Ascophyllum, the only repre-
sentative of the ‘‘browns’’ investigated, gave negative re-
sults. Where such enzymes were formed, they compared more
favorably with enzymes of fungi and higher plants than do
any of the other algal ferments.

None of the ‘‘browns’’ studied showed the presence of
oxidative enzymes, while in the ‘‘reds’’ and the ‘‘greens’’ but
one form gave the characteristic reactions. It is interesting
to note that these algae, Agardhiella and Ulva, were the most
enzymatically active forms studied. The oxidase reactions
with guaiacum, alpha naphthol, and phenylphthalin were very
positive, both directly, and indirectly with hydrogen peroxide.

In all cases where enzymes were demonstrated, the action
was very slow, being with the exception of nuclease, much less
rapid than in the higher plants. The reason for this is not
clear, but it cannot in all instances be due to inhibiting sub-
stances set free upon the death of the cell. Arber (’01), as
has been mentioned before, found that Ulva, Cladophora, and
Enteromorpha, placed in the dark but under otherwise pre-
sumably normal conditions, required from two weeks with
Ulva, to two months and more in the case of Enteromorpha
for destarching. This indicates the presence of a very slowly
acting diastase in the cells of these algae. The metabolism

[VoL. 2
830 ANNALS OF THE MISSOURI BOTANICAL GARDEN

of the algae is also probably slower than that of the higher
plants and one might expect, a priori, the enzymes also to be
less rapid in their action. Although the algal enzymes may
be inherently slow, it seems that there may also be substances
set free on the death of the cell which either partially or en-
tirely inhibit enzyme action. The writer has found evidence
in some preliminary experiments, that the action of taka
diastase upon starch is directly proportional to the amount
of free tannin present. In connection with this, it was also
found that Ascophyllum had a ‘‘tannoidal’’ content of 1.1
per cent of the dry weight. It is possible that such tannoids,
if in an uncombined state, may after the death of the cell unite
with an enzyme to throw it out of the sphere of action. That
diastases are demonstrable in tissues having a high tannin
content may perhaps be explained on the basis that they are
bound up in such a way as to render them incapable of uniting
with the ferments. Still other organic inhibiting compounds
may be present, and the point opens up a very interesting
problem concerning inhibition, not only in algal tissues, but
in those of many higher plants as well.

SuMMARY

1. Using standard methods of enzyme isolation and de-
termination, the following enzymes have been found in fresh
or dried algal tissue:

a. Carbohydrases hydrolysing the polysaccharides,
starch, dextrin, glycogen, and laminarin, but not those
hydrolysing the several disaccharides employed as
substrates.

b. Lipases acting upon neutral fats but not upon the
esters of the lower fatty acids.

ce. Proteinases (tryptic and ereptic) acting best under
neutral and alkaline conditions.

d. Nucleases.

e. Oxidases and peroxidases (in but two forms—Agard-
hiella and Ulwa).

f. Catalases.

1915]
DAVIS—ENZYME ACTION IN MARINE ALGAE 831

2. Negative results were obtained for cellulase, cytase,
maltase, lactase, sucrase, amidase, and esterase.

3. The action of all the enzymes isolated was very slow.

It is a pleasant duty to acknowledge indebtedness to those
who in various ways have lent support to this investigation.
Such acknowledgments are due Dr. George T. Moore for the
privileges of the Missouri Botanical Garden, especially its
library and laboratories; to Dr. Benjamin M. Duggar, at
whose suggestion the problem was undertaken, and under
whose direction and constant kindly interest it has been pur-
sued; to Dr. Phillip A. Shaffer, of the Biochemical Labora-
tories of the Washington University Medical School, for sug-
gestions and advice concerning biochemical methods, and for
the courtesy of his laboratory from time to time; and finally,
to the Woods Hole Biological Laboratory, for the privileges
extended during the summers of 1913-14.

Graduate Laboratory, Missourt Botanical Garden.

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1915]
DAVIS—ENZYME ACTION IN MARINE ALGAE 835

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[VoOL. 2, 1915]
836 ANNALS OF THE MISSOURI BOTANICAL GARDEN

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