Extracellular cellulolytic enzyme system of Aspergillus japonicus: 3. Isolation, purification and characterization of multiple forms of endoglucanase

Extracellular cellulolytic enzyme system of Aspergillus japonicus: 3. Isolation, p.urification and characterization of multiple forms of endoglucanase Ramendra K. Kundu,* Syamalima Dube** and Dipak K. Dube? Department of Biochemistry, University of Calcutta, Calcutta 700 019, India

(Received 5 March 1987; revised 28 May 1987) The crude culture filtrate of Aspergillus japonicus exhibits marked cellulose and xylan degrading activities. Affinity chromatography of the crude enzyme preparation on concanavalin A (Con A )-Sepharose 4B resolves it into three fractions. The eluted fraction A contains CMCase ( CMCase I) and xylanase activities; fraction B shows only CMCase activity (CMCase II); and fraction C exhibits CMCase (CMCase Ill) and xylanase activities. On further purification by gel filtration, only fraction B imparts a homogeneous preparation that gives a single band on polyacrylamide gel electrophoresis at pH 8.3. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of this homogeneous form shows the molecular weight about 57000 daltons. Sephadex G-IO0 column chromatography also supports the molecular weight. A low molecular weight endoglucanase, of about 12 700 daltons, was obtained from fraction A by Sephadex G-75 column chromatography whereas the molecular weight of the endoglucanase from fraction C was higher (77000 daltons). The silver ion (Ag I+) strongly inhibits the endoglucanase I activity but has no effect on II and I I l . Hg 2+ inhibits all the three forms. The pH optimum for endoglucanase I, II and III is 4.5. The endoglucanase II shows the highest temperature optimum of 65°C whereas those of endoglucanase I and I l l are 50°C and 55 ° C, respectively. The first two forms have the least activity toward tamarind kernel polysaccharide ( T K P ) but the last form, endoglucanase III, is highly reactive to it.

Keywords:Aspergillus japonicus; endoglucanase;multiple forms; purificationand characterization Introduction Multiple forms of endoglucanase have been isolated and characterized from the crude culture filtrate of Trichoderma viride, 1"2 T. koningii 3 and Sporotrichum pulverulentum. 4 In this paper we now report the isolation and characterization of three forms of extracellular carboxymethyl cellulases (CMCase) produced by the fungus Aspergillus japonicus grown on wheat bran.

Uppsala, Sweden. DE-52 (Whatman's preswollen DEAEcellulose) was purchased from Whatman Chemicals Ltd., UK. Carboxymethyl cellulose (the sodium salt of CMC) was purchased from Loba Chemie Indoaustranal; xylan (from Larchwood), o-nitrophenyl-fl-D-glucoside (ONPG), o-nitrophenyl-fl-D-xyloside (ONPX), bovine serum albumin, ovalbumin, trypsin, myoglobin and RNAse from Sigma Chemical Co., St. Louis, MO, USA. Other chemicals used were of Analar grade.

Materials and methods Chemicals Con A-Sepharose 4B, Sephadex G-100 and Sephadex G-75 were the products of Pharmacia Fine Chemicals,

* To whom correspondenceshould be addressed ** Present address: Department of Medicine, Rm-10, University of Washington, Seattle, WA 98195, USA t Present address: Department of Pathology SM-30, University of Washington, Seattle, WA 98195, USA 100

G r o w t h conditions A. japonicus was grown on moistened wheat bran (40% of the solid), which served as sole carbon source. The growth of the organism was maintained in 250 ml conical flasks containing 20 g sterilized moistened wheat bran at 28°C for 6 days. An inoculum of spore suspension containing spores was sprayed on the medium. After incubation, the content of each flask was extracted with

Enzyme Microb. Technol., 1988, vol. 10, February

Extracellular cellulolytic enzyme system of Aspergillus japonicus, part 3: K. Kundu et aL 100 ml citrate buffer (0.05 M, pH 4.8) containing 0.05 %. Triton X-100 at 4°C. After about 30 min, the mixture was centrifuged at 8000 rev min -1 in a refrigerated centrifuge at 4°C. The clear supernatant served as a crude culture filtrate.

Enzyme assays fl-Glucosidase/fl-xylosidase activity was determined in a reaction mixture containing 0.025 ml either O N P G or O N P X (40 mM), 0.5 ml 0.05 M citrate buffer (pH 4.8) and 0.01 ml enzyme, made to a total volume of 1 ml with water, incubated at 50°C for 30 min, followed by addition of 3 ml of 0.1 N NaOH. The o-nitrophenol released was measured at 410 nm 5. One unit of enzyme is defined as the liberation of 1 mg O N P per minute at 50°C. CMCellulase activity (endo-fl-l,4-o-glucanase) was measured as reducing sugar by the method of Nelson 6 as modified by Somogyi. v The reaction mixtures containing 1.0 ml CMCellulose (10 mg m1-1 in 0.05 M citrate buffer pH 4.8), 0.25 ml of 0.1 M citrate buffer and 0.025 ml enzyme source in a total volume of 1.5 ml were incubated at 55°C for 30 min. The reaction was stopped by adding 1.0 ml alkaline copper reagent, boiled for 10 min in a water bath and cooled. The color was finally developed by the addition of 1.0 ml aresenomolybdate reagent followed by centrifugation and was read at 540 nm after diluting the supernatant threefold with water. One unit of enzyme was defined as the amount of enzyme that liberates one #mole of o-glucose per minute under the assay conditions. A calibration curve was prepared with a standard D-glucose solution. Xylanase activity was determined by the amount of liberated reducing sugar exactly as described above except that the assay mixture contained 0.01 ml enzyme fraction, 1.0 ml xylan suspension (1% xylan in 0.1 M acetate buffer, pH 5.0) and 1.0 ml of acetate buffer (pH 5.0); incubation was carried out at 40°C for 30 min. After color development, the mixture was centrifuged and the supernatant was diluted threefold before the absorbance was read at 500 nm. One unit of enzyme activity was expressed as the amount of enzyme that produces one /~mole of D-xylose per minute under the assay conditions. A calibration curve was also made using a standard D-xylose solution.

(10 x 0.5 cm) equilibrated with 0.02 M Tris-HC1 buffer (pH 7.4) containing 1 mM Mn 2 + and 1 mM Ca 2+. The column, loaded with the enriched enzyme sample, was first eluted with the starting buffer. The fractions collected were designated as Component A (Fr. No. 1-7). The column was then washed with initial buffer plus 0.5 M NaCI. The collected fractions were bulked and marked Component B (Fr. No. 12-18). Finally, the elution was made with 0.02 M Tris-HC1 buffer, 0.5 M NaC1 and 0.1 M methyl a-D-glucoside. The eluted fractions obtained by the desorbing substrate were designated as Component C (Fr. No. 22 25). Each fraction of 2.0 ml size was collected in fraction collector (Fractometer 200, Buchler, USA).

Step 3:DE-52 column chromatography. The active pooled Component A (Fr. No. 1-5) was charged onto a DE-52 column (20 × 1 cm) previously equilibrated with 0.01 M Tris-HC1 buffer (pH 7.0). After washing with initial buffer, the column was eluted with buffer having a salt concentration increasing from 0.05-0.4 M KCI in batches. Elution was performed at a flow rate of 30 ml per hour with a fraction size 7.0 ml. Fifteen fractions were collected in the fraction collector. Step 4: Sephadex G-75 column chromatography. The fractionation was carried out on a 100 x 1 cm Sephadex G-75 column previously equilibrated with 0.01 M TrisHC1 buffer, 0.2 M NaCI (pH 7.0). A 6.0 ml aliquot of active pooled fraction (6 and 7) from DE-52 column was loaded on the column, which had a flow rate of 12 ml per hour. The column was eluted with the same equilibrating buffer. Forty fractions, each containing 2.0 ml, were collected in an automatic fraction collector (Fractometer 200, Buchler, USA).

Isolation and purification of endoglucanases

Step 5: Sephadex G-100 column chromatography. The active fraction B (12-18) and the active fraction C (22-25) from Con A Sepharose 4B column were individually applied to Sephadex G-100 column ( 100 x 1 cm) previously equilibrated with 0.01 M Tris-HC1 buffer, 0.2 M NaC1 (pH 7.0). A constant flow rate of 10 ml per hour was maintained with a fraction size 2.0 ml. The column was eluted with the same buffer. Thirty fractions of 2.0 ml size each were collected in the fraction collector in both cases. The final enzyme preparations from Fr. A, Fr. B and Fr. C were then desalted on Sephadex G-25 column as previously equilibrated with 0.01 M Tris HC1 buffer (data not shown).

The entire work of purification was carried out at 4°C unless otherwise mentioned.

Polyacrylamide gel electrophoresis

Protein estimation Protein was measured by the method of Lowry et al.,8 taking bovine serum albumin as standard.

Step 1: concentration of the enzymes. The prepared crude culture filtrate (225 ml) was concentrated by ultrafiltration (Diaflo-membrane, PM-10) to 25 ml in an ultrafiltration system obtained from Amicon Corp., USA.

Step 2: Con A-Sepharose 4B chromatography. Affinity chromatography was carried out on a mini-column

The homogeneity of the purified endoglucanase was analyzed by polyacrylamide gel (7.5%) electrophoresis (PAGE), according to the method of Davis 9 at pH 8.3, using bromophenol blue as the tracking dye. A current of 3 mA/gel was applied. Protein(s) was located by staining with Coomassie blue and destained with methanol/ acetic acid/water. Sodium dodecyl sulphate-polyaerylamide gel (10%) electrophoresis (SDS-PAGE) of the

Enzyme Microb. Technol., 1988, vol. 10, February

101

Papers purified enzyme preparation was performed in a slab in accordance with the method of Weber and Osborn. a° Molecular weight standards used here were bovine serum albumin (67 000 daltons), ovalbumin (43 000 daltons) and myoglobin ( 17 000 daltons).

~to]

Frac/ion-A 7MCase I ko

Determination of molecular weight

x.a

Molecular weights of endoglucanases were determined by gel filtration in either Sephadex G-75 or Sephadex G-100 column using as standard proteins BSA (67000 daltons), ovalbumin (43000 daltons), trypsin (24000 daltons) and RNase (13 7000 daltons).

[/'schbn-B

Determination of pH and temperature optima The optimum pH of CMCase I, II and IlI was determined by incubating 25 #1 of respective purified enzyme with 0.25 ml of 0.1 M citrate or acetate buffer (pH 3.0-7.0) and 1.0 ml CMCellulose solution (10 mg ml- 1 of CMC prepared in buffer of respective pH) in a final volume 1.5 ml at 55°C for 30 min. The reducing sugar was then estimated according to the method described earlier (enzyme assay). For determination of temperature optimum of the three enzymes, the incubation of the assay mixture was carried at different range of temperatures (30 70°C) in thermostatic water baths for 30 min.

a.5

CMC,~se ll

;, i-;,-,~

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Fraction A/umbGr

Figure 1 Affinity chromatography of enriched crude preparation on Con A-Sepharose 4B column: x , CMCase activity eluted in Fraction A with starting buffer; in Fraction B with buffer containing 0.5 M NaCI and in Fraction C with same buffer, 0.5 M NaCI and 0.1 M methyl-~-o-glucoside; ©, appearance of xylanase activity in Fraction A and Fraction C

is expressed as a percentage of activity level in absence of any chemical agents.

Kinetics study Thermal stability A unit of C MCase I (9.74 x 10-2) was diluted in buffer so that the mixture contained 25 mM sodium acetate (pH 5.0)/25 mM Tris-HC1 (pH 7.0). The enzyme was preincubated at 55°C. At different time intervals, 0.20 ml aliquot was withdrawn and subsequently assayed for enzyme activity at the optimum temperature. For other two forms, 0.165 units of CMCase II and 0.146 units of CMCase III were separately diluted in buffer in the same way as described earlier. Here preincubations were done at 65 ° C. At different time intervals, 0.15 ml aliquots for CMCase II and 0.05 ml aliquots for CMCase III were taken out; each enzyme was assayed at the respective optimum temperature. The unit of each enzyme activity is defined as the liberation of 1 #mole of D-glucose per minute at respective optimum temperature.

Energy of activation The kinetics of CMCase catalysed hydrolysis of CMC was measured over different temperature range by estimating reducing sugar according to the method of Nelson 6 as modified by Somogyi. 7 The value of activation energy was calculated from the Arrhenius plot as being equal to (slope × 19.14) J m o F 1.

The kinetics of CMCase catalysed hydrolysis of substrates like tamarind kernel polysaccharide (TKP), CMC and methyl cellulose (MC) were measured by estimating the reducing sugar liberated in the reaction. Different aliquots of enzyme of CMCase I ( l A x l0 -2 unit), CMCaseII(5.2 x 10-3 unit) and CMCase III (7.0 × 10 -3 unit) were incubated with T K P (0.66 mg ml-1), CMC (0.60 mg ml- 1) and MC (0.68 mg ml - 1) in an incubation volume of 1.5 ml at their optimum temperatures. One unit of enzyme is defined by liberation of 1 ktmole of D-glucose liberated per 30 min at optimum temperature.

Results

Enzyme purification The production of cellulolytic and hemicellulolytic enzymes on moist wheat bran is summarized in Table 1. The results from purification procedure are given in Table 2. Con A Sepharose 4B primarily resolved the protein into three Components A, B and C (Figure 1).

Table 1 Production of different cellulolytic and hemicellulolytic enzymes by Aspergillus japonicus in wheat bran (solid) after six days incubation

Effect of various metal ions The effect of metal ions on the activities of CMCase I, II and III was tested by treating metal ion ( 1 mM) with individual enzyme (25 #1) and 0.25 ml of 0.1 M citrate buffer (pH 4.8) and preincubated at 37°C for 30 min. After that 1.0 ml ! % CMC solution was added and was assayed at optimum temperatures for 30 min. The activity 102

Enzyme Microb. Technol., 1988, vol. 10, February

Enzyme /t-Glucosidase

fl-Xylosidase CMCase Xylanase

Specific activity ( Units/mg protein) 0.46 0.32 0.48 0.66

Extracellular cellulolytic enzyme system of Aspergillus japonicus, part 3: K. Kundu et aL Table 2

Purification of endoglucanases (CMCases) from Aspergillusjaponicus grown in wheat bran medium (solid)

Step

Total protein (mg)

Total activity (Units)*

Specific activity ( U n i t / m g protein)

Purification (fold)

Crude buffer extract

337.50

192.85

0.57

1.0

Concentrated ultrafiltered fraction

100.00

136.36

1.37

2.4

6.52

1.63

2.8

1.94

2.78

5.0

4.67

1.37

2.4

2.18

2.18

4.0

0.93 0.42

4.70

8.2

5.10

9.0

5.10

8.91

Affinity chromatography on Con A-Sepharose 4B Charging material 16.0 (a) Recovery of CMCase I in Fraction A with starting buffer 4.0 (b) Recovery of CMCase II in Fraction B with buffer and 0.5 M NaCI 0.7 (c) Recovery of CMCase III in Fraction C with buffer, 0.1 M methyl-s-Dglucoside and 0.5 M NaCI 3.4 DE-52 column chromatography Charging material of Fraction A Recovery of CMCase I 1.0 Sephadex G-75 column chromatography Charging material of active fractions from DE-52 column 0.43 Recovery of CMCase I 0.09 Sephadex G-100 column chromatography Charging material of Fraction B 1.26 Recovery of CMCase II 1.02 Charging material of Fraction C 3.01 Recovery of CMCase III 0.55

21.81

0.45 0.20 4.0 2.80

* One unit is defined as the amount of enzyme that liberates 1 /~mole glucose per min at 55°C

Component A eluted with starting buffer contained only CMCase and xylanase activities. Further purification of DE-52 ion-exchanger (Figure 2) followed by Sephadex G-75 gel filtration (Figure 3) retained the two activities. This CMCase was designated as CMCase I. Component B showed only CMCase activity and was subsequently

, ~ 0"9 -

marked as CMCase II. In the final step of elution, CMCase as well as xylanase activities appeared together in Component C. Gel filtration through Sephadex G-100 column chromatography (figure not shown) did not separate xylanase activity from CMCase activity. This CMCase was termed as CMCase III.

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Figure 3 Sephadex G-75 column chromatography: x , CMCase I activity; O, xylanase activity

Enzyme Microb. Technol., 1988, vol. 10, February

103

Papers

column chromatography (Figure 5). The molecular weight of CMCase II was determined by Sephadex G-100 gel chromatography (Figure 6) and SDS-PAGE gel electrophoresis (Fioure 7); it lies between 54000 57 000 daltons. The molecular weight of CMCase III as determined by Sephadex G- 100 column chromatography was higher, ~ 77 000 daltons (Figure 6). ~i~~!

General properties of three endoglucanases

i ii:

pH Optima. Endoglucanase I, II and III possess the same pH optimum of pH 4.5 (Figure 8).

iiii?

Temperature optima. CMCase II exhibits the highest temperature optimum, 65°C, among the three. The temperature optima of CMCase I and III are 50°C and 55°C, respectively (Figure 9).

ii ....

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Stability. CMCase I denatures quickly at pH 5.0 and 7.0 during preincubation at 55°C (Figure 10). It lost 90% of its activity when preincubation was performed at pH 7.0. In contrast, CMCase II and III are more stable. They do not show any loss of activities at 60°C (data not shown). Even at a still higher temperature, 65°C, the loss of activity at pH 5.0 is minimum. However, at pH 7.0 the fall of activity is noticeable in both the cases. The half life (tl/2) for CMCase II at pH 7.0 is 13 min while that for III is about 8 min (Figure 11).

A

Figure 4 Polyacrylamide gel electrophoretic pattern of CMCase I1: A, enriched ultrafiltered fraction; B, dialyzed pooled active fractions of CMCase II from Sephadex G-100 column

Energy of activation. The energy of activation of individual endoglucanases was determined over different temperature ranges: 37°C 50°C for CMCase I; 37 °65°C for CMCase II; and 37 ° 55°C for CMCase III. The velocity Vmaxwas measured at optimum pH over the respective temperature range, using the sodium salt of CMC as substrate. The energy of activation of CMCase III is relatively higher (18.22 kcal mole-1) whereas the value for CMCase I is 5.34 kcal mole -1 and the

Enzyme purity Only the endoglucanase II (CMCase II) was obtained in a homogeneous form. This protein moved as a single band in polyacrylamide gel upon electrophoresis at pH 8.3 (Figure 4). Component A from the final G-75 step imparted two bands in polyacrylamide gel (figure not shown).

Determination of molecular weight CMCase I was found to possess a low molecular weight (about 12700 daltons), determined by Sephadex G-75 s'2-

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Figure 5 Determination of molecular weight of CMCase I by Sephadex G-75 column chromatography. Standard proteins used here were albumin (67000 daltons), ovalbumin (43000 daltons), trypsin (24 000 daltons) and RNAase ( 13 700 daltons)

104

Figure 6 Determination of molecular weight of CMCase II and III by gel filtration through Sephadex G-100 column. Standard proteins run in this column were alkaline phosphatase (140000 daltons), albumin (67000), ovalbumin (43000), trypsin (24000) and RNAase ( 13 700)

Enzyme Microb. Technol., 1988, vol. 10, February

Extracellular cellulolytic enzyme system of Aspergillus japonicus, part 3: K. Kundu et aL 1

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Figure 7 Estimation of molecular weight by SDS-polyacrylamide gel (10%) electrophoresis. A, Protein pattern on SDS gel: lane 1, three standard proteins, BSA, ovalburnin and myoglobin; lane 2, purified CMCase II from Sephadex G-100 column. B, Plot of the migration of three standard proteins against molecular weight. From the distance migrated, the molecular weight of CMCase II was calculated to be 57 000 daltons

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energy of activation of CMCase II is 9.11 kcal mole -1

(Figure 12). Effect o f various metal ions The effect of metal ions on the activities of CMCase I, II and III is represented in Table 3. A m o n g the metal ions tested, Ag 1÷ ion has been found to be strongly inhibitory (93%) to CMCase I but has practically no

Figure 8 Determination of pH optima of CMCases: O, citrate buffer; A, acetate buffer

effect on the other two endoglucanases. Hg 2+ inhibits all of the activities of the three endoglucanases. CMCase I activity is also found to be inhibited by Mn 2÷ ion, whereas no considerable decrease in activity of either CMCase II or CMCase III is observed.

Enzyme Microb. Technol., 1988, vol. 10, February

105

Papers 0"05 CA¢Ca~e I

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lli

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Determination of optimum temperature of C M C a s e I, C M C a s e II and C M C a s e Ill

Substrate specificity The relative rates of hydrolysis of various substrates by CMCase I, II and III are represented in Table 4. CMCase III has a very high affinity toward T K P in comparison with the other CMCases. The very low rate of hydrolysis of methyl cellulose by all the endoglucanases is evident from this table. The kinetic constants for CMC (sodium salt) hydrolysis were calculated from Lineweaver-Burk plots (Figure 13). K m values for CMCase I, Ii and III for this substrate are 0.25 mg m1-1, 0.66 mg m1-1 and 0.33 mg ml -~, respectively.

(CMCase) from A. niger after fractionation on Sephadex G-100 column. All of these were associated with #glucosidase activity. Subsequent studies were made on a wide range of microorganisms, e.g., Trichoderma

viride, 1'2'x2"~3 T. koningii, 3 Sporotrichum pulverulentum 4 and Fusarium avaneeeum. ~4 These organisms are also able to produce multiple forms of endoglucanase. Ng and Zeikus a5 have also reported the multiple forms of endoglucanase from crude extracellular cellulase of an anaerobic thermophilic bacterium, Clostridium thermo-

cellum. The multiple forms of CMCases are well differentiated by their molecular weight, temperature optima, heat denaturation, substrate specificity and electrophoretic

Discussion

A. japonicus, one of the less-studied organisms in the Asperyillus genus, produces three forms of endoglucanase during its growth on wheat bran. The multiplicity of endoglucanases in Aspergillus species was reported by Petterson ~1 who obtained four forms of cellulase

Effect of metal ions on the activity of C M C a s e I, C M C a s e II and C M C a s e III of Aspergillusjaponicus

100"4

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60-

Table 3

Relative activity Metal ions

CMCase I

C M C a s e II

C M C a s e III

None M n 2+ M g 2+ Cu~ + Hg 2+ Ag 1 + Pb 2+

100.0 28.5 92.2 108.0 1 6.1 7.2 82.7

100.0 88.8 1 33.3 82.0 1 6.2 108.5 57.2

100.0 61.1 87.7 57.7 6.2 82.2 60.3

~ 40-

20-

T/me Of pt'o/ncu6a/l'on(m/ns) Activity is expressed as a percentage of activity level in absence of any chemical agents. The individual enzyme was preincubated with metal ion (1 mM) at 3 7 " C for 30 min

106

Enzyme Microb. Technol., 1988, vol. 10, February

Figure 10 Thermal stability o f C M C a s e I at 55° C; the initial activity corresponds to 100%: 0 , CMCase I, pH 5.0; O, CMCase I, pH 7.0

Extracellular cellulolytic enzyme system of Aspergillus japonicus, part 3: K. Kundu et aL I00 9080.~

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Figure 11 Thermal stability of CMCase II and III at 65°C; the initial activity corresponds to 100%: O, CMCase II, pH 5.0; C), CMCase II, pH 7.0; A , CMCase III, pH 5.0; A , CMCase III, pH 7.0

1

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Z~oa / ¢ - /

mobilities in polyacrylamide gel. The molecular weights of CMCase I, II and III of A. japonicus as determined by Sephadex G-75 and Sephadex G-100 column chromatography are 12 700, 54000 and 77000 daltons, respectively. Furthermore, gel electrophoretic study revealed that purified CMCase II behaved as a single protein under native as well as denaturing condition (SDS-treatment). The molecular weight of this denatured protein (57000 daltons) was close to that of native enzyme (mol. wt. 54000). However, the three forms of CMCases exhibit the same pH optima (4.5). This feature is similar to that reported for T. viride, 2 where endoglucanases II, III and IV have similar pH optima (pH 4.0-4.5) but different molecular weights - 37 200, 52 000 and 49 500 daltons, respectively. Weber et al. 12 separated five forms of endoglucanase from T. viride by affinity chromatography on cross-linked cellulose; among them, four forms show the same pH optima (5.6) whereas the last form exhibits a pH optimum of about 5.0. The low molecular weight (about 12700 daltons) of CMCase I of A. japonicus is not unusual, as is evidenced by the fact that Berghem et al. 1 obtained two CMCellulases of low (13000) and high (50 000) molecular weights, respectively, which were separated by chromatography and isoelectric focusing. The molecular weight of an endoglucanase from C. thermocellum ~5 was also found to be high and varied between 83000 and 94000 daltons. In contrast five Table 4

7-

Figure 12 Energy of activation of CMCase I, II and II1: O, CMCase I activity; 0 , CMCase II activity; A , CMCase III activity

endoglucanases T1, Tza, T2b, T3a and T3t,, purified by Eriksson and Petterson 4 from the culture solutions of S. pulverulentum have their molecular weights in a close range, between 28 300 daltons and 37 500 daltons. Another characteristic feature is the different temperature optima of three forms. CMCase II and CMCase III are more heat-stable than CMCase I. Moreover, Ag 1÷ ion inactivates only CMCase I; CMCase II and CMCase III activities remain practically unaffected in the presence of this ion. The substrate specificities of the three forms are also different. While CMCase III has the ability to degrade T K P very effectively, CMCase I and CMCase II possess the least specificity. CMCase III hydrolyses T K P four times faster than CMC, while the exact reverse is true for enzymes I and II. Furthermore, the three enzymes are different in respect to the value of energy of activation. A comparative study of the three CMCases in respect to their properties is summarized in Table 5. The CMCases or endoglucanases isolated and purified in the present study reveal that the three forms are well distinguished in respect to their temperature optima, thermal stability, molecular weight, effects of metal ions, substrate specificity and energy of activation.

Substrate specificity of CMCase I, II and III of Aspergillusjaponicus at their respective optimum temperatures Glucose liberated in 30 min (/~mole)

Substrate

Substrate concentration ( m g / m l )

CM Case I

CMCase II

CMCase III

Tamarind kernel polysaccharide Carboxyl methyl cellulose (sodium salt) Methyl cellulose

0.66 0.60 0.68

0.12 0.53 0

0.04 0.16 0

0.79 0.18 0.06

Different aliquots of enzyme, i.e., 1.4 x 10 2 unit of CMCase I, 5.2 x 10 3 unit of CMCase II and 7.0 x 10 -3 unit of CMCase III were incubated with different substrates in an incubation volume of 1.5 ml at their optimum temperatures

Enzyme Microb. Technol., 1988, vol. 10, February

107

Papers V

CMCaso I

2!0

4!0

/~/ mg -/Xm/ I

CMCase l/

"~

II CMCaseE]

~"~

I

0"0-

~ ,!o

;!o

J'O-

~:o

;,,

2

/~/ mg"t X m/

3(/mg-/X m/

6!o

Figure 13 Lineweaver-Burk plots of CMCase I, II and II1. The assays were carried out using CMCellulose (sodium salt) as substrate at optimum pH and respective optimum temperature by incubating different aliquots of enzyme, 1.4 × 10 2 unit for CMCase I, 5.2 x 10 3 unit for CMCase II and 7.0 × 10 3 unit for CMCase III; the reaction rate was proportional to the enzyme concentration

Table 5

Comparison of CMCase from

Aspergillusjaponicus

Physico-chemical properties

CMCase I

CMCase II

CMCase III

pH Optima Temperature optima Molecular weight by Column chromatography on Sephadex G-75 Column chromatography on Sephadex G-100 SDS-PAGE Associated/contaminating activity Xylanase Cellobiase Homogeneity Polyacrylamide gel SDS-PAGE Thermal inactivation At 55°C and pH 5.0 At 55°C and pH 7.0 At 65°C and pH 5.0 At 65"C and pH 7.0 Substrate specificity Methyl cellulose Tamarind kernel polysaccharide Km using CMC-Na as substrate

4.5 50°C

4.5 65°C

4.5 55°C

12 700 daltons N.D. N.D.

N.D. 54000 daltons 57000 daltons

N.D. 76 000 daltons N.D.

Yes Nil

Nil Nil

Yes Nil

Not homogeneous N.D.

Single band Single band

Not homogeneous N.D.

tl/2 = 6 . 2 5 tl/2-2.75

min min Rapidly denatured Rapidly denatured

Stable Stable > 20 min = 12.25 min

tl/2 tl/2

Stable Stable No appreciable decrease tl/2 = 7.6 min

0 + K m - 0 . 2 5 mg m1-1

0 + Krn = 0 . 6 6 mg ml 1

0 +++ Km = 0.33 mg m1-1

N.D. = Not detected; + = active; + + + = highly active

108

Enzyme Microb. Technol., 1988, vol. 10, February

Extracellular cellulolytic enzyme system of Aspergillus japonicus, part 3: K. Kundu et aL Acknowledgement The authors are indebted to Dr. Ambica C. Banerjee, the Department of Biochemistry, Calcutta University, India, for valuable criticism in the writing of this manuscript.

References 1

3

6 7 8 9 10 11 12

Berghem, L. E. R., Petterson, L. G. and Axio-Fredriksson, O.-B. Eur. J. Biochem. 1976, 61, 621

2

4 5

Showemaker, S. P. and Brown, R. D. Biochim. Biophys. Acta 1978, 523, 147 Halliwell, G. and Griffin, M. Biochem. J. 1978, 169, 713

13 14 15

Eriksson, K. E. and Petterson, B. Eur. J. Biochem. 1975, 51, 193 Michell, K. H., Karonovsky, N. J. and Kornovsky, M. L. Biochem. J. 1970, 116, 207 Nelson, N. J. Biol. Chem. 1944, 153, 375 Somogyi, M. J. Biol. Chem. 1952, 195, 19 Lowry, O. H. et al. J. Biol. Chem. 1951, 193, 265 Davis, B. J. Ann. N.Y. Acad. Sci. 1964, 121, 404 Weber, K. and Osborn, M. J. Biol. Chem. 1969, 2,44, 4406 Petterson, G. Biochim. Biophys. Acta 1963, 77, 665 Weber, M., Foglietti, M. J. and Percherson, F. J. Chromat. 1980, 188, 377 Okada, G. J. Biochem. 1975, 77, 33 Zalewska-Sobezak, J. and Urbanek, H. Arch. Microbiol. 1981, 129, 247 Ng, T. K. and Zeikus, J. G. Biochem. J. 1981, 199, 341

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