Cloning, DNA sequence, and expression of Aeromonas caviae WS7b chitinase gene.
A chitinase-producing bacterium, designated WS7b, was isolated from a soil sample obtained from a black-pepper plantation on Bangka Island, Indonesia. Fatty-acid methyl-ester analysis indicated that the isolate was Aeromonas caviae. A chitinase gene from WS7b was cloned in a pUC19-based plasmid vector, but without its natural promoter. The complete nucleotide sequence of the gene was determined, and the structural gene consisted of a 2748-bp region encoding 864 amino acids. DNA sequence analysis indicated that the gene had been cloned without its promoter, and this was confirmed by chitinase-plate assay of the truncated version of the gene in Escherichia coli. The chitinase gene product showed amino-acid sequence similarity to chiA from A. caviae. Chitinase enzyme activity was determined spectrophotometrically, using colloidal chitin azure as substrate for extracellular and intracellular fractions. The ability of the chitinase cloned in E. coli to hydrolyze chitin was less than that of the enzyme in its indigenous host.
Malik
Amarila
A
South East Asian Regional Center for Tropical Biology (SEAMEO BIOTROP), Jl. Raya Tajur Km 6, Bogor, Indonesia.
Wenuganen
S
S
Suwanto
Antonius
A
Tjahjono
Budi
B
D000333:Aeromonas / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002478:Cells, Cultured; D002688:Chitinases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D003001:Cloning, Molecular; D004789:Enzyme Activation; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D008969:Molecular Sequence Data; D029222:Piper nigrum / Q000382:microbiology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein; D012988:Soil Microbiology; D013045:Species Specificity; D014170:Transformation, Genetic
D011994:Recombinant Proteins; D002688:Chitinases