title: Enhancing the Yield of Active Recombinant Chitobiase by Physico-Chemical and In Vitro Refolding Studies.
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 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.
authors.affiliation: Department of Microbial Biotechnology, Panjab University, Sector 14, Chandigarh, 160014, India. arun.dangi.001@gmail.com.
authors.forename: Arun Kumar
authors.initials: AK
authors.lastname: Dangi
authors.affiliation: Department of Microbiology, Panjab University, Chandigarh, India. rishipraveen@yahoo.com.
authors.forename: Praveen
authors.initials: P
authors.lastname: Rishi
authors.affiliation: Department of Microbial Biotechnology, Panjab University, Sector 14, Chandigarh, 160014, India. rupindertewari5@gmail.com.
authors.forename: Rupinder
authors.initials: R
authors.lastname: Tewari
mesh_terms: D000118:Acetylglucosaminidase / Q000032:analysis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018834:Chaperonin 60; D002686:Chitin; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002479:Inclusion Bodies / Q000378:metabolism*; D058849:Protein Refolding; D011994:Recombinant Proteins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D018834:Chaperonin 60; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D002686:Chitin; D000118:Acetylglucosaminidase
keywords: Chaperones; Chitin; Chitobiase; GroEL/ES; Inclusion bodies; Refolding
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