Molecular and Biochemical Characterization of an Endochitinase (ChiA-HD73) from Bacillus thuringiensis subsp. kurstaki HD-73 |
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Authors: | J Eleazar Barboza-Corona Dora M Reyes-Rios Rubén Salcedo-Hernández Dennis K Bideshi |
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Institution: | (1) Instituto de Ciencias Agrícolas, Departamento de Ingeniería en Alimentos, Universidad de Guanajuato, Apartado postal 311, Irapuato, Guanajuato, 36500, Mexico;(2) Colegio de Estudios Científicos y Tecnológicos del Estado de Guanajuato, Plantel Irapuato Valle Verde, Irapuato, Guanajuato, 36568, Mexico;(3) Department of Natural and Mathematical Sciences, California Baptist University, 8432 Magnolia Avenue, Riverside, CA 92504, USA;(4) Department of Entomology, University of California, Riverside, CA 92521, USA |
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Abstract: | An endochitinase gene (chiA-HD73) from the insecticidal bacterium Bacillus thuringiensis subsp. kurstaki HD-73 was cloned, sequenced, and expressed in Escherichia coli DH5αF′. 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°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. |
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Keywords: | Escherichia coli Bacillus thuringiensis HD-73 Chitinases Chitooligosaccharides |
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