close

Вход

Забыли?

вход по аккаунту

?

Патент USA US3097164

код для вставки
July 9, 1963
HIRAo sHlMAzoNo ETAI-
3,097,145
ACID PROTEASE AND THE PRODUCTION THEREOF
3 Sheets-Sheet 1
Filed March 30, 1962
FIG I
ha)2Y|DOE7(PNT5ISECAL
ACTIVITY
AGAINST CASEIN
EFFECT OF pH
O c»
ON
FIG 2
^ 30
l
l
l
I
2
3
4
è
5
6
7
8
Q 25
pH
N
ACIlvlTx
m
E20
AGAlNsT HAEMOGLOBIN
cn l5
EFFECT oF pH
Z
ä
1o
_1
FIG 3
3
5I: 5
D.
ACTIVITY
)2DlENO7/SPIT5LYC)AE.
O
|
2
3
4
5
5
AGAlNsT ALBUMEN
EFFEcToF pH
N
PH
FIG 4
$6
Èloo
>
E) 75
§TAB|L|TY
<x
^
_j 50
è
E
~ ë
g 25
w
ä
ll-I
C»
fr
ì
AQIlVlIY
g4
'-U
O
I:
5
l
l
|
|
l
|
2
s
4
5
e
J
7
EFFECT oF
TEMPERATURE
I
3
pH
AGAINST CA§E|N
a’ 2
INVENTORS
H/'rao ôfhl'mazano
9
E
EFFECT OF pH
Kafsum/ Tomada
l
i
. o
by WMM ¿M/¿??awgä
2o
3o
4o
5o
so
7o
TEMPERATURE (oC)
Aìtornays
July 9, 1963
HIRAo sHlMAzoNo ETAL
3,097,145
Acïn PROTEASE AND THE PRODUCTION THEREOF
Filed March 50, 1962
3 Sheets-Sheet 2
FIG 6
ë loo
>
egaal-_IH
eo
g
wlIHQLE SUESTRATE
E eo
EFFECT 0F TEMPERATURE
<1
_J
<1
:>
40
g zo
m
Lu
Ir
4o
5o
eo
7o
TEMPERATURE (°C)
FIG 7
ULTRAVIOLET
ABSOR PTION
DOEPNTSICAYL
SPECTRUM
5
0.5
230 240 250 260 270 280 290 300
3IO
WAVE LENGTH (MILLIMICRONS)
_
.
/N VENTORS
H/rao .S‘h/mazono
Kafsum/ Tomada
Afforneys
July 9, 1963
3,097,145A
HIRAO SHIMAZONO ETAL
ACID PROTEASE AND THE PRODUCTION THEREOF
3 Sheets-Sheet 5
Filed March 50, 1962
70
|
I4
750
B0
850
9|0H500
(cnFR/E1QU"N)CY
|
|20
IO
(MILCWERNOAGVST)HE
8
FIG
|3450 0
20 0
23540IO0 O
/N VEA/70H5
TRANSMITTANCE (°/°TRANSMIS 0N)
H/'roo S@ im ozono
Kofsum/ Tomado
by «JWM/W »L WHMMÉ,
Altar/lays
United States
ant
‘
ice
3,097,145
Patented July> 9, 1963>
2
l
As the classification of fungi is rather complicated and
a microorganism is sometimes called by two or more
3,097,145
ACID PROTEASE AND THE PRODUCTION
THEREOF
Hirao Shimazono, Suita, and Katsumi Tomoda, Toyo
naka, Japan, assignors to Takeda Chemical Industries,
Ltd., Osaka, Japan
Filed'Mar. 30, 1962, Ser. No. 183,919
5 Claims. (Cl. 19E-_62)
names which may be quite different from each other,
the descriptions of the microorganisms in this specifica
tion is based on the system set fort-h in “Mycological
Flora of Japan” by Seiya I-to, vol. Il, published :by
Yokendo, Tokyo, Japan, in 1955.
Thus, the principal object of the present invention is
to provide a process for producing novel protease, espe
cially the acid protease, which has stable and strong ac
This invention relates to a method for producing pro 10 tivity fio-r catalyzing the hydrolysis of peptider bonds in
tease, and more particularly, to a method for the produc
proteins at an acid pH.
»tion .of acid protease, which is useful in the -food industry
Another object of this invention is to provide a method
as well as for medicinal purposes such as a digestive.
for
the production of acid protease which can be etil
It is known that proteases, which have a catalyzing
activity on the hydrolysis of peptide bonds in various 15 ciently carried out on an industrial scale with very good
yield.
proteins, are contained in animal viscera or in the cells
It is `a further object of the invention to provide -an acid
and culture broth of microorganisms such as molds and
protease which is novel and useful in the `food industries,
bacteria, and some of these proteases can be industrially
and for medicinal purposes such as a digestive, etc. ow
produced and are commercially available. Proteases may
ing to its stability and strong activity to degrade proteins
be divided on the basis of the optimal pH at which they 20
at an acidic pH.
exhibit the said catalytic activity into three groups, i.e.
The first object is realized by incubating a fungus
neutral protease, -alkaline protease and acid protease.
selected from those belonging to the family Polypcraceae
commercially available proteases are almost exclusively
in an appropriate liquid medium containing assimilable
neutral proteases;
carbon sources, digestible nitrogen sources and other
25
One of the shortcomings of known protease .production
necessary nutrients for the growth of the fungus under
is the low yield «of protease inevitably caused by the poor
aerobic conditions, and by recovering from the resulting
capacity of the microorganisms employed for producing
broth the protease produced.
the protease. Another defect is that neutral protease
The microorganism used in the method of this inven
shows neither desirable catalytic action nor stability under
30 tion can be selected from those belonging to the family
acidic conditions of pH lower than 4.
Polyporaceae. Among tho-se microorganisms of this
In sharp' contrast, the microorganisms employed in the
family, Tramezes sanguínea (L. ex Fr.) Lloyd (=P0lypresent Iinvention can produce and accumulate acid pro
stictus sanguìneus Fr.), Trametes cinnabarìno (Jacq.) Fr.
tease in a high concentration in the culture broth. More
(=P0lyslictus cinnalbarinus Sacc.), Poria vaporaria (Fr.
over, the acid protease shows maximum catalytic activity
Pers.) Cooke, etc., for example, have been found
at an acidic pH ranging about from l to 4, and this char 35 non
to be especially suitable for the process of this invention
acteristic of the acid protease Iis especially suitable lfor a
because of their :properties not only to produce respec
digestive which acts against dietary protein in the stomach
tively a stable acid protease having very strong activity
where gastric fluid provides strongly acid conditions of
but also to accumulate a large amount of the -acid pro
about pH l to 3.
.
tease du-ring the cultivation in a liquid medium in a tank.
It is known that acid protease is produced by Asper 40 That
is to say, .an industrial production of 'acid protease
gilli, and that industrial production has -been tried, em
has become possible by the use of these microorganisms.
ploying one of these microorganisms, for rendering the
For the purpose of industrial production, it is in gen
useful acid protease commercially available. However,
eral lpreferable to use liquid culture media. Thus, the
these attempts have not yet yielded any desirable result
because of the fact that the cultivation of the micro 45 present invention now makes it possible to produce acid
protease industrially.
organism has been carried out lby solid culture, for ex
As the assimilable carbon sources, one or more of
ample according to the so-«called koji-process, and such
glucose, dextrin, soluble starch, sucrose, maltose, lactose,
a process cannot readily be scaled up to industrial magni
etc., for example, may be used, and various organic corn
tude.
The present invention is based on the use of micro
organisms belonging to the family Polyporaceae, these
being widely, capable of producing protease in good yield;
50 pounds or organic materials such as organic ammonium
salts, organic nitrates, urea, various amino acids, corn
steep liquor, peptone, polypeptone, casein, meat extract,
soybean cake, soybean flour, potato juice, etc. may be
more especially, the protease thus produced is usually
used not only as carbon source but also as digestible
acid protease showing a remarkably strong and stable
activity to catalyze Ithe hydrolysis of peptide bonds in 55 nitrogen source in the same way as the use of inorganic
nitrogen sources, for example, inorganic ammonium salts,
proteins.
such as ammonium sulfate, ammonium carbonate, am
Based upon the widely acknowledged classification of
monium phosphate, ammonium nit-rate, etc. or inorganic
microorganisms, the family Polyporaceae is placed in the
nitrates such as sodium nitrate, potassium nitrate, etc.
following situation:
60 In addition, mineral salts, phosphates, vitamins or growth
factors may desirably be used for the culture media as
accessory nutrients, e.g. water-soluble salts of vitamin B1.
The initial pH of the culture medium is adjusted to
Order _______________________ _- Agaricales
Family _______________ __ Polyporaceae
about 3 to 7. When Trametes sanguínea (L. ex Fr.)
Lloyd is used for the production of the acid protease, it
It is a novel feature of this invention that any of the 65 is preferred to incubate the microorganism in -the liquid
microorganisms belonging to even thewide scope of the
medium adjusted to and kept at a pH of about 3, espe
class Basidiomycetes, not to speak of those of the family
`cially Ifrom 3.0 to 3.2, because the resulting culture filtrate
Polyporaceae, can produce acid protease; more espe
contains more than two -times as much acid protease as
cially, it is a novel 'aspect of this invention that the pro
70 in the case where the ¿medium is adjusted to labout pH 5.
tease production is carried out by the use of said micro
Subdivision
Class
________________________ __ Eumycetales
________________ ..1 _____ _.-
Basidiomycetes
organisms belonging to the family Polyporaceae.
(The preparation' of acid protease is also possible even
3
3,097,145
by the means `so-‘called koji-process where solid materials
such a-s sawdust, rice fbuan, wheat bran, etc. are used for
the culture.)
The incubation is carried out desirably at a tempera
ture of Iabout 25-35° C. Iand the accumulated protease
in the culture broth reaches «the maximum usually be
tween the second and fthe fifteenth day of incubation.
4
ments was »acetone powder of enzymes extracted from
the iiltered broth obtained «by the cultivation of Trametes
sanguínea (L. ex Fr.) Lloyd.
(a) Action on beef-Ln 20 milliliters of an aqueous
suspension of 0.1 gram of chopped beef in a bulîer solu
tion of pH 2.8, «the protease preparation in a quantity cor
responding to 10 milligrams of protein was allowed to
Under such conditions, »the protease produced by the mi
react with the chopped beef at 55° C. After 20 minutes,
crooragnisrn is not accumulated within mycelia, but pene
no precipitate formed on the addition of trichloroacetic
trates out to the environment and is accumulated in the l0 acid to the mix-ture. The absorption of the suspension at
culture medium. Therefore, in the method of the pres
the wave length of 275 millimicrons-which corresponds
ent invention, the accumulated protease is mainly recov
to the characteristic absorption band of tyrosine--re
ered ffrom the liquid part of the culture broth, usually
markably increased and reached the maximum 80 min
from the filtered broth.
utes after the start of the reaction. After 100 minutes,
Generally-known means for recovering enzymes from 15 the
chopped beef lost its intrinsic stickiness as meat and
their solutions can ‘bepapplied to the recovery of the pro
vbecame brittle.
tease by utilizing -the properties of the same. The pro
On the other hand, another chopped beef suspension
tease obtained by the present invention can be 'adsorbed
having the same composition as above was allowed to
on various «adsorbents, or can be precipitated by precipi
stand `at 55° C. without addition thereto of the protease
tants. Moreover, generalmeans for recovery such as
preparation, whereupon' no change was observed during
precipitation near the ‘isoelectric point, saltin-g out, di
alysis, and combinations of these expedients may be
far longer period than 100 minutes.
Reaction time, minutes:
Absorption 1
effected for the purpose of recovery and purilication.
For example, an aqueous solution containing the pro
___________________________________ __ 0.000
20 __________________________________ __ 0.455
tease is subjected to salting out Iby the addition of :a salt 25
40 __________________________________ __ 0.460
such as sodium sulfate, ammonium sulfate, sodium chlo
60 __________________________________ __ 0.540
ride, or the like to the solution, or to fractional precipi
80 __________________________________ __ 0.680
tation by the addition of an appropriate hydrophilic or
ganic solvent such as methanol, ethanol, normal-pro
panol, acetone, dioxane, ttetrohydrofurane, or the like to 30
the solution.
As the acid protease obtained by the present invention
100 _________________________________ __ 0.680
1(E-value) of beef suspension (1 cm. layer) at the wave
length of 275 mu.
(b) Action on "waste yeast.”---“Waste Yeast,” which
is obtained as waste material from the extraction of ribo
nu-cleic acids from yeast and of course is not soluble due
concomitantly with its susbtrates, i.e. proteins-as well 35 to cell walls, is solubilized by the action of the acid pro
as at fairly strong acid pH, the filtered «broth wherein con
tease. In each of 5 milliliters of the enzyme solutions
is stable at a rather high tempera-ture such as 40 to 50°
C.--or 60 to 70° C. when the acid protease is placed
centration of the protease is still low, may `directly be
listed below in the concentration of 300 milligrams per
concentrated under reduced pressure. I-f desired, it may
cent, 100 milligrams of Waste yeast was suspended, and
be freeze-dried.
the suspensions were kept at 37° C. for 24 hours to give
When it is desired to employ lan «adsorption process, ad 40 the result shown Ibelow:
sorbents such as calcium phosphate, alumina, bentonite
_a colloidal native hydrated aluminium silicate (clay)
solubilized
_magnesium silicate, carboxymethylcellulose, weak acid
carboxylic acid-type cation exchange resin, and -the like,
may advantageously be employed.
Enzyrnes
- Acîdîty of the medium
As the cation ex 45
yeast (percent)
change resins, Ifor example, “Duolite CS~-10l”-manufactured and sold by Chemical Process Co., U.S.A.
“Amberlite lRC-50”-manufactured and sold by Rohm
& Haas Co., U.S.A.-etc., are especially suitable for this
purpose. These resins 'are commercially available and
can 'generally be produced by such methods as described,
for example, in “Ion Exchange Resins,” second edition,
pages 85-87, by Robert Kunin, published by John Wiley
& Sons, Inc., U.S.A., in 1958. Adsorption and elution
solid part rel
ative to the
original solid
part in waste
N/lOO-HCI acidic _______ __
11. 9
Trgpsin _________________ __ pH 7 (phosphate buüer)...
26.4
Acid protease preparation. N /100-HC1 acidic _______ ._
Pe sin __________________ __
61. 0
The following examples set forth presently-preferred
exemplary embodiments of the present invention; they
>are intended to be `solely illustrative, however, and not
at rall limitative of the invention. In the examples, per
may be carried out 'by column chromatography or in 55 centages are on the weight basis unless` otherwise noted.
batch process.
“ATCC” refers to American Type Culture Collection,
The protease may be precipitated from its aqueous so
Washington, D.C., U.S.A.
lution by the addition of `a proteín-precipitant such as
Example I
nucleic acids, tannic acid, phosphotungstic acid, or the
like.
Tnametes sanguínea (L. ex Fr.) Lloyd (IPO-7045) was
60
inoculatedin 5 liters of the culture medium of pH about
Precipitation of the protease is facilitated by adjust
5.2 which consisted of 5 percent of dext-rin, 2 percent of
ing the pH of the solution to .fthe isoelectric point. Elec
cornsteep liquor, 0.15 percent of potassium dihydrogen
trodialysis may also .be employed for the purpose of puri
phosphate, 0.05 percent of magnesium sulfate (7 hy
iication o-f the acid protease.
The ‘acid protease thus produced and recovered has 65 drate), 2 milligrams per liter of thiamine hydrochloride,
and water, and then incubated for 6 days under shaking
the outstanding properties of strongly catalyzing the hy
at la temperature between 25 and 30° C. At the end of
dolysis of peptide-bonds in proteins, and the catalytic
the incubation, mycelia were plentifully grown. The
action is independent of the kind of protein involved.
culture ibroth was centrifuged to separate a clear aqueous
Therefore, the protease can remarkably degrade, a Vari
layer from the mycelia. Ethanol was added to` the aque
70
ety of proteins such as casein, hemoglobin, albumin, etc.
ous layer to obtain 25 »grams of white enzyme powder.
to the same degree.
The enzyrnic activity as protease of the powder at pH
'I'he following illustrations show the activity of the
3.0 was:
acid protease and indicate the practical utilization of the
same. The protease preparation used in these experi 75
3,097,145
il
The Iabbreviation to show protease activity, i.e.
PUâiìâlyî" 2"’ m“
means that casein as substrate was exposed to the action
of the protease at 37° C. for 20 minutes and the increase
in the light absorbence at the wave length of 275 mil
lirnicrons during the reaction was measured; and that the
absorbence corresponding to 1 milligram equivalent of
tyrosine at »the same w-ave length was «taken as the unit
of enzymilc' activity. The abbreviation “/mg. P.” means
per milligram protein.
6
was gradually added, while cooling,y ca. 20 percent by
volume of `cooled acetone, and the mixture was allowed
to stand in an ice chest. After 24 hours, crystals began
to appear and the crystallization was completed after 4
to 5l days. The protease activity of the crystalline enzyme
thus obtained was:
m2563222' 275 m: ses/mg. P.
It was found that about 10 percent of the protease activi-ty
in the culture íiltrate was preserved in .the crystalline prod
uct. It was also observed that the speciiic activity of the
product was ca. 20' times as high as that of the culture
Tram-etes cínnabarína (Jacq.) Fr. (IPO-6139) was
ñltrate.
employed instead of Trametes sanguínea (L. ex Fr.)
In .the event that the dialyzed solution «is colored, the
Lloyd in the procedure precedingly set forth in this ex
color-imparting impurities can be eliminated by passing
15
ample, acid protease powder was again obtained. The
the colored solution through a tower packed with a Weak
strains of Trametes sanguínea (L. ex Fr.) Lloyd and
base anion exchange resin (eg. Duoli-te A-7, manufac
Tmm-etes cinnaibarina (Jacq.) Fr. used in the present
tured and sol-d by Chemical Process Co., U.S.A.) which
example are on deposit at ATCC under accession numbers
can be produced, for example, by »the method described
ATCC-14622 and ATCC-14623, respectively.
20 by Kunin, supra, pages 87-88 and 97. The thus decolored
Example 2
solution is then further processed as precedingly described.
The crystalline acid protease obtained in this example
Poria vaporaria (Fr. non Pers.) Cooke (IPO-7044)
shows ythe following properties:
was inoculated in 5 liters of the culture medium of pH
(1) Its optimum pH is about 2.3 to 2.5 as shown in
5.2 which consisted of 5 percent of glucose, 0.3 percent of
polypepton-e, 0.15 percent of potassium dihydrogen phos 25 FIG. l of the accompanying drawings and its optimum
temperature is about 55 to 60° C. as shown in FIG.
phate, 0.05 percent of magnesium sulfate (7 hydrate),
5-when the activity is tested against casein as substrate.
0.2 percent of yeast extract, 2 milligrams per liter of
It
also shows the optimum »pH 2.8 against hemoglobin
thiamine hydrochloride and water and then incubated
as shown in FIG. 2 and the optimum pH 3.4 against egg
for 6 days under shaking at a temperature from 28 to
30° C. After the incubation, the culture broth was ñlte-red 30 albumin as shown in FIG. 3;
(2) `It is stable in the pH range from 2.0 to 6.5 as
to obtain filtered broth. The protease activity of the
shown in FIG. 4, and loses the activity by heating at
iiltered broth was determined through a modiñcation of
70° C. lfor 10 minutes without substrate as shown in
Kunin’s method by the use of lactate buffer solution of
FIG. 6;
pH 2.8 to be:
35
(3) Its ultraviolet absorption spectrum shows the curve
of .a typical protein which has the maximum absorption
at the wave length of 277-280 millimicrons, as shown in
Ethanol W-as added to the filtered broth to obtain 20
FIG.
7;
grams of enzymic powder preserving the acid protease
activity.
The strain of Porìa vaporaría (Fr. non Pers.) Cooke
used in the present example is on deposit at ATCC under
number ATCC-14624.
Example 3
(4) I-ts uniformity is coníirmed through electrophoresis
40 by Tiselius’ apparatus, where 15 percent of the enzymic
protein in phosphate buifer (,u=0.1) of pH 6.08 is sub
jected to an electric field of 72 volts at the current of l0
mil-liamperes for 3 hours;
(5) The uniformity of the protease was ¿further estab
Trametes sanguínea (L. ex Fr.) Lloyd (ATCC-14622; 45 lished through the analysis by ultracentrifuge in acetate
buffer (n=0.l) of pH 5.0 at a speed of 9.667><10'2 rounds
.the same strain as used in Example l) Was inoculated in
30 liters of the culture medium of a pI-I of 3.0‘ to 3.2 in
per second for 75 minutes.
a tank, «the medium consisting of 5 percent of dextrin, 2
9.75><10“13, Vfrom which the molecular weight of the
sedimentation constant was
percent of cornsteep liquor, 0.15 percent of potassium
acid protease was calculated to be about 3.0)(104;
(6) The elementary analysis of the protease:
dihydrogen phosphate, 0.05‘ percent of magnesium sul 50
fate (7 hydrate), 2 milligrams per liter of thiamine hy
dnochloride, and water, and then incubated for 6 days
under agitation and aeration at a temperature of 25 to
C,
percent
H,
percent
N,
percent
30° C. After the incubation, \ he culture broth was filtered`
to obtain filtered broth. Acetone was added to the ñltered 55
broth to give 150 grams of white enzyme powder. The
(1) ____________________________________ ._
(2) ____________________________________ __
(3)__-_
___
44 18
44 16
__...
7. 12
7. 12
_\
__
14. 2
12. 78
13.37
enzyme powder was dissolved in a small amount of
hydrochloric acid-sodium acetate bulfer solution of pI-I
3.5 in the molar concentration `of 0.02 mole, and the solu
(7) The infrared absorption spectrum of the protease
tion was allowed to pass through a column of weak acid 60 is shown in FIG. 8 and the significant absorption bands
carboxylic yacid-type cation exchanger (“Duolite CS
101”), which had been pretreated with the buffer solu
t-ion of the same constitution as above-used, and then the
protease absorbed in the resin was eluted with the hy
drochlori‘c acid-Sodium acetate buffer solution of pH 65
5.5 in the molar concentration of 0.5 mole. From the
eluate, fractions showing protease activity were collected
and submitted to fractional precipitation by ammonium
in microns are as follows:
3.0 (strong)
3.38 (middle)
6.105 (broad, strong)
6.55 (broad, strong)
6.88 (weak)
7.15 (broad, middle)
8.12 (middle), and
9.3 (broad, weak)
sulfate. Precipitates obtained at between 40 and 80 per
ment saturation of ammonium sulfate were collected and 70
(8) It is easily soluble in water but insoluble in or
dissolved in a small amount of said buffer solution of pH
ganic solvents such as methanol, ethanol, acetone, ether,
3.5 in the molar concentration of 0.02 mole, and the solu
benzene, petroleum ether, etc.; and
tion was subjected `to dialysis for 24 hours against the
(9) Crystal form of the protease is usually needle or
buffer solution of the same constitution as the solvent.
Into the solution remaining Within the diaphragm, there 75 -platelet shape.
3,097,145
Having thus disclosed the invention, what is claimed is:
1. A method for producing acid protease, which com
(4) sta-bility in the pH range Ifrom 2.0 to 6.5, as indi
cated in FIG. 4 of the drawings;
prises incubating a member selected from the group con
(5) maximum absorption, in its ultraviolet absorption
sisting of Trametes sanguínea (L. eX Fr.) Lloyd, Trametes
cinnabarz'na (Jacq.) Fr. and Porìa vapomria (Fr. non 5
Pers.) Cooke in an aqueous medium containing assimi
lable carbon sources, digestible nitrogen sources and other
nutrients necessary for the growth of the microorganism
at a temperature of from 20 to 35° C. under aerobic
conditions until the acid protease is substantially accumu 10
lated in the culture broth, ‘and recovering the accumulated
acid protease ‘from the liquid part of the culture broth.
ATCC-14623.
4. The method as claimed in claim 1, wherein the
microorganisrn is Poria vapora‘rìa (Fr. non Pers.) Cooke, 20
ATCC~14624~
5. A crystalline acid protease, metabolite of an acid
protease-producing microorganism belonging to the family
Polyporaceae, which has the following properties:
(l) optimum activity, when tested against casein as 25
substrate, at a pH of about 2.3 to 2.5, as indicated in
=as substrate, at a pH of about 2.8, as indicated in
as substrate, at a pH ‘of about 3.4, as indicated in
FIG. 3 of the drawings;
(8) infrared absorption spectrum as shown in FIG. 8
3.0 (strong),
3.38 (middle),
6.05 (broad, strong),
6.55 (broad, strong),
6.88 (weak),
7.15 (broad, middle),
8.12` (middle), and
9.3 (broad, weak);
(9) soluble in water but insoluble in organic solvents
methanol, ethanol, acetone, ether, benzene and petro
leum ether;
(10) elementary analysis being between 44.16 and
44.18% by weight carbon, about 7.12% by weight
hydrogen and between 12.78 and 14.2% by weight
nitrogen, the latter as determined by the Dumas
method.
FIG. 1 of the drawings;
(2) `optimum activity, when tested against hemoglobin
»
of the drawings;
(7) molecular weight .of about 3.0)(104;
are as follows:
microorganism is Trametes sanguínea (L. ex Fr.) Lloyd,
ATCC-44622.
15
3. The method as claimed in claim 1, wherein the
microorganism is Trametes cìnnabarina (Jacq.) Fr.,
FIG. 2 of the drawings;
microns, as indicated in FIG. 7 of the drawings;
(6) 'loss of activity upon being heated at 70° C. for
10 minutes without substrate, as indicated in FIG. 6
wherein the significant ‘absorption bands in microns
2. The method as claimed in claim 1, wherein the
(3) optimum activity, when tested against egg albumin
l spectrum, at the wave length of 277-280 milli
30
References Cited in the ñle of this patent
Gonshvili: Chem. Abst., 46, 4591d (1952).
Levine et al.: Journal of Bacteriology, 1953, pages
10-15, page 11 particularly relied on.
Документ
Категория
Без категории
Просмотров
0
Размер файла
689 Кб
Теги
1/--страниц
Пожаловаться на содержимое документа