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Applied and Environmental Microbiology, February 2003, p. 1023-1029, Vol. 69, No. 2
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.2.1023-1029.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Cloning, Sequencing, and Expression of the Chitinase Gene chiA74 from Bacillus thuringiensis

J. Eleazar Barboza-Corona,1* Elizabeth Nieto-Mazzocco,1 Rocio Velázquez-Robledo,1 Rubén Salcedo-Hernandez,1 Mayela Bautista,1 Beatriz Jiménez,2 and Jorge E. Ibarra3

Instituto de Ciencias Agrícolas, Universidad de Guanajuato,1 Departamento de Ingeniería Genética ,2 Departamento de Biotecnología y Bioquímica, Centro de Investigación y de Estudios Avanzados del IPN, 36500 Irapuato, Guanajuato, México3

Received 9 August 2002/ Accepted 4 November 2002

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°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.

* Corresponding author. Mailing address: Instituto de Ciencias Agrícolas, Universidad de Guanajuato, Apartado Postal 311, 36500 Irapuato, Gto., México. Phone and fax: 52-462-624-2484. E-mail: josebar{at}dulcinea.ugto.mx.

Applied and Environmental Microbiology, February 2003, p. 1023-1029, Vol. 69, No. 2
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.2.1023-1029.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.





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