Cloning, sequencing, and expression of the chitinase gene chiA74 from Bacillus thuringiensis

Appl Environ Microbiol. 2003 Feb;69(2):1023-9. doi: 10.1128/AEM.69.2.1023-1029.2003.

Abstract

The endochitinase gene chiA74 from Bacillus thuringiensis serovar kenyae strain LBIT-82 was cloned in Escherichia coli DH5 alpha F'. A sequence of 676 amino acids was deduced when the gene was completely sequenced. A molecular mass of 74 kDa was estimated for the preprotein, which includes a putative 4-kDa signal sequence located at the N terminus. The deduced amino acid sequence showed high degree of identity with other chitinases such as ChiB from Bacillus cereus (98%) and ChiA71 from Bacillus thuringiensis serovar pakistani (70%). Additionally, ChiA74 showed a modular structure comprised of three domains: a catalytic domain, a fibronectin-like domain, and a chitin-binding domain. All three domains showed conserved sequences when compared to other bacterial chitinase sequences. A ca. 70-kDa mature protein expressed by the cloned gene was detected in zymograms, comigrating with a chitinase produced by the LBIT-82 wild-type strain. ChiA74 is active within a wide pH range (4 to 9), although a bimodal activity was shown at pH 4.79 and 6.34. The optimal temperature was estimated at 57.2 degrees C when tested at pH 6. The potential use of ChiA74 as a synergistic agent, along with the B. thuringiensis insecticidal Cry proteins, is discussed.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Bacillus thuringiensis / enzymology*
  • Bacillus thuringiensis / genetics
  • Chitinases / chemistry
  • Chitinases / genetics*
  • Chitinases / metabolism*
  • Cloning, Molecular*
  • DNA, Bacterial / analysis
  • DNA, Bacterial / genetics
  • Escherichia coli / enzymology
  • Escherichia coli / genetics
  • Molecular Sequence Data
  • Sequence Alignment
  • Sequence Analysis, DNA

Substances

  • DNA, Bacterial
  • Chitinases

Associated data

  • GENBANK/AF424979