A constitutively expressed 36 kDa exochitinase from Bacillus thuringiensis HD-1.
A 36 kDa chitinase was purified by ion exchange and gel filtration chromatography from the culture supernatant of Bacillus thuringiensis HD-1. The chitinase production was independent of the presence of chitin in the growth medium and was produced even in the presence of glucose. The purified chitinase was active at acidic pH, had an optimal activity at pH 6.5, and showed maximum activity at 65 degrees C. Of the various substrates, the enzyme catalyzed the hydrolysis of the disaccharide 4-MU(GlnAc)(2) most efficiently and was therefore classified as an exochitinase. The sequence of the tryptic peptides showed extensive homology with Bacillus cereus 36 kDa exochitinase. The 1083 bp open reading frame encoding 36 kDa chitinase was amplified with primers based on the gene sequence of B. cereus 36 kDa exochitinase. The deduced amino-acid sequence showed that the protein contained an N-terminal signal peptide and consisted of a single catalytic domain. The two conserved signature sequences characteristic of family 18 chitinases were mapped at positions 105-109 and 138-145 of Chi36. The recombinant chitinase was expressed in a catalytically active form in Escherichia coli in the vector pQE-32. The expressed 36 kDa chitinase potentiated the insecticidal effect of the vegetative insecticidal protein (Vip) when used against neonate larvae of Spodoptera litura.
Arora
Naresh
N
International Center for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, PO Box 10504, New Delhi 1100 67, India.
Ahmad
Tarannum
T
Rajagopal
R
R
Bhatnagar
Raj K
RK
D000595:Amino Acid Sequence; D000818:Animals; D001413:Bacillus thuringiensis / Q000201:enzymology*; D002688:Chitinases / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D003001:Cloning, Molecular; D004357:Drug Synergism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D007306:Insecticides / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D018411:Spodoptera; D013379:Substrate Specificity
D007306:Insecticides; D002688:Chitinases