Studies on enhanced production of biologically active recombinant chitinase and chitobiase

Abstract

In the present study, an attempt has been made to create an eco-friendly biotechnological process for the conversion of chitin, an organic surplus waste, into NAG (a monomer of chitin biopolymer). Chitin was converted into NAG by the use of microbial enzymes i.e. chitinase (CHI) and chitobiase (CHB). The over-expression of these two enzymes in E. coli resulted in the production of inclusion bodies (IBs), an inactive form of enzymes. In the present study, successful efforts were made to achieve large amount of soluble and functionally active enzymes by (a) optimizing incubation conditions for the growth of recombinant E coli strains and (b) protein refolding studies. The percentage of active rCHI was enhanced from 28% to 44% by optimizing culture conditions (Luria Bertani broth, 37oC, 200 rpm, IPTG - 0.25 mM, sorbitol - 500 mM). No effect on the levels of rCHI was observed by the introduction of a plasmid (pGro7) containing groEL/ES gene coding for a molecular chaperonin, GroEL/ES. Further, 20% of active rCHI was recovered from the IBs by in vitro solubilization (50 mM Tris-HCl, pH 8.5 containing 7 M urea) followed by refolding studies (50 mM Tris-HCl, pH 7.5 + 0.75 M L-arginine). Similarly, the recovery of active rCHB was enhanced, though marginally, from 42% to 45% by optimizing the culture conditions (Luria Broth media, 37oC, 200 rpm, IPTG - 0.5 mM). The percentage of active rCHB was increased significantly to 70% by the introduction of a recombinant plasmid, pGro7, to recombinant CHB + E. coli strain and supplementing the growth medium with L-arabinose (13.2 mM). Further, 10% of active rCHB was recovered from the IBs by in vitro solubilization (50 mM Tris base, pH 12.5 containing 2 M urea) followed by refolding studies (50 mM Tris-HCl, pH 7.5 + 1.0 M L-arginine). Using combinatorial approach (culture conditions, IB solubilization and refolding studies), ~64% of rCHI and ~80% of rCHB could be recovered from cells grown in one litre of LB medium.