FEMS Microbiology Letters 79 (1991) 99-104 © 1991 Federation of European Microbiological Societies 0378-1097/91/$03.50 Published by Elsevier ADONIS 0378109791001738

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FEMSLE 04372

Physical characterization of isozymes of endo-fl-l,4-glucanase and fl-l,4-glucosidase from Aspergillus species

Suni t a Sha rma , D h a n w a n t K. S a n d h u a n d P.S. Bagga

Microbiology Unit, School of Life Sciences, Guru Nanak Dev University, Amritsar, India

Received 7 June 1990 Revision received 22 November 1990

Accepted 27 November 1990

Key words: endo-fl-l ,4-Glucanase; fl-l,4-Glucosidase; Isozyme; Aspergillus; Characterization

1. S U M M A R Y

Electrophoretic data revealed the presence of various isozymes of endoglucanase and fl-gluco- sidase, the number of which varied from one to three in various species of the genus Aspergillus. p H 5.0 was op t imum for all the isozymes whereas metal ion treatment showed complete inhibition of almost all the isozymes by Hg 2+ and partial in- hibition by Ca 2+ and Co 2+ of isozymes of both the enzymes. An alteration in the electrophoretic mobility of isozymes of fl-glucosidase was also noticed in some species with Hg z+ treatment.

sidase, are responsible for stepwise hydrolysis of cellulose into glucose by their synergistic action. In order to understand the mechanism of action of each individual enzyme activity it is important to know the number of isozymes of each component of the cellulase enzyme complex and their physico-chemical properties. The number of iso- zymes of the endoglucanase and fl-glucosidase components has been established with the help of electrophoretic studies, and varies from 1 to 3 for each enzyme in various species of the genus Aspergillus [1,2]. The present paper describes the physical characterization of these enzymes.

2. I N T R O D U C T I O N

Cellulolytic enzymes of microbial origin com- prising exoglucanase, endoglucanase and fl-gluco-

Correspondence to: K. Sandhu, Microbiology Unit, School of Life Sciences, . Guru Nanak Dev University, Amritsar 143005, India. * Present address: Department of Microbiology and Molecular Genetics, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, NJ 07103-2757, U.S.A.

3, MATERIALS A N D M E T H O D S

3.1. Microorganisms

Fourteen species and varieties of the genus Aspergillus were selected (Table 1). Delineation and classification of various species within the genus were done on the basis of differences in cultural and morphological characters [3]. After single spore isolation, these species were preserved in soil at 4 ° C. Subculturing was done on Vogel's complete medium whenever required.

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3.2. Enzyme production Vogers [4] complete medium supplemented with

1% pectin as the sole source of carbon was em- ployed for enzyme-production cultures. Each 250- ml Erlenmeyer flask containing 50 nil of the above medium was inoculated with seven mycelial discs (7 m m diameter) cut from the periphery of ac- tively growing colonies. The flasks were incubated at 3 7 ° C for 8 days as stationary cultures. The contents were centrifuged at 22 000 x g for 30 min at 4 ° C. The supernatant was used as extracellular crude enzyme extract. For electrophoretic studies, the crude enzyme was concentrated using a 100% ammonium sulphate precipitation.

3.3. Electrophoresis For detection of endoglucanase activity, en-

zyme electrophoresis was performed according to the method of Beguin [5] with some modifications

[6]. The samples were run for 5 h at 250 V on a polyacrylamide (6%) slab gel measuring about 110 × 90 × 2 m m and containing 0.1% carboxymethyl cellulose (CMC) (medium viscosity, Sigma). The gel was incubated at 5 0 ° C in sodium acetate buffer (pH 5.0) for 45 rain followed by washing with distilled water. The gel was stained with Congo red (0.1%) for 30 min and destained by flooding it with 1 M sodium chloride until clear bands were visible against a red background. /3- Glucosidase isozymes were visualized using p- nitrophenyl-D-glucopyranoside (PNPG). A hori- zontal polyacrylamide gel prepared with 6% cyanogum (Sigma) in Tris-glycine buffer, p H 8.3. was employed for electrophoresis using a continu- ous buffer system. After running, the gel was incubated in 1 mM solution of p-nitrophenyl-/3- D-glucopyranoside (PNPG) (Sigma), pH 5, at 37°C for 30 min, and later dipped in 0.2 M

T a b l e 1

E f f e c t o f d i f f e r e n t p H ( 3 . 0 - 7 . 0 ) o n i s o z y m e s o f e n d o - ~ 8 - 1 , 4 - g l u c a n a s e i n v a l a o u s s p e c i e s a n d v a r i e t i e s o f g e n u s Aspergillus ( p H o f t h e

c o n t r o l is 5.0)

Aspergillus Si t e o f N u m b e r o f p H

s p e c i e s c o l l e c t i o n b a n d s 3.0 4 .0 5 .0 6 .0 7 .0

A. aculeatus P u n j a b E G I - + + + + + + + +

E G I I I - + + + + + + + + + +

A. brevipes P u n j a b E G I - + + + + + + + + + +

E G I I I - + + + + + + + + . + +

A. carneus P u n j a b E G I - + + + + + + + + +

E G I I I - + + + + + + + + + + +

A. flavus P u n j a b E G I + + + + + + + + + + + + -

E G I I I + + + + + + + + + + + + -

A. flavus v a r . P u n j a b E G I + + + + ' + + + + + + + + -

colurnnaris E G I I I 4- + + + + + 4- + + + + + -

A. fumigatus P u n j a b E G I + + + + + + + + + + + +

E G I I I + + + + + + + + + + + +

A. fumigatus v a r . P u n j a b E G I + + + + + + + + + + + +

ellipticus E G I I I + 4- + + + + + + + + + +

A. niger P u n j a b E G I - + + + + + + + + + + +

A. oryzae P u n j a b E G I + + + + + + + + + +

A. parasiticus P u n j a b E G I - + + + + + + + + + +

A. spectabilis P u n j a b E G I - + + + + + + + + +

A. terreus var . P u n j a b E G I - + + + + + + +

aureus E G I I I - 4- 4- + 4- + 4- 4- + +

A. terreus P u n j a b E G I + 4- + + + + + 4- + + + 4- +

E G I I I - + + + + + + + + +

E G I - + + + + + + + + + +

A. nidulans P u n j a b E G I I - + + + + + + + + + +

E G I I I - + + + + + + + + + +

I n t e n s i t y o f b a n d s : + + + , m a x i m u m ; + + , m e d i u m ; 4-, m i n i m u m ; - , a b s e n t .

NaOH/glyc ine buffer, pH 10.6. This resulted in the appearance of yellow-coloured bands of re- leased p-nitrophenol. For both endo-fl-l,4- glucanase and fl-glucosidase activities, the bands were examined by visual inspection of the gels. In the former the clearing of the bands and in the latter the yellow colour of the bands were noted and correlated to the enzyme activity.

3.4. Physicochemical characterization of endo- glucanase and fl-glucosidase isozymes

3. 4.1. Effect of metal ions. For an investigation of the effect of metal ions HgCI2, CoC12 and CaC12 (1 mM and 5 mM) were selected on the basis of earlier reports [7,8]. Equal volumes of these solutions and the enzyme extracts were in- cubated at 50°C and 37°C for endoglucanase

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and fl-glucosidase respectively f o r 15 min and later used for electrophoretic studies, Enzyme ex- tracts without metal ions served as controls.

3.4.2. Effect o fpH. ~ Equal amounts (0.1 ml) of the enzyme extracts and different buffer systems of varying pH ranging from 3.0 to 7.0, were mixed and processed in the same manner as mentioned above for metal ions.

4. RESULTS AND DISCUSSION

4.1. Endoglucanase In A. nidulans three isozymes of endoglucanase

i.e. EG I, II and III were detected. In all the other species only EG I and EG II were detected except- ing A. niger, A. spectabilis, A. oryzae, and A.

Table 2

Effect of different pH (3.0-7.0) on control is 5.0)

isozymes of fl-l,4-glucosidase in various species and varieties of genus Aspergillus (pH of the

Aspergillus Number of species bands

pH

3.0 4.0 5.0 6.0 7.0

d. aculeatus fl-Glu II fl-Glu III

A. brevipes fl-Glu III A. carneus fl-Glu II

fl-Glu III A. flavus fl-Glu III A. flavus vat.

columnaris B-Glu III d. fumigatus fl-Glu II

fl-Glu III A. fumigatus var. fl-Glu II

ellipticus fl-Glu III A. niger fl-Glu II

fl-Glu III A. oryzae fl-Glu II

fl-Glu III A. parasiticus fl-Glu III A. spectabilis fl-Glu I

fl-Glu II fl-Glu III

A. terreus fl-Glu III A. terreus var. fl-Glu II

aureus fl-Glu III .4. nidulans fl-Glu I

fl-Glu II fl-Glu III

_ _ + + + + + + +

_ _ + + + + + + + + +

_ _ + + + + + + + + +

_ _ + + + + + + +

_ _ + + + + + + + + + + + + + + + + + + -

+ + + + + + + + + + -

_ _ + + + + + + + + _ _ + + + + + + + +

_ _ + + + + + + +

_ _ + + + + + + +

- + + + + + + + -

+ + + + + + + + + + -

- + + + + + + + -

- + + + + + + + + + -

- + + + + + + + +

_ _ + + + + + + + +

_ _ + + + + + + + +

_ _ + + + + + + -

_ _ + + + + + + + +

_ _ + + + + + +

_ _ + + + + + + + +

_ _ + + + + + -

_ _ + + + + + -

_ _ + + + + + -

Intensity of bands: + + + , maximum; + + , medium; + , min imum; - , absent.

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I 2 3 4 5 6 7 B 9 10 Fig. 1. Effect of d i f ferent meta l ions and p H on isozymes of

endo- f l - l ,4 -g lucanase of A. niger. Effect of meta l ion: 1, con-

trol; 2, Ca2+; 3, Co2+; and 4, H g 2+ ( there was no effect of

me ta l ions in this species). Effect of p H : 6, control ; 7, p H 7.0

(no effect of p H ) ; 8, 4.0 ( m e d i u m intensi ty of EG1); 10, 3.0

( comple te inhibi t ion of E G I ) ; a nd 5, 9 isolates of A. nidulans control , p H 5.

parasiticus where only E G I was present. Ex- amination of the effect of different p H values on isozymes of various species and varieties showed

that p H 5.0 was opt imum for all the lsozymes in all the species studied in the present investigation.

A differential effect of pH 6.0 was observed" in A. aculeatus, A. carneus, A. terreus and A. terreus var. aureus where it partially inhibited EG I only. Complete inhibition of both the isozymes in A. flavus and A. flavus var. columnaris was observed with pH 7.0. However, pH 7.0 did not show any effect on isozymes of A. fumigatus, A. fumigatus var. ellipticus and A. niger (Fig. 1). Complete or partial inhibition of all the isozymes of all the species studied was seen at pH 3.0 except for A. flavus and A. flavus var. columnaris, and p H 4,0 also exhibited a differential effect on various iso- zymes (Table 1).

In the case of fl-glucosidase in the control extracts, three isozymes of fl-glucosidase, viz. fl- Glu I. fl-Glu II and fl-Glu III were detected only in A. nidulans and A. spectabilis, while in other

Tab le 3

Effect of three me ta l ions Ca 2+, Co 2+ a n d H g 2+ on isozymes of endo- f l - l ,4 -g lucanase in genus Aspergillus

Aspergillus N u m b e r of Meta l ions

species ba nds Ca 2 + Co 2 + H g 2 + Cont ro l

A. aculeatus E G I + + + + + + + + + + + +

E G I I I + + + + + + + + + + + +

A. brevipes E G I + + + + + + - + + +

E G I I 1 + + + + + + + + + + +

A. carneus E G I + + + + + + - + + +

E G I I I + + + + + + - + + +

A. flavus E G I + + + + - + + +

E G I I I + + + + - + + +

A. flavus vat . E G I + + + + - + + +

columnaris E G I I I + + + + - + + +

A. fumigatus E G I + + + + + + + + + +

E G I I I + + + + + + + + + +

A. fumigatus va t . E G I + + + + + + + + + +

ellipticus E G I I l + + + + + + + + + +

A. oryzae E G I + + + + + + + + + +

A. niger E G I + + + + + + + + + + + +

A. parasiticus E G I + + + + + + - + + +

A. terreus E G I + + + + + +

E G I I I + + + + + + + + + + + +

A. terreus var. E G I + + + + + + + + +

aureus E G I I I + + + + + + + + + + + +

A. spectabilis E G I + + + + + + - + + +

A. nidulans E G I + + + + + + - + + +

E G I I + + + + + + - + + +

E G I l i + + + + + + - + + +

In tens i ty of bands : + + + , m a x i m u m ; + + , m e d i u m ; + , m i n i m u m ; - , absent .

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s p e c i e s l ike A. aculeatus, A. carneus, A. fumigatus, A. fumigatus var . ellipticus, A. niger, A. oryzae a n d

A. terreus var . areus o n l y f l - G l u I I a n d f l - G l u I I I

w e r e d e t e c t e d , a n d i n A. brevipes, A. f lavus va r .

columnaris, A. parasiticus, A. f lavus a n d A. terreus o n l y f l - G h i I I I w a s o b s e r v e d ( T a b l e 2). I n t h e c a s e

o f t h i s e n z y m e , p H 5.0 w a s a l s o o p t i m u m f o r all

t h e i s o z y m e s w h i l e p H 6.0 s h o w e d a d i f f e r e n t i a l

e f fec t . I t p a r t i a l l y i n h i b i t e d f l - G l u I I i n A. aculea- tus, A. niger, A. oryzae, A. terreus var . aureus a n d

A. nidulans, a n d f l - G l u I I I w a s i n h i b i t e d in A.

flavus, A. f lavus va r . columnaris a n d A. nidulans. I n m o s t o f t h e s p e c i e s p H 3.0 w a s f o u n d t o b e

i n h i b i t o r y f o r all t h e i s o z y m e s e x c e p t f o r A. f lavus

a n d A. f lavus va t . columnaris. T h e o t h e r p H va l -

u e s s h o w e d t h e i r d i f f e r e n t i a l e f f e c t o n v a r i o u s

i s o z y m e s o f f l - g l u c o s i d a s e . F o r b o t h t h e e n z y m e s

p H 5.0 w a s o p t i m u m , w h i l e p H 3,0 w a s i n h i b i t o r y

in m o s t o f t h e c a se s . T h e s e r e s u l t s c o r r o b o r a t e

e a r l i e r f i n d i n g s i n v a r i o u s f u n g i l ike Trichoderma s p e c i e s [9], Chaetomium erraticum [8], a n d Chae- tomium fusisporale [10]. I t w a s s e e n t h a t i s o z y m e s

o f b o t h t h e e n z y m e s w e r e s t a b l e a t h i g h e r p H

v a l u e s in m o s t o f t h e Aspergillus s p e c i e s , h o w e v e r ,

i n A. f lavus a n d A. f lavus va r . columnaris, t h e

i s o z y m e s w e r e s t a b l e a t l o w e r p H v a l u e s .

4.2. Effect o f metal ions T h e e f f ec t o f t h r e e m e t a l i o n s C a 2+, C o 2+ a n d

H g a+ o n i s o z y m e s o f e n d o g l u c a n a s e is s h o w n in

T a b l e 3. O v e r a l l i t w a s s e e n t h a t H g 2+ i n h i b i t e d

all t h e i s o z y m e s in d i f f e r e n t s p e c i e s e x c e p t i n g E G

I I I i n A. aculeatus, A. terreus a n d A. terreus v a t .

aureus, a n d E G I in A. niger ( F i g . 1). T h e o t h e r

t w o m e t a l i o n s , i.e. C a 2+ a n d C o 2+ p a r t i a l l y in -

Table 4

Effect of three metal ions Aspergillus

C a 2+, C o 2+ and Hg 2+ on isozymes of fl-l,4-glucosidase in various species and varieties of genus

Aspergillus Number of Metal ions

species bands C a 2 + Co 2 + Hg 2 + Control

A. aculeatus fl-Glu II + + + + + + + + + + + + fl-Glu III + + + + + + + + + + + +

d. breoipes fl-Glu III + + + + + + + + + A. carneus fl-Glu II + + + + + + + + + +

fl-Glu III + + + + + + + + + + + + A. flavus fl-Glu III + + + + - + + + A. flavus var

columnaris fl-Glu III + + + + - + + + A. fumigatus fl-Glu II + + - + + +

fl-Glu III + + - + + + A. fumigatus var fl-Glu II + + - + + +

ellipticus fl-Glu III + + - + + + A. niger fl-Glu II + + + - - + + +

fl-Glu III + + + - - + + + A. oryzae fl-Glu II + + + + + + - + + +

fl-Glu III + + + + + + + + + + + + A. parasiticus fl-Glu III + + - + + + A. spectabilis fl-Glu I + + + + + + + + + + + +

fl-Glu II + + + + + + + + + + + + fl-Glu III + + + + + + + + + + + +

A. terreus fl-Glu III + + + + + + + + A. terreus var. fl-Glu II + + + + + + + + + + + +

aureus fl-Glu III + + + + - + + + A. nidulans ]3-Glu I + + + + + + + + + + + +

,8-Glu II + + + + - + + + fl-Glu III + + + + - + + +

Intensity of bands: + + + , maximum; + + , medium; + , minimum; - , absent.

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hibi ted both the isozymes (EG I and EG III) in A. flavus and A. flavus var. columnaris. In other species these metal ions did not show any effect. In the case of ]3-glucosidase, Hg 2+ inhibi ted all

the isozymes in A. flavus, A. flavus var. colum- naris, A. fumigatus, A. fumigatus var. ellipticus and A. niger (Fig. 1). In A. oryzae it inhibi ted /3-Glu II while in A. parasiticus, A. terreus var. aureus it inhibi ted fl-Glu III. In A. nidulans it inhibi ted both /3-Glu II and /3-Glu III isozymes. The other two metal ions did no t affect the in tensi ty of the bands in A. aculeatus, A. carneus, A. oryzae and A. spectabilis. However, in other species the metal ions reduced the in tens i ty of one or bo th the isozymes. In A. nidulans the intensi ty o f /3 -Glu II and /3-Glu I I I was reduced whi le /3-Glu I was no t affected (Table 4). In our previous studies also these metal ions have been shown to inhibi t the activity of the purif ied forms of fl-glucosidase II.

Similar studies in other fungi have also indi- cated the inhib i tory effect of heavy metal ions like Hg 2+ on various isozymes of different compo- nents of the cellulase enzyme complex [7,8,10,12-

141. Another impor tan t observat ion was that an al-

tera t ion in the mobi l i ty of f l-Glu I I I was observed after Hg 2÷ t rea tment in A. brevipes and A. carneus. This change in mobi l i ty may be due to the b ind ing of the heavy metal ion with the en- zyme polypept ide and hence changing its mobil i ty, thereby creating a new conformat ional isozyme. This may be one of the epigenetic causes of iso- zyme polymorphisrn.

A C K N O W L E D G E M E N T

The authors acknowledge the receipt of fellow-

ship from the council of Scientific and Industr ial Research, New Delhi, dur ing the course of this study.

R E F E R E N C E S

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[3] Raper, K.B.. and Fennel, D.I. (1965) The Genus Aspergil- lus. Robert E. Krieger, New York.

[4] Vogel, H.J. (1956) Microbial. Genet. Bull. [5] Beguin, P. (1983) Anal. Biochem. 131, 333-336. [6] Sharma, S. and Sandhu, D.K. (1986). Ind. J. Exp. Biol. 24,

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Appl. Bacteriol. 61. 73-80. [8] Sandhu. D.K. and Purl. R. (1988) Can. J. Bot. 66. 2162-

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Progress in Microbial Ecology (Agnihotrl. V.P. and Singh, R.P.. Eds.), pp. 525-547, India Print House. Lucknow.

[10] Sandhu, D.K. and Purl. R. (1989) J. Basic Microbiol. 29, 519-525.

[11] Bagga, P.S., Sandhu. D.K. and Sharma. S. (1989) J. Appl. Bacteriol. 67. 1-8.

[12] Otsuka. K.. Yadomae. T. and Miyazaki, T. (1979) Chem. Pharm. Bull. 27. 2042.

[13] Garg, S.K. and Neelakantan. S. (1982) Biotechnol. Bio- eng. 24. 737-742.

[14] Ferchek. J.D. and Pye, E.K. (1983). Biotechnol. Bioeng. 25. 2865.