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

• Published in: FEMS microbiology letters Vol. 277; no. 2; pp. 133 - 141
• Main Authors: Lee, Han-Seung, Jang, Jun Sung, Choi, Soo-Keun, Lee, Dong-Woo, Kim, Eui-Joong, Jung, Heung-Chae, Pan, Jae-Gu
• Format: Journal Article
• Language: English
• Published: Oxford, UK Oxford, UK : Blackwell Publishing Ltd 01.12.2007; Blackwell Publishing Ltd; Blackwell; Oxford University Press
• Subjects: Amino Acid Sequence; Bacillus; Bacillus cereus; Bacillus thuringiensis; Bacillus thuringiensis - enzymology; Bacillus thuringiensis - genetics; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Bacteriology; Biological and medical sciences; Chitosan; Chitosan - metabolism; chitosan-oligosaccharides; Chitosanase; Chromatography, High Pressure Liquid; Cloning, Molecular; DNA, Bacterial - chemistry; DNA, Bacterial - genetics; E coli; Enzymatic activity; Enzyme activity; Enzymes; Escherichia coli; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Gene Expression; Genetics; GH-8; Glycoside hydrolase; Glycoside Hydrolases - genetics; Glycoside Hydrolases - metabolism; High performance liquid chromatography; High-throughput screening; Hydrolase; Liquid chromatography; Metabolism. Enzymes; Microbiology; Microorganisms; Molecular Sequence Data; Mutagenesis; Mutant Proteins - metabolism; Nucleotide sequence; Nucleotides; Oligosaccharides; Open Reading Frames; Phylogeny; Polymerase Chain Reaction; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Screening; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Thin layer chromatography; Trimers; chitosan-oligosaccharides; GH-8; chitosanase; high-throughput screening; High throughput screening; Enzyme; Phylogenetic tree; Bacillus thuringiensis; Sequence alignment; Identification; high- throughput screening; Bacillaceae; Glycosylases; Bacillales; Enzymatic activity; Glycosidases; Chitosanase; Bacteria; Hydrolases; Chitosan; Molecular cloning; chitosan- oligosaccharides
• ISSN: 0378-1097; 1574-6968
• DOI: 10.1111/j.1574-6968.2007.00944.x

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.