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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

Authors 

H S LeeJ S JangSoo Keun ChoiDong-Woo LeeE J KimHeung Chae JungJae Gu Pan

Publisher 

Wiley-Blackwell

Issue Date 

2007

Citation 

FEMS Microbiology Letters, vol. 277, no. 2, pp. 133-141

Keywords 

bacillus thuringiensischitosan-oligosaccharideschitosanaseGH-8high-throughput screeningbacterium identificationhigh performance liquid chromatographyhigh throughput screeningprotein expression

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.

ISSN 

0378-1097

Link 

http://dx.doi.org/10.1111/j.1574-6968.2007.00944.x

Appears in Collections

1. Journal Articles > Journal Articles

Registered Date

2019-05-02


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