Production and optimization of cellulase from Fusarium oxysporum by submerged fermentation

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1 Journal of Scientific & Industrial Research 454 Vol. 69, June 2010, pp J SCI IND RES VOL 69 JUNE 2010 Production and optimization of cellulase from Fusarium oxysporum by submerged fermentation G Ramanathan*, S Banupriya and D Abirami Department of Microbiology, V H N S N College, Virudhunagar , India Received 27 July 2009; revised 25 February 2010; accepted 03 March 2010 Fusarium oxysporum, isolated from infected tomato plant parts, produced maximum cellulase at optimum parameters (ph, 6.0; temp., 50 C; and incubation period, 12 d) in cellulase enzyme production broth having 1% CMC (carboxy methyl cellulose) as a cellulose substrate. Activities of purified cellulases (mol wt, 24, 29 and 45 kda) were stimulated by concentrations (0-70 mm) of Na + and Mg ++, while EDTA inhibited enzyme activity at all concentrations. Keywords: Cellulose, CMC, Fermentation, Fusarium oxysporum, Glucosidase Introduction Cellulose is most common carbohydrate on earth 1. Utilization of cellulose as a nutrient source requires enzymes that cleave β- 1, 4- glycosidic bonds between constituent sugars 2. Cellulase is associated with pathogenicity of a number of microorganisms 3. Fusarium oxysporum, a cosmopolitan soil borne filamentous fungus, is an anamorphic species that includes numerous filamentous plant pathogenic strains causing wilt disease of a broad range of agricultural and ornamental host plant species. It produces several enzymes that act upon pectic and cellulose components of cell walls 4. This study presents production and optimization of cellulose enzyme from F. oxysporum by submerged fermentation. Experimental Section Sample Collection and Assessment of Cellulase Activity Infected parts (leaf, stem) of diseased tomato plant were collected and aseptically transfered to laboratory for further isolation of fungal pathogen. Fungal colonies were isolated in potato dextrose agar (PDA) plates. Cellulose degradation potential was assessed of selected strains by growth and zone formation in carboxy methyl cellulose (CMC) agar medium. Liquid State Fermentation Cellulase enzyme production medium and inoculated spore suspensions of F. oxysporum were prepared. *Author for correspondence san_than2002@yahoo.co.in Broth cultures were incubated on a rotarty shaker (150 rpm) at 30 C. Fermented samples were withdrawn at two days (2, 4, 6, 8, 10, 12 days) interval. Reducing sugar was estimated by DNS (Dinitro Salicylic acid) method 5 and protein content by reported method 6. Assay of Cellulase Enzyme Activity of cellulase was assayed by reported method 7. Fermented sample was centrifuged at 3000 rpm for 10 min. Supernatant was used as enzyme source. For CMCase activity, suitably diluted enzyme solution (1 ml diluted equally with 0.2 M acetate buffer, ph 4.8) was incubated with 1 ml CMC (10 mg/ml) in 0.2 M acetate buffer at 60 C for 30 min. For FPase activity, suitably diluted enzyme solution (1 ml diluted equally with 0.2 M acetate buffer) was incubated with 50 mg filter paper (What man No.1) in 1 ml of 0.2 M acetate buffer (ph 4.8) for 1 h at 60 C. For β-glucosidase activity, suitably diluted enzyme solution (1 ml diluted with 0.2 M acetate buffer) was incubated with 1 ml of salicin (10 mg/ml) solution in 0.2 M acetate buffer (ph 4.8) at 60 C for 30 min. Amount of reducing sugars released in CMCase, FPase, β-glucosidase assay after incubation was estimated by DNS method 5. Cellulase was estimated 8 and its activity was expressed in International Unit (IU). Effect of Cations and EDTA on Cellulase Activity Effect of cations on cellulase activity was determined by using two cations (Na + and Mg ++ ; conc., 0, 10, 20, 30, 40, 50, 60 and 70 mm). Substrate cation mixture was incubated at room temperature (RT) for 1 h before it was

2 RAMANATHAN et al: CELLULASE FROM FUSARIUM OXYSPORUM 455 used in enzyme assay. Effect of ethylene diamine tetra acetic acid (EDTA) at various molar concentrations (0, 10, 20, 30, 40, 50, 60 and 70 mm) on activity of cellulase was determined. Substrate chemical compound mixture was incubated at RT for 1 h before it was used in enzyme assay. Optimization of Culture Conditions Optimum culture conditions [ph (4, 5, 6, 7, 8 and 9), temperature (30, 40, 50, 60, 70 and 80 C), incubation period, carbon (glucose, lactose, sucrose and cellulose; 1%w/v) and nitrogen (peptone, yeast extract, urea, diammonium sulphate; 1%w/v) source requirements] were determined for maximum growth of F.oysporum and cellulase activity were recorded. Crude Protein Extraction by Ammoniumsulphate Precipitation Ammonium sulphate (60%) was added to filtrate slowly with continuous stirring at low temperature (in an ice bath/beaker) for 5-10 min and left overnight in refrigerator. Mixture (salt+filtrate) was centrifuged at rpm for 20 min at 4-6 C, then pellet was collected and dissolved in 0.2 M sodium phosphpate buffer (ph 7.0) using two volumes of buffers. During collection of enzymes, tubes and funnel were washed with same buffer and collected; process was repeated thrice. Volume should not be 20% of total volume of filtrate. Enzyme salt solutions were dialysed with same buffer, which was changed 3-5 times for removing ammonium salt 9. Double-Layer Plate Assay To detect crude enzyme activity, a bottom layer [15 ml of 0.7% (w/v) agarose and 50 mm potassium phosphate-citric acid buffer (ph 5.2)] was overlaid with 5 ml of CMC (0.2%; w/v) and agarose (0.5%; w/v). Plates were inoculated with protein extract (40 µl) at 30 C for 24 h. To detect CMCase activity, plates were flooded with congored solution (1%; w/v) for 30 min and then rinsed several times with 1 M NaCl. This procedure revealed distinct hydrolysis regions 10. Purification by Ion- Exchange Chromatography on DEAE - Sephadex A - 50 DEAE sephadex A 50 column was packed into a vertically mounted column (1.5 cm x 40 cm) at a flow rate of 30 ml/h. Column was equilibrated with 3 bed volumes of 0.02 M sodium phosphate buffer, ph 7.0 containing 1 mm 2-mercaptoethanol. Enzyme concentrate obtained from ammonium sulphate precipitation was redissolved in minimal amount of buffer and dialysed for 24 h against 0.02 M sodium phosphate buffer, ph 7.0 containing 1 mm EDTA and 1 mm 2-mercaptoethanol with two changes of buffer per hour. Undissolved precipitate was removed by centrifugation and clear supernatant was layered on prepared column. Column was washed to remove all unbound proteins and a linear gradient of M NaCl in 0.02 M sodium phosphate buffer (ph 7.0) was used to elute any bound proteins. Fractions containing cellulase activities were pooled together and precipitated with ammonium sulphate. Precipitate was collected by centrifugation at 4000 g in a cold centrifuge at 4 C for 30 min, redissolved in 2.5 ml of 0.02 M sodium phosphate buffer (ph 7.0) and dialysed against buffer (ph 7.0) for 6 h 11. Molecular Mass Determination Molecular weight of cellulase was determined by SDS- PAGE using selectable markers 12. Results and Discussion Assessment of Cellulase Activity in CMC Agar Medium Selection of over producing cellulase strains of F. oxysporum were based on diameter of clearing zone surrounding small well in plate screening medium. Size of clearing zone diameter for selected strain was recorded for different incubation period as follows: 2 days, 12 mm; 4 days, 24 mm; 6 days, 32 mm; and 8 days, 36 mm. Enzyme activity of F. oxysporum was found as follows: CMCase, 1.92 U/ml; FPase, 1.34 U/ml; and β-glucosidase, 1.78 U/ml. Cellulose Cellulose degradation was assessed on 8 th day of fermentation by selected fungal strains. F. oxysporum utilized 32.68% of cellulose on 8 th day of incubation (Fig. 1a). Similar results have been reported 13 with studies on cellulosic substrates. Reducing Sugar Initial content of free reducing sugar in cellulase production broth was found to be 2.38 mg/ml. A gradual and steady increase in reducing sugar content was observed in cellulase production broth supplement with CMC. A tremendous (1.28 fold) increase in content of reducing sugar was observed during 8 th day of fermentation period by F. oxysporum (Fig. 1b). Amount of reducing sugar increased with time of incubation with presence of enzyme when cellulose rich agro waste supplemented as a substrate 14.

3 456 J SCI IND RES VOL 69 JUNE a) a) 40 Protein content, mg/ml Reducing sugar, mg/ml Cellulose utilization, % b) b) c) C) Fermentation period, days Fermentation period, days Fermentation period, days Fig. 1 Liquid state fermentation media inoculated with F.oxysporum and supplemented with CMC: a) Cellulose utilization; b) Reducing sugar content; and c) Protein content

4 RAMANATHAN et al: CELLULASE FROM FUSARIUM OXYSPORUM 457 Fig. 3 Effect on cellulase activity of F.oxysporum during 8 th day of fermentation of: a) EDTA; b) Mg concentration; and c) Na concentration Protein Initial protein content of cellulase enzyme production broth was 0.35 mg/ml. Protein content gradually increased till 8 th day of fermentation. F. oxysporum could produce 0.71 fold increase in protein content in same period. After incubation, protein content declined (Fig. 1c). Enzyme Assay Fermentation time (8 days) had optimum effect of F. oxysporum on enzyme production as follows: CMCase activity, 1.92 ± 0.005; filter paper degradation activity, 1.34 ± 0.003; and β -glucosidase activity, 1.78 ± (Table 1). Fig. 2 Effect on cellulase activity of F.oxysporum during 8 th day of fermentation of: a) ph; b) Temperature; c) Carbon sources; and d) Nitrogen sources Effect of ph, Temperature, C & N Sources on Cellulase Enzyme Production At ph 6 and 7, selected fungal strains of F. oxysporum showed heavy growth and higher cellulase activity

5 458 J SCI IND RES VOL 69 JUNE 2010 Table 1 Cellulase enzyme activity of Fusarium oxysporum in liquid state fermentation system supplement with carboxy methyl cellulose Enzymes activity, Duration of fermentation, days U/ml CMCase FPase β-glucosidase (Fig. 2a). Maximum growth and enzyme production were recorded at 50 C liquid state fermentation (Fig. 2b). At 50 C, confluent growth and better enzyme activity was noticed 15. β-glucosidase activity (0.769 U/ml), observed in F. oxysporum when lactose supplemented as a sole carbon source, was higher than FPase (0.654 U/ml) and CMCase (0.542 U/ml) activity (Fig. 2c). Cellulase biosynthesis required residual inducer in culture medium for 2 days, when cellulose and lactose were used as growth substrate 12. In F. oxysporum, enzyme activity got influenced when urea supplemented as a nitrogen sole source (Fig. 2d). Effect of Cations and EDTA on Cellulase Enzyme Production CMCase, FPase and β-glucosidases activity were inhibited with increasing concentration of EDTA (Fig. 3a). Cellulase activity was observed with decreasing concentration of Mg ++ ions by F. oxysporum (Fig. 3b). Maximum cellulase activity was observed without addition of sodium for selected fungal strains 16. Drastic reduction of enzyme activity was observed with increasing concentration of sodium ions (Fig. 3c). Extraction of Crude Cellulase Enzyme Crude cellulase enzyme of F. oxysporum, recovered following 60% saturation of culture supernatant with ammonium sulphate, showed an increase of specific activity. Molecular Weight Molecular weight of partially purified cellulase seemed to be 24, 29 and 45 kda in F. oxysporum. Purified enzymes of F. oxysporum (three distinct bands) have been observed with the molecular weight of 24, 29 and 45 kda 17. Conclusions Production and optimization of cellulase has been found suitable for F. oxysporum and optimization of ph, temperature, incubation time, carbon and nitrogen sources, EDTA and cations are limiting factors for maximum cellulase enzyme production as well as enzyme activity. Therefore, F. oxysporum is a potential fungal strain for production of cellulase enzyme. Acknowledgement Authors thank management and Department of Microbiology, V H N S N College, Virudhunagar, to carry out this study. References 1 Good E S & Good E P, Who needs cellulase? J Biol Edu, 27(1993) Updergraff D M, Semi-micro determination of cellulose in biological material, Anal Biochem, 32 (1969) Jan H D & Chen K S, Production and characterization of thermostable cellulases from Streptomyces transformant T 3-1, World J Microbiol Biotec, 19 (2003) Apel Brikhold P C & Walton J D, Cloning, disruption and expression of two endo - ² - 1,4 - xylanase genes, XYL2 and XYL3, from Cochliobolus carbonum, Appl Environ Microbiol, 62 (1996) Miller L, Use of dinitro salicylicacid reagent for determination of reducing sugars, Anal Chem, 31 (1959) Lowry O H, Protein measurement with Folin phenol reagent, J Biol Chem, 193 (1951) Ray L A, Pal A K & Dhyay P, Cellulases and β - ghluosidases from Aspergillus niger and saccharification of some cellulosic waste, J Micorb Biotechnol, 8 (1993) Selby K & Maitland C C, The cellulase of Trichoderma viride separation of the components involved in the solubilization of cotton, Biochem J, 104 (1996) 716.

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