Implementation of Response Surface Methodology for Enhanced Production of Endoglucanase by Thermophilic Aspergillus Fumigatus
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Enzymes are biocatalysts which play key roles in the body of living organisms. Cellulose is major source of plant biomass. Its β-1,4-glucosidic bonds are hydrolyzed by cellulases. These cellulases can be produced by a variety of microorganisms including fungi, bacteria and actinomycetes which are used today for the industrial applications. The current study was aimed to optimize the cultural conditions for maximum production of endoglucanase by Aspergillus fumigatus through solid state fermentation of sugarcane bagasse. Response Surface Methodology (RSM) was employed under Central Composite Design (CCD) for the optimization of growth including pH, temperature, time period & inoculum size and nutritional parameters including glucose, fructose and (NH₄)₂SO₄. The effect of different metal ions on endoglucanase production was also monitored. It was partially purified by (NH₄)₂SO₄ precipitation and gel filtration chromatography. Finally, edogucanase was characterized for optimum pH, temperature and determination of kinetic parameters. Maximum enzyme activity was found as 0.9 IU/mL/min in the presence of 6 g substrate, 3.5 mL inoculum, 4.5 pH, 40 °C temperature at incubation time of 84 hrs. After addition of carbon and nitrogen sources enzyme activity increased to 1.4 IU/mL/min. It was further increased to 1.56 IU/mL/min with 0.42% of CaCl2. Maximum purification was achieved at 50% saturation by ammonium sulphate (NH₄)₂SO₄. Optimum temperature and pH were 40 °C and 5 respectively, whereas the values for Km and Vmax were 5.37 mM and 696 uM/mL/min., respectively. These findings suggested that endoglucanase by Aspergillus fumigatus could be suitable for various industries.
Keywords: Aspergillus fumigatus, Central Composite Design (CCD), endoglucanase, Response Surface Methodology (RSM), Solid State Fermentation (SSF)
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Dashtban M, Schraft H, Qin W. Fungal bioconversion of lignocellulosic residues; opportunities & perspectives. Int J Biol Sci.2009;5(6):578-595.2.Silvestre M, Carreira R, Silva M, Corgosinho F, Monteiro M, Morais H. Effect of pH and temperature on the activity of enzymatic extracts from pineapple peel. Food Bioprocess Technol. 2012; 5:1824-1831. 3.Shahzadi T, Mehmood S, Irshad M, Anwar Z, Afroz A, Zeeshan N, Rashid U, Sughra K. Advances in lignocellulosic biotechnology: a brief review on lignocellulosic biomass and cellulases. ABB. 2014; 5:246-251.
Macedo EP, Cerqueira CLO, Souza DAJ, Bispo ASR, Coelho RRR, Nascimento RP. Production of cellulose-degrading enzyme on sisal and other agro-industrial residues using a new Brazilian Actinobacteriastrain Streptomyces sp. SLBA-08. Braz J Chem Eng. 2013; 30(04):729-735. 5.Acharya PB, Acharya DK, Modi HA. Optimization for cellulase production by Aspergillus niger using saw dust as substrate. Afr J Biotechnol. 2008; 7(22):4147-4152. 6.Kuhad RC, Gupta R, Singh A. Review article: Microbial cellulases and their industrial applications. Enzyme Res. 2011. 7.Sadhu S, Saha P, Sen S, Mayilraj S, Maiti T. Production, purification and characterization of a novel thermotolerant endoglucanase (CMCase) from Bacillusstrain isolated from cow dung. Singerplus.2013; 2(10).8.Jasani H, Umretiya N, Dharajiya D, Kapuria M, Shah S, Patel J. Isolation, optimization and production of cellulase by Aspergillus niger from agricultural waste. J Pure Appl Microbiol. 2016; 10(2):1159-1166. 9.Srivastava N, Srivastava M, Mishra PK, Singh P, Ramteke PW. Application of cellulases in biofuels industries: an overview. J Biofuel Bioenergy. 2015; 1(1):55-63. 10.Cohen R, Suzuki MR, Hammel KE. Processive endoglucanase active in crystalline cellulose hydrolysis by the brown rot basidiomycete Gloeophyllum trabeum. Appl Environ Microbiol.2005; 71(5):2412-2417.11.Li Y, Wilson DB. Chitin binding by Thermobifida fuscacellulase catalytic domains. Biotechnol Bioeng. 2008; 100(4):644-652. 12.Mejia-Castillo T, Hidalgo-Lara ME, Brieba LG, Ortega-Lopez J. Purification, characterization and modular organization of a cellulose-binding protein, CBP105, a processive beta-1,4-endoglucanase from Cellulomonas flavigena. Biotechnol Lett. 2008; 30(4):681-687.13.Parsiegla G, Reverbel C, Tardif C, Driguez H, Haser R. Structures of mutants of cellulase Cel48F of Clostridium cellulolyticum in complex with long hemi-thio-cello-oligosaccharides give rise to a new view of the substrate pathway during processive action. J Mol Biol. 2008; 375(2):499-510.14.Yoon JJ, Cha CJ, Kim YS, Kim W. Degradation of cellulose by the major endoglucanase produced from the brown-rot fungus Fomitopsis pinicola. Biotechnol Lett. 2008; 30(8):1373-1378.
Zverlov VV, Schantz N, Schwarz WH. A major new component in the cellulosome of Clostridium thermocellum is a processive endo-beta-1,4-glucanase producing cellotetraose. FEMS. Microbiol Lett. 2005; 249(2):353-358. 16.Soares I, Vora ZT, Barcelos RP, Baroni S. Microorganism produced enzymes in the food industry. In: Valdez B, editor. Scientific, Health and Social Aspects of the Food Industry. InTechOpen. 2012. 17.Moretti MMS, Martins DAB, Silva RD, Rodrigues A, Sette LD, Gomes E. Selection of thermophilic and thermotolerant fungi for the production of cellulases and xylanases under solid-state fermentation. Braz J Microbiol. 2012;1062-1071. 18.Bhargav S, Panda BP, Ali M, Javed S. Solid-state fermentation: An overview. Chem Biochem Eng. Q.2008;22(1):49-70.19.Sinnott M. Study of hemocellulytic enzymes from the fungi Phanerochaete chryosparium and Trichoderma reesei. J Bacteriol. 1997; 133:465–471. 20.Mahmood RT, Asad MJ, Mehboob N, Mushtaq M, Gulfraz M, Asgher M, Minhas NM, Hadri SH. Production, purification, and characterization of exoglucanase by Aspergillus fumigatus. Appl Biochem Biotechnol. 2013; 170(4):895-908.21.Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem. 1951; 193(1):265-275. 22.Mehboob N, Asad MJ, Asgher M, Gulfraz M, Mukhtar T, Mahmood RT. Exploring thermophilic cellulolytic enzyme production potential of Aspergillus fumigatus by the solid-state fermentation of wheat straw. Appl Biochem Biotechnol. 2014; 172:3646-3655. 23.Ahmed S, Bashir A, Saleem H, Saadia M, Jamil A. Production and purification of cellulose degrading enzymes from a filamentous fungus Trichoderma harzianum. Pak J Bot. 2009; 41(3):1411–1419. 24.Deshpande MV, Eriksson KE, Pettersson LG. An assay for selective determination of exo-1,4-β-glucanases in a mixture of cellulolytic enzymes. Analy Chem. 1984;138(2):481-487.25.Asadi N, Zilouei H. Optimization of organosolv pretreatment of rice straw for enhanced biohydrogen production using Enterobacter aerogenes. Bioresour Technol. 2017; 227:335-344. 26.Garrett RH, Grisham CM. Biochemistry. 4th ed. University of Virginia Brooks/Cole –Cengage Learning. 2010; 1059.
Speight JG. Industrial Inorganic Chemistry. Environmental Inorganic Chemistry for Engineers. Butterworth-Heinemann. 2017; 111-169. 28.Stylianopoulos CL. Carbohydrates: chemistry and classification. Encyclopedia of human nutrition. 3rd ed. Academic Press. 2017; 265-271. 29.Masood M, Mahmood RT, Asad MJ, Zia N, Safder A, Nasreen S, Ahmed D, Iqbal R. Production and characterization of endoglucanase from Phaeolus spadiceus. Pak J Biochem Biotechnol. 2021; 2(1):27-37.30.Youssef GA. Physiological studies of cellulase complex enzymes of Aspergillus oryzaeand characterization of carboxymethyl cellulase. Afr J Microbiol Res. 2011;5(11):1311-1321.31.Sherief AA, El-Tanash AB, Atia N. Cellulase production by Aspergillus fumigatusgrown on mixed substrate of rice straw and wheat bran. Res J Microbiol. 2010;5(3):199-211.32.Zia N, Mahmood RT, Asad MJ, Hadri SH, Javaid B, Masood M, Nasreen S, Safder A. Fermentation of sugarcane bagasse for the production and characterization of exoglucanase by Phaeolus spadiceus. Pak J Biochem Biotechnol. 2021; 2(1):38-48. 33.Abo-State MAM, Hammad AI, Swelim M, Gannam RB. Enhanced production of cellulase(s) by Aspergillus spp. isolated from agriculture wastes by solid state fermentation. American-Eurasian J Agri Environ Sci. 2010; 8(4):402-410.34.Shobana P, Maheshwari NU. Production of cellulase from Aspergillus fumigatus under submerged and solid state fermentation using agricultural waste. Int J Adv Pharma Biol Chem. 2013; 2(4).35.Farinas CS, Scarpelini LM, Miranda EA, Neto VB. Evaluation of operational parameters on the precipitation of endoglucanase and xylanase produced by solid state fermentation of Aspergillus niger. Braz J Chem Eng. 2011; 28(01):17-26.36.Bagewadi ZK, Ninnekar HZ. Production, purification and characterization of endoglucanase from Aspergillus fumigatus and enzymatic hydrolysis of lignocellulosic waste.Int J Biotech Biomed Sci. 2015; 1:25-32.
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