Heterologous expression, purification and biochemical characterization of endochitinase ChiA74 from Bacillus thuringiensis

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 5′ sequence coding for its secretion signal peptide (chiA74Δsp) 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 ChiA74Δsp ORF was fused to an artificial sequence of 28 amino acid, including a 6× histidine tag for purification of recombinant 6×His tagged-ChiA74Δsp (rChiA74, ∼74 kDa). Along with a protein of ∼74 kDa, we co-purified its ∼55 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 ∼30%, and Hg+2 (10 mM) drastically inhibited ChiA74 activity by ∼75-100%. The Vmax, Km and kcat for rChiA74 were 0.11 ± 0.01 nmol/min, 2.15 μM ± 0.45 and 3.81 s−1, 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.