JACKFRUIT SEED AS A NOVEL SUBSTRATE FOR THE PRODUCTION OF AN ACIDOPHILIC AND ACID-STABLE α-AMYLASE FROM BACILLUS SP.4
Keywords:alpha-Amylase, Acidophilic, Jackfruit seed, Agro-industrial residue, Bacillus sp.4
Objective: The objective of the current study is to do a comparative analysis of the ability of a strain of Bacillus to grow and produce α-amylase on various agro-residues under solid state fermentation (SSF), as amylases comprise one of the most important enzymes in industries.
Methods: Bacteria were isolated from various soil samples by serial dilution method, screened for amylase production by rapid screening method on starch agar plates and the best amylase producer was chosen. The best isolate was cultured on different agro-residues such as wheat bran, watermelon outer rind, Avarekai seed coat (Dolichos lablab), coconut endosperm, and jackfruit seeds for maximum amylase production. The pH and temperature optima of the enzyme were determined by culturing the bacteria under different pH and temperatures. The crude enzyme was purified by ammonium sulfate precipitation followed by ion-exchange chromatography methods.
Results: The best isolate chosen was Bacillus sp.4, which produced an acidophilic and acid-stable α-amylase with maximum enzyme production at the acidic pH of 5.5 and 6.5 (21.11 and 21.62 U/mg protein, respectively) and maximum stability at pH 5.5. Jackfruit seed was found to be the most suitable agro waste for α-amylase production by our isolate. Purification of the enzyme by ammonium sulfate precipitation followed by ion-exchange chromatography resulted in 23.17-fold increase in its activity (86.67 U/mg protein).
Conclusion: Considering its acid-stable and highly promising enzyme activities, the enzyme from this bacterial isolate can be further characterized for future applications in starch and other food industries.
Bobbio FO, El-Dash AA, Bobbio PA, Rodrigues LR. Isolation and characterization of the physicochemical properties of the starch of jackfruit seeds (Artocarpus heterorphyllus). Cereal Chem 1978;55:505-11.
Kumar S, Singh AB, Abidi AB, Upadhyay RG, Singh A. Proximate composition of jack fruit seeds. J Food Sci Technol 1988;25:308-9.
Krishana C, Chandrasekaran M. Banana waste as substrate for a amylase production by Bacillus sublitis (CBTK 106) under solid state fermentation. Appl Microbiol Biotechnol 1996;46:106-11.
Miller GL. Use of dinitro salicylic acid reagent for determination of reducing sugar. Anal Chem 1959;31:426-9.
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin-Phenol reagents. J Biol Chem 1951;48:17-25.
Gomori G. Preparation of buffer for use in enzyme active studies. In: Colwick SP, Kaplan NO, editors. Methods in Enzymology. Vol. 1. New York: Academic Press. Inc.; 1955.
Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680-5.
Shukla J, Kar R. Potato peel as a solid state substrate for thermostable α-amylase production by thermophilic Bacillus isolates. World J Microbiol Biotechnol 2006;22:417-22.
Mukherjee AK, Borah M, Rai SK. To study the influence of different components of fermentable substrates on induction of extracellular α-amylase synthesis by Bacillus subtilis DM03 in solid-state fermentation and exploration of feasibility for inclusion of α-amylase in laundry detergent formulations. Biochem Eng J 2009;43:149-56.
Kokab S, Asghar M, Rehman K, Asad MJ, Adedyo O. Bio-processing of banana peel for amylase production by Bacillus subtilis. Int J Agric Biol 2003;5:36-9.
Tanyildizi MS, Ozer D, Elibol M. Optimization of α-amylase production by Bacillus sp. using response surface methodology. Process Biochem 2005;40:2291-6.
Syu MJ, Chen YH. A study on the-amylase fermentation performed by Bacillus amyloliquefaciens. Chem Eng J 1997;65:237-47.
Haq I, Ashraf H, Qadeer MA, Iqbal J. Pearl millet, a source of alpha amylase production by Bacillus licheniformis. Bioresour Technol 2005;96:1201-4.
Mishra S, Noronha SB, Suraishkumar GK. Increase in enzyme productivity by induced oxidative stress in Bacillus subtilis cultures and analysis of its mechanism using microarray data. Process Biochem 2005;40:1863-70.
Mendu DR, Ratnam BVV, Purnima A, Ayyanna C. Affinity chromatography of α-amylase from Bacillus licheniformis. Enzyme Microb Technol 2005;37:712-7.
De Silva SN. Purification and Characterization of Raw Starch Hydrolysing α-Amylase from Aspergillus spp. M.Sc Thesis. Colombo: Faculty of Medicine, University of Colombo; 1997.
Mohammed MA, Neelgund S, Gurumurthy DM, Rajeshwara AN. Identification, characterization of novel halophilic Bacillus cereus Ms6: A source for extra cellular α-amylase. Adv Environ Biol 2011;5:992-9.
Afifi AF, Kamel EM, Khalil AA, Foaad MA, Fawziand EM, Houseny M. Purification and characterization of α-amylase from Penicillium olsonii under the effect of some antioxidant vitamins. Glob J Biotechnol Biochem 2008;3:14-2.
Alva S, Anumpama J, Savla J, Chiu YY, Vyshali P, Shruti M, et al. Production and characterization of fungal amylase enzyme isolated from Aspergillus sp. JGI 12 in solid state culture. Afr J Biotechnol 2007;6:576-81.
Varalakshmi KN, Kumudini BS, Nandini BN, John S, Suhas R, Mahesh B, et al. Production and characterization of Alpha Amylase enzyme from Aspergillus niger JGI 24 isolated from Bangalore. Pol J Microbiol 2009;58:29-36.
Rengsutthi K, Charoenrein S. Physico-chemical properties of jackfruit seed starch (Artocarpus heterophyllus) and its application as a thickener and stabilizer in chilli sauce. LWT Food Sci Technol 2010;44:1309-13.
Mulimani VH, Patil RG. α-Amylase production by solid state fermentation: A new practical approach to biotechnology courses. Biochem Educ 2000;28:161-3.
Haq I, Ashraf H, Iqbal J, Qadeer MA. Production of alpha amylase by Bacillus licheniformis using an economical medium. Bioresour Technol 2003;87:57-61.
Gangadharan D, Swetha S, Nampoothiri KM, Pandey A. Solid culturing of Bacillus amyloliquefaciens for alpha amylase production. Food Technol Biotechnol 2006;44:269-74.
Soni SK, Kaur A, Gupta JK. A solid state fermentation based bacterial α-amylase and gluco amylase system and its suitability for the hydrolysis of wheat starch. Process Biochem 2003;39:185-92.
Ramesh MV, Lonsane BK. Ability of a solid-state fermentation technique to significantly minimize catabolic repression of α-amylase production by Bacillus licheniformis M27. Appl Microbiol Biotechnol 1991;35:591-3.
Sodhi HK, Sharma K, Gupta JK, Soni SK. Production of a thermostable α-amylase from Bacillus sp. PS-7 by solid state fermentation and its synergistic use in the hydrolysis of malt starch for alcohol production. Process Biochem 2005;40:525-34.