Solid-state fermentation of waste cabbage by Penicillium notatum NCIM NO-923 for production and characterization of cellulases

Transcription

1 Journal of Scientific & Industrial Research 714 Vol. 68, August 29, pp J SCI IND RES VOL 68 AUGUST 29 Solid-state fermentation of waste cabbage by Penicillium notatum NCIM NO-923 for production and characterization of cellulases Arpan Das 1 and Uma Ghosh 2 * 1 Department of Microbiology, Vidyasagar University, Midnapore , India 2 Food Technology and Biochemical Engineering Department, Jadavpur University, Kolkata7 32, India Received 23 February 29; revised 17 April 29; accepted 24 April 29 Solid-state fermentation (SSF) of waste cabbage was carried out by Penicillium notatum NCIM NO-923 for production of extracellular enzymes. Highest activities of carboxymethyl cellulase () ( ±1.2 IU/gds) was obtained at 3 C and 48 h fermentation time and that of filter paper activity () (67.49 ±1.33IU/gds) was obtained at 3 C and 24 h fermentation time. SSF of mixed substrate (cabbage and bagasse; 3:2 w/w) resulted in increase in activity by ±1.22 and by12.76 ±1.33. Kinetic parameters were determined from Linewaver Burk plot. Both enzymes exhibited significant thermo stability up to 5 C. Keywords:,, SSF, Mixed substrate, Penicillium sp. Introduction Cellulases degrade cellulose to produce glucose and other soluble sugars, which can be converted to food 1, fuel 2 and other chemicals 3. High saccharification efficiency, mild operating conditions with respect to ph and temperature, absence of by-products and avoidance of pollution makes enzymatic hydrolysis 4,5 superior over chemical processes. However production of cellulase enzymes has been widely studied in submerged culture fermentation, but relatively high cost of enzyme production has restricted its application in large-scale processes. Solid-state fermentation (SSF) is a promising alternate to produce cellulases economically using cellulosic waste as substrate rather than expensive pure cellulose. Various agricultural wastes including wheat straw 6, sawdust, bagasse, corncob 7, willow, spruce, oat bran, wheat bran and rice straw 8 have been used successfully in SSF for cellulase production 9. This study assesses suitability of cabbage as substrate of SSF for production of cellulase enzymes by Penicillium notatum NCIM NO-923, optimizes fermentation condition for yield of enzymes, and presents characterization of enzymes and enzyme kinetics to analyze reaction kinetics. *Author for correspondence ughoshftbe@yahoo.co.in Materials and Methods Microorganism and Substrates Strain P. notatum NCIM NO-923 was maintained on Czapek Dox agar medium by monthly sub culturing and stored at 4 C. Cabbage and bagasse, obtained from local market, were sun dried and milled to pass through a.5 mm screen. Dry substrates were stored for further use. Enzyme Production and Extraction Dry substrate (1 g) was taken in Erlenmeyer flask (25 ml) and supplemented with distilled water to obtain required moisture prior sterilization. Growth medium was inoculated with 5 ml spore suspension ( 1 8 spores /ml), and incubated at 3 C under static condition. Moldy substrates produced by SSF was mixed with distilled water (1:1 w/v), stirred slowly at 3 C for 3 h and filtered followed by centrifugation at 1 rpm (C-24 REME INDIA) for 3 min. Clear supernatant was used as crude enzyme. Optimization of Fermentation Condition Optimization of fermentation condition regarding composition of substrate, time of incubation, growth temperature, substrate bed depth, growth of ph and hydration were conducted. To study ph effect on enzyme production, addition of water to medium was replaced by buffer at appropriate ph.

2 DAS & GHOSH: CELLULASES PRODUCTION FROM CABBAGE WASTE USING PENICILLIUM NOTATUM 715 Enzyme Assay Cellulase concentration 1 was estimated for carboxymethyl cellulase () and filter paper activity (). Enzyme assay was done with sodium acetate buffer (ph 4.6). Carboxymethyl cellulose solution (1%) and Whatman No-1 filter paper (5 mg) were used as substrate for and activities respectively. One unit of enzyme activity is defined as 1 micromole of glucose released per minute of reaction and calculated for 1gds (gram dry solid). Analytical Methods Moisture content of substrate was determined by AOAC method 11. Proteins were determined using bovine serum albumin as standard 12. Reducing sugars were estimated colorimetrically with dinitrosalicylic acid 13 using glucose as standard. Characterization of Cellulases Enzyme activities were determined by carrying out assays at several temperatures and ph to evaluate optimum temperature, ph and heat stability. For optimum temperature, enzyme was incubated with substrate for 1 h at various temperatures (3- C). For optimum ph, enzyme was mixed with substrates of different ph levels. For temperature stability, enzyme was incubated in standard buffer at 35- C for 1 h, ice cooled and then residual cellulase activity was measured in each case. Enzyme Kinetic Parameters Initial velocity kinetics studied influence of enzyme characteristics on production of reducing sugars. Reciprocal of Michelies- Menten equation, [ S] V Vo = max, gives Line-Weaver Burk Km + [S] equation 1 Km 1 = + (1) Vo Vmax[S] Vmax Kinetic parameters, Km (Michelies constant) and Vmax (Maximum velocity of enzyme catalyzed reaction), were calculated from Lineweaber-Burk plot by plotting reciprocal of substrate concentration with reciprocal of enzyme velocity. Filter paper and carboxymethyl cellulose were used as substrate. Results and Discussion Optimization of Fermentation Condition Enzyme production by P. notatum NCIM NO-923 on cabbage by SSF appeared to reach activities within 48 h of incubation (Fig. 1). Yield of was higher than that of, might be because these components show different response to substrate for their production. Organism is superior to cellulolytic fungi as productivities in this organism are achieved after 2 days while in fungi yield occur normally after 6-12 days of fermentation. Maximum yield of and were obtained when fermentation was carried out at 3 C (Fig. 2). These results are in agreement with earlier report 14 that filamentous fungi prefer acidic ph (3.8-5.). Proper aeration in SSF depends on air spaces available in substrate and thus depends on thickness of substrate layer. Using 5 g cabbage in 25 ml Erlenmeyer flask (Fig. 3), was produced. Under same fermentation conditions, reduction of enzyme activity for (28%) and (%) with substrate (1 g) was observed. In SSF, aeration not only supplies O 2 but also removes metabolic heat, and gaseous and volatile products from fermenting mass 9. Thus higher bed depth of substrate may result adverse effect on cellulase production, lowering yield of enzyme with higher amount of solid substrate used for fermentation. Effect of Mixed Solid Substrate Mixed solid substrates are viable medium for SSF process. Sugarcane bagasse, which is cheap, easily available, and contains sufficient nutrients, was used with cabbage for SSF. This is first report on utilization of mixed solid state substrate using bagasse and cabbage for production of cellulases by P. notatum NCIM NO-923 and each combination produces better yield than any cabbage or bagasse (Table 1). Table1 Effect of mixed substrate SSF on production of and by P. notatum NCIM NO-923 Substrate ratio (w/w) Enzyme activity, IU/gds Bagasse: cabbage 1: ±1.21 1: ± ±1.22 1:4 153 ± ±1.24 3: ± ±1.42 2: ± ±1.33 1: ± ±1.24 3: ± ±1.16 7: ± ±1.35 4: ± ±1.22 9: ± ±1.24 : ± ±1.33 Data was mean of three observations

3 716 J SCI IND RES VOL 68 AUGUST Time, days Fig. 1 Time course of enzyme production in SSF by P. notatum NCIM NO Temperature, o C Fig. 2 Effect of environmental temperature on cellulase production in SSF by P. notatum NCIM NO-923 Effect of Hydration Inter particle mass transfer 15 of oxygen, nutrients and enzymes are dependent on substrate characteristics and moisture content of fermenting media. Maximum yield was obtained at following ratios (Fig. 4): mixed substrate and water, 4:3 (w/v); and cabbage and bagasse, 3:2 (w/w). Higher and lower hydration results less enzyme activity, may be because low moisture content is unfavorable for microorganisms growth and at higher moisture content, void space within solid phase is filled

4 DAS & GHOSH: CELLULASES PRODUCTION FROM CABBAGE WASTE USING PENICILLIUM NOTATUM 717 (% of ) Amount of cabbage, g g) H ph Fig. 3 Effect of substrate amount on production of cellulase enzymes in SSF by P. notatum NCIM NO-923 Fig. 5 Optimum ph for cellulase enzyme produced in SSF by P. notatum NCIM NO /V Temperature, C o C : 4:1 2:1 5:3 4:3 1:1 Substrate to moisture ratio R 2 =.997 Fig. 4 Effect of hydration on production of cellulase enzymes in SSF by P. notatum NCIM NO-923 Fig. 6 Optimum temperature for cellulase enzymes produced in SSF by P. notatum NCIM NO Temperature, ) o C Fig. 7 Thermal stability of and produced in SSF by P. notatum NCIM NO /[S] Fig. 8 Enzyme kinetics for cellulases produced in SSF by P. notatum NCIM NO-923

5 718 J SCI IND RES VOL 68 AUGUST 29 with water and air is driven out, affecting growth of organisms and also enzyme production. Enzyme Characteristics Both and were optimally active at ph 4 (Fig. 5) and temperature (5 C) (Fig. 6). Maximum and thermo stability was observed at C (Fig. 7). Beyond C, thermo stability decreased, may be due to thermal denaturation of enzyme. At 5 C, was fairly stable (stability % of ), whereas stability of was about %. At C, stability of was 78% of, but stability of decreased to 42%, indicating that was more stable than. Enzyme Kinetics Kinetic studies were made with multienzyme system including and. From Linewaver Burk plot, Km and Vmax values were (Fig. 8).33 M/l and IU/gds respectively. Conclusions Bioconversions of cabbage mixed with bagasse (3:2) are potential raw materials for production of industrial important enzymes and could be used with great commercial significance. References 1 Lawford H G & Rousseau T D, Cellulosic fuel ethanol alternative fermentation process designs with wild type and recombinant Zymomonas mobilis, Appl Biochem Biotechnol, 15 (23) EI-Hawary F L, Mostafa Y S & Laszlo E, cellulase production and conversion of rice straw to lactic acid by simultaneous saccharification and fermentation, Acta Aliwerst Hung, 3 (23) Taguchi F, Yamada K, Hasegawa K, Taki Saito T & Hara K, Continious hodrogen production by Clostridium sp. Strain no. 2 from cellulose hydrolysate in an aqueous two phase system, J Ferm Bioeng, 82 (1996) Curreli N, Agelle M, Pisu B, Resigno A, Sanjust E & Rinaldi A, Complete and efficient enzymatic hydrolysis of pretreated Wheat straw, Process Biochem, 37 (22) Kubicek, C P, Messner R, Guber F, Mach, R L & Kubicek- Pranz E M, Trichoderma cellulase regulatory puzzle: from the interior life of a secretory fungus, Enzyme Microb Technol, 15 (1993) Chahal P S, Chahal D S & Le G B B, Production of cellulase in solid state fermentation with Trichoderma resie MCG on wheat straw, Appl Biochem Biotechnol, 57 (1996) Jumu O, Solomon T V, Ogbe B, Eriola B, Stephen L, Amigun K & Bamikole, Cellulase production by Aspergillus flavus isolate NSPR11 fermented in sawdust, bagasse and corncob, African J Biotechnol, 2 (23) Kalogeris E, Christakopoulos P, Katapodis P, Alexious A & Valchou S, Production and characterization of cellulolytic enzymes from the thermophilic fungus Thermoascus aurantiacus under solid state cultivation of agricultural waste, Process Biochem, 38 (23) Jeeu I, Solid-state fermentation of agriculture waste for endoglucanase production, Ind Crops Prod, 36 (2) Mandel M, Andreoni R & Roche C, Measurement of saccarifying cellulase, Biotechnol Bioeng Symp, 6 (1976) AOAC methods of analysis, 1 th edn (AOAC, Washington DC) Lowry O H, Rosebrough N J, Farr A L & Randall R J, Protein measurement with the Folin phenol reagent, J Biol Chem, 143 (1951) Miller G l, Use of dinitrosalicylic acid reagent for determination of reducing sugar, Anal Chem, 31 (1957) Khalil A I, Production and characterization of cellulolytic and xylanolytic enzymes from the lignocellulosic white-rot fungus Phanerochaete chrysosporium grown on sugarcane bagasse, World J Microbiol Biotechnol, 18 (22) Moo-Young M, Moreire A R & Tengardy R R, Filamentous Fungi, vol 4 (Oxford/IBH Publications, New Delhi) (1983) 117.