Optimization for high level expression of cold and pH tolerant amylase in a newly isolated Pedobacter sp. through Response Surface Methodology

Abstract

Amylase is one of the most widely used enzymes in the industry. Cold environments are the most ubiquitous environments in the world that have been occupied by cold tolerant microorganisms. The enzymes of these microorganisms have a wide range of applications in various areas of biotechnology. The aim of this study was to isolate cold-active amylase producing bacteria. A total of 64 cold-tolerant bacteria producing amylase were isolated from Binaloud Mountain soil, Iran. An isolate (<em>Pedobacter</em> sp. BTR84) registered under accession number KM459538 with the highest enzyme productivity was selected for production optimization. The production of amylase was evaluated via One-factor-at-a-timemethod and RSM (Response Surface Methodology). The enzyme production was optimized at 20°C, pH 9, starch 2% (w/v), and inoculation level 3% (v/v) by One-factor-at-a-timemethod. Then, in order to investigate the interaction between these variables and determine the final optimal conditions, optimization was carried out through the response surface methodology (RSM). Four variables were evaluated at three levels using the Box-Behnken design. Starch concentration and inoculation level variables had a significant effect on amylase production (<em>p</em><0.05). The final optimum conditions for amylase production in the isolates were temperature 25°C, pH 7, starch concentration 2.5% (w/v), and inoculation level 3% (v/v). The current study suggests that Psychrotrophic local bacteria are capable of producing extracellular hydrolytic enzymes that have a good potential to be applied in biotechnological industries.