Cloning, sequencing, and expression of the chitinase gene chiA74 from Bacillus thuringiensis.
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.
Barboza-Corona
J Eleazar
JE
Instituto de Ciencias Agrícolas, Universidad de Guanajuato, 36500 Irapuato, Guanajuato, México. josebar@dulcinea.ugto.mx
Nieto-Mazzocco
Elizabeth
E
Velázquez-Robledo
Rocio
R
Salcedo-Hernandez
Rubén
R
Bautista
Mayela
M
Jiménez
Beatriz
B
Ibarra
Jorge E
JE
D000595:Amino Acid Sequence; D001413:Bacillus thuringiensis / Q000201:enzymology* / Q000235:genetics; D002688:Chitinases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
D004269:DNA, Bacterial; D002688:Chitinases