Identification and expression of GH-8 family chitosanases from several Bacillus thuringiensis subspecies = Bt 균주로부터 GH-8 키토산아제의 분리 및 발현

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Title
Identification and expression of GH-8 family chitosanases from several Bacillus thuringiensis subspecies = Bt 균주로부터 GH-8 키토산아제의 분리 및 발현
Author(s)
H S Lee; J S Jang; Soo Keun Choi; Dong-Woo Lee; E J Kim; Heung Chae Jung; Jae Gu Pan
Bibliographic Citation
FEMS Microbiology Letters, vol. 277, no. 2, pp. 133-141
Publication Year
2007
Abstract
The Gram-positive spore-forming bacterium, Bacillus thuringiensis, a member of the Bacillus cereus group, produces chitosanases that catalyze the hydrolysis of chitosan to chitosan-oligosaccharides (COS). Although fungal and bacterial chitosanases belonging to other glycoside hydrolase (GH) families have been characterized in a variety of microorganisms, knowledge on the genetics and phylogeny of the GH-8 chitosanases remains limited. Nine genes encoding chitosanases were cloned from 29 different serovar strains of B. thuringiensis and they were expressed in Escherichia coli. The ORFs of the chitosanases contained 1359 nucleotides and the protein products had high levels of sequence identity (>96%) to other Bacillus species GH-8 chitosanases. Thin-layer chromatography and HPLC analyses demonstrated that these enzymes hydrolyzed chitosan to a chitosan-trimer and a chitosan-tetramer as major products, and this could be useful in the production of COS. In addition, a simple plate assay was developed, involving a soluble chitosan, for high-throughput screening of chitosanases. This system allowed screening for mutant enzymes with higher enzyme activity generated by error-prone PCR, indicating that it can be used for directed chitosanase evolution.
Keyword
Bacillus thuringiensisChitosan-oligosaccharidesChitosanaseGH-8High-throughput screening
ISSN
0378-1097
Publisher
Oxford Univ Press
DOI
http://dx.doi.org/10.1111/j.1574-6968.2007.00944.x
Type
Article
Appears in Collections:
Division of Research on National Challenges > Infectious Disease Research Center > 1. Journal Articles
Division of Bio Technology Innovation > SME Support Center > 1. Journal Articles
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