Functional Expression and Refolding of Recombinant Chitinase, Chi21702 from Antarctic Bacterium Sanguibater sp. KCTC 10714

Abstract

A psychrotrophic Sanguibater sp. strain KCTC 10714 has been previously isolated from sea sand on King Sejong Station in Antarctica. An endochitinase gene, designated chi21702, from KCTC 10714 was PCR- amplified, cloned into pEXP5-CT/TOPO vector, and overexpressed in Eschrichia coli BL21(DE3). The production of recombinant chitinase (Chi21702) was detected in the heterologous expression system, the size of which was determined to be approximately 56 kDa. However, most of Chi21702 was converted to inclusion body which lost the chitinase activity against chitin. In next step, refolding experiment for the inert chitinase was carried out using a various refolding methods. As a result, the refolding conditions, such as temperature (4 ℃) and buffer components (EPPS, a mixture of reduced and oxidized gluthationes

pH8.0), for the chitinase in inclusion body were determined. The refolded Chi21702 restored its chitinase activity (16.5 U/mg/hr), which was almost same as that of the original form in KCTC 10714.