Identification and expression of GH-8 family chitosanases from several Bacillus thuringiensis subspecies

Han Seung Lee, Jun Sung Jang, Soo Keun Choi, Dong Woo Lee, Eui Joong Kim, Heung Chae Jung, Jae Gu Pan

Research output: Contribution to journalArticle

17 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)133-141
Number of pages9
JournalFEMS Microbiology Letters
Volume277
Issue number2
DOIs
Publication statusPublished - 2007 Dec

All Science Journal Classification (ASJC) codes

  • Microbiology
  • Molecular Biology
  • Genetics

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