BIOSYNTHESIS AND PROPERTIES OF BETA-1,3-GLUCANASES OF TRICHODERMA HAMATUM

Agnieszka Maj, Danuta Witkowska, Małgorzata Robak

ABSTRACT

The strain of Trichoderma hamatum C-1 is able to produce extracellular lytic enzymes i.e. ß-1,3- glucanases, chitinases, ß-1,6-glucanases in the shaked cultures in the base medium enriched with fodder yeast supplemented with Avicel cellulose, as well as with glucose. Glucose had a repressive effect on ß-1,3- glucanase activity only when the fungus was cultivated in a high quantity (3%) of glucose medium. The enzymes were precipitated from liquid culture medium by ethanol and then optimal conditions for the enzymatic activity (pH 3.0-5.0; 50°C) and stability (pH 5.0; 50°C) of ß-1,3-glucanases were determined. The enzyme activity was strongly inhibited in the presence of HgCl₂ , EDTA and stimulated by cations such as Mg⁺² and Ca⁺². After enzymatic hydrolysis (120 min.)of laminarin products of reaction were measured using HPLC and about 90% of glucose and a little laminaribiose and laminaritriose were obtained. The enzymatic preparation was used for Yarrowi

Key words: lytic anzymes, beta-1,3-glucanases, biosynthesis, biochemical properties, Trichoderma hamatum.

INTRODUCTION

The extracellular complex of ß-glucanases produced by fungi of Trichoderma hamatum is able to hydrolyse ß-1,3, ß-1,4 and ß-1,6 bonds of ß-glucanes of plant or microorganism cell wall biopolymers. The ß-glucanases from Trichoderma spp. are important enzymes used in genetic manipulation (DNA separation, protoplast formation) and in releasing protein and pigments from cells. The ability of ß-1,3-glucanases to degrade the yeast cell wall seems to indicate that this enzyme may be helpful in the future as a factor increasing the digestibility of protein feed of microbiological origin. In recent years the cell wall degrading enzymes have often been used for biological control of soilborne pathogenic fungi.

MATERIALS AND METHODS

Microorganism and culture conditions. The strain Trichoderma hamatum C-1 from our own collection was used in these studies. The strain was stored on potato-dextrose slants at 4°C. Duplicate shaking cultures were carried out on a mineral Saunders medium (Witkowska, Stempniewicz, Maj, 1999) enriched with yeast extract (0.1%) and wheat germs (0.1%). As carbon source, addition of fodder yeast (with or without Avicel cellulose) or glucose was used. The medium was inoculated with conidial suspension of 1x10⁷ cells in 1ml of 1% Tween 80. The cultures were conducted in 500ml flasks (100ml of medium) at 28°C for 7days. Samples for enzymatic determination were collected after centrifugation every 24h.

The enzymatic preparation (powder) was obtained after ethanol (4vol.) precipitation of enzymatic protein from postcultural liquid.

Enzyme assays Activities of beta-1,3-glucanases, beta-1,6-glucanases and beta-1,3(1,4) glucanases (Santos et al., 1977) were determined using laminaryn (6.5g/L), (Sigma) or pustulan (5g/L), (Sigma) or lichenan (5.0g/L), (Sigma), respectively as a substrate in 0.05M/L acetic buffer, at 50°, pH4.8. Activities of cellulases (Mandels et al.) were determined using NaCMC (10g/L) as a substrate in 0.05M acetic buffer, pH 4.8, at 50°C. Activities of chitinases (Witkowska et al., 1999) were determined using colloidal chitin (6.0g/L), (Sigma) as a substrate in0.05M acetic buffer at 40°C. The reducing substances – products of enzymatic reaction, were determined colometrically by the Miller`s method (DNS) using dinitrosalicylic acid (Sigma), The activities of examined enzymes were expressed in µmol of reducing substances/ml/min. (U/ml or U/mg of protein).The Lowry method was used to determine the protein content.

The effect of pH on the activity of beta-1,3-glucanases: solutions of 0.2% of enzyme preparation in 0.05M acetic buffer of various pH (3.0; 4.0; 5.0; 6.0; 7.0; 8.0) were used (50°C).

The effect of pH on the stability of beta-1,3-glucanases: solutions of 0.2% of enzyme preparation of pH 3.0; 4.0; 5.0; 6.0; 7.0; 8.0 were preincubated for 24h in 4°C, then activities of enzymes were determined at pH 4.8, 50°C.

The effect of temperature on the activity of beta-1,3-glucanases: solutions of 0.2% of enzyme preparation in 0.05M acetic buffer at various temperatures (40; 50; 60; 70; 80°C) were used.

beta-1,3-glucanases thermostabilty: solutions of 0.2%of enzyme preparation in 0.05M acetic buffer were preincubated at various temperatures (40; 50; 60; 70; 80°C) for 60min. and then activities of enzymes were determined at pH 4.8, 50°C.

The effect of activator and inhibitor solutions of 0.2% of enzyme preparation in 0.05M acetic buffer were preincubated for 15min. in 50°C in the presence of 2mM of each of the following substances: EDTA, HgCl₂, CaCl₂, ZnSO₄, CuSO₄, MgSO₄ and the activities of enzymes were determined by the standard assay procedure.

The laminarine (1%) hydrolysis process: was carried at 50°C. pH 4.8 for 30 and 120min. Reducing substances and glucose as final products of hydrolysis were determined with DNS and enzymatic methods (with glucoxidase and peroxidase), respectively. HPLC was used to determine the products of hydrolysis, too (Aminex HPX 87H column; 0.01M H₂SO₄ as eluent; flow - 0.3ml/min, detection refractometrical RF).

In production of yeast cell protoplasts, the cells of Yarrowia lipolytica A101.1.31. and Saccharomyces cerevisiae from 14h of culture (old) in YM medium were used. After cultivation, the culture was centrifuged (3500xg), the cells were washed twice with distilled water, suspended in a pre-treatment solution (1% ß-mercaptoethanol and 1% EDTA), (Sakai et al., 1986) and incubated at 28°C for 30min. Yeast suspension was standardised at 10⁸ cell/ml (Campbell and Duffus, 1988). After incubation, the cells were centrifuged, washed twice with 0.6M/L KCl and suspended in lytic medium (60mM/L phosphate buffer pH7.5 and 10mM/L of ß-mercaptoethanol), (Sakai et al.,1986). The lytic medium contained enzymatic preparation from Trichoderma hamatum or commercial preparation (Sigma), (150mg/ml). The samples were incubated at 28°C. The degree of protoplast production was controlled using microscopy.

RESULTS AND DISCUSSION

The selection of a suitable carbon and energy source has a particular importance in the process of extracellular production of hydrolases by filamentous fungi. Very often this carbon and energy source plays the role of an inductor of enzymes. Many authors, in their works, stress induction feature of extracellular hydrolases of Trichoderma fungi (beta-1,3-glucanases, cellulases, chitinases) and positive influence of the added stimulators present in waste fungi mycelium, in yeasts biomass , and in cell walls of fungi and yeasts, etc. on those enzymes [15,17,20]. In this work, the addition of fodder yeast (D) specially in 4% concentration (together with 2% of AC) to the cultures of T. hamatum C-1 was the most suitable for biosynthesis process of beta-1,3-glucanase( 12U/ml), (Fig.1). Biopolymers of cell walls present in yeast biomass have a stimulating influence on synthesis of those enzymes (Fig.1). The simultaneous addition of ye ast and Avicel cellulose (AC) has a stimulating influence especially in case of addition of 4% of yeasts and of 2% of AC (Fig.1). That effect was not observed in the other combinations of inductors which were tested, and the maximal beta-1,3-glucanase activities did not excess 10U/ml. Rudawska & all.[10] in their works on induced biosynthesis of beta-1,3-glucanase, have obtained the activity not exceeding 0,9U/ml. Sandahu [12] obtained the beta-1,3-glucanases with activity of 0.28U/ml in the culture of T.harzianum with addition of mycelium of Trichoderma sp., and Targoński & Wójcik [16] obtained those enzymes with activity of 2.17 U/ml in case of T.reesei M.-7 growing in the presence of cellulose. In the authors’ own studies it was proved that T. hamatum C-1 was able to produce beta-1,3-glucanases, not repressed by the presence of glucose in the medium (fig.2). The highest values of activities 7.7 U/ml were obtained in the presence of 1.5 % glucose after 7 days of culture, however an increased amount of glucose in the medium, in the range of 2% and 3% inhibited the biosynthesis of the studied enzymes, in~50% and~70 %, respectively.

Further studies on the properties of beta-1,3-glucanases were made on an enzymatic preparation obtained from the culture medium by ethanol precipitation of proteins. Some biochemical properties of beta-1,3-glucanases present in so obtained preparation were studied, such as pH and temperature range activity, and the action of some inhibitors or inductors on the activity and enzyme stability. Moreover, activities of other associated enzymes were measured. In those enzymatic preparations, besides the activity of beta-1,3-glucanases (1034U/g), the activity of cellulases (38.3 U/g) , beta-1,6-glucanases (9.0U/g) and beta-1,3(1,4)-glucanases (1482U/g) was present (Table 1). Beta-1,3-glucanases, present in enzymatic preparation were characterised by the highest activity at pH of 3.0-5.0, at the temperature of 50°C, and were stable at pH of 5.0 and temperature of 50°C (Fig. 3.). In many publications, the difference between the optimal values of pH and temperature of activity of beta-1,3-glucanases from different sources were reported. In Kitamoto [3] studies on properties of egzo-beta-1,3-glucanases from T. harzianum the optimal activity of those enzymes was at pH of 4.6 and 45°C. Maximal activity values of beta-1,3-glucanases from T.viride, Merc & Galas [7] obtained at pH 6.5 and temperature of 45°C. Norhona & Ulhoa [9] obtained beta-1,3-glucanases from T. harzianum, at pH 4.4 and temperature of 45°C, and Sharma & Nakas [14] observed the highest activity of beta-1,3-glucanases from T.longibrachiatum at pH 3.0-5.0 and temperature range of 30-70°C. The biochemical properties, i.e. inhibitors and activators are also important in the characteristic of enzyme, because they make it possible to establish such conditions of application that do not decrease the action of lytic enzymes. In the present work beta-1,3-glucanases from T.hamatum have shown about 20% increased activity in the p resence of Ca⁺² and Mg⁺², an insignificantly increased activity in the presence of Cu⁺² and Zn⁺², and were inhibited by HgCl₂ and EDTA (Fig.4). That results implied the presence of SH containing amino acid residues in the active site and the role of metal ions in enzymatic catalysis. Totsuka & Usui [18] in the study on enzymes from Rhizoctonia solani evaluated the influence of metal ions on activity of beta-1,3-glucanases and an important inactivation by Hg⁺² (decrease of activity by 87%), and a small effect of Mg⁺²,Ca⁺², Zn⁺², Fe⁺², Pb⁺² and Cu⁺² were observed.

In the present work, characteristics of hydrolytic process of laminaryn as the substrate by beta-1,3-glucanases from T.hamatum C-1 was studied. Degradation products were analysed by HPLC and colorimetric measure (glucose was measured enzymatically). After 30 min of hydrolysis, about 30 % of glucose was detected and a small amount of laminarytriose, and after 120 min of hydrolysis near 90% of glucose and a small amount of laminarybiose and laminarytriose (Fig. 5. Table 2). Those results have shown the synergetic action of egzo and endo glucanases that conducted the process to glucose. De la Cruz [1] detected in enzymatic hydrolyses mainly the presence of glucose, laminarybiose and laminaryterose after the hydrolysis process of laminaryn by enzymes from T.harzianum CECT 2413 culture.

The enzymatic preparation obtained from T.hamatum C-1 was successfully used in the process of protoplast formation of Yarrowia lipolytica cells (70% of protoplasts after 1 h; 100% after 2 h) and of S. cerevisiae (60% after 2 h; 100% after 4h), (Table 3).

Literature data show the different ability of yeast cells to be hydrolysed by lytic enzymes, not only among species, but also in the relation to age of cells used in protoplast formation. Kopecka [4] obtained the protoplasts of Schizosaccharomyces versatilis and S.pombe in 30 min only in the presence of a mixture of lytic enzymes from the stomach of a snail and an enzymatic preparation from the culture of T.viride. Evans [2] obtained after 1h 95% of protoplast of S. cerevisiae in the presence of Zymolase (from Arthrobacter luteus), while with enzymes from the culture of Rhodotorula rubra at the same time only 15% of protoplasts were obtained. In this work, the commercial preparation of lytic enzymes from Sigma company used for comparison was more effective in the protoplast formation of Y.lipolytica and S.cerevisiae (respectively 100% of protoplast after 0.5h and 100% after 1h), but the value of enzyme activity in the used dose was about twi ce higher in case of Sigma preparation than in case of own preparation from T.hamatum (Table 4) So, after some purification steps, the studied enzymatic preparation can be more effective in the protoplastisation process. The presented results suggest that the study on enzymes produced by the strain T. hamatum C-1 is important.

1. The strain of Trichoderma hamatum C-1 is able to produce beta-1,3-glucanases on mineral medium enriched with fodder yeast supplemented with Avicel cellulose as well as on glucose.

2. Beta-1,3-glucanases were characterized by optimal activity at pH 3.0-5.0 and 50°C and stability at pH 5.0 and 50°C.

3. The enzyme activity was inhibited in the presence of HgCl₂, EDTA and stimulated by cations such as Mg⁺² and Ca⁺².

4. 90% of glucose and a little laminaribiose and laminaritriose were obtained after enzymatic hydrolysis using HPLC analysis.

5. The enzymatic preparation was effective in protoplast production of Yarrowia lipolytica A101-1.31 cells (100% of protoplast after 4h) and Saccharomyces cerevisiae cells (100% after 2h).

1. de la Cruz J., Pintor-Toro J.A., Benitez T., Llobel A., Romero L.D., 1995. A novel endo-beta-1,3-glucanase, BGN 13.1, Involved in the mycoparasitism of Trichoderma harzianum. J.Bacteriol., 177, (23), 6937-6945.

2. Evans C.T., Conrad D., 1987. An improved method for protoplast formation and its application in the fusion of Rhodotorula rubra with Saccharomyces cerevisiae. Arch.Microbiol., 148, 77-82.

3. Kitamoto Y., Kono R., Shimotori A., Mori N., Ichikawa Y., 1987. Purification and some properties of an exo-beta-1,3-glucanase from Trichoderma harzianum. Agric.Biol.Chem., 51(12), 3385-3386.

4. Kopecka M., 1975. The isolation of protoplast of the fission yeast Schisosaccharomyces by Trichoderma viride and snail enzymes. Folia Microbiol., 20, 273-276.

5. Lowry O.H., Rosebrough N.J., Farr A.C., Randall R.J., 1951. Protein measurements with the Folin phenol reagent. J.Biol.Chem., 193, 265-275.

6. Mandels M., Andreotti R., Roche C., 1976. Measurement of saccharifying cellulose. Biotechnol. Bioeng. Symp., 6, 17-34.

7. Merc M., Galas E., 1984. Beta-1,3-Glucanase from Trichoderma viride . Purification and characterization. Acta Biotechnol., 4,(1), 67-74.

8. Miller G.L., 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal.Chem., 31, 426-428.

9. Noronha E.F., Ulhoa C.J., 1996. Purification and characterization of an endo- beta-1,3-glucanase from Trichoderma harzianum. Can.J.Microbiol., 42, 1039-1044.

10. Rudawska M., Kamoen O., 1992. Regulation of beta-1,3-glucanase synthesis in Trichoderma harzianum. Arboretum Kórnickie., R37, 51-59.

11. Sakai T., Koo K., Saitoh K., Katsuragi T., 1986. Use of protoplast fusion for the development of rapid starch fermenting strains of Saccharomyces diastaticus. Agr.Biol.Chem., 50, 297-306.

12. Sandhu D.J., Wadhwa V., Bagga P.S., 1989. Use of lytic enzymes for protoplast production in Trichoderma reesei QM9414. Enzyme Microb.Technol., 11, 21-25.

13. Santos T., Villanueva J.R., Nombela C., 1977. Production and catabolite repression of Penicillium italicum. J.Bacteriol., 129, 52-58.

14. Sharma A., Nakas J.P., 1987. Preliminary characterization of laminarinase from Trichoderma longibrachiatum. Enzyme Microb. Tech., 9, (2), 89-93.

15. Schickler H., Haran S., Oppenheim A., Chet I., 1998. Induction of Trichoderma harzianum chitinolitic system is triggered chitin monomer, N-acetyloglucosamine. Mycol. Res., 102, (10), 1224-1226.

16. Targoński Z., Wójcik W., 1993. Biosynthesis of extracellular enzymes by isolates, mutants and recombinant strains of Trichoderma spp. Biotechnologia., 1(20), 14-19.

17. Thrane C., Tronsmo A., Jensen D.F.,1997. Endo-beta-1,3-glucanase and cellulase from Trichoderma harzianum: purification and partial characterization, induction and biological activity against plant pathogenic Pythium spp. Europ.J.Plant Pathol., 103, (4) 331-344.

18. Totsuka A., Usui T., 1986. Separation and characterization of the endo-beta-(1,3)-D-glucanase from Rhizoctonia solani. AgricBiol.Chem.: 50(3),543-550.

19. Ulhoa C.J., Peberdy J.F., 1992. Purification and some properties of the extracellular chitinase produced by Trichoderma harzianum. Enzyme Microbiol.Technol., 14, 236-240.

20. Wayman M.,Chen S., 1992. Cellulase production by Trichoderma reesei using whole wheat flour as a carbon source. Enzyme Microb.Technol., 14, 825-831.

21. Worasatit N., Sivasithamparam K., Ghisalberti E.L., Rowland C., 1994. Variation in pyrone production, lytic enzymes and control of Rhizoctonia root rot of wheat among single-spore isolates of Trichoderma koningii. Mycol.Res., 98, (12), 1357-1363.

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