Cloning, sequencing, and expression of a chitinase-encoding gene from Bacillus circulans No. 4.1.
A chitinase encoding gene from Bacillus circulans No. 4.1 was cloned in Escherichia coli by using pBluescript II SK. The recombinant plasmid containing the 2.6-kb chitinase gene was designated as pCHIB43. The nucleotide sequence revealed a single open reading frame containing 1794 bp and encoding 598 amino acids with a molecular mass of 65.78 kDa. The gene was sequentially deleted; the deletion clones were designated as pC66, pC6S, pSS6, and pEVS. The clones pC6S, pSS6, and pEVS hydrolyzed soluble chitin, but the ability to hydrolyze colloidal chitin was lost. The deduced amino acid sequence was investigated and found to be a chitin-binding domain and a catalytic domain containing 40 and 57 amino acid residues, respectively. A HindIII-SacI fragment of pSS6 was subcloned into pBluescript SK to reverse the orientation of the gene, and the resulting plasmid pSK43 did produce chitinase. Thus, the cloned gene was expressed under the control of a self-promoter.
Wiwat
Chanpen
C
Department of Microbiology, Faculty of Pharmacy, Mahidol University, 447 Sri-Ayudhya Rd., Bangkok 10400, Thailand. pycww@mahidol.ac.th
Thepouyporn
Apanchanid
A
Siwayaprahm
Patcharaporn
P
Bhumiratana
Amaret
A
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D002688:Chitinases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid
D002688:Chitinases