International Research Journal of Engineering and Technology (IRJET)
e-ISSN: 2395-0056
Volume: 04 Issue: 07 | July -2017
p-ISSN: 2395-0072
www.irjet.net
ISOLATION, OPTIMIZATION, PRODUCTION AND PURIFICATION OF ALPHA AMYLASE FROM SOIL BACTERIA Ram Kumar T1, Ramkumar J2, Karthikeyan S3, Ramesh Babu N G4, Manivasagan V5 1,2,3,4,5Department
of Biotechnology, Adhiyamaan College of Engineering (Autonomous)-Hosur 635109 ---------------------------------------------------------------------***---------------------------------------------------------------------
Abstract - Amylase is a hydrolytic enzyme that is used
widely in industries. It is easier to produce enzymes from bacteria than any other organism. The present work focused on the production of amylase enzyme from bacteria isolated from soil samples. The amylase producing bacterial strain screening was performed using starch agar plates. Various biochemical tests were carried out to confirm the strain and was identified as B.subtilis. Fermentation conditions such as temperature and PH were optimized for maximum enzyme production from B.subtilis. The optimum PH was found to be 7.0 and the temperature was found to be 37° C. The enzyme was partially purified by ammonium sulfate precipitation method and it was purified by dialysis. The SDS-PAGE was also performed to determine the molecular weight of amylase enzyme produced. Key Words: Amylase, Bacillus subtilis, ammonium sulfate, dialysis, SDS-PAGE
1. INTRODUCTION α-Amylase is a protein enzyme E(.3.2.1.1) that hydrolyses alpha bonds of large, polysaccharides, such as starch and glycogen , yielding glucose and maltose[1]. They can specifically cleave the O-glycosidic bonds in starch. Starch depolymerization by amylases is the basic mechanism for used in the preparation of glucose syrups, Bread making and brewing. Thus it is a key enzyme in the production of starch derivatives and is also used in desizing fabrics, in pharmaceuticals and detergents. Submerged fermentation (SmF) has been traditionally used for the production of industrially important enzymes because of the ease of handling and greater control of environmental factors such as temperature and pH[2]. Solid-state fermentation dominates over submerged fermentation in aspects such as better yield, simple technique, low capital investment, lower levels of catabolite repression, high stability and better product recovery [3]. Agro-residues are generally considered as the best substrate for the solid-state fermentation processes[4]. Many agro-industrial by-products such as wheat bran, rice bran, molasses, barley bran, maize meal, soybean meal, potato peel and coconut oil cake have been screened as low cost solid substrates for microbial production of αamylase in solid-state fermentation[5]. The major factors that © 2017, IRJET
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affect microbial synthesis of enzymes in a solid-state fermentation system include the selection of a suitable substrate, microorganism, inoculum concentration, particle size and moisture level of the substrate. There are different sources from which the enzymes can be produced. Plants, animals and microbial sources can produce amylases[6]. Microorganisms produce different kinds of industrial enzymes. Because of their biochemical diversity and the case with environment and genetic manipulation, they have replaced enzymes, which traditionally have been isolated from complex eukaryotes[7]. Microorganisms utilize various substrates as a nutrient source for their growth and metabolic activities and subsequently produce metabolism related products. However, fine tuning of nutrient concentrations regulate the microbial metabolism and associated metabolic product formation. Balancing of nutrient concentration with minimum experimentation and other cultural parameters is an art in microbial metabolism to optimize enzyme production[8]. Enzyme production is dependent on a number of factors that include strain type, culture medium composition and condition of fermentation, availability of carbon, nitrogen, mineral salts etc., The growth and enzyme production of the organisms are strongly influenced by medium composition. Thus, optimization of media components and cultural parameters is the primary task in a biological process [9]. The main strategy used is media engineering for which the optimal operating condition of a parameter is standardized by changing one parameter at a time and keeping the others at a constant level[10]. The optimization studies do not consider the interaction effects among the variables as any process is influenced by several variables .The present study focused on the isolation, optimization, production and purification of α-amylase from soil bacteria.
2. MATERIALS AND METHODS 2.1. Isolation of microorganisms The soil samples were collected from Jai Nagar Park, Arumbakkam, Chennai, Tamil Nadu. The soil samples were subjected to serial dilutions and 10-2 and 10-3 dilutions were swabbed on agar plates. They were incubated at 37° C for 48 hours. ISO 9001:2008 Certified Journal
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