General Information
Author: Nguyen Huu Hoang, Amit Kumar Chaudhary, Nguyen Thi Lan Huong, Anil Shrestha, Jae Kyung Sohng, Je Won ParkIssued date: 31/12/2013
Issued by: J. Biomolecule Reconstruction 2013, Vol. 10, No. 1, 36-45
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Abstract
The kanamycin biosynthetic gene cluster in Streptomyces kanamyceticus ATTC 12853 contains a gene encoding for the second glycosyltransferase KanM2 (also known as KanE) which acts as both glucosyltransferase and kanosamyltransferase required for the formation of pseudotrisaccharides. However, a detailed biochemical analysis of KanM2 including substrate specific remain unclear, leading to requirement of purified enzyme for detailed biochemical studies. In this work, we have tried to heterologously express KanM2 protein in E. coli BL21 (DE3) at low temperature and characterized by in vitro reaction using crude enzyme. Nevertheless, to further improve the expression of KanM2 in soluble form, D-sorbitol and Betain was added in LB medium (LBSS media). Furthermore, combined use of molecular chaperones and cultivation at low temperature in LBSS media significantly improved the solubility of protein in E. coli BL21 (DE3).
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