Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
2020386 | Protein Expression and Purification | 2015 | 7 Pages |
Abstract
ChiA74 is a secreted endochitinase produced by Bacillus thuringiensis. Previously we have partially characterized the physical parameters that affect enzymatic activity of ChiA74 in crude preparations of bacterial secretomes. In the present study, we cloned the chiA74 open reading frame (ORF) lacking the 5â² sequence coding for its secretion signal peptide (chiA74Îsp) into a cold shock expression vector (pColdI) for production of the enzyme in Escherichia coli BL21-Rosetta 2. As a result, the N-terminal end of ChiA74Îsp ORF was fused to an artificial sequence of 28 amino acid, including a 6à histidine tag for purification of recombinant 6ÃHis tagged-ChiA74Îsp (rChiA74, â¼74 kDa). Along with a protein of â¼74 kDa, we co-purified its â¼55 kDa processed form which was confirmed by Western blot analysis. Optimal endochitinase activity of purified rChiA74 occurred at pH 7 and 40 °C. Most divalent cations (e.g. Ba+2, Ca+2, Mn+2, Mg+2, Zn+2 and Cu+2) at concentration of 10 mM reduced chitinase activity by â¼30%, and Hg+2 (10 mM) drastically inhibited ChiA74 activity by â¼75-100%. The Vmax, Km and kcat for rChiA74 were 0.11 ± 0.01 nmol/min, 2.15 μM ± 0.45 and 3.81 sâ1, respectively, using 4-MU-GlcNAc3 as substrate. Using purified rChiA74 and colloidal chitin as substrate, chitin-derived oligosaccharides with degree of polymerization of 2 and 1 were detected.
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Authors
Luz Edith Casados-Vázquez, Salvador Avila-Cabrera, Dennis K. Bideshi, J. Eleazar Barboza-Corona,