Enhancing the Yield of Active Recombinant Chitobiase by Physico-Chemical and In Vitro Refolding Studies Arun Kumar Dangi 1 • Praveen Rishi 2 • Rupinder Tewari 1 Published online: 30 January 2016 Ó Springer Science+Business Media New York 2016 Abstract 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 sys- tem led to formation of inclusion bodies. The production and soluble yield of active CHB was enhanced by co-ex- pression 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 pro- duced 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. Keywords Chaperones Á Chitin Á Chitobiase Á GroEL/ES Á Inclusion bodies Á Refolding Abbreviations CHB Chitobiase IPTG Isopropyl b-D-1-thiogalactopyranoside SDS Sodium dodecyl sulfate NAG N-acetyl-D-glucosamine E. coli Escherichia coli 1 Introduction Chitobiase (CHB; b-N-acetylglucosaminidase: EC 3.2.1.52) is one of the important enzymes of chitinase family and converts chitobiose (a major dimeric product of chitin hydrolysis by chitinase) into a monomer, N-acetyl-D- glucosamine (NAG), which has many biomedical appli- cations including a pain killer for the treatment of arthritis [1]. Currently, the commercial production of NAG is mediated by a chemical process in which chitin, a marine waste of crustaceans, is broken down to NAG with the involvement of strong acids/alkalis and organic solvents. An alternate eco-friendly biotechnological route is being explored involving chitinase and CHB [2, 3]. In our labo- ratory, a gene (nagZ) coding for chitobiase from E. coli K12 strain has been cloned and the recombinant enzyme hyper expressed in E. coli M15 host, which showed very high catalytic efficiency [3]. However, over-expression of CHB resulted in the formation of insoluble aggregates i.e. & Rupinder Tewari rupindertewari5@gmail.com; rupinder@pu.ac.in Arun Kumar Dangi arun.dangi.001@gmail.com Praveen Rishi rishipraveen@yahoo.com 1 Department of Microbial Biotechnology, Panjab University, Sector 14, Chandigarh 160014, India 2 Department of Microbiology, Panjab University, Chandigarh, India 123 Protein J (2016) 35:72–79 DOI 10.1007/s10930-016-9648-z