US3655570A - Detergent containing alkali protease - Google Patents

Abstract

Alkaline protease if produced by cultivating an alkaline protease-producing microorganism from the genus Fusarium or Giberella in a proper growth supporting medium and the alkaline protease is recovered from the medium. The alkaline protease degrades protein under conditions of high pH and thus is useful in the formulation of detergent and other cleanser compositions.

Description

United States Patent Isono et al.

[is] 3,655,570 1451 Apr. 11,1972

DETERGENT CONTAINING ALKALI PROTEASE lnventorsi Masao I sono, Nishinomiya; Katsumi Tomoda, Toyonaka, Osaka; Kouichi Miyata, Takatsuki, Osaka; ,Kazutaka Maejima, Nishinomiya; Keisuke Tsubaki, Suita, Osaka, all of Japan Assignee: Takeda Chemical Industries, Ltd., Osaka,

- Japan Filed: Feb. 3, 1969 Appl. No.: 795,951

Foreign Application Priority Data Feb. 8, 1968 Japan ..43/7863 Mar. 15, 1968 Japan ..43/l6970 U.S. Cl ..252/132, 252/89, 252/135 Int. Cl. ..Clld 7/42, Cl ld 9/40 [58] Field ofSearch 195/62, 66, 63, 68; 252/135, 252/DIG. 12

References Cited UNITED STATES PATENTS 3,451,935 6/1969 Roald et al ..252/1 35 Primary Examiner-Lionel M. Shapiro Attorney-Wenderoth, Lind & Ponack [5 7] ABSTRACT 5 Claims, 6 Drawing Figures PATENTEDAPRH 1912' I 3,655,570

'SHEET 2 OF 5 (70) HDNVLLIWSNVHL MASAO ISONO KATSUMI ToMbDA,

KOUICHI MIYATA,

KAZUTAKA MAEJIMA &

KEISUKE TSUBAKI INVENTORS Attorneys PATENTEDAPR n 1912 3,555,570

' SHEET u 0F 5 Fig. 5

MASAO I SONO KA'ISUMI TOMODA,

KOUI CHI MIYATA KAZUTAKA MAEJIMA AND KEISUK E TSUBAKI Inventors Bywwm: AM

Attorneys PATENTEDAPR 11 191? 3. 655,570

SHEET 5 BF 5 Fig. 6

TEMPERATURE ('C) All/\LLOV 'IVOOISHU MASAO ISONO,

KATSUMI TOMODA,

KOUICHI MIYATA,

KAZU'IAKA MAEJIMA' AND KE'ISUKE TSUBAKI,

Inventors By, MMM,%LL

Attorneys DETERGENT CONTAINING PROTEASE The present invention relates to a novel alkali protease and to a process for producing the protease as well as to detergents and other cleansers containing the enzyme.

in the course of study for obtaining alkali proteases, it'has been found that certain microorganisms belonging to the genus Fusarium or the 'genusGibberella produce an alkali protease which exhibits a potent proteolyticactivity around It has been found that the said protease actively degrades various kinds of protein under conditions of highpH even in the presence of'surfactantsand/or chelating agents,'suggesting its applicabilityin the field of the detergent industry. 'l-leretofore, although certain enzymes'havebeen applied to' the laundry process, satisfactory results have not=always been obtained. One of the possible causes-therefor is th'at most cleansing solutions have a'pH over pl-l8. Another cause is that the enzymic activity 'is sometimes inhibited'by' surfactants and/or chelating agents contained in the-detergents.

"In thisconnection, further extensive studies-have been carried out'and a process established forjproducing a potent al 'kali protease on a'cornmercial'scale and manufactured detergents and other clean'sers containing' the enzymes'lhe enzyme is referred toherein as alkali protease.

"The principal object of the'present'invention is therefore-to provide an alkali proteasewhich exhibitspotent activityin the p'l-l r'arlge'from 8:0 to 1210, particularly, from 10.0 to 1 1 .5.

Another object is toprovidedetergents and other cleansers containing' thealkaliprotease.

ln order 'to' realize the objects of the present invention,'-a "strain of alkali protease-producing microorganisms belonging to the genus Fusariumorthe genus Gibberellais cultivated in a culture medium. Some ;typical-=microoganisms' producing the alkaliprotease are as'follows:

Fusariumoxysporum (ll- 05942) ('ATCC659) Fusari'umoxysporum fibatatas (IFO 4468) 'Fusarium oxysporum f. gladioli- "(IFO 5894) Fusarium oxysporumfllini (IFO 5880) Fusariumoxysporumf; neveum IFO 4471) Fusarium-bxysporumf.*liriilvasinfectum :(IFO4472) Fusarium solani (lFO 5232) Fusarium-spl s:1'9:5 (IFO8884) ATCC 20l92) {Gibberlldfujikuroi n= o 5268) I I I Gibberella saubin'etii 1 (lF'6608) (ATCC 20193 lFO numbers in the parentheses are the accession numbers at the institute for Fermentation, Osaka, Japan; ATCCnumb'e'rs-areaccession numbers at American Type Culture Collec- "'tion Rockville', Maryland.

* Amongthese microogranisms,"Fusarium sp'.' S'-19-5-is one of the "most useful strains for the production of jthe alkali protease. This strain was isolated from a soil-sample-"by the inhyphae; wrinkled on reverse; a scarlet-purple pigment r 'in themedium. 3'. Czapeckssolution:

On each of the above media, conidiophores develop from aerial hyphae, unbranched, 10 to along, 1.0 to 1.5 uwide, hyaline. Conidia are produced apically on conidiophore and grouped in a slimy cluster, ovate or kidnay-shaped, one to two-celled, rarely three-celled, 5 to 8 I by l to 2 p), hyaline. -No sexual state develops. Chlamydospore usually absent, sometimes present. 3. Physiological characteristics: 1. Condition for growth:

Hydrogen ion'concentration: Preferably grows in alkaline media,optimal at between pH 8.0 to 9.0 Temperature; Optimal at about 24 C. Allow to grow'between -l5 and 28 C.,-although better growth is observed around at 15 C. rather than at 28 C. Oxygen requirement: Aerobic. 2. Gelatin: liquefaction:

Slight. 3; Utilization of ethyl-alcohol:

Negative. 4. Degradation of pectin:

Very slight. 5 Degradation of tannin:

Negative. 6 Degradation of fats and oils:

Positive. 7 Utilization of carbohydrate:

Utilizes maltose, r'galactose, melibiose, sucrose, trehalose, fructose, .mannose, raffinose, dextrin, starch,1glucoseand lactose. Slightly utilizes inulin, xyloseparabinose and cellulose. According to Dictionary of theFungi (G.C. Ainsworth, 5th edition), the said microbial characteristics indicate that this particular strain'belongs to the genus Fusarium, the family 'luberculariaceae, the order Moniliales, the subclass Deutermycetes, the class Fungi Imperfecti.

Based on the Snyder&Hansen classification,

i-Eusarium is divided into Section 1fifl 2 follows:

: A rmicroconidia develop, usually one-celled.

A no microconidia develop or rarely produced, spindle,

comma'or kidney-shaped, one to'several-celled. The organism, Fusarium sp.-S-l9--5,' produces microconidia --dominantly and can be included in th Elegans Group of SectionA. The Group Elegans'isitypified by F usarium oxysporum and includes 36 species which are all plant pathogens producing macroconidia in abundance.

"The organism, Fusarium sp. S-l9-5, however, produces only microconidia in mostcases andthe formation of macroconidiais very rare. This characteristic indicates the or- #:ganism cannot be assigned'to any of the hitherto known species of'this group. 7 v

In order to cultivate analkali protease-producing microorganism, per se conventional culture media and conditions are used.

-itis more advantageous to adopt shake culture or aerobic cul- -ture methods.

a The medium may be usedin either liquid or solid form. The culture maybe effected under stationary conditions, however,

"ple, it may contain, as carbon sources, such materials as glucose, sucrose, dextrin, starch, cellulose glycerol, sorbitol and Good growth,- white; flocculant pellicle formed; wrinkled on reverse; substantially no pigment. 4YCz apecks agar:

* Good growth, white and-flocculant; wrinkled onreverse;

substantially no pigment. 5. Gelatin:

Good growth, white and flocculant; dark blackish purple pigment onreverse; weak liquefaction. 6." Potato dextrose:

" Goodgrowth, white and flocculant;-the surface uneven,

raised; wrinkled on reverse,- white tolight purple; pur- 'ple pigment liberated in the medium. 2; Microscopic characteristics:

the-like, and, as nitrogen sources, such materials as peptone,

meatextract, yeast extract, dried yeast, soybean meal,

soybean cake, rice'bran, wheat bran, potato extract, casein,

gluten, casein hydrolysate, corn steep liquor, urea, ammonium salts, nitrates and other organic or inorganic nitrogenous compounds. As inorganic salts, various phosphates, sulfates and hydrochlorides, for example, may be incorporated. Under certain circumstances, for the purpose of promoting growth of "the microogranism, various vitamins, amino acids, nucleic acids and their related compounds, etc. may be added. Depending upon the culture methods and conditions to be employed, the addition of a natural or synthetic defoarning agent, e.g., soybean oil or silicone oil, may be effective to increase accumulation of the enzyme.

ln cultivating the microorganisms, it is preferable to prepare a small scale preculture which is, in turn, inoculated into a main culture medium.

Culture conditions such as incubation temperature and time, pH of the medium, aeration rate, etc., should vary with the microorganism and medium compositions to be used.

The requisite is that the conditions should be selected and controlled so that the accumulation of the alkali protease is maximal. In many instances, the preferred conditions include the incubation temperature of from 20 to 30Co., an incubation time from 2 to 7 days, a medium pH of near 7, and an aeration rate of 0.5 to 1.5 liters per minute per liter of the medium.

Thus, the microogranism accumulates a large amount of the alkali protease in the culture. When a liquid medium is employed, the object enzyme occurs mostly in the liquid phase of the culture. Therefore, it is preferable to follow the steps of removing mycelia by filtration or centrifugation and, then, of recovering and isolating the enzyme from the filtrate or the supernatant fluid as the case may be.

When a solid medium is employed, it is usually preferable to subject the culture to extraction with water or an aqueous solution of an inorganic salt and, then, to recover the enzyme from the resulting extract.

In order to recover and isolate the enzyme from the culture filtrate, supernatant or extract, any of per se conventional isolation and purification means may be employed. These include salting-out of enzyme with an inorganic salt such as sodium sulfate, ammonium sulfate or sodium chloride as well as fractional precipitation of enzyme by adding a suitable hydrophilic organic solvent such as methanol, ethanol or acetone. In addition, an enzyme solution may be concentrated under reduced pressure and/or demineralized by dialysis. It is also possible to employ means such as adsorption and desorption on calcium phosphate gel, alumina, bentonite, ion exchange resin, etc., chromatography using a cellulose derivative, e.g. diethylaminoethyl cellulose, gel-filtration, precipitation, electrodialysis, electrophoresis and removal of impurities as heavy metal complex.

The alkali protease produced and prepared in the foregoing manner is active over a broad pH range between 8.0 and 12.0, particularly in the pH region of 10.0 to l 1.5.

The optimal temperature for enzyme activity lies somewhere between about 20 and about 60 C., particularly between about 40 and about 50 C. This temperature range for the activity is in quite good accord with actual laundry conditions. Moreover, the activity of the alkali protease is not inhibited by surfactants and/or chelating agents which are principal ingredients in detergent products or cleansers.

In this invention, the alkali protease may be employed not only as a highly purified preparation but as a crude enzyme product and thus the product is obtained in a desired purity in accordance with the purpose; either pure or crude preparation of the enzyme may be used as an ingredient of detergent products or cleansers.

As surfactants contained in the detergent products, there may be mentioned for purpose of exemplification various compounds including anionic surfactants of the fatty acid salt type, sulfate type or sulfonate type, such as natural fatty acid soap (NS), alkylsulfate (DAE), olefine sulfate, n-a-olefine sulfonate (AOS), tetrapropylbenzene sulfonate (ABS) and n-alkylbenzene sulfonate (LAS); and non-ionic surfactants of the ether type or ester type, such as polyoxyethylene alkyl ethers, polyoxyethylene alkylphenol ethers, polyhydric alcohol alkyl esters, polyoxyethylene alkyl esters, sugar esters and the like.

The detergent products or cleansers may contain builders such as tri-polyphosphate, sulfates, carbonates, borates, as well as carboxymethyl cellulose, fluorescent dyes, scents, bleaching agents, e.g. perborates, chelating agents, e.g., N(CH COONa);;, skin-protective agents, e.g. dimethyllaurylaminoxide, disinfectants, e.g. tertiarily amine, and

the like. he

The alkali protease is mixed with other components of the product which may be a powdery, granular or liquid form and,

when the resulting mixture is a liquid, it may be dried to a powdery or granular product by conventional means such as p y y s- Since there is no international system of unit for this kind of enzyme, it is difficult to prescribe the portion of the enzyme in detergent products or cleansers in general.

For purposes of the present invention, activity of the alkali protease is assayed by the following modified Kunitz method (J. Gen. Physiol. 30, 291, 1947). 1.0 ml. of a 2 percent auqeous solution of casein (I-Iammarsteins) and 0.5 ml. of an NaOI-I buffer, pH 11.0, are mixed with 0.5 of an enzyme solution. 2.0 ml. of the resulting mixture is allowed to incubate at 37 C. for 20 minutes, and then the reaction is stopped by the addition of 3.0 ml. of a 5 percent aqueous solution of trichloracetic acid. The mixture is allowed to stand at 37 C. for a further 30 minutes, whereby the undigested casein is thoroughly precipitated. The precipitated casein is filtered off and the resulting filtrate is subjected to optical density determination at 275 mu, from which the amount of the digested casein is calculated as the amount of tyrosine. One unit of protease activity (PU) is defined as the amount of the enzyme which dissolved an amount of casein equivalent to 1.0 pg. of tyrosine per minute under the assay conditions,. The specific activity of an enzyme sample is expressed as PU/mg.

The portion of the alkali protease to be incorporated in detergent products will vary with the type of product. In the case of a detergent to be used for washing cloth, the preferred amount will generally range from 5 to 10,000 in terms of units per gram of the detergent and for practical purposes from 10 to 5,000 units per gram.

An alkali protease-containing detergent prepared in the foregoing manner shows exceedingly powerful cleansing action against proteinous stains atributable to sweat, blood, broth and milk, which are hard to wash away with a conventional detergent.

Further, the detergent is employed in prewashing as well as in mainwashing.

In prewashing, soiled materials are usually soaked in a detergent solution at a room temperature for several hours, while the mainwashing is preferably carried out at an elevated temperature between 40 and 60 C. for 1 hour or so.

Presently preferred embodiments of the invention are shown in the following examples, but they are not intended to be construed as a limitation on the present invention.

Throughout the specification, the abbreviations ml, mg., p.g,p., mu and C." mean milliliter(s), milligram(s), microgram(s), micron(s), millimicron(s) and degree centigrade, respectively. Percentages are calculated on the weight per volume basis. In the following examples, parts by weight bear the same relation to parts by volume as do gram(s) to milliliter(s).

EXAMPLE I 500 parts by volume of a liquid medium, composed of 5 percent defatted soybean meal, 5 percent glucose, 2 percent sodium dihydrogen phosphate, and adjusted to pH 7 is dispensed in a fermenter (its capacity being 2,000 parts by volume), sterilized, inoculated with Fusarium sp. S-l9-5 (IFO 8884) (ATCC 20192) and incubated at 28 C. for 5 days under aeration and agitation to prepare a seed culture.

The seed culture is inoculated to a fermenter (its capacity being 50,000 parts by volume) containing 30,000 parts by volume of the same liquid medium as above, and the fermenter is incubated at 25 C. for 144 hours with the aeration rate of 45,000 pars by volume per minute under agitation of 500 rev./min. During the incubation, foaming is suppressed by the addition of a suitable amount of soy bean oil from ime to im Changes of the pH value and the protease activity course of the cultivation are shown as follows:

Time of culture (hours) 18 30 42 54 66 78 90 122 144 pH of culture 7.30 6. 40 6.30 6.12 6.35 6.72 7. a0 7. 09 7. 50 1,50 Enzyme activity (PU/ml.) 67 141 1,430 2,250 2. 270 2, 520 2, 520 2. 760 2,540

EXAMPLE 2 Soluble in water and aqueous mineral salt solutions hav- The culture obtained after 144 hours, as in Example 1, is $25235 s figfifi iogginzerlnsoluble 1n cooled to about C. and, then, passed through a filter press 9 pH activity (See FIG with the filter aid, Hyflo Super-Ce] (Johns-Manville Products optimal PH at about 11 Corp. U.S.A.), whereby the mycelia is removed. To the result- Temperature activity s FIG ing 20,000 parts by volume of the filtrate is added 0.6-satu- Optimal temperature about 5 rated ammonium sulfate, and the salted-out precipitate is col- 1 1 pH Stability (See FIG. 5 lected by filtration with the filter aid. The resulting ammonium N i tivation in the pH range from 5 to 8; 10 percent sulfate precipitate containing the filter aid is dissolved in i ti i n t H 11.5 (1 hour-incubation at 37C.). about 6,000 parts by volume of cold water and insoluble 12. Temperature stability (See FIG. 6): materials are removed by filtration. 0.6-Saturated ammonium No inactivation below 55 C; percent inactivation at sulfate is then added to the filtrate, as to precipitate the en- 65 and 80 percent at 70 C. (10 minute-incubation at zyme again, which is, in turn, collected by centrifugation, dispH 5). solved in 1,000 parts by volume of cold water, dialyzed against 20 EXAMPLE 4 cold water by means of fish-skin diaphragm for 4 days and lyophilized to give a crude enzyme powder. By the above procedure, 30 parts by weight of the crude enzyme powder with brownish color is obtained. The specific activity of this sample is bout 980 PU/mg.

EXAMPLE 3 1n 3,000 parts by volume of cold water is dissolved 30 parts by weight of the crude enzyme powder prepared in Example 2, and insoluble materials in the enzyme solution are filtered off with the filter aid to obtain a clear solution. 0.6-Saturated ammonium sulfate is added to the clear filtrate to give precipitate of the enzyme which is, then, re-dissolved in 1,500 parts by volume of cold water, decolorized with charcoal, dialyzed against cold water at 45 C. for 4 days and liphilized to a powder. The above procedure yields 4.5 parts by weight of a partially purified enzyme powder with a specific activity of 1,580 PU/mg.

4.5 parts by weight of the enzyme powder, dissolved in 450 parts by volume of 0.01 M Tris-HCl buffer, pH 9.0, are dialyzed against 0.001 M Tris-HCl buffer, pH 9.0, for 3 days put on a column of diethylaminoethyl cellulose previously equilibrated with the Tris-HCl buffer and eluted with the same buffer. About 675 parts by volume of the enzyme rich fraction are collected, dialyzed against cold water for 3 days and lyophilized to a powder. The above procedure yields 0.35 part of purified enzyme in a powdery state with a specific activity of 5,400 PU/mg.

0.35 Part by weight of the purified enzyme powder, dissolved in 335 parts by volume of 0.01 M Tris-HCl buffer., pH 8.0, is chromatographed through a column of Sephadex G-100 previously washed with the TrisHCl buffer. 62 parts by volume of the active fraction is collected, dialyzed against cold water for 3 days and lyophilized, whereupon 0.15 part by weight of a highly purified enzyme powder is obtained. The specific activity of this sample is 6,500 PU/mg.

The enzymological characteristics of this example are as follows:

1. Color and shape:

White powder. 2. A typical elementary analysis C, 42.72; H, 6.59; N, 15.03. 3. Sedimentation constant (S about 3.19 X 10. 4. Molecular weight (by Archibalds method): 3.65 X 10 (15%). 5. Ultraviolet absorption spectrum (see FIG. 1):

Maximal absorption at 275-280 mu; 280nm,tcm 6. Infrared absorption spectrum (See FIG. 2):

Significant absorption bands in microns at 3.38, 6.05,

6.55, 6.88, 7.15, 8.12, 9.30. 7. lsoelectric point (by paper electrophoresis):

About pH 11. 8. Solubility:

Influence of several surfactants on the alkali protease is examined as follows:

According to the formula given in Table l, five different detergent compositions are prepared where sodium salt of n-C natural fatty acid (NS), sodium n-C -alkylsulfate (DAS), sodium n-a-C olefmesulfonate (AOS), sodium tetrapropylbenzenesulfonate (ABS) and sodium n-C -alkylbenzenesulfonate are used as the surfactants, and these result- 5 Mg. each of the respective detergents is dissolved in 2 ml. of an enzyme solution prepared by dissolving 2 mg. of the crude enzyme powder obtained in Example 2 into ml. of 0.05 M Tris-HC1 buffer, pH 1 1.0, and the enzyme activity of the resulting solution is assayed according to the specified assay method. The results are shown in Table 2 which indicates that none of these detergents inhibits the enzyme activity.

Enzyme-containing detergent compositions are prepared by blending 0.05 g. of the crude enzyme powder obtained in Example 2 with 100 g. each of the above different detergents.

TABLE 3 Prepared by the method of Japan Oil and Fat Chemical Association, using a 1:1 mixture of air cleaner dust and casein in place of carbon black.

Soiled Cloth (5 cm X cm) The cleaning activity is evaluated by a panel monitation. Five pieces of the soiled cloth are assigned to the test of each detergent and, after the sequence of washing, rinsing and drying under the specified laundry test, the resulting washed cloths are scored by five judges according to the following standards of score.

Standards of score:

+2 Cleanliness of the cloth washed with an enzyme-containing detergent is definitely superior to that with the corresponding enzyme-free detergent.

+1 Cleanliness of the cloth washed with an enzyme-containing detergent is slightly superior to that with the corresponding enzyme-free detergent.

0 No significant difference is observed in cleanliness between the cloths.

The results of scoring are shown in Table 4, where the relative cleansing effects of the enzyme-containing detergents versus the corresponding enzyme-free detergents are presented, as to the respective types of detergent.

In the same manner as in Example 1, several microogranisms belonging to the genus Fusarium and the genus Gibberel- 1a are cultivated for 6 days.

The cultures are then centrifuged to give supernatant fluids which are used as enzyme solutions. To 2,000 parts by volume each of the enzyme solutions are added 5 parts by weight of the LAS-detergent described in Example 4, and protease activity of the resulting solution is determined by the specified assay method. The results are set forth in Table 5 indicating that the LAS-detergent does not inhibit any activity of the enalkali zymes produced by those protease-producing microogranisms.

TABLE 5 Enzyme Activity (PU/ml) Mircoogranism with without detergent detergent Furan'um vxysporum (ll-O 5942) (ATCC 659) 200.8 199.2 Furarium uxyrporumf. lini (ll-O 5880) 550.5 553.8 Furarium oxyrporum f. niveum (IFO 4471) 315.8 307.2 Fusarium .rvlani (IFO 5232) 230.2 235.0

Gibberella fujikuroi (IFO 5268) 73.2 75.6 Gibberella .ruubinelti (II-O 6608) (ATCC 20193) 530.2 523.4

EXAMPLE 7 A liquid detergent for kitchen use: In 55 parts by volume of hot water at 60 65 C. are dissolved 18 parts by weight of sodium tetrapropylbenzenesulfonate, 12 parts by weight of sodium n-C alkylphenolether-sulfate, 5 parts by weight of lauryldiethanolamide and 10 parts by weight of sodium xylenesulfonate. After allowing to cool, the solution is supplemented with 0.5 part by weight of the crude enzyme powder prepared in Example 2 and a small quantity of a scent, to give a liquid detergent composition for kitchen use.

EXAMPLE 8 Hair shampoo: In 64 parts by weight of hot water at 60-65 C. are dissolved 5 parts by weight of acetylated lanolin, 6 parts by weight of Alkylolarnine (American Alcolac Corp.) and 25 parts by weight of Duponol XL E. I. duPont de Nemours Company). After allowing to cool, the solution is supplemented with 0.2 part by weight of the crude enzyme powder prepared in Example 2 and a small quantity of a scent, to give a hair shampoo.

What is claimed is:

l. A detergent or cleanser composition comprising at least one surfactant and alkali protease selected from the group consisting of Fusarium sp. S-19-5 (ATCC 20192), Fusarium oxysporum f. lini (IFO 5880) and Gibberella saubinetti (ATTC 20193), characterized by the following properties:

1. sedimentation constant (s of about 3.19 X 10",

2. molecular weight of about 2.65 X 10 (by Archibalds method,

3. elementary analysis about 46.72 percent by weight carbon, about 6.59 percent by weight hydrogen and 15.3 percent by weight nitrogen,

4. maximum absorption, in its ultraviolet absorption spectrum at the wavelength of 275 to 280 u, as indicated in FIG. 1,

5. infrared absorption spectrum as shown in FIG. 2, wherein the significant absorption bands in microns are as follows: 3.0 (strong), 3.38 (medium), 6.05 (strong), 6.55 (strong), 6.88 (weak), 7.15 (medium), 8.12 (medium), 9.30 (weak),

6. isoelectric point about pH 1 l,

7. optimal activity at a pH from 8 to 12 as indicated in FIG. 3, and at a temperature from about 40 to about 50 C. as indicated in FIG. 4,

8. stable in the pH range from 5 to 8 in 1 hour-incubation at 9. 20 and percent loss of activity upon being heated at 65 and 70 C., respectively, in 10 minute-incubation at pH 5.0.

2. A composition as in claim 1, wherein the amount of the said alkali protease is from about 5 to about 10,000 PU per gram of the final product by the modified Kunitz method.

3. A composition as in claim 1, wherein the said surfactant is selected from an anionic surfactant and a nonionic surfactant.

4. A composition as in claim 3, wherein the said anionic surfactant is from the group of a fatty acid salt, alkyl sulfate, olefine sulfate, alkyl sulfonate, olefine sulfonate, and alkylaryl sulfonate.

5. A composition as in claim 3, wherein the said nonionic surfactant is from the group of polyoxyethylene-alkyl ether, polyoxyethylene alkyl phenol ether, polyoxy-ethylene alkyl ester, polyhydric alcohol alkyl ester and sugar ester.

Claims (12)

1. 2. molecular weight of about 2.65 X 104 (by Archibald''s method,
2. 2. A composition as in claim 1, wherein the amount of the said alkali protease is from about 5 to about 10,000 PU per gram of the final product by the modified Kunitz'' method.
3. 3. A composition as in claim 1, wherein the said surfactant is selected from an anionic surfactant and a nonionic surfactant.
4. 3. elementary analysis about 46.72 percent by weight carbon, about 6.59 percent by weight hydrogen and 15.3 percent by weight nitrogen,
5. 4. A composition as in claim 3, wherein the said anionic surfactant is from the group of a fatty acid salt, alkyl sulfate, olefine sulfate, alkyl sulfonate, olefine sulfonate, and alkylaryl sulfonate.
6. 4. maximum absorption, in its ultraviolet absorption spectrum at the wavelength of 275 to 280 Mu , as indicated in FIG. 1,
7. 5. infrared absorption spectrum as shown in FIG. 2, wherein the significant absorption bands in microns are as follows: 3.0 (strong), 3.38 (medium), 6.05 (strong), 6.55 (strong), 6.88 (weak), 7.15 (medium), 8.12 (medium), 9.30 (weak),
8. 5. A composition as in claim 3, wherein the said nonionic surfactant is from the group of polyoxyethylene-alkyl ether, polyoxyethylene alkyl phenol ether, polyoxy-ethylene alkyl ester, polyhydric alcohol alkyl ester and sugar ester.
9. 6. isoelectric point about pH 11,
10. 7. optimal activity at a pH from 8 to 12 as indicated in FIG. 3, and at a temperature from about 40* to about 50* C. as indicated in FIG. 4,
11. 8. stable in the pH range from 5 to 8 in 1 hour-incubation at 37* C.,
12. 9. 20 and 80 percent loss of activity upon being heated at 65* and 70* C., respectively, in 10 minute-incubation at pH 5.0.

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