High-level synthesis of endochitinase ChiA74 in Escherichia coli K12 and its promising potential for use in biotechnology
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In the present study, we expressed the chiA74 gene of Bacillus thuringiensis in Escherichia coli K12 and demonstrated that the active ChiA74 enzyme was produced at a high level in this strain. The ChiA74 enzymatic activity (in units per milliliter) was approximately 500 % greater in E. coli K12 when compared to that produced in E. coli DH5α. Moreover, we showed that, when using our protocol, ChiA74 preparations obtained from recombinant E. coli K12 did not contain live bacteria, although transformable DNA (erm, bla genes) was detected. Nucleic acids were subsequently easily eliminated when samples were treated with magnesium. Importantly, ChiA74 was secreted by E. coli K12 and the active enzyme was shown to generate chitin-derived oligosaccharides (C-OGS) with degrees of polymerization of 2, 3, 4, 5, and 6. From an applied perspective, the C-OGS showed activity against various pathogenic bacteria. In addition, we demonstrated that ChiA74 was not toxic to Hek 293 and 3T3 L1 cells, i.e., the enzyme did not induce apoptosis or affect normal cellular cycle and also did not produce abnormal changes in cell morphology. The potential biotechnological use of producing endochitinase of B. thuringiensis in a microorganism recognized as safe (i.e., E. coli K12) is discussed.
KeywordsChitinase Colloidal Chitin Chymosin chiA74 Gene Endochitinase Activity
This research was supported by grants from the University of Guanajuato (67/10) and CONACyT (5568), Mexico to J.E. Barboza-Corona. We thank Dr. Mayela Bautista-Justo and Ma. Cristina del Rincón-Castro for their advice during the process of this work and also Dr. Gustavo Hernández-Guzmán for designing the primers to detect the bla gene. Cristóbal Castañeda-Ramírez is a graduate student supported by a fellowship from CONACyT, Mexico.
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