Production, partial purification and characterization of amylase from sphingomonas sp. isolated from artic soil Universiti Teknologi Malaysia

Abstract

Amylase is an important enzyme that is responsible for hydrolysis of starch based materials into simple sugars. To date, extensive researches had been done on the characteristics of amylase produced by microorganisms isolated from different sources. However, most of the reported amylases do not have a wide range of thermostability. In this project, a total of 14 bacteria isolated from artic regions were screened for their capability of amylase production. The Sphingomonas sp. was found to be the best candidate for amylase production among the rest, it had the lowest Hc value (0.44). Production of amylase was subsequently quantified in submerged liquid fermentation (SmF). The Sphingomonas sp. showed highest amylase activity of 0.23 U at 18th hour of incubation in amylase production medium, pH 7, containing 0.1% (w/v) of soluble starch. Optimization of amylase production was conducted using One-Factor-at-a-Time (OFAT) method. Medium with pH 9 containing 0.87% (w/v) autoclaved baker’s yeast cells, and 3% (w/v) soluble starch was found to have increased the amylase activity by 27.6 fold (6.31U). For partial purification of amylase, three methods: ammonium sulphate precipitation, 60% (w/v), centrifugal concentrator and the combination of both were used. The highest specific activity was obtained in ammonium sulphate precipitation, 60% (w/v) which increased the specific activity by 2.5 fold (23.43 U/mg) as compared to the activity of crude enzyme (9.41 U/mg). Characterization of amylase further revealed that the enzyme was stable at temperature ranging from 4º C to 95°C (72.3% relative activity remained) with the optimum temperature of 20°C. It was most stable at pH8 with optimum activity at pH7. The SDS-PAGE and zymogram analysis revealed that the amylase having molecular weight between 50 to 60 kDa.