Molecular and biochemical characterization of an endochitinase (ChiA-HD73) from Bacillus thuringiensis subsp. kurstaki HD-73.
An endochitinase gene (chiA-HD73) from the insecticidal bacterium Bacillus thuringiensis subsp. kurstaki HD-73 was cloned, sequenced, and expressed in Escherichia coli DH5alphaF'. The chitinase activity of the encoded protein was studied in assays with different fluorogenic substrates. The chiA-HD73 gene contained an open-reading frame that encoded an endochitinase with a deduced molecular weight and an isoelectric point of, respectively, 74.5 kDa and 5.75. A putative signal peptide with cleavage sites for both Gram-positive and Gram-negative bacteria was identified. Comparison of ChiA-HD73 with other chitinases revealed a modular structure composed of a catalytic domain and a putative chitin-binding domain. ChiA-HD73 hydrolyzed both tetrameric and trimeric fluorogenic substrates, but not a chitobiose analog substrate, suggesting that the activity of ChiA-HD73 is mainly endochitinolytic. In addition, ChiA-HD73 showed high enzymatic activity within a broad pH range (pH 4-10), with a peak activity at pH 6.5. The optimal temperature for enzymatic activity was observed at 55 degrees C. Its activity in a broad range of temperatures and pH suggests ChiA-HD73 could have biotechnological applications in insect control, particularly in synergizing the insecticidal crystal protein toxins of B. thuringiensis.
Barboza-Corona
J Eleazar
JE
Instituto de Ciencias Agrícolas, Departamento de Ingeniería en Alimentos, Universidad de Guanajuato, Apartado postal 311, Irapuato, Guanajuato, 36500, Mexico. josebar@dulcinea.ugto.mx
Reyes-Rios
Dora M
DM
Salcedo-Hernández
Rubén
R
Bideshi
Dennis K
DK
D000595:Amino Acid Sequence; D001413:Bacillus thuringiensis / Q000201:enzymology*; D020134:Catalytic Domain; D002688:Chitinases / Q000737:chemistry / Q000378:metabolism*; D003001:Cloning, Molecular; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D014170:Transformation, Genetic
D011994:Recombinant Proteins; D002688:Chitinases