PH26128A - Alkaline cellulases and micro-organisms capable of producing same - Google Patents

Description

Es

BACKGROUND OF THE INVENTION

1, ald of the Invention

This invention relates to novel alkaline cellulases and also $0 microorganisms which axe able to produce the seme, belong to the genus Bagillus, and grow up dn a neutral medium, 2, Desoxipkiaon of the Prior Ark

The development of cellulases, which sre oellulose~ decomposing enzymes, has been made fox the purpose of effectively utilising biomass resources and particularly, cellulose resources, A diversity of strains have been isolated as cellulase-producing fungi or bacteria inolud- ing, for example, not only molds of the genera Aspergillus,

Paxdadllium, Irichoderms, Pussriim, Hmicols, Aaremondum and the like, but also bacteria of the genera Pagudcmonas, gallnlomonas, Rmingcocous, Bacillus snd the like and actinamycetes of the genera Hirsptomyocas, Tharsossiinomyoss and the like, At present, however, cellulsses for biomass have not been frequently utilised on em industrial soale.

On the other hand, studies have been made on no- novel infustrial utility of cellulases as sn ingredient for detergents for clothes, to which attention hes now been paid (Japanese Patent Publication Nos. 5359-49279, 60-23158 and 60-36240). Most cellulases produced by niorcorganisms in the natural fields are classified es

Co.

Lrg 26128 80-called neutral or acidie cellulases which exhibit optimum and stable enyymatic aotivity in a neutral to acidic range, Only a few cellulases are so-called alka~ line cellulases which meot the requirements for formula tion in detergent compositions for clothes or oman exhijyit 8 meximm activity in en alkaline pH Tenge and so-called alkali-resistant cellulases "hich have en alkali resis- tance, The term "alkaline cellulases” used herein is in- tended to mean one whose optimm pH 4s 4n mm alkaling range, and the tem "alkali resistant oellulase™ moms one whose optimm pH im 4n a neutral or mn aside range, but which has a satisfactory activity as compared with an aotivity at an optimm PH and 4» maintained stable in nn alkaline ranges, The tern "neutral means a PH range of 13 from § $0 8) and the term "alkaline" means s higher pi range,

For the production of alkaline cellulases amd al- i kali -resiwtont cellulases usable in detergent compositions for olothes, only several methods have been Jroposed;

These methods include, for exemple, a method of collecting osllulase A by cultivation of alkalophilic baci114 belong- ing to the gems Bacillup (Japenese Patent Publioation No, 3028515), a method of producing alkaline celluloses 301-4 by cultivation of alkalophilic baoteria belonging to the gerus Gellhulononas (Jep enese Patent Application laid-Open a -

? (0)? £.

No. 58-224686), a method of producing carboxymethyl cel-

Iulase by cultivation of alkslophilie Bacillus Fo, 1139 (Pulamors, ¥,, Kudo 7, amd Horikoshi, K., J, Gen, Miero~ bol., 131, 3339, (1985), ant a method of producing mm $ alkaline cellulase Ly the use of one wirain belonging to

She genus Streptomyces (Japenese Patent Application ledd~

Open No, 61-~19483), However, these methods sre all wn suitable for the industrial fermentation production,

In recent years, we have found that Bacillus mp.

KEM-635 (PERM P-8872), which is one of alialophilie bac~ teria, cam efficiently product alkaline cellulase X which is suitable as an ingredient for detergents for elothes and that proper selection of cultivation conditions enables one to enhance the productivity emd conduct industrial fore mentation production of the alkaline cellulase;

However, the cultivation conditions of the Bandllus 8p. KR-635 are not always advantageous from san infugtrdal point of view, More particularly, sm alkalophilic strain should be cultivated under alkaline PH conditions during the cultivation, A so-called alkaline fermentation process using alkalophilic strains has Just deen started, and a full knowledge on the physiologieal end diochemioal pro- perties of these alkalophilic miercorganisms has not been obtained, Ts, Aifficulties have been involved in the

Preparation of media and the mamer of cultivation suff) -

Clee 26128 cient to effect the industrial production by fermenta- tion,

Moreover, the trus alkaline cellulases of the afore-described documents which have an optimm pH in en alkaline region, are ensymes which are produced by

Bacillus Nl strain, E2 strain and N3 strain (Japmese

Patent Fublieation No. 50-2851%) end have optimm pHs of 8 to 9y 9 and 8 to 9, respectively, =n ensyme produced dy

Bagdllus No. 1139 end having en optimm pH of 9, and alka line cellulame K produced by Bacillus sp. XZM-635 amd have ing en offtimm pH of 10 (Japaneses Patent Application Wo. 61-25TT76)s Now, there is a demand for alkaline cellule vhich have en optimum pH in an alkaline region and ean be suitably formulated in detergent compositions snd which have 1% a wide working pH range.

SUMMARY OF THE INVENTICH

Under these ciroumstences, the present inventors

Rade extensive studies in order to obtain strains whieh grow in neutral media and are capable of producing alkaline cellulases having good effects.

In order to solve the prior art rrodlens, a gene re- combination technique may be used in which a strain which grows up in a neutral region is employed as a host snd cor-

‘Ce tog responding cellulase genes are cloned, In this commeotion, however, it is more effective to sexreh for neutral microorganism, in the natural field, which im» able ¥o produce sn alkaline cellulase having en op¥imm pH in mn alkaline region and to isolate it. Accordingly, the pre- sent inventors have sought such a microorganism in the natural field and, as a result, found that a series of nioroorgenisms belonging to the gems Bagillus grow in neutral media and produce certain types of alkaline cel~ lulases,

Typical alkaline cellulases according to the in- vention have the following enzymatic properties: (1) having a broad optimum pH range of from 8 $0 10 with a maximm activity at pH of approximately 10; (2) the activity being inhibited by the presence of rt, (3) the activity being rarely ivhibited with proteinases, surface active agents and chelating agents; md (4) having the OMCase activity (Gx activity) as a medn sotivity with additional filter paper-disine tegrating activity and Avicelase sovivity (0; eotivity).

BRIEF DESCRIPTION OF THE IRAWINGS

Fig, 1 1s a graph showing the relation between a

Ks é | ’6Q 26128

PH for the enzyme reaction of alkaline cellulase X-380 and a relative activity;

Pig. 2 is a graph showing the relation between a treating pH for the above ensyme and a residual activity

Pig. 3 18 a graph showing the relation between a reaction temperature for the shove muyme md a relgtive activity;

Fig. 4 ie a graph showing activities at 15%; em 20°C when the activity of the above enxyne at 30°C, 1s taken as 100;

Fig. 5 18 a graph showing the relation between a treating temperature for the above enzyme end a residual activity) pi Pig. 6 18 a graph showing the relation betwosn a pH for the enzyme reaction of alkaline cellulase K=42% and a relative activity

Tig. 7 18 a graph showing a treating pi for X-425 and a residual activity;

Pig. 8 18 a graph showing a reaction temperature for X-425 and a relative activity;

Mg, 948 a graph showing a treating temperature for F425 ad a residual activity;

Fig. 10 18 a graph showing the relation between o 2s PH for the ensyme reaction of alkaline cellulase X-521

Cela end a relative activity

Meg. 11 is a graph showing the relation between a treating pH for X-521 and a residual motivityy

Mg. 12 4s a graph showing the relation between a reaction temperature for X-521 and a relative activity;

Fig. 13 ie a graph showing the relation between a treating temperature for K-521 and a residual activity;

Hg. 14 i8 a graph showing the relation between a pH for the ensmyme reaction of alkaline cellulase X~%22 and a relative activity:

Fig. 15 is a graph showing the relation between a treating pH for K-522 and a resifual activity;

Mg, 16 48 a graph showing the relation between a reaction temperature for K-522 smd a relative setivity)

Mg, 17 48 a graph showing the relation between a treating temperature for X-522 end n residual activity

Tg. 18 48 a graph showing the relation between a pH for the enzyme reaction of alkaline cellulase E-IT md a relative activity;

Figs 19 48 a graph showing the relation between a treating pH for E-II and a residual activity;

Mg. 20 48 a graph showing the relation between a reaction temperature for E~II and a relative activity;

Pig. 21 i8 a graph showing the relation between a treating temperature for E~I1 and a residual aotivity;

Ce 28 . 26128 1

Pig. 22 418 =n ion-exchange chromatogram obtained in the third purification step;

Fig. 23 18 a chart showing the results of Sps/ polyacrylamide gel electrophoresis of alkaline gellu= lasep E~II and E-11I1}

Fig. 24 is a UV absorption spectrum of alkaline cellulase EIT;

Pig. 25 18 a graph showing the relation between a pH for the enzyme reaction of alkaline cellulase PITT and a relative activity;

Fig. 26 18 a greph showing the relation between o treating pH for BP-II and a residual sctivity;

Fig. 27 1s a graph showing the relation between a reacting temperature of B-~III and a relative activity;

Fig. 28 is a graph showing the relation between a treating temperature for E-ITI end a residual activity; ad

Fig. 29 4s a UV absorption spectrum of alkaline e6llulase B-III,

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

Beemples of the microorganisms capable of producing the alkaline cellulases of the invention inelude strains 1solated from the soils of Haga-gun smd Nilko-whi in

‘C( 128

Tochigi, Japen,

These strains have the following mycological properties, It will be noted that the olassifyeation of the strains is carried out using the following medivm

Hoss 1 to 25 (in which values are by wif),

Medium 1: meat extract, 1,0) Bacto peptone, 1.0; NaOl, 0s55 Bacto ager, 1.5 (pH 7.2)

Medivm 21 meat extract, 1.0; Bacto peptons, 1,0; NaOl, 0:5 (pH 7.2)

Medium 3: meant extraot, 1,0; Bacto peptone, 1,03 NaCl, 0.5) gelatin, 1,0 (pH 7.2)

Medium 4: Baoto litmus milk, 10,0

Medium 5:1 Bagto peptone, 1,0 Kxo,, 0.1

Medium 6t Bacto peptone, 1,0; NaNoy, 0.1

Medium 7: Bagto peptone, 0,7; NaCl, 0,5; glucose, 0,% (pH 7.0)

Medium 8: Bacto peptone, 1,0

Medium 91 TSI agar (by Eiken Chem, Co., Itdey Japan), indicated emount

Medium 10: meat extract, 1,0; Paoto peptone, 1,05 NaCl, 0.53 moluble starch, 0,2; agar, 1.5%

Medium 11: HeaNH, HPO, .45, 0, 0,1%3 KH, PO, , Oly MgSO, TH, 0, 0,02 sodium citrate, 0,25 (pH 6.8)

Cle)

Y

26128 . A 25, \ i

Medium 12¢ Christensen's medium (Eiicen Chem, Go. Itd,,

Japan), indicated amount

Medium 13 glucose, 1,0; KH, 0, 0.13 MgS0, + TH, 0, 0.08;

KCl, 0,02; mi trogen sources, 0,1 (pH 7,2) ™he nitrogen sources used were sodium mi trate and emondum sulfate:

Medium 14+ Xing A medium "Biken" (Eiken Chem, Cosy IRd,,

Japen), indicated emoumt

Medtwm 15: Xing B medium "Riken" (Eiken (hem, Cosy Id.

Jp en), indloated mount

Medium 16: urea medivm "Eiken" (Eiken Chem, Co., Its,

Jepen), indicated mount

Medium 17: Miter Paper for cytochrome oxidase test (Nisei Mmarm Coey I%4,, Japan)

Medium 18: 3% hydrogen peroxide aqueous solution

Medium 191 Of basal medium (Pifoo Tab. ), indicated mount : Medium $0: (rE, ), Bro, , 0.1; KC1, 0,02; HgS0, .M,0, 0.02

Feast extract, 0,02; Bacto agar, 2,03 BOP (0.2% solution), 0,4

Medium 21s Bacto Saboursud's dextrose ager mediwm (Difeo

Lab, ), indicated emount

Medium 22: meg extract, 0,3; Bacto peptone, 0,3; yest extract, 1,04 glycerin, 2,0

Medium 23: phenyl alanine Balomio acid salt medium (Mig- . Ye

Calg

Shui Pharm, Co., Itd,, Jepan), indicated

Medium 24: skim milk, 5,0; Bacto agar, 1.5

Medium 25: meat extract, 0,3; Bacto peptone, 0,5; I= tyrosine, 0.5; Bacto agar, 1.5 (Mycologioal properties)

Bacillus sp. X.580; (2) Bacillus sp, K®L580 has a size of the body of 0,4 « 008 micrometers x 1.5% ~ 5,0 micrometers, amd has a oy- lindrical or elliptical endospore (0.4 - 0.8 micrometers

X 0,8 ~ 1,2 micrometers) at the terminal of the body.

The bdeoillus has merginal flogellpes and is mobile, The

Orem's stedming is positive, Not acidurie, (b) Growing state in Various Media: (1) Meat troth agar plate oulture (medium 1)

The growing state is weak, The shape of the colonies is in a round or irregular form with a mooth sure face end a smooth or leaf-like margin, The color tome of the colonies is light yellow, pemi-tramspsrent snd glossy, (2) Moat troth ager slant culture (medium 1)

The growth is weak with the state being in a cloth-spreading form and being glossy, light yellow in color and semi-trsmsparent, (3) Meat broth liquid culture (medium 2) -l? = oo Clg 26128

Growing. Especially, the upper layer be- comes turbid, (4) Meat broth gelatin stab culture (medium 3)

Growing in the murfave layer with the gelatin being liquefied, (5) 14tmus milk medsum (Mediwm 4)

The liquefaction of the milk is recognised.

The litmus does not change its color, (e) mMysiologleal properties: (1) ™e reduction md derdtrifdoation reactions of nitretes (media 5 and 6) are both negative: (2) MR test (medium 7)

Whether the test is negative or positive is not olear (pH 5.2). 1s (3) YP test (medium 7)

Vhether the test is negative or positive is not clear (pH 5.2). (4) Formation of indole (medirm 8)

Negative. (5) Pormation of hydrogen sulfide (medium 9)

Negative, (6) Hydrolysis of starch (medium 10)

Positive. (7) vt1li4y of citric acid (media 11, 12)

Negative in a Komer's medium snd positive in - IR o

Chlvg a Chriptensen's medivm, (8) U111ty of inorganic nitrogen sources (medium 13)

Positive with respect to the nitrate emd ma- nondum melt, (9) Pormation of pigment (media 14, 15)

Negative, (10) Urease (medium 16)

Negative, (11) oxidase (medsm 17)

Positive, (12) vatalase (medivm 18)

Tositive, (13) Temperature and pH ranges for growth (medimm 2)

The temperature range for growth is 15 = 50%¢ and sn optimm temperature range is 25 - 40°0,

The pH range for the growth 18 S = 11 and an optimm pH renge 48 6 ~ 10 (14) Behavior to coxygen

Factultatively mmasrobic. (15) OF test (medium 19)

Although growing up, the growth is poor either eerodicelly or emaerobically. (16) Ut114ty of eugars (41 utilising, -1 not utilising) 1. I~arabinose 4 _ 12 _

CGI 8, 26128 2. .::ylose + 3. D-glucose + 4. D-mannose + 5. fructose 4 6+ Dgalactose +

T. maltose + 8, sucrose + 9. lactose + 10. trehalose - 11, Desord tol + 12, D-mermitol 4! 13. inositol + 4. glycerin $ 15, starch +: (17) pH in VP medium (medtvm 7)

PH 5.2 (18) orowth in a salt-contedning mediim (modified medivm 1)

Growing at 5%,

Growing at 7,

Not growing at 10%, (19) crown at a pH of 5.7 (medtum 21)

Growing, (20) Pormation of dihydroxyacetone (mediim 28)

Negative,

erp (21) Desmination of phenylalanine (medium 23)

Negative, ‘ (22) Decomposition of casein (medium 24)

Positive, (23) Decomposition of tyrosine (medium 2%)

Regative.

Hacillus sp, KSM-425, (a) Results of microscopic observation

Baalllne sp. KSM-425 neg a size of the body of

O¢4 = 0.8 micrometers x 1.5 - 4.0 micrometers, making em ellipsoidal endospore (0.8 - 1.2 micrometers x 1,2 - 1.5 micrometers) at one end of the body. It has marginal flagella and is mobi: a, The Crem staining is indefinite,

Not aciduric, (v) crowing state in various media (1) Meat broth agar plate culture (medium 1)

The growing state is poor, The shape of the oolordes is circular with a smooth surface end a anooth margin, The color tons of the colonies is white, and the colonies ere semi-tremsparent and glossy, (2) Meat broth egar slant culture (meds 1)

The growth is poor amd its state is in a eloth~ spread form and is glossy, white in color amd sei ~trens-~ parent, (3) Meat roth 11quid culture (medtwm 2) we

G12 ~~ 26128

Growing snd becoming turbid, (4) Meet tro * gelatin stab culture (mediwm 3)

Growing but in a poor state, The liquetan- tion of gelatin ip not recogrined, (5) dtms milk culture (medium 4)

The coagulation and liquefaction of milx is not recognised, The 1itmus does not undergo my change in color, (e) Mysiologioal properties (1) Reduotion and denitrification reactions of nitrates (media 5, 6)

Both negative, (2) MR test (meatum 7)

Whether the test 1s negative or positive is not olear (pH 5,2), (3) VP test (medsim 7)

Negative (pH 5,2), (4) Formation of indole (medtim 8)

Negative, (5) Pormation of hydrogen sulfide (medium 9)

Negative, (6) Rydrolysis of starch (medium 10)

Positive, (7) ¥#114ty of ettric acta (medtn 1, 12) 26 Negative both in a Koser's medium ed in

BAD ORIGINAL

RE,

Christensen's medium, (8) Utility of inorgemic nitrogen sources (medium 13)

Negative with respect to the nitrate smd am- nonlum salt, (9) Pormation of pigment (media 14, 15)

Negative, (10) Ureese (medium 16)

Negative, (11) aridase (medirm 17)

Positive. (12) Catalase (medium 18)

Positive, (13) Temperature amd pH ranges for growth (medium 2)

The temperature rmnge for the growth is 15 = 37°C, end an optimum temperature range is 25 - 30° o;

The pH range for the growth 4s 5 —- 10 end an optimm pH renge is 6 - 9, (14) Behavior to oxygen

Facultatively snasrobiec, (15) O-P test (meatvm 19)

Although growing up, the growth is poor either aerobically or anmerobically. (16) Utility of sugers (4: utilising, -: not utilizing) 1, I~arabinose + pias + - 1A = oo <Q (128 26128 2, Dxylowe 4 3+ D-glucose 4 4, D-mannose $ 5. fructose 4 €. D-galactose + 7. maltome + 8, sucrose 4 9, lactose $ 10, trehalose + 11, D-sorbitol + 12, D-mgmitol + 13, inositol - 14. glycerin 3 15. Starch : 1s (17) pH in VP moddum (medium 7) pH 5,2 : (18) Orowth in a salt-conteining medium (modified medium 1)

Not growing at 9%,

Fot growing at TH.

Not growing at 10%. (19) crowth at a pH of 5,7 (mediwm 21)

Not growing, (20) Formation of athydroxyacetene (medium 22)

“ed 2q

Whether the formation is negative or positive it not clear, (21) Deaminstion of phenylalanine (medium 23)

Negative, (22) Decomposition of cesein (medium 24)

Negative. (23) Decomposition of tyrosine (medirm 25)

Negative,

Bacillus sp. KSM 5211 (a) Results of microscopic observation

Begiilus spe KSM=521 hes a size of the body of 0.6 - 0.8 micrometers x 1,0 = 2,0 micrometers, making a oylin- dricel or ellipsoidal endospore (0.4 = 0.8 micrometers ® 1.0 = 2,0 micrometers) at the center of the body, I hes flegelll and is mobile, The Gram staining is positive,

Not scidurie. (b) Orowing state in various medin (1) Moat broth agar plate culture (medium 1)

The growing state is good, The shape of the colonies is circular with a smooth surface and a mmooth mergin or a leaf-like form. Tho color tone of the colonies is light yellow, ed the colonies are semi-trmmsparent ad glosmy. (2) Meat broth ager slat culture (medium 1) 2% Growing. The growing state is in a clothapread

Cee, 26128 form and is light yellow end sed «transparent, (3) Meat broth liquid culture (medium 2)

Growing tut becoming turbid; (4) Meat broth gelatin stab enlture (medium 3)

Urowlng in the surface portions. The lique- faction of gelatin is recognized, (5) 1dtmus milk culture (medium 4)

The liquefaction of milk is recognized. The litmus does not undergo my change in color, (c) Physiological properties (1) Reduction and denitrification resections of nitrates (media By 6)

Both negative, (2) MR test (medium 7) 13 Positive, (3) VP test (medium 7)

Popi tive, (4) Pormation of indole (medivm 8)

Regative, (3) Pormation of hydrogen sulfide (medium 9)

Fegative, (6) Hydrolysis of starch (medium 10)

Negative, (7) Utility of oAtric acid (media 1, 12)

Positive in Christensen's medium, md whether

”) els a negative or positive is not clear in Koser's medium, (8) Ut11ity of inorganic nitrogen sources (medium 13)

Negative with respect to the nitrate and mm mondvm salt, (9) Pormation of pigment (media 14, 15)

Positive. (10) Vresse (medium 16)

Hogative, (11) Oxidase (medium 17)

Yhether negative or positive is not clear. (12) Catalase (medium 18)

Positive, (13) Temperature mnd pll ranges for growth (medium 2)

The temperature range for the growth is 10 - 50°0, and an optimmn temperature range is 20 - 40°c,

T™e pH renge for the growth 48 5 « 10 end an op=- ¥imm pH range is 6 - 10, (14) Behavior to oxygen :

Aerobic, (15) OF test (medium 19)

Oxidation, (16) Formation of An pcdd nnd e ges from sugers (medium 20) (41 formed, -1 not formed)

CoiIrg 26128

Formation of aodd Formation of gas 1, Irarabinose $ - 2. D-xylose 4 - 3. D=glucose + - 4, D-marmose 4 - 5. fructose 4 - 6. D-galmsotose 4 -

T. maltose - - 8. sucrose + - 9. lactose - ——- 10, trehalose + - 11, D-sorbitol - - 12, D-marmitol 4 - 13, Inositol - - 14, glyeerin 4 - 18, starch - - (17) pH in VP medium (medium 7)

PH 5.0 (18) Growth in a salt-conteining medium (modified medium 1) Growing in 5%, 7% end 10% Nal’ (19) Growth at a pH of 5.7 (medium 21)

Growing, (20) Formation of aihydraxyacetons (medium 22)

Negative, - 2% uw

Colngy (21) Deamidation of phenylalanine (medium 23)

Negative. (22) Decomposition of casein (medium 24)

Positive, (23) Decomposition of tyrosine (medium 25)

Negative,

Bacillus sp. KSM-522y (a) Results of microscopic observation

Bacillus sp. KSM=522 has a sise of the dody of 0,5 ~ 0.8 micrometers x 1,0 = 2,0 micrometers, making sn oval of a cylindrical endospore (0.5 = 0,8 micrometers x 1,0 = 1.2 micrometers) at the end of the center of the body, I% has marginal flagelli and is mobile, The Oram staining is positive, Not acidurie. (b) oOrowing state in various media (1) Meat broth agar plate culture (medium 1)

Growing well, The shape of the colonies is oir- cular with a coarse surface snd a smooth or wavy margin.

The color tone of the colonies is light yellow, and the colonies are semi-transparent with a resin hardness, (2) Meat broth agar slant culture (medium 1)

Growing. The growing state is in a cloth spread from and glossy, with milky white or light yellow in color snd msemi-transparency. 28 (3) Meat broth liquid culture (medium 2)

Vhlog ~~ 26128

Growing nnd hecoming turbid. (4) Meat broth gelatin stab culture (medium 3)

Grewing in the top surface portions. The liquefaction of relatin is recognized. (5) Litmus milk culture (medium h4)

The liquefaction of milk is recognized but the litmus dces not undergo any change in color. (¢) Physiological properties (1) Reduction and denitrification reactions of nitrates (media 5, 6)

Both negative, (2) MR test (medium 7)

Positive, (3) VP test (medium 7)

Fositive, (4) Formation of indole (medium 8)

Negative, ‘ (5) Formation of hydrogen sulfide (medium 9)

Negative, (6) Hydrolysis of starch (medium 10)

Nagative, (7) Utility of citric acid (media 11, 12) + Fositive in Christensen's medium, but it is not clear in Koser's medium as to whether positive or negative,

, Chie g (8) Utility of inorganic nitrogen Sources (medium 13)

Negative with respect tq the nitrate and ammonium galt, (9) Formation of pigment (media 14, 15)

A water-soluble Yellow pigment ie formed in the King B medium, (10) Urease (medium 16)

Negative, (11) Oxidase (medium 17)

Whether negative or positive jg not clear, (12) Catalase (medium 18)

Positive, (13) Temperature ang PH ranges for growth (medium 2)

The temperature range for the growth is 10 . 50°C and an optimum temperature range ia 20 . 4g° Ce

The pH range for the 8rowth is 5 . 10 and : an optimum py range ig 6 . lo, (14) Behavior to oxygen

Aerobic, (15) o-p test (medium 19)

Oxidation, 6) Formation of an acid and g88 from sugarg (Medium 20) (+: formed, .: not formed)

Cele, 26128

Formation of acid Formation of gas 1. L=~arabinose + - 2. D-xylose + - 3, D-glucose + - 4, D-mannose + - 5. fructose + - 6. DL-galactose + - 7. maltose - - 8, sucrose +> - 9, lactose - - 10. trehalose + - 11. p-sorbitol - - 12. p-mannitol *> - . 13. Inositol - - 14, glycerin + - 15. starch - - (17) pH in VP medium (medium 7) pH 5.0 = Sed (seventh day)e (18) Growth in & salt-containing medium (modified medium 1) growing in 5%, 7% and 10% NaCl. (19) Growth at a pH of 5.7 (medium 21)

Qrowinge (20) pecomposition of casein (medium 2k) positive.

ZC ¢ 128

The above mycological properties are com- pared by reference to Bergey's Mannual of Determi- native Bacteriology, 8th edition and "The (Genus

Bacillus", in Agriculture Handbook No. L427 written by

Auth E. Gordon, Agricultural Research Service, U.S.

Department of Agriculture, Washington D.C. (1973). As a result, it has been found that all the strains of the invention are considered to be microorganisms be« longing to the genus Bacillus. The strains of the invention are apparently different from so-called alkalophilic microorganisms which have been recently reported oy Horikoshi and Akiba ("Alkalophilic Micro- organism", Japan scientific Society Fress (Tokyo), 1982). This is because the alkalophilic microore ganisms grow in alkaline media having a pH not less than 8 and cannot grow up in a neutral or lower pH region, whereas the strains of the invention are able to grow in a weakly acidic to alkaline region (pH 5 - 10). Thus, the strains of the invention can be determined as ordinary microorganisms belong to the genus Bacillus, which grow under neutral con- ditions.

More detailed studies on the strains of the invention reveal that the species which is most ana- logous to the strain of Bacillus sp. KSM-580 may be ~n

Bacillus licheniformis. However, the comparison between the strain of the present invention and known strains belonging to the Bacillus licheni- formis reveals that the KSM=580 strain is different from those known strains with respect to the re- ducibility of nitrates. In addition, the above known strains cannot produce at least alkaline cele lulases, and thuo the strain of the present invention is considered as a novel strain.

The species which is most analogous to Bacilk lus sp. K5M=-425 may be Bacillus circulans. The comparison between known strains belonging to the circulanital and the strain of the invention demona- trates a differente in the capability of ths hydroly- sis of gelatin. Moreover, the above known strains do not produce alkaline cellulases, Thus, the KSM- 425 is considered as a novel strain. . The species which is most analogous to the

Bacillus sp. KSM-521 and KSM-=522 strains may be Bacil- lus pumilus. However, known strains belonging to

Bacillus pumilus do net produce at least alkaline cellulases. Thus, the K5M-~521 and KSM=522 are con= sidered as novel strains.

The present inventors deposited these strains to Fermentation Research Inatitute of Japan as follows, w 20 =

CGI? 8

Bacillus sp. KSM=580 as FERM BP=1511

Bacillus sp. KSM=-U25 as FERM BP-1505

Bacillus sp. KSM-=521 as FERM BP-1507

Bacillus sp. KSH~522 as FLRIl BP-1512

For obtaining alkaline cellulases of the in- vention using these strains, the strain is inoculated into media and cultivated by a usual manner. The me- dimm should prefsrably have suitable amounts of care bon and nitrogen sources to be utilized. These car- bon and nitrogen sources are not critical. Examples of the nitrogen sources include corn gluten meal, soybean flour, corn steep liquor, casamino acid, yeast extract, Pharmamedia, sardine meal, meat exe tract, peptone, Hypro, Ajipower, corn soybean meal, coffee grounds, cotton seed oil cake, Cultivator,

Amiflex, Ajipron, Zest, AJix and the like. The car=- wr bon sources include, for example, plant fibers such as chaff, wheat-gluten bread, filter paper, ordinary papars, sawdust and the like, wasted theriac, invert sugar, CMC, Avicel, cellulose cotton, xylan, pectin and the like. In addition, utilizable carbon sources include, for example, arabinose, xylose, glucose, mannose, fructose, maltose, sucrose, lactose, tre- halose, mannitol, sorbitol, inositol, glycerin, so=- luble starch and the like, and utilizable organic le/ep 26128 acids include, for example, citrine acid, acetic acid and the like. Besides, phosphoric acid and inorganic salts such as of Mgt, cat, Mn2*, zn,

Co, Na‘, kK* and the like, and inorganic and ore ganic trace nutrient sources may be appropriately added,

An intended alkaline cellulase can be col- lected from the thus obtained culture product and puri fied according to ordinary techniques of collect ing and purifying enzymes. More particularly, the fungus bodies can be removed from a culture broth or solution by ordinary solid-liquid separation tech- niques such as centrifugal separation, filtration and the like, thereby obtaining a crude enzyme solution,

This crude enzyme solution may be used as it is, or may be separated by salting-out, precipitation, ultrae filtration or the like to obtain a crude enzyme, The crude enzyme is subsequently purified by crystalli- zation by any known methods to obtain a purified enzyme,

Among the thus obtained alkaline cellulases, the alkaline cellulase K~522 may be further sepa- rated into novel alkaline cellulases E-II and E-III.

For the preparation of the alkaline cellulases E-II and E-III, the alkaline cellulase K-522 is frac bo VR IRY: tionally purified by a suitable combination of a hydroxyapatite chromatography, an ion exchange chro- matography using DHAE-Sephadex (Pharmacia Inc.),

DEAE-cellulose or the like, and a molecular sieve gel chromatography using Sephadex, Biogel (BioeRad

Laboratories Inc.) and the like.

The thus obtained alkaline cellulases of the present invention have the following enzymatic pro perties. It will be noted that the enzymatic activity is measured according to the following procedure use ing the following buffer solutions, pH 3 - 8 : McIlvaine buffer solution pH 8 - 11: glycine-sodium hydroxide buffer so- lution

PH 12-13: potassium chloride-~sodium hydroxide buffer eolution

Enzymatic activity measurement: ’ (1) CMCase activity 0.1 ml of an enzyme solution was added to 0.9 ml of a base solution comprising 10 mg of CMC (A-OlL, by Sanyo Kokusaku Pulp Co., Ltd., Japan) and 100 umols of each of the buffer solutions (McIlvaine, phosphoric acid, glycine-NaOH and the like), fol lowed by reaction at 30% for 20 minutes. After - 32 a

Cai1?g 26128 completion of the reaction, (the resulting) reduce~ ing sugar was quantitatively determined according to the 3,5-dinitrosalicylic acid (DNS) method. More particularly, 1.0 ml of the DNS reagent was added to 1.0 ml. of the reaction solution and heated at 100°C for 5 minutes for color development. After cooling, 4.0 ml of deionized water was added for di- lution. The diluted solution was sub jected to colorimetry using a wavelength of 535 nm. The en=- gyme strength was expressed as one unit which was an amount of the enzyme sufficient to produce a re- ducing sugar corresponding to 1 pmol of glucose under } the above conditions for 1 minute. (2) Decomposition activity of PNPC

A suitable amount of CMCase was acted on 1.0 ml of a reaction solution containing 0.1 Jmol of PNPC (Sigma Coli, Ltd.) and 100 Jimols of a phos- phate buffer solution (pH 7.0) at 30°C., to which 0.3 ml of 1M Na,CO5 and 1.7 ml of deionized water were added successively, followed by subjecting the resultant released p-nitrophenol to colorimetry at 400 nm, The enzyme strength was expressed as one unit which was an amount of the enzyme sufficient to re- lease 1 pmo! 62 p-nitrophenol under the above condie tions for 1 minute.

(3) Decomposition activities of Avicel, cel= lulose powder and filter paper

A suitable amount of an enzyme solution was added to 240 ml of a reaction solution contain- ing 20 mg of Avicel (Merck Inc.) and 200 pr mols of a phosphate buffer solution (pH 7.0), followed by shaking for reaction at 30°¢. at 250 r.pe.m. After completion of the reaction, the solution was cooled and cantrifugally separated (5°C., 3000 r.p.m., 20 minutes), and 1.0 ml of the resultant supernatant } liquid was subjected to quantitative determination of reducing sugar according to the 3,5-dinitroe salicylic acid (DNS) method. The above procedure was repeated for a cellulose powder decomposition activity using cellulose powder (Toyo Filter Paper

Co., Ltd.) and for a filter paper decomposition activity using a filter paper (filter paper for exa= ’ mination of the cellulase activity, Toyo No. 51= specific). The enzyme strength was expressed by one unit which was an amount of the enzyme sufficient to produce reducing sugar corresponding to 1 Jmol of glucose under the above conditions for 1 minute. (4) Cellobiase activity

A suitable amount of CMCase was acted on a 1.0 ml reaction solution containing 10 mg of cel-

“Cr? & 26128 lobiose (Kanto Chem. Cosy Ltd.) and 100 J{ mols of a phosphate buffer solution (pH 7,0) for an appro priate time, and then treated at 100%, for 2 minutes, thereby inactivating the enzyme. Thereafter, the amount of the resultant glucose was measured by the

Mutarotase-gop method (glucose C-test, Wako Junyaku

Ind. Coe, Ltde). The enzyme strength was expressed by one unit which was an amount of the enzyme suffi. cient to produce 2 J mole of glucose under the above conditions for 1 minute, (Enzymatic properties)

Alkaline cellulase K~580: (1) Action

Acting well on cellulosic materials such as CMC, cellulose , filter paper, Avicel and the like and causing them to be dissolved, thereby producing reducing sugars such a8 glucose, (2) Substrate specificity

This enzyme haa activity not only on CMC, but also on cellulose powder, Avicel and filter paper. (3) Working PH and optimum pH

The working PH ranges very widely from 3 to 12.5 and the optimum pH is in the wide range of 7 to 10. In a range of 4.5 to 10.5, the relative activity is not less than 50% of the activity in the

Ce 28 optimum pH range. Accordingly, this enzyme is be- lieved to exhibit a satisfactory activity at the most alkaline side among known alkaline cellulases studied up to now (Fige 1). (4) pH Btability

The residual activity was measured after keeping the enzyme at different pHs at 30% for 1 hour to determine the pH stability. As a result, it was found that the enzyme was very stable and was not inactivated at a pH of 4.5 to 12. In a pH of from 3.5 to 12.5, an activity of about 50% or over was maintained. Thus, the present enzyme is satis=- factorily stable in a high alkaline region (Fig. 2). (5) Optimum temperature

The working temperature was in a wide range of from 15 to 80°C. and the optimum temperature was found to be 65°C. In a temperature range of from 50 to 75°C, the activity was 50% or higher of the activity at the optimum temperature (Fig. 3).

At 15°¢c, the activity was not less than 40% of the activity at 30°C (Fig. 4). (6) Temperature stability

After treatment at the optimum pH for 30 minutes at different temperatures, the residual activity was measured. As a result, it was found lo C128 26128 that it was stable at 55°C. and a residual activity of about S0% was obtained at 65°¢ (Fig. 5). (7) Molecular weight

The molecular weight of the present en- zyme was measured according to the gel filtration me~ thod using Sephadex G-100, with the result that it was about 18,000 and 50,000, (8) Influences of metal ions

The present enzyme was subjected to deter mination of influences of various metal ions (a1,

Feo, ca2*, cd®*, co?*, crt, cu? Fe°*, ng?*, Mn2*,

M02, Mi2*, Pot, zn, Lit, k*, and Na’) by permit- ting the ions to coexist at the time of the measure- ment of the activity (in which the Concentration of k* or Na’ was 50 mM and the concentration of other ions was 1 mM), As a result, it was found that the activity was inhibited with Hg>*, but was more en- hanced with BaZ*, ca®*, co? and ca2*, (9) Influences of surface active agents

Influences of various surface active agents (e.g. LAS, AS, ES, AOS, alpha-SFE, SAS, soap and polyoxyethylene secondary alkyl ether) on the enzyme activity were determined. The present enzyme was treated with a 0.05% solution of each surface active

C12 © agent at 30°%¢ for 15 minutes and subjected to the measurement of activity. As a result, the activity was not inhibited by any surface active agents. In addition, the inhibition of the activity was not re- cognized when using sodium dodecylsulfate which was a potential detergent. (10) Proteinase resistance

Proteinases for detergents such as, for example, API-21 (Showa Denko Co., Ltd.), Maxatase (Gist Co., Ltd.) and Alkalase (Novo Co., Ltd.), were allowed to coexist at the time of the measurement of the activity (0.1 mg/ml) to determine their ine fluences. It was found that the enzyme had a high resistance to these proteinases. (11) Influences of chelating agents

Chelating agents such as EDTA, EGTA, sodium tripolyphosphate, zeolite and citric acid were allowed to coexist at the time of the measurement of the activity, with the result that no inhibition was re cognized,

Alkaline cellulase K=425: (1) Action action well on cellulosic materials such as

CMC, cellulose, filter paper, Avicel and the like and cuusing them to be dissolved, thereby producing ree

C17 o 7 ' 26128 ducing sugars such as glucose, (2) Substrate specificity

This enzyme has activity not only on CMC, but also on cellulose powder, Avicel, filter paper,

PNFC and cellobiose. (3) working pH and optimum pH

The working pH ranges very widely from 3.5 to 12.5 and the optimum pH is in the wide range of 8 to 10, In a range of 5.5 to 10.5, the relative activity is not less than 50% of the activity in the optimum pHi range. Accordingly, this enzyme is be=- lieved to exhibit a satisfactory activity at the most alkaline side among known alkaline cellulases studied up to now (Fig. 6), (4) pH Stability

The residual activity was measured after keeping the enzyme at different pls at 30% for 1 hour to determine the pH stability, As a result, it was found that the enzyme was very stable and was not inactivated at a pH of 5 to ll. In a pH of from 3 to 12, an activity of about 50% or over was maintained. Thus, the present enzyme is satisfac torily stable in a high alkaline region (Fig. 7). (5) Optimum temperature

The working temperature was in a wide range

C6128 of from 15 to 75°C. and the optimum temperature was found to be 50°¢. In a temperature range of from 35 to 55°C., the activity was 50% or higher of the activity at the optimum temperature (Fig. 8). (6) Temperature stability

After treatment at the optimum pH for 30 minutes at different temperatures, the residual acti- vity was measured. As a result, it was found that it was stable at 30%. and & residual activity of about 50% was obtained at 50°¢ (Fig. 9). (7) Molecular weight

The molecular weight of the present enzyme was measured according to the gel filtration method using Sephadex G-100, with the result that it was about 35,000. (8) Influences of metal ions

The present enzyme was subjected to deter- mination of influences of various metal ions (a1>*, root, cat, ca?*, co?*, crt, cut, Fe?*, ug?*, m2, mot, Nit, 12%, un%", 11%, k*, and Na’) by permit- ting the iona to coexist at the time of the measure= ment of the activity (in which the concentration of

KY or Nat was 50 mM and the concentration of the other jons was 1 mM). As a result, it was found that the activity was inhibited by Hg>* and Bas? but was more

0 7 on 26128 enhanced by go", (9) 1nfluences of surface active agents

Influences of various surface active agents (e.g. LAS, AS, 1S, AOS, alpha-SFE, SAS, soap and polyoxyethylene secondary alkyl ether) on the enzyme activity were determined. The present enzyme was treated with a 0.05% solution of each surface active agent at 30°. for 15 minutes and sub jected to the measurement of activity. As a ree sult, the activity was not inhibited by any surface active agents. In addition, the inhibition of the activity was not recognized when using sodium dedecyle sulfate which was a potential detergent. (10) Proteinase resistance

Proteinases for detergents such as, for example, API-21 (Showa Denko Co., Ltd. ), Maxatase (Giet Co., Ltd.) and Alkalase (Novo Co., Ltd.), were allowed to coexist at the time of the measurement of the activity (0.1 mg/ml) to determine their ine fluences. It was found that the enzyme had a high resistance to these proteinases, (11) Influences of chelating agents

Chelating agents such as EDTA, EGTA, so=- dium tripolyphosphate, zeolite and citric acid were allowed to coexist at the time of the measurement of

C128 the activity, with the result that no inhibition was recognized.

Alkaline cellulase K-521: (1) Action

Acting well on cellulosic materials such as CMC, cellulose powder, filter paper, Avicel and the like and causing them to be dissolved, thereby producing reducing sugars such as glucose. (2) Substrate specificity

This enzyme has activity not only on CMC, but also on cellulose powder, Avicel, filter paper, p-nitrophenyl cellobioside and cellobiose. (3) working pH and optimum pH

The working pH ranges very widely from 3 to 12.5 and the optimum pH is in the wide range of 7 to 10, In a range of 4,5 to 10.5, the relative activity is not less than 50% of the activity in the optimum pH range. Accordingly, this enzyme is believed to exhibit a satisfactory activity at the most alkaline side among known alkaline cellulases studies up to now (Fig. 10). (4) pH stability

The residual activity was measured after keeping the enzyme at different pHs at 30°C for 1 hour to determine the pH stability. As a result, it

Coie 26128 was found that the enzyme was very stable and was not inactivated at a pH of 5 to 12. In a pH of from 4.5 to 12.5, an activity of about 50% or over was maintained, Thus, the present enzyme is satisfac= torily stable in a high alkaline region (Fig. 11), (5) Optimum temperature

The working temperature was in a wide range of from 15 to 80°C. and the optimum temperature was found to be 60°C. In a tempsrature range of from 45 to 68°C, the activity was 50% or higher of the activity at the optimum temperature (Rig. 12). (6) Temperature stability

After treatment at the optimum pH for 30 minutes nt different temperature, the residual activity was measured. As a result, it was found that it was stable at 40°C. and a residual activity of about 50% wes obtained at 55°C. (Fig. 13). (7) Molecular weight

The molecular weight of the present enzyme was measured according to the gel filtration method using Sephadex (3-100, with the result that it was about 31,000, (8) Influences of metal ions

The present enzyme was subjected to deter- mination of influences of various metal ions (a1,

‘C128 reo, Bat, cat, ca®’, co’t, ert, cu’? Foot, gst, m2’, moot, n12*, mt, z®*, 11°, k*, and

Na’) by permitting the ions to coexist at the time of the measurement of the activity (in which the concentration of K* or Na' was 50 mM and the con- centration of the other idns was 1 mM0. As a re=- sult, it was found that the activity wan inhibited by gt, but was more enhanced by ca”. (9) Influences of surface active agents

Influences of various surface active agents (e.g. LAS, AS, ES, AUS, alphe-~GFE, SAS, soap and polycxyethylene secondary alkyl ether) on the enzyme activity were determined. The nresent enzyme was treated with a 0,05% snlution of each surface active agent at 30°¢ for 15 minutes and subjected to the measurement of activity. As a result, the activity was rarely inhibited LY any surface active agents.

In addition, the inhibition of the activity was not recognized when using sodium dodecylsulfate which was a potential detergent, (10) Proteinase resistance

Proteinases for detergents such as, for example, API-2) (Showa Denko Co., Ltd.), Maxatase (Gist Co., Ltd.) and Alkalase (Novo Co., Ltd.), were allowed to coexist at the time of the measurement of - bh oo 2¢128 . 26 128 the activity (0.1 mg/ml) to determine their ine fluences. It was found that the enzyme had a high resistance tc these proteinases. (11) Influences of chelating agents

Chelating agents such as EDTA, EGTA, 80- dium tripolyphosphate, zeolite and citric acid were allowed to coexist at the time of the measurement of the activity, with the result that little inhibi- tion was recognized.

Alkaline cellulase K=522: (1) Action

Acting well on cellulosic materials such as CMC, cellulose powder, filter paper, Avicel and the like and causing them to be dissolved, thereby producing reducing sugars such as glucose. (2) Substrate specificity

This enzyme has activity not only on CMC, but also on cellulose powder, phosphoric acid-swol- len cellulose, alkali-swollen @llulose, Avicel, fil- ter paper and PNPC. (3) Working pH and optimum pH

The working pH ranges very widely from 3 to 12.5 and the optimum pH is in the side range of 7 to 10. In a range of 4.5 to 10.5, the relative activity is not less than S0% of the activity in the

Ci 128 t optimum pH range. accordingly, this enzyme is be- lieved to exhibit a satisfactory activity at the most alkaline side anong known alkaline cellulases studied upto now (Fig. 14). (4) pH stability

The residual activity was measured after keeping the enzyme at different pHs at 30°¢ for 1 hour to determine the pH atability. As a result, it was found that the enzyme was very stable and was not inactivated at a pH of 5 to 12. In a pH of from L.5 12.5, an activity of about 50% or over was maintained.

Thus, the present enzyme is satisfactorily stable in a high alkaline region (Fig. 15). (5) Optimum temperature

The working temperature was in a wide range of from 15 to 80%°c. and the optimum temperature was . found to be 60°c. 1n a temperature range of from Ls to 65°Ce the activity was 50% or higher of the acti- vigy at the optimum temperature (Fig. 16). (6) Temperature stability :

After treatment at the optimum pH for 30 minutes at different temperatures, the residual activity was measured. As a result, it was found that it was stable at 40°C. and a residual activity of about 50% was obtained at 55°C (Fig. 17).

LL

‘CL 26128 (7) Molecular weight

The molecul ar weight of the present enzyme vas measured According to the gel filtration method using Bio-Gel P~150 (Bio-Rad Laboratories Co., Ltd.), with the result that it was about 35,000, (8) Influences of metal ions

The present enzyme was subjected to determina. tion of influences of various meta ions (a3, ret,

Ba2*, ca?*, ca?*, co?*, cr, cu*, Fe2*, Hg2*, un2*,

Mo*, ng2+ Pht, n+ 11%, K*, and Na*) by permitting the ions to eu2xist at the time of the Reasurement of the activity (in which the concentration of x* or Na' vas 50 mM and the concentration of the other ions vas 1 mM). Ag a result, it yas found that the activity was inhibited by yg2*. (9) Influences Of surface active agents

Influences of various surface active agents . (e.g. LAS, As, ES, A0s, alpha~SFE, 8A8, soap and poly- oxyethylene secondary alkyl ether) on the enzyme activity were determined, The present enzyme wan treated with 5 0.05% solution of each surface active agent at 30% for 15 minutes ang Bubjected to the measurement of activity, as & result, the activity vas rarely inhibited by any surface active agents,

In addition, the inhibition of the activity yams not recognized when using sodium dodecylsulfate which

Was a potential detergent. (10) Proteinase resistance

Proteinases for detergents such as, for example, API-21 (Showa Denko Co., Ltd.), Maxatase (Gist Co., Ltd.) and Alkalase (Novo Co., Ltd.), were allowed to coexist at the time of the measurement of the activity (0.1 mg/ml) to determine their influences.

It was found that the enzyme had a high resistance to these proteinases. (11) Influences of chelating agents

Chelating agents such as EDTA, EGTA, sodium tripolyphosphate, zeolite and citric acid were allowed to coexist at the time of the measurement of the activity, with the result that little inhibition was recognised, . Alkaline cellulase E-II: (1) Action

Acting well on cellulosic materials such as

CMC and phosphoric acid-swollen celludose and cause ing them to be dissolved, thereby producing reducing sugars such as glucose. (2) Substrate apecificity

This enzyme has activity not only a main activity on CMC, but also an activity on cellulose

C612 8 26128 swollen with 4% phosphoric acid which is about 0.4% of CMCase activity. Moreover, it has a slight de~ composition activity on xylan, inulin and lichenan, but has little activity on cellulose powder, Avicel, filter paper, PNPC and cellobiose. (3) working pH and optimum pH

The working pH ranges very widely from 4 to 12.5 and the optimum pH is in the wide range of 7 to 10. In a range of 5.5 to 11, the relative activity is not less than 50% of the activity in the optimum pH range. Accordingly, this ensyme is believed to exhibit a satisfactory activity at the most alkaline side among known alkaline cellulases studied upto now (Fig. 18). (4) pH stability

The residual activity was measured after keep- ing the enzyme at different pHs at 0°C for 24 hours ’ to determine the pH stability. As a result, it was found that the enzyme was very stable and was not in- activated at a pH of 6 to 11. In a pH of from 5.5 to 11.5, an activity of about 50% or over was main- tained. Thus, the present enzyme is satisfactorily stable in a high alkaline region (Fig. 19). (5) Optimum temperature

The working temperature was in a wide range

CC ClGlrg of from 10 to 80°¢c and the optimum temperature was found to de 50°C. In a temperature range of from 30 to 65°C, the activity was 50% or higher of the activity at the optimum temperature (Fig. 20). (6) Temperature stability

After treatment at a pH of 7 for 30 minutes at different temperatures, the residual activity was measured. As a result, it was found that it was stable at 50°C. and a residual activity of about 50% was obtained at 55°C. (Fig. 21). (7) Molecular weight

The molecular weight of the present enzyme was measured according to the gel filtration method using Bio-Gel P-100 (Bio-Rad Laboratories Co., Ltd.), with the result that it was about 34,000. With an 8DS-polyacrylamide gel electrophoresis, the molecular weight was about 61,000 (Fig. 23). (8) Influences of metal ions

The present enzyme was subjected to deter mination of influences of various metal ions

The present enzyme was subjected to (at,

Foot, Ba2t, ca2*, ca®*, co2*, cu’, re2*, Hg2*, mn?*,

Mest, Ni2*, poet, zn2*, Lit, K' and Na*) by permite ting the ions to coexist at the time of the measure~ ment of the activity (in which the concentration of 20 o

/

K* or Nea' waa 50 mM and the concentration of the other ions was 1 mM). As a result, it was found that the activity was inhibited with Hg>* and enw hanced with o>. (9) Influences of surface active agents

Influences of various surface active agents (e.g. LAS, AS, ES, AOS, alpha-SFE, SAS, soap and poly- oxyethylene secondary alkyl ether) on the enzyme acti~ vity were determined. The present enzyme was sub jected to the measurement of the activity in 0,05% of a sur-~ face active agent. As a result, any significant in- fluences of the surface active agents were recognized as shown in Table 1.

TABLE 1 eames

Surface Active Agent Residual Activity (%)

Nil 100

LAS 79

AS 108

ES 98

AOS 100

Alpha-SFE 129

SAS 93

Soap 100

Polyoxyethylene secondary 86 alkyl ether

In addition, the inhibition of the activity was not recognized when using sodium dodecylsulfate which was a potential detergent. (10) Proteinase resistance

Proteinases for detergents such as, for example API-21 (Showa Denko Co., Ltd.), Maxatase (Gist

Co., Ltd.) and Alkalase (Novo Co., Ltd.), were allowed to coexist at the time of the measurement of the sotivity (0.1 mg/ml) to determine their influences.

It was found that the enzyme had a high resistance to these proteinases as shown in Table 2.

TABLE 2

Proteinase Residual Activity (%) em ———— arr erate nil 100

Alkalase 111 . API=21 115

Maxatase 120 ee eer Ameer ree eerie (11) Influences of chelating agents

Chelating agents such as EDTA, EGTA, sodium tripolyphosphate, zeolite and citric acid were allowed td.6oexist at. the time of the measurement of the activity, with the result that little inhibition was recognised,

Ce28 26128 (12) UVssorption spectrum

The present enzyme was subjected to measurement of UV absorption spectrum. As a result, it was found that it had a maximum absorption at about 280 nm with a shoulder absorption being shown at 290 nm by a differential absorption spectrum (Fig. 2h),

Alkaline cellulase E-IIX: (1) Action

Acting well on celluloses such as CMC and phosphoric acid-swollen cellulose and causing them to be disaoclved, thereby producing reducing sugars such as glucose. (2) Substrate specificity

This enzyme has not only a main activity on CMC, but also an activity on cellulose swollen with about 4.5% phosphorie acid. Moreover, it has a } slight decomposition activity on xylan, lichenan and the like, but has little activity on cellulose pOve der, Avicel, filter paper, PNPC and cellobiose. (3) Working pH and optimum pH

The working pH ranges very widely from 4 to 12.5 and the optimum pH is in the wide range of 7 to 9. In a range of 6 to 10.5, the relative activity is not less than 50% of the activity in the optimum pH

°C G2 8 range. Accordingly, this enzyme is believed to exhibit a satisfactory activity at the most alkaline side among known alkaline cellulases studies up to now (Fig. 25). (4) pH stability

The residual activity was measured after keeping the enzyme at different pis at 5% for 24 hours to determine the pH stability. As a result, it was found that the encyme was very stable and was not inactivated at a pH of 6 to 10. In a pH of from 3.7 to 11.5, an activity of about 50% or over was maine tained, Thus, the present enzyme is satisfactorily stable in a high alkaline region (Figl 26). (3) Optimum temperature

The working temperature was in a wide range of from 10 to 80°¢c and the optimum temperature was . found to be 50°¢c. In a temperature range of from 30 to 62°C, the activity was 50% or higher of the actie vity at the optimum temperature (Fig. 27). (6) Temperature stability

After treatment at a pH of 7 for 30 minutes at different temperaturea, the residual activity was measured. As a result, it was found that it was stable at 50°C and a residual activity of about 50% was obtained at 55°C. (Fig. 28).

CCI? i

I

26128 (7) Molecular weight

The molecular weight of the present enzyme was measured according to the gel filtration method using Bio-Gel P-100 (Bio-Rad Laboratories Co., Ltd.), with the result that it was about 35,000, With an 8DS~-polyacrylamide gel electrophoresis, the molecular weight was about 61,000 (Fig. 23). (8) Influences of metal ions

The present enzyme was sub jected to deter- mination of influences of various metal ions (a2,

Fe? cal, coZ*, crt, cult, Foot, ug2*, Mg2*, MnZ*,

N12*, pb2*, zn%*, k*, and Na*) by permitting the ions to coexist at the time of the measurement of the acti- vity (in which the concentration of K* or Na* was 50 mM and the concentration of the other ions was 1 mM).

As a result, it was found that ths activity was ine hibited with Hg>' and enhanced with Co>'. €(9) Influences of surface active agents

Influences of various surface active agents (e.ge LAS, AS, ES, AOS, alpha-SFE, SAS, soap and polyoxyethylene secondary alkyl ether) on the ensyme activity were determined. The present enzyme was sub- jected to the measurement of the activity in 0,05% of a surface active agent. As a result, any signi- ficant influences of the surface active agents were elo recognized as shown in Table 3.

TABLE 3

Surface Active Agent Residual Activity (%) nil , 100

LAS 78

AS 107

ES 100

AOS 101 alpha~SFE 104

BAS 9?

Soap 101

Polyoxyethylene secondary 96 alkyl ether ee eee ert eee eee erent

In addition, the inhibition of the activity was not recognized when using sodium dodecylsulfate : which was a potential detergent. (10) Proteinase resistance

Proteinases for detergents such as, for example API-21 (Showa Denko Co., Ltd.), Maxatase (Gist Co., Ltd.) and Alkalase (Novo Co., Ltd.), were allowed to coexist (0.1 mg/ml) at the time of the measurement of the activity to determine their ine fluences, It was found that the ensyme had a high

Gl? 6 ~~ 26128 resistance to these proteinases as shown in Table 4,

TABLE &4

Proteinase Residual Activity (%) nil 100

Alkalase 94

API=21 106

Maxatase 103 (11) Influences of chelating agents

Chelating agents such as EDTA, EGTA, sodium tripolyphosphate, zeolite and citric acid were allowed to coexist at the time of the measurement of the acti- vity, with the result that little inhibition was ree cognized, (12) UV absorption spectrum

The present enzyme was subjected to measure= ment of a UV absorption spectrum. As a result, it was found that it had a maximum absorption at about 280 nm with a shoulder absorption being shown at 290 nm by a differential absorption spectrum (Fig. 29).

The alkaline cellulases of the invention have an optimum pH at a higher level (pH 10) than known alkaline cellulases and are very stable over a wide pH range. For example, alkaline cellulase K-580 has, in a widé pH range of from 4.5 to 10.5, an activity

CCI? s ! not less than 50% of the activity at the optimum pH, and is very stable in a pH range of from 4,5 to 12.

Alkaline cellulase K-425 has also, in a wide pH range of from 5.5 to 10,5, an activity of not less than 50% of the activity at the optimum OH, and is very stable in a pH range of from 5 to 11. Moreover, alkaline cellulases K-521 and K=522 have, respective= ly, an optimum pH in a wide range of from 7.0 to 10 and is very stable in a wide range.

The alkaline cellulases E~II and E~III dee rived from the alkaline cellulase K«522 have, res= pectively, higher optimum pHs (of 10 and 9) than known alkaline cellulases. In addition, they have, respectively, wide optimum pH ranges of 7.0 to 10 and 7.0 to 9 are very stable in such wide ranges.

These alkaline cellulases are rarely inhi. bited with ingredients ordinarily formulated in de- tergents such as, for example, surface active agents, proteinases, chelating agents and the like. Accore dingly, the present enzymes can be conveniently used in detergent compositions.

The microorganisms of the invention grow under neutral conditions, so that it is possible to indus- trielly produce alkaline cellulases more easily than in the case using alkalophilic strains.

Cel? a 26128

The present invention is described in more detail by way of the following examples.

Example 1

A spoonful (about 0.5 g) of the soil obtained at Ichikai-machi, Haga-gun, Tochigi~ken, Japan was suspended in a sterilized physiological saline solue tion and thermally treated at 80°C. for 10 minutes,

The supernatant liquid of the thermally treated soe lution was appropriately diluted and applied to an agar medium for isolation (medium 1), followed by cultivation at 30°C. for 3 days to form colonies. :

Colonies around which a transparent zone was formed on the basis of the dissolution of CMC were selected to collect CMCase~producinf microorganisms belonging to the genus Bacillus. The thus collected microore ganisms were inoculated into a liquid medium as me- dium 2 and sub jected to shaking culture at 30° c, - for 3 days. After completion of the culture, a centrifugally separated supernatant liquid was obe tained and subjected to measurement of the CHCase activity in a pH range of from 3 to 13 and also ¥%o screening of alkaline cellulase~producing microor- ganisms belonging to the genus Bacillus,

By the above procedure, there could be ob- tained Bacillus sp. KSM-580 strain, Bscillus Spe KSM=-

Cal 2 425 strain, and Bacillus sp. KSM=521 strain.

Medium 1

CMC 2%

Polypeptone 0.5

Yeast extract 0.05

KH,POy, 0.1

Na, HPO. 12H,0 0.25

MgS0,,« 7H,0 0.02

Agar 0.75 pH 6.8

Medium 2

CMC 1%

Polypeptone 1

Yeast extract 0.5

KH, PO), 0.1

Na, HPO, .12H,0 0.25 . : MgSO, 7H,0 0.02 pH 6.8

Example 2

The Bacillus sp. KSM-580 strain obtained in

Example 1 was inoculated into the liquid medium 2 of

Example 1, followed by shaking culture at 30°¢ for 3 days. After completion of the culture, the bacil- lus cells were centrifugally removed to obtain a crude enzyme solution. 3 liters of ethanol was

Zc

L126 26128 added to 1 liter of the crude enzyme solution in dry ice/ethanol and the resultant precipitate was centrifugally removed, followed by freeze-drying to obtain 11 g of alkaline cellulase K-580 (specific - activity® 33 units/g) as a dry powder. *The enzyme activity was a value at a pH of 9.

Exmmple 3

The Bacillus sp. KSM=580 strain was inoculated into a medium of the same composition as the liquid medium 2 of Example 1 except that CMC was replaced by sucrose and polypeptone was replaced by 7% of a corn steep liquor (CSL), followed by shaking culture at 30°c. for 2 days. The resultant culture product was sub jected to centrifugal separation and the resulting supernatant liquid was, in turn, subjected to measure- ment of the CMCase activity. As a result, the acti~ vity was 60 units/liter.

Example 4

The Bacillus sp. KSM-425 strain obtained in

Example 1 was inoculated into the liquid medium 2 of

Example 1 and shake-cultured at 30°C for 3 days.

After completion of the culture, the bacillus cells : were centrifugally removed to obtain a crude enzyme - fl

7c 128 solution. 3 liters of ethanol was added to 1 liter of the crude enzyme solution in dry ice/ethanol, and the resultant precipitate was centrifugally ree moved and freeze-dried to obtain 10 g. of alkaline cellulase K-425 (mpecific activity® 10 units/g) as a dry pdwder. *The enzyme activity was a value measured at a pH of 9.

Example 5

The Bacillus sp. KSM=U425 strain was inoculated into a medium of the same composition as the liquid medium 2 of Example 1 except that CMC was replaced by sucrose and polypeptone was replaced by 7% CSL, fol- lowed by shaking culture at 30°¢. for 2 days. The culture product was centrifugally separated and the resultant supernatant liquid was subjected to mea= . surement of the CMCase activity. As a result, the activity was 160 uhits/liter.

Exanple 6

The Bacillus sp. KSM=-521 strain obtained in

Example 1 was inoculated into the liquid medium 2 of

Example 1 and shake-cultured at 30%. for 3 days.

After completion of the culture, the bacillus cells were centrifugally removed to obtain a crude enzyme

Choe © 26128 .- (J solution. 3 liters of ethanol was added to 1 liter of the crude enzyme solution in dry ice/ethanol, and the resultant precipitated was centrifugally se-~ parated and freeze-dried to obtain 9 g of alkaline cellulase K-52) (specific activity® 20 units/g) as a dry powder, *The enzyme activity was a value measured at a pH of 9 herein and hereinafter.

Example 7

The KSM-521 strain was inoculated into a mee dium of the same composition as the liquid medium 2 of Example 1 except that CMC was replaced by 1% sucrose and polypeptone was replaced by 7% CSL, fol- lowed by shaking culture at 30% for 2 days. The culture product was centrifugally separated and the resultant supernatant liquid was subjected to measure- ment of the CMCase activity. As a result, it was found that the activity was 100 units/liter.

Example 8

A spoonful (0.5 g) of the soil obtained at

Nikko-shi, Tochigi-ken, Japan was taken and suspended in a sterilized saline solution, followed by repeate ing the procedure of Example 1, thereby obtaining

KSM=-522 strain (FERM BP=1512) of the invention.

2¢)28

Example 9

The Bacillus sp. KSM-522 strain obtained in

Example 8 was inoculated into the liquid medium of

Example 1 and shake-cultured at 30°¢. for 3 days.

After the culture, the bacillus cells were centri- fugelly separated to obtain a crude solution. 3 liters of ethanol was added to 1 liter of the crude enzyme solution in dry ice/ethanol, and the re- sultant precipitate was centrifugally separated and freeze-dried to obtain 8 g of alkaline cellulase K-522 (specific activity® 23 units/g) as a dry powder. * The enzyme activity was a walue measured at a pH of 9.

Example 10

The KSM=522 strain was inoculated into a mee dium of the same composition as the liquid medium 2 . of Example 1 except that CMC was replaced by 1% sucrose and polypeptone was replaced by 7% CSL, fol= lowed by shaking culture at a temperature of 30°C. for 2 days. The culture product was centrifugally separated and the resultant supernatant liquid was subjected to measurement of the CHCane activity, with the result thst the activity was 150 units/ liter.

Sr v 26128

Example 11 liters of the supernatant obtained in Exam- ple 10 was purified according to the following proe cedure, 5 (1) Concentration by ultrafiltration (Amicon Co.

Ltd., fractionating molecular weight of 10,000). (2) Treatment with Streptomycin. (3) Chromatography using DEAE-~-BioGel A (Bio-

Rad Laboratories Co., Ltd.)e. (4) Chromatography using hydroxyapatite (Wako

Junyaku Ind. Co., Ltd.). (5) Chromatography using DEAE-BioGel A. (6) Chromatography using DEAE-BioGel A.

In the third step of the above procedure, the enzyme was adsorbed in a column having a size of 3.2 x 33 cm (equilibrated with a 10 mM phosphate buffer } solution with a pH of 7), followed by linearly ine creasing the concentration of NaCl from O to 300 mM for eluation. This permitted neutral cellulase E-I, alkaline cellulase E-II and alkaline cellulase E-III to be eluted in this order (Fig, 22). The fractions of from 533 to 580 from which the fraction of E-I had been removed were collected and further purified.

In the fourth step, the fractions were adsorbed in a column with a size of 2.5 x 13 cm (equilibrated with a 10 mM phosphate buffer solution with a pH of 7), after which the concentration of the phos~- phate was increased from 10 to 200 mM linearly to ob=- tain an alkaline cellulase fraction (a mixture of E=

II and E-III). In the fifth step, the procedure of (3) was repeated (in which the concentration of NaCl was incressed linearly from 70 to 200 mM) to collect alkaline cellulase E-II with the residue being sub jected to the sixth step. The sixth step was effected similar to (3) or (5) but the gradient in concentra tion of NaCl was further lowered by changing from 90 to 150 mM for elution, thereby isolating the alka- line cellulase E-III. The thus purified alkaline cellulases E-II and E-11I were sub jected to poly~ acrylamide electrophoresis by a usual manner and further to Coomassie Brilliant Blue dyeing and sile ver staining, by which it was confirmed that the respective cellulases gave a single band.

Example 12

The alkaline cellulases E-II and E-III obe tained in Example 11 were subjected to SDS=~poly~ gorylamide gel electrophoresis by a usuall manner.

The results are shown in Fig. 23. From the results, it was found that the alkaline cellulases E-II and

E-III each had a molecular weight of about 61,000,

C128 26128 : 8

According to the gel filtration method using

Bio-Gel P-100, the alkaline cellulase E-II had a molecular weight of about 34,000 and the alkaline cellulase E-III had a molecular weight of about 35,000.

Claims (1)

1. ei g WHAT IS CLAIMED 1S:

   1. A microorganism belonging to the genus Bacillus SpKSM - 580 deposited as FERM, which is capable of producing alkaline cellulase K~580 have S ing the following enzymatic properties: (1) Aotion = «= Acting well on cellulosic materials including care boxymethyl cellulose (CMC), cellulose, filter paper and Avicel and causing them to be dissolved, thereby forming reducing sugars} (2) Substrate specificity - having activity not only on CMC, but also on cellu~ lose powder, Avicel and filter paper} (3) working pH and optimum pH~ the working pH range is from 3 to

   12.5 and the optimum pH is from 7 to 10 with a rela- tive activity of not less than 50% of the activity at an optimum pH being shown in the range of 4.5 to 10.5% (4) pH stability =~ very stable and no inactivated at a pH of U.5 to 12 and an activity of not leas than about 50% being maintained at a pH of 3.5 to

   12.5; (5) optimum temperature - the working teme perature is in a wide range of 15 to 80° Cc. and an optimum temperature is 65°C in the range of 50° to 75°C, the activity is not less than 50% of the acti- vity at the optimum temperature; (6) Molecular weight « Peaks of the molecular weight care at about 18,000 LR]

   ° t 26128 and about 50,000 (when determined by a gel filtra- tion method using Sephadex G100)3; (7) Influences of metal ions ~ inhibited by Hg>* and activated by Bal, cast, coZ* and ca2*; (B) Influences of sur=-

   face active agents LAS, AS, ES, ADS, alpha«SFE, SAS and soaps and polyoxyethylene secondary alkyl ethers do rarely inhibit the activity; (9) Proteinase re-~ sistance-Resistant to proteinases; (10) Influences of chelating agents EDTA, EGTA, citric acid, sodium tri-

   polyphosphate and zeolite do not inhibit the activity.

   SHUJI KAWAI HIROMI OKOSHI SHITSUW SHIKATA HAJIME MORI ’ SUSUMU ITO Inventors