Identification and expression of GH-8 family chitosanases from several Bacillus thuringiensis subspecies.
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 1,359 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.
Lee
Han-Seung
HS
Jang
Jun Sung
JS
Choi
Soo-Keun
SK
Lee
Dong-Woo
DW
Kim
Eui-Joong
EJ
Jung
Heung-Chae
HC
Pan
Jae-Gu
JG
D000595:Amino Acid Sequence; D001413:Bacillus thuringiensis / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D048271:Chitosan / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D050505:Mutant Proteins / Q000378:metabolism; D016366:Open Reading Frames; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D050505:Mutant Proteins; D011994:Recombinant Proteins; D048271:Chitosan; D006026:Glycoside Hydrolases; C019868:chitosanase