Purification and properties of an endoglucanase from a Bacillus isolate

Prince Sharma * J. K. Gupta,* D. V. Vadehra*

• Department o f Microbiolo~,,y, Panjab University, Chandigarh, India

and D. K. D u b e t

+ Institute o f Microbial Technology, CSIR. 177G, Phase 11, lnd,strial Area, Chandigarh, India

The thermophilic soil isolate, Bacillus sp. PDV, produced endogluvanase when grown on glucose and could not utilize carboxymethyl cellulose (CMC) or Avicel as growth substrates. The CMCase was purified on a DEAE-Sephacel ion exchange column Jbllowed by Biogel-A-O.5m and Superose-6 gel filtration columns. The purified CMCase was found to be homogeneous on polyacrylamide gel electro- phoresis with silver staining and had a molecular weight of 33,000 (SDS-PAGE and Sephadex G-lO0 gel flhration). It showed activity towards crystalline Jbrms of cellulose such as cotton and A vicel and was stable in the pH range of 4-10 and temperature up to 60°C. The enzyme hydrolysed CMC with pH and temperature optima of 5.0 and 60°C and a Km of 0.588 mg m l i . The enzyme activity was completely inhibited by Hg 2+, while other metal ions, chelators, various oxidizing agents, and repeated freezing and thawing had no marked effect on activity. Reducing agents, in general, increased the activity.

Key words: Endoglucanase (E.C. 3.2.1.4); Bacillus sp. PDV: CMC: enzyme purification: stability; substrate spe- cificity

Introduction

Cellulases are commercially important enzymes with a potential application for obtaining energy, chemicals, and single cell protein from the abundant renewable resource cellulose. Crystalline cellulose is hydrolysed to glucose by the synergistic action of cellobiohydro- lase (E.C. 3.2.1.91), endoglucanase or CMCase (E.C. 3.2.1.4), and fl-glucosidase (E.C. 3.2.I.21). Recent studies on the mode of action of cellulase system have focused mainly on the purification, characterization, and substrate specificity analysis of each of the cellu- lase components and synergism between them. From the industrial point of view, the need for highly active cellulases, stable over wide pH and temperature ranges, feedback inhibition resistant, and active over multiple substrates, has drawn the attention towards thermophilic, rapidly growing, cellulolytic bacterial genera. Among the well-studied are species of Ther- momonospora, l Acetivibrio,2 Cellulomonas,] Clostrid- ium, 4,5 and Bacteroides. 6 Some Bacillus strains have

Address reprint requests to Dr. Sharma at the Department of Micro- biology, Panjab University, Chandigarh-160014, India The present address of D. K. Dube is Department of Pathology, University of Washington, Seattle, WA 98195, USA Received 26 August 1987; revised 2 November 1988

also been reported to degrade noncrystalline cellu- lose. v,8 A soil isolate Bacillus strain DLG 9 produced constitutive CMCase in the stationary phase, while some of the alkalophilic bacilli 1°,1~ produced CMCase active under alkaline pH conditions.

We have isolated from soil a thermophilic bacte- rium, Bacillus sp. PDV, which elaborated a very stable cellulase and xylanase constitutively and maximally within 8-12 h of the onset of growth. The extracellular CMCase was active over broad pH and temperature limits and its activity was not affected by the presence of glucose in the assay mixture. This paper describes the purification and some properties of this purified CMCase.

Materials and methods

Chemicals and other materials used were of analytical grade: sodium salt of carboxymethyl cellulose (CMC, low viscosity, Sigma 8758), bovine serum albumin (BSA), larchwood xylan, ceilobiose, methyl cellulose, SDS, Tris, TEMED, Acrylamide, N, N-methylene bis acrylamide, and ammonium persulfate were from Sigma Chemical Co., USA. Sephadex G-100, DEAE- Sephacel, Superose-6 column (FPLC), and reference proteins of standard low molecular weight were

1:32 Enzyme Microb. Technol., 1990, vol. 12, February © 1990 Butterworth Publishers

bought from Pharmacia Fine Chemicals, Sweden. Other materials used were Biogel-A-0.5m (Bio Rad), PM-10 and PM-30 ultrafiltration membranes (Diaflo, Amicon Corp., USA), Avicel-RC-581 (FMC Corp., Pennsylvania).

Isolation of the organism

Fifty different soil samples were collected from the environments of the pulp and paper industry and wood rotting areas and were screened for the isolation of thermophilic and cellulolytic Bacillus species. The soil suspensions, in sterile distilled water, were heated at 80°C for 10 min and plated on YT agar (yeast extract, 0.5%; tryptone, 0.8%; NaCI, 0.5%; and agar, 1.5%, pH 7.0). After overnight incubation at 55°C the colonies were replicated to fresh YT agar before being overlaid with 0.8% agar containing 0.5% CMC. The plates were incubated at 55°C for a further 4 h and the cellulolytic colonies were detected by staining with Congo Red.12 In this process a thermophilic and cellulolytic isolate Bacillus sp. PDV was selected. The morphological and taxonomic characters were studied by established methods 13 to characterize and identify the genus. The pure culture was maintained on YT agar containing 0.5% CMC and was transferred every month.

Enzyme production

Shake flask experiments were carried out in the nutri- ent broth or in minimal M9 medium t4 containing 0.5% glucose as carbon source. The organism was grown at 37°C for 18 h on a rotary shaker (200 rev min -1) and the culture broth was centrifuged at 9,000g for 15 min at 4°C. The supernatant was used as a crude preparation of CMCase.

Enzyme assays

CMCase activity was measured by incubating 0.5 ml of appropriately diluted enzyme with 0.5 ml of 1% CMC, prepared in 0,1 M citrate-phosphate buffer, pH 5.0, for 10 min at 60°C. The reducing sugars liberated were quantitated according to Somogyi. ~5 One unit of CMCase activity was defined as the amount of enzyme that released 1/x mol reducing sugar as glucose equiva- lent rain -j. Hydrolytic activities towards xylan and methyl cellulose were measured similarly and the ac- tivity towards Whatman filter paper no. 1, Avicel, ab- sorbent cotton, ECTEOLA, and microcrystalline cel- lulose was determined according to Moreira et al. 16 The results were taken as the mean of triplicate values.

Gel electrophoresbs

Polyacrylamide slab gel electrophoresis, with or with- out SDS, was performed according to LaemmlP 7 to check the homogeneity of CMCase and for its molecu- lar weight determination. The 0.7-mm thick gel con- tained 10% (w/v) acrylamide monomer and 0.1% SDS at pH 8.6. The gels were stained with silver nitrate according to Merril. 18

Endog/ucanase from Bacillus iso/ate: P. Sharma et al.

Activity gel

The purified CMCase was electrophoresed in 10% polyacrylamide gel under nondenaturing conditions and the gel was then blotted on to CMC-agar gel, containing 0.5% CMC and 1% agar in 0.05 M citrate- phosphate buffer, pH 5.0, for 20 min at 55°C. The acrylamide gel was then silver stained for protein while the CMC-agar replica was stained with Congo Red. J2

Molecular weight determination

The molecular weight of the purified CMCase was de- termined by SDS-PAGE using phosphorylase b (94,000), BSA (67,000), ovalbumin (43,000), carbonic anhydrase (30,000), soybean trypsin inhibitor (20,100), and o~-lactalbumin (14,400) as standard proteins for ref- erence mobilities and also by molecular sieve chroma- tography j9 on a column (1 × 100 cm) of Sephadex G- I00 equilibrated with 10 mM Tris/HC1 (pH 7.0). The marker proteins used were ribonuclease A (13,700), chymotrypsin A (25,000), and BSA (67,000).

Results

Characteristics of the isolate

The soil isolate was a Gram-positive, motile rod (0.6- 0.8 × 3.0-4.0 tzm) with central elliptical endospores not distending the sporangium distinctly and was found to belong to the genus Bacillus. The organism showed a maximum growth temperature of 60°C and optimal growth (O.D. 600 nm) was at 55°C, although CMCase production was slightly better at 37°C. The Bacillus sp. PDV did not produce any pigment and was positive for citrate, catalase, Voges-Proskeur reac- tion, and starch hydrolysis. The growth was confluent in 5%, sluggish in 7%, and negative in 9% NaCI. The organism was negative for indole, nitrate, and arginine and did not produce acid from glucose, mannitol, xy- lose, and sucrose. The production of proteases by Ba- cillus sp. PDV started after 8 h and reached its maxi- mum level after 18 h of the onset of growth.

CMCase production

The Bacillus sp. PDV produced cellulase constitu- tively and maximally at 12 h after the onset of growth, and further incubation decreased the level of activity. The organism secreted CMCase completely into the medium and synthesized CMCase maximally in the presence of 0.5% glucose in M9 medium, while there was no growth in this medium containing CMC or Avi- cel as sole carbon (Table 1). In nutrient broth, CMC and holocellulose increased the enzyme production while Avicel showed decreased levels.

Purification of CMCase (Table 2)

Five liters of culture supernatant was ultrafiltered us- ing a pellicon cassette system (Millipore Ltd.) equipped with a 10,000-dalton molecular weight cutoff

Enzyme Microb. Technol., 1990, vol. 12, February 133

Papers Table 1 Effect of cellulosic substrates and glucose on the production of CMCase by Bacillus sp. PDV grown for 18 h in nutrient broth (NB) or in minimal M9 medium (M9)

Reducing sugar concentration Sp. activity in medium Dry weight of culture

Medium (units mg ~ protein) (/~g ml 1) (mg per 50 ml)

NB 0.88 10 24.5 NB + CMC (0.5%) 1.42 95 30.5 NB + Avicel (0.5%) 0.72 55 26.0 NB + holocellulose (0.5%) 1.60 120 32.0 NB + glucose (0.5%) 1.84 - 44,5 M9 ND ND 4,0 M9 + CMC (0.5%) ND ND 6,0 M9 + Avicel (0.5%) ND ND 6.0 M9 + holocellulose (0.5%) ND 10 7.0 M9 + glucose (0.25%) 2.34 - 17.0

(0.50%) 3.58 - 29.5 (0.75%) 3.14 - 40.5 (1.00%) 2.62 23.5

ND, Not detectable

Table 2 Purification of CMCase from thermophi l ic Bacillus sp. PDV

Volume Total enzyme activity Total protein Specific activity Purification Yield Purification step (ml) (units) (mg) (units mg ~ protein) (folds) (%)

1. Ultrafi ltration of 500 5378 1105 4.87 1 100 crude extract (Pellicon PM-10)

2. (NH~)2SO4 precipitate 40 844 90.8 9,29 1.90 15.69 (50-80% cut)

3. UItrafiltration of 10 484 14.9 32.48 6.67 8.99 DEAE-Sephacel eluate (Amicon PM-10)

4. Ultrafiltration of 4 302 3.48 86.78 17.82 5.60 BiogeI-A-0.5m eluate (Amicon PM-30)

5. Superose-6 eluate 1 11 0.075 146.66 30.11 0.20 (fraction 21 )

membrane at l0 lb pressure until 90% of the volume had passed through the membrane. At this point all subsequent steps were carried out at 4°C and with 10 mM Tris/HCl buffer (pH 7.0). The 50-80% ammonium sulfate fraction of the pellicon concentrate was di- alyzed exhaustively and applied to a DEAE Sephacel ion exchange column (2.2 x 14.5 cm) equilibrated and eluted first with 200 mi of 10 mM Tris/HCl (pH 7.0) and then with 200 ml of the same buffer containing 0.5 M NaC1. However, the enzyme did not bind to the ion exchanger and was eluted in breakthrough and wash through fractions. But this step removed many extra- neous proteins. The breakthrough and wash through fractions were pooled and concentrated I 1-fold in an Amicon cell equipped with a PM-l0 membrane and the concentrate was charged on a Biogel-A-0.5m gel filtra- tion column (1.9 x 74 cm). Fractions showing endo- glucanase activity were pooled and concentrated 10- fold in Amicon-PM-30. The resulting concentrate was applied to Superose-6 (standard gel filtration Pharma- cia FPLC column of cross-linked agarose with a bed

volume of 25 ml) and the active fraction was collected. PAGE of this fraction (Figure 1) showed that the en- zyme was homogeneous.

The purified CMCase showed a single band of activ- ity on CMC-agar replica (Figure 2), thus indicating that CMCase is one of the major proteins in extracellu- lar enzyme preparation from Bacillus sp. PDV.

Molecular weight

The molecular weight of the purified CMCase was esti- mated to be 32,500 when its mobility was compared with the relative mobilities of marker proteins in the SDS-PAGE, while it was 33,000 when elution vol- umes (VJVo) versus log molecular weights of standard proteins were plotted.

Substrate specificity

The purified CMCase was assayed with various cellu- losic substrates and xylan. The enzyme showed the capability to degrade crystalline forms of cellulose but

134 Enzyme Mic rob . Techno l . , 1990, vol . 12, February

b

Endoglucanase from Bacillus isolate: P. Sharma et al.

a b c

K

- -43 K

-CMCase (3310

--20.1 K

Figure 1 SDS-polyacrylamide (10%) slab gel electrophoresis of purified CMCase (7.5/~g, lane b) and marker proteins (lane a). The gel was silver stained

Figure 2 Electrophoretic characterization of the Bacillus sp. PDV purified CMCase (7.5/~g) under nondenaturing conditions. The CMC-agar replica was stained with Congo Red after 10 min (lane a) and 20 min (lane b) of blotting. The protein in acryl- amide gel was silver stained (lane c)

to a lesser extent than CMC (Table 3). The enzyme showed no activity toward larchwood xylan but had varied activity on Avicel, filter paper, and cotton.

Effect of temperature on activity and stability of CMCase

The enzyme showed a thermal optimum at 60°C, al- though it was quite (92%) active even at 70°C. At 80°C the activity was reduced to 50%. For the thermal sta- bility of CMCase, 100/xl of enzyme (20/xg ml J) in 0.1 M citrate phosphate buffer, pH 5.0, was heated at the desired temperature for I0 rain and the residual activ- ity was measured. The enzyme was stable up to 60°C, retained 80% activity at 70°C, and only 14% at 75°C. Repeated freezing and thawing (three times) had no effect on the CMCase activity.

Effect of pH on activity and stability of CMCase

The pH optimum of CMCase was determined using 0.1 M citrate-phosphate buffer (pH 3-7), 0.1 M Tris/HCl buffer (pH 7-9), and 0.1 ~ carbonate-bicarbonate buffer (pH 9-11). Other conditions were the same as for the standard assay. The CMCase was almost equally active in the pH range of 4.5-7.0, although showing most activity at pH 5.0. The enzyme retained 77% activity at pH 8.0, 63% at pH 9.0, and only 9% at pH 10.0.

The stability of CMCase at various pH values was investigated. One hundred microliters of enzyme (20 /,g ml -~) was mixed with 100/xl of appropriate buffer

and incubated for 24 h at 4°C. Residual activity was measured by standard assay. The CMCase was stable over the pH range of 4-I0 under these conditions, showing maximum stability at pH 5.0.

Kinetics

With CM-cellulose as substrate and using 375 mU CMCase, the Lineweaver-Burk plot gave a Km value of 0.588 mg m1-1 and graax value of 148.15 /xmol glu- cose mg -l CMCase rain -~. With methylcellulose, the Km was 2.28 mg m1-1 and V,na× was 98 /xmol glucose mg -l CMCase rain -j.

Table 3 Activity of purified CMCase on different cellulosic sub- strates a

Specific activity Substrate (kilounits mg -~ enzyme)

Carboxymethylcellulose 4.600 Methylcellulose 1.120 Xylan 0.000 ECTEOLA Cellulose 0.091 Avicel RC-581 0.377 Microcrystalline cellulose 0.086

(chromatography grade) Filter paper (Whatman No. 1) 0.011 Absorbent cotton 0.006

a 10 mg of each substrate was assayed with 1.5/~g of purified CMCase

Enzyme Microb. Technol., 1990, vol. 12, February 135

Papers

Effect o f metal ions and certain reagents on CMCase activity

The chloride salts of Na +, Mn z+, Mg 2+, Fe 3+, NH4 ~, Co 2+, Zn 2+, and Cd 2+ had no effect, while Hg 2+ com- pletely inhibited the activity. Ca 2+ and Ag + (AgNO3) inhibited the activity by 36% and 30%, respectively, at 10 mM concentration, while 1 mM of them had no ef- fect. The activity increased by I5% at 1 mM ofCu + and Ba 2+ and by 25% at 10 mM Ba 2+ concentration.

Various other reagents, such as PMSF (pheny[- methylsulfonylfluoride), sodium arsenate, EDTA, glu- cose, cellobiose, ascorbic acid, and certain oxidizing agents, e.g. H202, sodium perchlorate, hydrazine sul- fate, and potassium iodate, had no effect on activity, although ammonium molybdate and certain reducing agents, viz. cysteine-HC1, glutathione, and sodium thioglycollate, enhanced the CMCase activity by 12- 16% at 10 mM concentration. Ammonium molybdate and 2-mercaptoethanol at 1 mM and SDS at 0.1% con- centrations increased the activity by 34%, 43%, and 15%, respectively.

Discussion

There are only a few reports on the cellulases pro- duced by Bacillus species. The cellulolytic bacilli lack the complete cellulase system and produce/3-1,4-en- doglucanase but not cellobiohydrolase, thus hydrolys- ing only the noncrystalline forms of cellulose. 7.8 Pre- liminary work with the soil isolates, Bacillus sp. PDV, showed the presence of CMCase, avicelase, filter pa- per, and xylanase activities in culture supernatant and /3-glucosidase in intracellular milieu. We also observed that the organism produced all these enzymes consti- tutively when grown in nutrient broth or on a variety of mono- or disaccharides or sugar alcohols, but could not utilize CMC or crystalline cellulose as sole carbon source unless provided with some easy carbon source, e.g. yeast extract or limiting amounts of glucose in the medium for the start of the growth. The increased en- zyme activity on addition of CMC or holocellulose to the nutrient broth and detection of increased amounts of reducing sugar in the medium suggest the require- ment of preformed CMCase for further growth on CMC and for its degradation. The Bacillus sp. DLG 9 and B. cereusZ° also exhibited CMCase production in the presence of sugars, while the alkalophilic Bacillus sp. ~° No. 1139 stopped the synthesis of CMCase on addition of glucose to medium. Thus the production of cellulase by the strain PDV is not subject to catabolite repression by glucose or other sugars that has been found with other fungal and bacteriaP ,21-23 cellulases. The decrease in the levels of CMCase after 12 h of the onset of growth corresponded well with the increase in protease level from 8 to 18 h of growth.

In this study we observed that the purified CMCase of the strain PDV showed activity towards crystalline forms of cellulose such as Avicel, cotton, and filter paper, although this activity was quite small compared to the activity against CMC. Thus the purified enzyme is endoglucanase in nature and seems to have the capa-

bility to adsorb to and cleave the crystalline cellulose molecules from the nonreducing ends, although the efficiency was quite small. The ability to degrade car- boxymethylcellulose more than methylcellulose indi- cates the affinity of the enzyme for the carboxyl group. The CMCase from other Bacillus species 9,11 has not been reported to degrade cystalline cellulose.

The Bacillus sp. PDV CMCase showed stability over a wide pH range (4-10) and tolerated a tempera- ture of 60°C, thus making this enzyme suitable for cel- lulose saccharification at elevated temperatures and under acidic and alkaline environments. The CMCase from most Bacillus species 9,j~,2° showed stability up to 40-45°C.

The inability of glucose and cellobiose to inhibit CMCase activity is in agreement with endoglucanases of Bacillus sp. DLG, 9 B. cereus, 2° T. viride, 24 and Clos- tridium thermocellum. 5 Most of the metal ions, oxidiz- ing, and chelating agents had no marked effect on CMCase activity, with a few exceptions, e.g. Hg 2~ completely abolished the activity while Cu + and Ba 2. increased the activity slightly. The enzyme activity increased by 5-43% in general in the presence of re- ducing agents. Increase in activity by 2-mercapto- ethanol and complete inactivation of enzyme by Hg 2+ suggested that reduced thiol groups are required for activity and these groups are present in the active site of the enzyme. Increase in CMCase activity by mer- captoethanol and glutathione may also be attributed to the enzyme-stabilizing effect of these agents. The lack of any effect of chelators and sodium azide showed that CMCase is not a metalloenzyme and did not re- quire any cation as cofactor for activity. PMSF did not inhibit CMCase, thus showing that the enzyme is not a protease in nature. The heavy metals have been re- ported to potentially inhibit the CMCase activity in Bacillus species. 9,11

The lack of catabolite repression by glucose or cel- lobiose on the synthesis and activity of CMCase has the distinct advantage of using this physicochemically stable cellulase, in conjunction with other enzymes, for the bioconversion of cellulose to energy and chemi- cal feed stocks. The overproduction of such stable en- zymes by cloning 25 of their genes or by mutagenesis can help achieve this target.

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