title: Heterologous expression, purification and biochemical characterization of endochitinase ChiA74 from Bacillus thuringiensis.
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 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, ∼74kDa). Along with a protein of ∼74kDa, we co-purified its ∼55kDa 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 10mM reduced chitinase activity by ∼30%, and Hg(+2) (10mM) drastically inhibited ChiA74 activity by ∼75-100%. The Vmax, Km and kcat for rChiA74 were 0.11±0.01nmol/min, 2.15μM±0.45 and 3.81s(-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.
authors.affiliation: Universidad de Guanajuato Campus Irapuato-Salamanca, Life Science Division, Graduate Program in Biosciences, Irapuato, Guanajuato 36500, Mexico; Department of Food, Irapuato, Guanajuato 36500, Mexico.
authors.forename: Luz Edith
authors.initials: LE
authors.lastname: Casados-Vázquez
authors.affiliation: Department of Food, Irapuato, Guanajuato 36500, Mexico.
authors.forename: Salvador
authors.initials: S
authors.lastname: Avila-Cabrera
authors.affiliation: California Baptist University, Department of Natural and Mathematical Sciences, 8432 Magnolia Avenue, Riverside, CA 92504, United States; Department of Entomology, University of California, Riverside, Riverside, CA 92521, United States.
authors.forename: Dennis K
authors.initials: DK
authors.lastname: Bideshi
authors.affiliation: Universidad de Guanajuato Campus Irapuato-Salamanca, Life Science Division, Graduate Program in Biosciences, Irapuato, Guanajuato 36500, Mexico; Department of Food, Irapuato, Guanajuato 36500, Mexico. Electronic address: josebar@ugto.mx.
authors.forename: J Eleazar
authors.initials: JE
authors.lastname: Barboza-Corona
mesh_terms: D001413:Bacillus thuringiensis / Q000201:enzymology*; D001671:Biochemistry / Q000379:methods*; D015153:Blotting, Western; D002413:Cations, Divalent / Q000494:pharmacology; D002686:Chitin / Q000378:metabolism; D002688:Chitinases / Q000302:isolation & purification* / Q000378:metabolism*; D002855:Chromatography, Thin Layer; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D013696:Temperature
chemical_list: D002413:Cations, Divalent; D011993:Recombinant Fusion Proteins; D002686:Chitin; D002688:Chitinases
keywords: Bacillus thuringiensis; Characterization; Endochitinase ChiA74; Purification