Enhancing the Yield of Active Recombinant Chitobiase by Physico-Chemical and In Vitro Refolding Studies

Chitobiase (CHB) is an important enzyme for the production of N-acetyl-D-glucosamine from the chitin biopolymer in the series of chitinolytic enzymes. Majority of over-expressed CHB (58%) in E. coli expression system led to formation of inclusion bodies. The production and soluble yield of active CHB was enhanced by co-expression with GroEL/ES chaperonin, optimizing culture conditions and solubilization followed by refolding of remaining inactive chitobiase present in the form of inclusion bodies. The growth of recombinant E. coli produced 42% CHB in soluble form and the rest (~58%) as inclusion bodies. The percentage of active CHB was enhanced to 71% by co-expression with GroEL/ES chaperonin system and optimizing culture conditions (37 °C, 200 rpm, IPTG--0.5 mM, L-arabinose--13.2 mM). Of the remaining inactive CHB present in inclusion bodies, 37% could be recovered in active form using pulsatile dilution method involving denaturants (2 M urea, pH 12.5) and protein refolding studies (1.0 M L-arginine, 5% glycerol). Using combinatorial approach, 80% of the total CHB expressed, could be recovered from cells grown in one litre of LB medium is a step forward in replacing hazardous chemical technology by biotechnological process for the production of NAG from chitinous waste.