Journal of Applied Bacteriology 1995, 78, 370-383

Chitinase production by Sirepiomyces viridificans : its potential in fungal cell wall lysis

Rani Gupta, R.K. Saxena, Prlya Chaturvedi and J.S. Virdi Department of Microbiology, University of Delhi South Campus, New Delhi, India

4835/02/94: received 7 February 1994, revised 16 November 1994 and accepted 22 November 1994

R . G U P T A , R . K . S A X E N A , P . CHATURVEDI AND J.S. V I R D I . 1995. Streptomyces viridlfcans was found to be a good chitinase producer among nine species of Streptomyces screened. Minimum levels of constitutive enzyme were observed with both simple and complex carbon substrate. Arabinose doubled the enzyme production amongst the various pentoses and hexoses used with chitin. However, with glucose end-product inhibition and catabolite repression were observed. T h e enzyme tolerated a wide range of temperature (3(rSS°C) and pH (3-7.5). Among various divalent cations Mn2 + and Hg2 + completely inhibited the purified enzyme while /I-mercaptoethanol stimulated its activity. Crude and purified enzyme had potential for cell wall lysis of many fungal pathogens tested.

INTRODUCTION parvies, S . venezuelae and S . vtrtdijicans, from Institute of

Chitin is an unbranched polysaccharide with a composition of j-1-Clinked N-aCetyl-D-glUCOSamine (NAG) residue. I t is hydrolysed by two separate enzymes categorized as chi- tinases: an endo-chitinase (EC 3.2.1.14) which produces soluble low molecular weight multimers of NAG, the dimer N,N-diacetylchitobiose being dominant, and chitobiase (EC 3.2 1.30) which hydrolyses the intermediate to NAG (Jeuniaux 1966; Monreal and Reese 1969; Reid and Ogryd- ziak 1981 ; Vyas and Deshpande 1989). Microbial chitinases are also commercially available, purified from Streptomyces spp. (Berger and Reynolds 1958; Jeuniaux 1966) and Ser- ratia marcescens (Monreal and Reese 1969).

During the last decade, chitinases have received increased attention because of their wide range of applica- tions (Carroad and Tom 1978; Boller 1986; Gooday 1986; Jones et al. 1986; Ordentlich et al. 1988; Rast et al. 1991; Sherief et al. 1991; Oppenheim and Chet 1992). As the fungal cell wall is rich in chitin (Peberdy 1990), its potential application in biocontrol of fungal phytopathogens is prom- ising. The enzyme could be used directly in biological control on micro-organisms (Ordentlich et a / . 1988) or indi- rectly using purified protein or through gene manipulation (Shapira et al. 1989; Oppenheim and Chet 1992).

MATERIALS AND METHODS

Nine cultures of Streptomyces spp., viz. S . aureofaciens, S. coelicolor, S . glaucescens, S . kanamyceticus, S. lividans, S .

Correspondence to : Dr Rani Gupta. Department of Mil robtology, University of Delhi South Campus. Benito Juarez Road, New Delhi-110021, Indla.

Microbial Technology, Chandigarh, India, were maintained on yeast extract maltose agar slants. Screening for chitinase production was by plate agar assay followed by tests in broth (Berger and Reynolds 1985; Vasseur et a/. 1990). The basal medium contained (g 1- l ) : colloidal chitin 15; yeast extract 0.5; (NHJ2S0, 1; MgSO, . 6 H 2 0 0.3, KH,PO, 1.36; and agar 15, as required. Colloidal chitin was pre- pared by the procedure of Berger and Reynolds (1958). Enzyme production was carried out in shake culture (50 ml medium/250 ml Erlenmeyer flask) incubated at 30°C in a New Brunswick incubator shaker at 250 rev min ~ ' for 6 d. Spores were inoculated to a concentration of 2 x lo5 ml-' . Physiological conditions, viz. temperature, rev min ', sub- strate concentration, carbon source and additives were studied to obtain maximum enzyme production from the best chitinase-producing strain S. viridzjicans.

Enzyme assay

Colloidal chitin was used as a substrate to assay chitinase activity: 0.2 g in 2 ml of citrate buffer (pH 6.0) was incu- bated with 1 ml of enzyme at 50°C for 60 min. The product was measured in 1 ml of filtrate by the dinitro- salicylic acid (DNSA) method (Miller 1959). One unit of enzyme activity was defined as the amount of enzyme that catalysed the release of 1 pmol of N-acetylglucosamine ml- ' in 60 min.

Enzyme purification and kinetics

The crude enzyme sample was precipitated by 60% ammonium sulphate and further purified by Sephadex

C H l T l N A S E PRODUCTION B Y STREPTOMYCES VlRlDlF lCANS 379

Table 1 Screening of different Streptomyces spp. for production of chitinases using plate assay and in broth using colloidal chitin

Streptomy ces Enzyme organism 24 h 36 h 48 h 72 h (U m1-l)

vzridijicans - + + + + + + + + 0.23 venezuelae - + + + + + + + 0.20 aureofaciens - + + + + + + + + 0.21 fradiue - - - - Not tested

+ + + + + + 0.20 parvies kunamyretrcus - - + + + + + 0.14

- + 0.09 lividans glaucescens - + + + + + + + + 0.20 coelicolor - + + + + + + 0.15

-, Absence; +, + +, etc., extent of enzyme production.

- -

- -

G-75 column chromatography. The fractions with chitinase activity were pooled for biochemical characterization by incubation at different temperatures and pH. The effect of various divalent cations (Pb", M n 2 + , CaZ+, Fe2+, ZnZ+, Cd2+, Ni2+, Co2+, Cu2+ and Hg2+) and other inhibitors such as 2-mercaptoethanol and EDTA on enzyme activity was investigated. Purified enzyme was incubated at 30 f 2°C with 5 mmol 1-' of each inhibitor for 10 min, and then the enzyme was assayed.

The enzyme chitinase obtained from S. vir idzjcans was tested for its potential to act as a biocontrol agent against important fungal phytopathogens by virtue of its lytic action on the chitin component of the cell walls.

Preparation of fungal cell wall

The fungi Rhizoctonia solani, Aspergillus sp., Fusarium oxy- sporum, Curvularia lunata and Sclerotinia oryzae , were grown in potato dextrose broth, incubated at 30°C and then harvested after 72 h growth. Cell walls were obtained from the mycelia of the above species as described by Skujins et a l . (1965). Mycelium of each fungus was autoclaved and homogenized at 4000 rev min-' for 3 min in a tissue homogenizer (Yorco, New Delhi, India). The homogenate was further subjected to ultrasonic disintegration in ice bath for 6 min in 12 cycles each of 30 s in an ultrasonicator (SoniPrep 150 MSE, USA). The pellet obtained by cen- trifugation at 5000 rev min-' for 25 min at 4°C in a refrigerated centrifuge (Remi C30, Bombay, India) was washed four times with distilled water and dried.

Colloidal chitin along with chitin present in the fungal cell wall in a 1 : 3 ratio was used for enzyme production in the medium. Flasks containing medium consisting of (g I - ' ) : fungal cell wall preparation 11.3; colloidal chitin 3-7; yeast extract 0.5; (NH,)2S0, 1; MgSO, . 7 H 2 0 0.3; KH2P04 1.36; pH 7, were inoculated with S. viridzjicans and incubated at 30°C in an incubator shaker at 250 rev

min-' for 6 d. The contents of the flasks were then filtered through Whatman No. 1 filter paper and the culture filtrate was centrifuged in a refrigerated centrifuge at 4°C for 20 min at 5000 rev min-' to remove any remaining cell debris. The culture filtrate thus obtained was tested for enzyme activity as described above.

Dissolution of fungal cell wall

The ability of the culture filtrate to lyse different fungal cell walls and colloidal chitin, and thereby release N- acetylglucosamine, was tested in a reaction mixture contain- ing 10 mg of fungal cell wall and 50 ml of culture filtrate in a 250 ml flask. The flask containing the reaction mixture was incubated for 12 h at 30°C. Reducing sugars were mea- sured in l ml of the mixture and enzyme units were expressed as a standard enzyme assay. Heat-inactivated culture filtrate plus fungal cell wall were used as control.

Inhibition of fungal growth by crude and purified enzyme extract

A spore suspension of fungal pathogen was uniformly spread on plates of complete medium: NaNO, 6.0; KCl 0.54; MgS0, . 7 H 2 0 0.54; KH2P0, 1.52; FeSO, , 7 H 2 0 trace; Cu(NO,), . 5 H 2 0 trace; ZnSO, . 7 H 2 0 trace; dex- trose 10; yeast extract 1 ; peptone 2; Casein hydrolysate 1.5 g 1- ' ; pH 6.2 with streptopenicillin (commercial analytical grade), to inhibit bacterial growth. Discs soaked in crude and partially purified enzyme extract were laid on the seeded plates; controls were discs soaked in boiled enzyme extract. Fungal spore germination and subsequent growth were observed over 4 d of incubation at 30°C.

RESULTS

Among the nine species of Streptomyces, S. viridijicans was found to be the best producer of chitinase (Table 1). All subsequent experiments used S. v ir id t jcans only.

Physiological optimization of chitinase production

Chitinase of S. viridijieans is an inducible enzyme and maximum levels are achieved by addition of 1.5% colloidal chitin (Fig. Id), at 30°C with an agitation of 200 rev min-' after 6 d incubation (Fig. la, b, c). A direct decrease in enzyme production was observed with increased concentra- tion of reducing sugars in culture filtrate (Fig. lc).

The effect of various sugar additions along with 1.5% colloidal chitin on chitinase production was tested (Fig. le). There was a decrease in chitinase production with all sugars tested, except arabinose. Arabinose plus chitin doubled production but arabinose alone failed to induce the

380 R A N I G U P T A E T A L .

0.30 r

- c 0 . 2 0 0'25"e)

0.24

0.18

0.15

0.10 - 100 200 300

Temperature PC) Agitation (rev min-l)

0 .25 1.00 2.00 3.00 Incubation time ( d ) Colloidal chitin (%)

0 .- V

1 2 3 4 5 6 Additives Chitin Arabinose%

Fig. 1 Parametric optimization of chitinase production (U ml- ') by Sireptomyces virid$cuns. Effect of: (a) temperature ("C); (b) agitation (rev min I ) ; (c) incubation period (+ enzyme; reducing sugars pg ml-I); (d) different concentrations of colloidal chitin (%); (e) sugar additives (0,5'%)) (1) chitin, (2) xylose, (3) ribose, (4) raffinose, (5) glycose, (6) arabinose, + 1% colloidal chitin; (f) different ratios of colloidal chitin and arabinosc

CHlTlNASE PRODUCTION B Y STREPTOMYCES VlRlDlFlCANS 381

0.10 /

0 . 2 0 1 ~ ; , 0.18

I $ 0.16 g 0.14

0 12 9 010

0 0 6 I I

q I I \

o . w t , 0.02 ,; I l I , I I -I -- 0 I 2 3 4 5 6 7 8 9 10 I / 12 13 14 15 16 17 18

0.36 0.33 0.30 0.27 = 0.24 0.24 2

0.15

0.09 0.06 i - 19 0.03

0.20 2 0.18 5 0.12 k

Fractions

Fig. 2 Chitinase activity (U ml- ') (----) of different protein fractions (a, b, c) obtained on Sephadex G-75 gel chromatography

Table 2 Production of Streptomyces viridrjcans chitinase on mixed substrate containing 1 : 3 colloidal chitin and chitin derived from different fungal cell walls to a total of 1.5%

Source of cell Amount of enzyme wall chitin produced (U ml-')

Curvularia lunata 0.22 Pythium aphanidermatum 0.22 Fusarium oxysporum 0.19 Rhizoctonra solani 0.22 Colletotrichum dematium 0.20

Colloidal chitin (1.5Y0) 0.19 Colloidal chitin (0.5%) 0.05

I .oo 0.90

- 0.80

0.70

0.60

S 0.50

0.40 E ," 0 - 3 0

- I

3 2. c

c V

I: 0.20

0.10

I ' 00

0.90

0.80

/ /+

0.30

0.20

Temperature ("C)

enzyme (Fig. If). Glucose showed complete suppression of production (Fig. le).

PURIFICATION AND BIOCHEMICAL CHARACTERIZATION

The enzyme was concentrated by precipitation with 60% (NH,),SO, and was further subjected to Sephadex G-75 column purification. The purification fraction showed three protein peaks. Of these peaks, a and b showed maximum chitinase activity. The third protein peak was devoid of enzyme activity (Fig. 2). Purified enzyme showed a broad range of temperature (10-55"C) and pH activity (3-7.5) with optima at 50°C and pH 6 (Fig. 3a, b). The K , of the enzyme was 0.19 mg ml- ' and V,,, was 9.5 pmol min-' p g - protein. Mercaptoethanol enhanced the activity of both the crude and purified samples (Fig. 3c). No signifi- cant inhibition was found with divalent cations except MnZ + and Hg2 + .

The crude enzyme extract could digest only colloidal chitin and chitin derived from the fungal cell walls: no other substrates could be digested. Improved levels of chi- tinase production were observed when fungal cell wall chitin was used along with colloidal chitin in a 3 : 1 ratio (Table 2).

The culture filtrate thus obtained had only chitinase activity; no other enzyme activity was detected with various polysaccharide substrates. Only colloidal chitin and chitin from fungal walls were digested at detectable rates.

The maximum digestion of fungal cell walls by the crude enzyme extract occurred at pH 6.0 after 12 h incubation. The residue accounted for 8% of the weight of the original purified walls and no further weight loss was observed with increased incubation. The lytic components in the culture

I30

I

+ I I I I 1 1 3 4 5 6.5 7 7 .5

0

PH

Fig. 3 Biochemical characterization of purified chitinase of Streptomyces viridrficans. (a, b) Enzyme activity (U mi- I ) at different temperatures (a) and pH values (b). (c) Relative YO enzyme activity in crude (w) and purified (m) enzyme sample in the presence of 5 mmol 1- ' different divalent cations as (1-1 1) and mercaptoethanol(12), all at 5 mmol I- ' . (1) Control; (2) CaCI,; (3) PbCI,; (4) MnCI,; (5) HgCI,; (6) NiCI,; (7) CdCI,; (8) ZnCI,; (9) PbSO,; (10) CoCI,; (11) CuCI,

382 R A N I GUPTA ET A L .

Table 3 Activity of Streptomyces viridificans chitinase produced on specific Source of enzyme chitinase

Cell wall S. viridy5cans + fungal culture no.* fungal cell wall and colloidal chitin (3 : 1)

on dissolution of cell wall of different fungi substrate 1 2 3 4 5 6 7 for dissolution Enzyme activity (U m1-l)

1 Rhizoctonia solani 0.01) 0.08 0.08 0.08 0.00 0.09 0-07 2 Colletotrrchum 0.08 0.07 0.07 0.07 0.08 0.08 0.08

dcmartum 3 Aspergillus sp. 0.08 0.08 0.07 0.08 0.07 0.08 0.08 4 Fusarrum oxysporum 0.08 0.08 0.08 0.08 0.07 0.08 0.08 5 Sclerotinia nryzae 0.08 0.08 0.08 0.08 0.08 0.08 0.07 6 Curvularra lunata 0.07 0.07 0.08 0.08 0.07 0.09 0.08 7 Pythium 0.08 0.07 0.08 0.08 0.08 0.08 0.08

ap hanrdermatum

Numbers represent S. vircdificans and cell walls of different fungi listed in first vertical column.

filtrate of each of the fungi were effective against all the seven fungi tested (Table 3).

FUNGAL INHIBITION

Inhibition of fungal growth was observed on an agar plate with discs coated with chitinase enzyme.

DISCUSSION

Streptomyces vjridz~cans exhibited much lower enzyme activity when chitin powder was used. However, a fourfold increase in the level of enzyme was observed with colloidal chitin. The ratio of the activities was found to vary between 1 : 3 and 1 : 4, which is much higher than reported earlier for actinomycetes (Monreal and Reese 1969). Carroad and Tom (1978) have suggested that the chemical modification of colloidal chitin provides more surface area for the activ- ity of the chitinase. Like the cellulolytic system, chitinase was also reported to be a two component enzyme complex, one responsible for action on the crystalline form and the other on the soluble form (Monreal and Reese 1969; Tiunova et al. 1976). Our results support the above views.

Maximum enzyme production at 30°C is consistent with results reported by Skujins et al. (1965) for Streptomyces. Chitinase production gradually decreased from the seventh day with no activity on the tenth day, which may be due to accumulation of sugars (Monreal and Reese, 1969). Negligi- ble levels of enzyme in the absence of chitin indicates con- stitutive levels which may help in inducing the enzyme in the presence of chitin (Vasseur et al. 1990). Most of the chitinolytic systems reported in the literature are inducible (Monreal and Reese 1969; Vyas and Deshpande 1989; Ulhoa and Peberdy 1991). Among the various hexoses and

pentoses as additives to chitin, only arabinose did not affcct enzyme production. Furthermore, a synergistic effect of arabinose with chitin (1 : 1) was observed. This synergistic effect suggests a possible relationship with the ara operon or its product in induction of the chitinolytic system. Strep- tomyces viridificans chitinase is an inducible enzyme with low constitutive levels and it shows catabolite repression. I t has been reported that chitinases are like cellulases in induction and repression (Monreal and Reese 1969).

As the molecular weight of the enzyme is known to be in the range of 30-60 kDa (Tiunova et al. 1976; Pegg 1982; Rowena and Cabib 1982), a G-75 Sephadex column was used giving a threefold concentration. The protein profile of the fraction revealed two peaks, a and b, with good chi- tinolytic activity, confirming the findings of Tiunova et al. (1976).

Purified enzyme was completely inhibited by 5 mmol I ~

divalent cations. The increase in activity with mercapto- ethanol indicates presence of sulphydryl groups on the active site of the enzyme, confirmed by total inhibition by Hg2 + . Similar inhibition and mercaptoethanol enhance- ment has been reported by Pegg (1982) and Ueno et al. (1990).

L Y S l S OF THE FUNGAL CELL WALL

As the fungal cell contains chitin as the major component, chitinases are well known to lyse fungal cell walls (Henis and Chet 1975; Morrissey et al. 1976; Beyer and Diekmann 1985; Ueno et al. 1990). In the present study, it has been shown that S. uiridzjicans is able to produce a high level of

CHlT lNASE PRODUCTION B Y STREPTOMYCES VlRlDlF lCANS 383

chitinase on fungal cell wall chitin. Chitinase produced using wall material of one fungal genera was active against all other fungi. Beyer and Diekmann (1985) and Ordentlich et a l . (1988) have reported that incubation of Streptomyces chitinase with several fungi could release sugars from cell walls. The results of the present investigation using enzyme-soaked discs in controlling fungal growth are a promising method of biocontrol of plant pathogens.

ACKNOWLEDGEMENTS

The authors sincerely thank Mrs Meena Singh and M r Mustafa Hussain for their technical assistance during the course of this present investigation and Mr Rajiv Chawla, Junior Technical Personal Assistant, University of Delhi South Campus, New Delhi, for his help in the preparation of the manuscript.

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