Computational re-design of Streptomyces griseus Chitinase C and analysis of biochemical, biophysical and antifungal properties of designed variants

Despite the importance of chitinases as potential biocontrol agents, their applications have not attracted significant attention due to the lack of stable enzyme formulations, high production costs and low yields from both wild and recombinant sources. However, through protein engineering techniques, these limitations can be overcome. Engineered mini-proteins for instance, could possess; enhanced stability, low production costs and simplified structural and functional mechanisms. Thus, this study was focused on developing miniaturized variants of Streptomyces griseus HUT6037 chitinase C that could serve as simpler yet potentially stable antifungal agents. Through computational techniques involving sequence analysis, docking and molecular dynamics simulation important residues and/or motifs within the catalytic cleft of SgChiC were identified as essential for recognition and binding to complex or crystalline chitin. Residues outside the catalytic clefts were thus considered targets for miniaturization. Five (5) SgChiC variants namely: M159, M140, M139, M109 and M101 were designed in silico, their genes were subsequently synthesised and cloned into pET-22b(+). The variants were then expressed in Escherichia coli BL21(DE3) and purified through on-column refolding. Biochemical assays revealed that all variants although had lower activities towards colloidal chitin when compared with the wild-type, retained the optimum temperature at 40 C. The optimum pH for activities of variants however varied with M101 and M139 drifting towards acidic pH of 5.0 and 6.0 respectively, while M159 and M109 had optimum activities at pH 8. Interestingly, all variants retained 40-50% of the specific activity of the WT towards colloidal chitin, with M159 displaying the highest specific activity at 31.6 Umg-1 compared to the WT with 52.3 Umg-1. Contrastingly, with chitosan, the smallest variant M101, displayed high chitosanase activity comparable with the WT with 59.6 and 61.4 Umg-1 respectiv