#halophilic.

Abstract

There is a burgeoning demand for amylase production due to wide range applications of amylase in different industrial processes like saccharification of starchy materials, food, detergents and textile industries. But high cost of fermentation media is one of the technical barriers in amylase production from microbial sources. Extremophiles microorganisms (thermophilic and halophilic) could be potential source for thermostable amylase. Present study deals with isolation of extremophilic amylase-producing bacteria from soil samples in starch agar medium and their subsequent identification through 16sRNA analysis. Different parameters like pH, temperature, and metal ions concentration (Ca+2, Mn+2, Fe+2, Zn+2) were optimized for amylase production. Five thermophilic strains and one halophilic strain were found positive for amylase production. Phylogenetic analysis showed that the amylase producing thermophilic strains includes Bacillus spp., Rheinheimera spp., Alishewanella spp., Pseudomonas spp., Microbacterium spp. while halophilic strain includes Bacillus spp. Thermophilic strains showed optimum amylase production at pH of 8 & 60°C, while maximum amylase activity for halophilic strains was observed at pH 7 and 40°C. Divalent ions Ca+2 and Mn+2 enhanced the amylase production while Zn and Fe did not a have any significant effect. Current research revealed that use of extremophile bacteria could be an important step towards the development of environmental friendly and cost effective process for thermostable amylase production.

Introduction

Starch is a main part of our diet and is being used in food as well as in industry as gelling agent, bulking agent, staining agent, thickener and colloidal stabilizer (Jaspreet et al., 2007). Soil inhabiting microorganisms are the sources of many enzymes including amylase. Amylase breaks down starch and glycogen into smaller molecules. It contributes about 25% in total sale of industrially used enzymes and has many potential industrial applications. It is also extensively used in treatment of digestive disorders (Burhan et al., 2003). Starch comprises of amylase and amylopectin. Alpha-amylase is able to cleave α1,4 glycosidic bonds present in the inner part of the amylose or amylopectin chain. Alpha-amylase belongs to a family of endo-amylases that catalyze the initial hydrolysis of starch into shorter oligosaccharides through the cleavage of α-D-(1-4) glyosidic bonds (Stamford et al., 2001; Whitcomb and Lowe, 2007).

Chemical technologies are becoming popular due to the generation of dangerous by-products that contaminate our environment and require high cost for energy input at large scale manufacturing (Gomes et al., 2003). Microorganisms using their diverse enzyme system efficiently perform their metabolic processes with higher specificity under ambient conditions. Due to the high catalytic efficiency and more specificity, enzymes produced from microorganisms are good alternative to harsh chemical technologies and therefore the research in the field led to explore microbial diversity to discover enzymes for pollution free “dream technology” in the future (Gomes et al., 2003;Prakash and Jaiswal, 2009).

Extremophile microorganisms are structurally adapted at the molecular level to withstand harsh conditions (Stamford et al., 2001) and are capable of producing different thermostable enzymes. Such organism have been isolated and characterized from various environments such as soil, spring, food material and wastes containing carbohydrate in different studies (Gupta et al., 2003). They have

modified themselves to flourish in extreme environments. These properties make them to survive in harsh conditions and in addition, help them to grow in bio-industrial processes designed basically on the optimal conditions of these biomolecules (Kamekura, 2011). Moreover, thermostable amylases produced from these microorganism enzymes have been currently investigated to improve industrial processes of starch degradation and are of great interest for the production of valuable products like glucose, crystalline dextrose, dextrose syrup, maltose and maltodextrins (Prakash and Jaiswal, 2009).

The main objective of the study was to isolate and identify amylase producing bacteria from Halophilic sites, their partial characterization for enzyme production and properties in regard to the effect of temperature, metal ion-dependency and pH.