J. Basic Microbiol. 36 ( 1996) 4,289-296

(Department of Microbiology, Guru Nanak Dev University, Amritsar- 143005, India)

Isozyme polymorphism of cellulases in Aspergillus terreus S. SINGH, J. K. BRAR, D. K. SANDHU and A. KAUR

(Received 12 May IY9YAccepted IS January 1996)

Maximum cellulase production in Aspergillus terreus was obtained at a temperature of 28 "C, pH 4.0 and a substrate concentration of I % CMC. Variability in cellulase enzyme production and isozyme polymor- phism of endo-p- 1,4-glucanase and p- 1,4-glucosidase was studied in 45 natural isolates of A. terreus. Different electrophoretic patterns were evident for endoglucanase. Three zones of activity viz EG I , EG I I and EG I I 1 were observed showing different electrophoretic mobilities. Some of the isolates exhibited the presence of null alleles for EG 1. During development EG 1 and EG 1 I were observed throughout while EG I 1 1 appeared on the eighth day. For /3-1,4-glucosidase two zones of activity viz p-glu 1 and p-glu I I were observed. p-glu 1 showed variable electrophoretic mobilities. p-glu 1 appeared throughout during development while p-glu 11 appeared on the twelfth day.

The phenomenon of isozyme polymorphism has been widely documented with organisms varying from microbes to mammals. This multiplicity has been found to reflect the existence of different genes. The isozymes provide a natural marker system for a wide range of investigations in biochemistry, genetics and developmental biology. The electrophoresis of soluble enzymes has proven to be a useful technique for studying the genetic variation among natural populations of various organisms including fungi (BUTH 1984). Any change occurring in electrophoretic pattern has been related to developmental changes (COOPER er al. 1985). The protein profiles from various fungal components of different species have been used as taxonomic criterion (KULIK and BROOKS 1970, KAOSIN and ZENTMEYER 1980, MERQUIOR et al. 1994). But there is little information about the isozyme polymorphism of cellulases among fungal populations and their developmental regulation (SHARMA et al. 1990).

The purpose of the present investigation is to explore the patterns of electrophoretic variation among natural populations ofA. terreus and to reveal the isozyme pattern of endo-b- I ,4-gluca- nase and p- 1 ,4-glucosidase in growth profile.

Materials and methods

Different isolates of A. terreus were isolated from soil collected from different places of India using the soil plate method for isolation of fungi (SINGH 1983). Isolation was done on CZAPEK'S solution agar with WARCUP'S modification (addition of 0.5% yeast extract and acidification up to pH 4.0 with phosphoric acid). Identification was done on the basis of morphological and cultural characteristics (RAPER and FENNEL 1965).

Optimization of cellulase production: Fifty ml Of VOGEL'S CM supplemented with I % CMC was taken in 250 ml ERLENMEYER flasks. Standard inoculum of six discs (7 mm diameter) taken from the periphery of a growing colony was used for each flask. The flasks were incubated at different temperatures and were assayed for enzyme activity on 8th day of stationary cultivation. Various parameters standardized included temperature, pH and substrate concentration.

For study of isozymes pectin was used as a substrate as it is a better substrate for isozymes (SHARMA 1990). For development studies the flasks were analysed in triplicate on alternate days for 24 days. After the desired time of incubation under optimum conditions, the culture filtrate was centrifuged at 22,000 g for 30 minutes at 4 "C. All the three fractions extracellular, intracellular and cell debris were obtained and

290 S. SINGH et al.

dry weight of the mycelium determined. The supernatant obtained as above was taken as extracellular enzyme fraction. The mycelium was washed with 0.1 M acetate buffer (pH 5.0 and dried between the folds of filter paper. It was frozen and crushed in pestle and mortar with chilled acid washed sand and centrifuged as above, after adding 5 ml of acetate buffer. The supernatant so obtained was taken as cytosolic or intracellular fraction. For obtaining the cell debris fraction, the mycelium was crushed in pestle and mortar but sand was not used. After adding 5 ml of acetate buffer the mixture was centrifuged as above and the pellet was resuspended in 5 ml of fresh buffer. All the extracts were stored at -15 "C.

Enzyme estimation was done by the method of SANDHU and KALRA (1982). Isozyme studies of endo-P-l,4-glucanase (EC 3.2.1.4) and /3-glucosidase (EC 3.2.1.74) were done following the method of SHARMA and SANDHU (1986) using partially purified extracts.

Partial purification: The pH of the crude extract was set at 5.0 with sodium acetate and acetic acid, followed by addition of ammonium sulfate (100% saturation) with continuous shaking. The precipitates were collected after centrifugation at 12000 x g for 30 minutes at -4 "C and redissolved in minimum volume of chilled 0.1 M sodium acetate buffer (pH 5.0). The concentrated extracts were dialysed (dialysis tubing: m w cut off 6,000-8,000) against 0.1 M (pH 5.0) sodium acetate buffer for 24 hrs with changes of buffers at various intervals. The concentrated extracts were stored at -15 "C for further studies.

- - m B ~ ~ ~ ~ ~ ~ ~ ~ ~ E G - n

Results

Maximum production of cellulases in A. terreus was obtained on 1 % CMC at a temperature of 28 "C and pH 4.0.

Isozyme expression during growth The fungus was grown on pectin for 24 days. Extracellular and intracellular fractions were electrophoretically analysed.

Expression of endo-/3-1,4-glucanase During the initial stages of growth two zones EG 1 and EG 11 were observed and a third zone EG 11 1 appeared on the 81h day. All the three isozymes persisted upto 24Ih day. Intensity of bands increased with development. In the intracellular fraction same pattern was observed but the intensity of bands was very low (Fig. 1 A and B).

Expression of /3-1,4-glucunase During cultivation p-glu 1 was present throughout the cultivation with same intensity in the extracellular fraction while /3-glu 11 appeared after 121h day and persisted till the termination

E G - U l

Isozyme polymorphism of cellulases 29 1

of the experiment. Intensity of p-glu 11 was better than that of p-glu 1. In the intracellular fractions only p-glu I 1 was observed throughout the development (Fig. 2A and B).

Qualitative studies

Polyacrylamide gel electrophoresis was performed to investigate the isozyme polymorphism of endoglucanase and p-glucosidase in wild isolates of A. terreus. About 45 wild isolates were surveyed electrophoretically. Three zones of activity were observed for endoglucanase viz EG 1, EG 1 1 and EG 1 1 1 starting from most anodal to least anodal migration zone. Similarly two zones were observed for p-glu 1 and p-glu 1 1. At each zone various electromorphs were observed. Each electromorph was designated with a number in accordance with the migration in mms from the point of origin.

Endoglucanase polymorphism

Different isolates showed variable expression of the three endoglucanase forms. At EG 1 zone total eight electromorphs were observed designated as ET 17, ET 18, ET 19, ET 20, ET 21, ET 22, ET 23, ET 24 and ET 25. EG 1 was not detected in four isolates from Amritsar. In rest of the isolates most common forms were ET 19 and ET 25. ET 17 was of rare occurence and detected only in one Darjeeling isolate. Each electromorph was of variable intensity among different isolates. At ET I 1 zone seven electromorphs were observed viz ET 9, ET 10, ET 11, ET 12, ET 13, ET 14 and ET 15. The most common form was ET 10 detected in 13 isolates. ET 12 was observed among 11 isolates. ET 14 and ET 15 were of rare occurence. At EG 11 1 seven electromorphs evident were ET 2, ET 3, ET 4, ET 5, ET 6, ET 7, and ET 8. The most common forms were ET 3 and ET 5 detected among 12 and 13 isolates respectively, while ET 4 and ET 8 were of rare occurence (Table I and Fig. 3A).

Effect of heat treutment on endoglucanase isozymes

The isolates showing variable electromorphs were selected. The enzyme extracts from these isolates were treated at 65 "C for about 20 minutes. The temperature was selected after a preliminary treatment at 40 "C, 50 "C, 60 "C, 70 "C and 80 "C.

Differential effect was observed at 65 "C. The electromorphs were further differentiated on the basis of thermal resistant and thermal sensitive forms. P-8 and P-27 showed thermoresistant forms for all the three zones while P-3 exhibited all the three, thermosensitive forms. Some isolates had one thermoresistant form while others were sensitive (Fig. 3 B).

t

tl A

t

8 t P-GIU-II 1 1 - ~~~~~

- 2 4 6 8 10 12 14 16 18 20 22 24 DAYS

Fig. 2 Zymograms showing the developmental patterns of isozymes of /3-glucosidase in wild type A. terreus. A: Extracellular; B: intracellular.

292 S. SINGH et al.

Table I Distribution of electromorphs of endo-p- 1,4-glucanase zones in the natural populations of A. terreus.

Zones of electromorphs

EG I

17 18 19 20 21 22 23 24 Null Aliwal - - - - I - - _ _ Arnritsar - 1 4 2 1 1 1 4 4

- - - - 2 - - - _ Batala Braharnanwala - I - - - - - - - Chandigarh - - I I - - - - - Darjerling I - - - - - - - -

- 2 - _ _ _ - - - Delhi

Harchowal - - - 3 I - - - - Jalandhar - I 2 - - - - - -

- 1 - - - - _ _ - Moga - 1 - - - - - - - Muchike

Pdthankot - - - - 2 - 3 - - Patiala - - I - - - - - - Taragarh - - - - - I - I -

Dinanagar - - - - - - - I -

Totdl 1 7 8 6 7 2 4 6 4 4 1 3 4 1 1 7 3 3 5 1 2 3 1 3 3 6 3

Effect of urea treatment on endoglucanase isozymes

8 M urea was incorporated in the polyacrylamide gel. Different enzyme samples were run electrophoretically in this gel. It was found that EG 11 1 zone was sensitive to urea treatment in all the isolates selected (Fig. 3C).

Effect of substrate on endoglucanase isozymes

Isozyme pattern was changed when the isolates were grown on carboxymethyl cellulose instead of pectin for enzyme production. In some isolates the number of zones remained the same but the mobility differed. However in P-3, P- 16- 1, P- 16-2 three zones instead of the regular two were observed whereas P-27 and P-79-1 showed two zones of activity instead of three (Fig. 3D).

p- 1,4-glucosidase polymorphism

Two zones of activity observed were p-glu 1 and p-glu 1 1. At p-glu 1 five electromorphs were observed ET 3, ET 4, ET 5, ET 7 and ET 8. At p-glu 11 two electromorphs ET 1 and ET 2 were observed. p-glu 11 was a very slow moving zone. In most of the isolates p-glu 1 was absent. The most common form was ET 4. Intensity of p-glu 1 was very low (Table 2, Fig. 4).

Discussion

The present work indicates that A. terreus is a good producer of cellulases and isozyme polymorphism exists in the natural population. Various cultural conditions significantly effect cellulase production. The optimum pH and temperature were observed to be 4.0 and 28 "C. A number of cellulolytic fungi also show optimum pH around 4.0 to 5.0 (SIDHU et al. 1984, BAGGA et al. 1988). The optimum concentration of CMC was found to be 1 % for the production of endoglucanase and exoglucanase but B-glucosidase showed maximum production with 2 %

Isozyme polymorphism of cellulases 293

i i

- P-8 P-27 P-3 P-79-1 P16-1 P-16-2 P-14 PH3-1 PH3-2

bl

+

t l I

e m - - - e r n - - - - - n o o o - = ~ I e z I d

0 0

- P-8 P-27 P-3 P-79-1 P-16-1 P-16-2 P-14 P H r l P H s 2

+

1

EG-I EG-II EG-III

- P-8 P-27 P-3 P-79-1 P-16-1 P-16-2 PHS-1 Isolates

d )

Fig. 3 Zymograrns showing: a) various isozyrnes and electrornorphs of endoglucanases; b) effect of heat treatment on isozyrnes of endoglucanases; c) effect of urea treatment on isozyrnes of endoglucanases; d) effect of substrate (CMC) on isozymes of endoglucanases. P-8, P-27. P-3, P-16-1, P-16-2, P-14 - Amritsar isolates; P-79-1 - Taragarh isolate; PH3-I, PH3-2 - Harchowal isolates; PD - Darjeeling isolate; P-84 - Delhi isolate.

CMC. Higher substrate concentration results in the increased levels of reducing sugars which repress the synthesis of exoglucanase and endoglucanase.

The population studies indicated polymorphism for isozymes and variability in enzyme production. A total of forty seven isolates ofA. terreus analysed showed a significant variability in the enzyme yield. The electrophoresis analysis revealed the existence of isozyrne polymor- phism of endoglucanase and P-glucosidase. Isozyme polymorphism of cellulases has also been demonstrated in Trichoderma, Aspergillus, Chaetomium, Streptomyces (SHARMA et al. 1990, PURI 1990, BAWA 1992, HARCHAND 1993). Three zones of activity were observed for endo- glucanase viz EG 1, EG 1 1 and EG 1 11, all of them showing a number of electromorphs. The existence of various electromorphs can be explained on the basis that the electrophoretic

294 S . SIKH et al.

Table 2 Distribution of electromorphs of p- 1 ,4-glucosidase in the natural populations of A. terreus.

Zones of electromorphs

j3-glu 1 p-glu 1 1

3 4 5 7 8 N u l l I 2

Aliwal - - - - - 1 1 1 Amritsar 2 2 1 - - 13 7 1 1

1 1 Batala Brahamanwala - - - - - 1 - 1 Chandigarh - - - - - 2 2 - Darjeeling - - - - - I 1 - Delhi - - - - - 2 2 - Dinanagar - - - - - 1 1 - Harchowal 1 - - - - 3 4 -

1 2 1 Jalandhar - - - 1 1 Moga - - - - - 1 1 - Muchike - - - - - 1 - I Pathankot - - - - - 1 - I Patiala - 1 - - - 4 5 - Taragarh - 2 - - - - 2 -

Total 3 5 1 1 1 2 4 29 16

2 - - - - -

mobility is the function of charge, conformation, molecular weight and polymeric complexity. The electrophoretic technique relies on the existence of genetic polymorphism resulting in the amino acid substitutions responsible for the differences in the relative mobilities among the allelic forms of an electrophoretic gel (LEWONTIN 1974). JOHNSON (1974) indicated the correlation between the degree of polymorphism at a locus and its physiological function. This is known as an adaptive role of enzyme polymorphism. The high frequencies of some electromorphs may be the result of high adaptive values of these forms in different heteroge- nous environments (ROCWOOD-SLUSS et al. 1973) and further it can be presumed that electro- morphs showing low frequencies are either of recent origin or they do not confer any selective advantage. Since a culture is derived from a single conidium and is presumably haploid, one could expect the production of single protein bands due to the expression of single alleles, but multiple bands are observed for an enzyme. These additional bands are examples of secondary isozymes which have arisen from post translational processing, incomplete binding of cofactors

t

- P-27 P-1 P -2 P-5 P - 6 PO P-14 P-21 P-55 P-38-1 P-38-2

Isolates

Fig. 4 Zymogram showing various isozymes and electromorphs of /?-glucosidase. P- 1, P-2, P-3, P-6, P-14, P-21, P-27 - Amritsar isolates; P-D - Darjeeling isolates; P-55 - Brahamanwala isolates; P-38-1, P-38-2 - Jalandhar isolates.

Isozyme polymorphism of cellulases 295

by the enzymes or existence of differential transformational state of the enzymes. According to OHNO (1970) various isozymes can be formed by gene duplication followed by structural and positional divergence. Occurrence of rare electromorphs could be considered as the result of low intragenic recombination frequency (KOEHN and EANES 1979) or due to differential sensitivity of these enzymes to electrophoretic techniques. The missing bands is a consequence of the presence of null alleles (MICALES et al. 1987, SWRMA et al. 1990). According to BEWLEY and LUCCHESI (1975) the missing bands are due to the fact that the physiological function of these enzymes is not well understood.

In the population studies EG 1 is variably detected so it could be considered under separate genetic control. This gene is active in some wild isolates while in others null alleles were detected corresponding to this gene. EG 11 and EG 11 1 were detected in all the isolates but heat and urea treatment indicated their variable sensitivity. Further during development EG 11 1 made its appearance during the later stages of growth whereas EG 11 was present during the early stages. From the above data it can be concluded that EG 11 and EG 11 1 are not under same genetic control and there are three structural genes for endoglucanase in A. terreus. The multiple forms of various components of cellulase have already been reported to be under separate genetic control. Two genes have been identified for endoglucanase in Clostridium thermocellulam (BEGUIN et al. 1983, CORNET et al. 1983). MILLET et al. (1985) reported seven genes for endoglucanase and 2 genes were reported in A. nidulans by SHARMA et al. (1990). Similary b-glu 1 and b-glu 1 1 could be considered under separate gene control. SHARMA et al. (1990) reported three segregating zones being coded by three presumptive loci for /3-glucosi- dase in A. nidulans.

The isozyme pattern of growth profile of endoglucanase showed the presence of two bands EG 1 and EG 11 during the earlier stages while EG 11 1 appeared during the eighth day. All these enzymes persisted upto 24th day. Intensity of bands increased with age which was in accordance with the quantitative studies. This may be attributed to enhanced expression of genes during development. EG 1 and EG 11 remined fairly constant during development indicating that some genes directly associated with the production of cellulase enzymes are operative from the very early stages of development (CHAUHAN and NANDA 1987). b-glu 1 appeared during later stages of development i.e. after 12th day. It could be attributed to the changes in the growth conditions, however no important function could be assigned to it as most of the wild isolates showed null alleles forb-glu 1. Exact conclusions can be drawn only after mutant studies. Developmental regulation of EG 11 1 has already been reported (SHARMA et al. 1990, BAGGA et al. 1990). Isozyme specific to various developmental stages have been reported in Physarum polycephalum (FUJISHITA et al. 1984), Chaetomium species (PUN et al. 1990) and Candida albicans (RICHARD et al. 1990).

Heat treatment and urea treatment showed the presence of sensitive and resistant forms resulting in the increase in the number of electromorphs. This may be due to the hidden genetic variability as already reported in various organisms (SINGH et al. 1974). SHARMA et al. (1990) reported hidden genetic polymorphism in Aspergillus supporting the above observations.

The above studies well document the isozyme polymorphism in A. terreus, but it would be necessary to analyse more isolates electrophoretically and to perform genetic crosses to determine the exact genetic control of these enzymes.

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Mailing address: Prof. Dr. S. SINGH, Department of Microbiology, Guru Nanak Dev University, Amtitsar- 143005, India

589-594.