title: Characterization of recombinant pectate lyase refolded from inclusion bodies generated in E. coli BL21(DE3).
abstract: Pectate lyase (EC 4.2.2.2) gene from Bacillus subtilis RCK was cloned and expressed in Escherichia coli to maximize its production. In addition to soluble fraction, bioactive pectate lyase was also obtained from inclusion body aggregates by urea solubilization and refolding under in vitro conditions. Enzyme with specific activity ∼3194IU/mg and ∼1493IU/mg were obtained from soluble and inclusion bodies (IBs) fraction with recovery of 56% and 74% in terms of activity, respectively. The recombinant enzyme was moderately thermostable (t1/2 60min at 50°C) and optimally active in wider alkaline pH range (7.0-10.5). Interaction of protein with its cofactor CaCl2 was found to stimulate the change in tertiary structure as revealed by near UV CD spectra. Intrinsic tryptophan fluorescence spectra indicated that tryptophan is involved in substrate binding and there might be independent binding of Ca(2+) and polygalacturonic acid to the active site. The recombinant enzyme was found to be capable of degrading pectin and polygalacturonic acid. The work reports novel conditions for refolding to obtain active recombinant pectate lyase from inclusion bodies and elucidates the effect of ligand and substrate binding on protein conformation by circular dichroism (CD) and fluorescence spectrofluorometry.
authors.affiliation: Lignocellulose Biotechnology Lab, Department of Microbiology, University of Delhi South Campus, New Delhi 110021, India.
authors.forename: Sandeep
authors.initials: S
authors.lastname: Kumar
authors.affiliation: Lignocellulose Biotechnology Lab, Department of Microbiology, University of Delhi South Campus, New Delhi 110021, India.
authors.forename: Kavish Kumar
authors.initials: KK
authors.lastname: Jain
authors.affiliation: Product Development Cell, National Institute of Immunology, New Delhi 110067, India.
authors.forename: Anupam
authors.initials: A
authors.lastname: Singh
authors.affiliation: Product Development Cell, National Institute of Immunology, New Delhi 110067, India.
authors.forename: Amulya K
authors.initials: AK
authors.lastname: Panda
authors.affiliation: Lignocellulose Biotechnology Lab, Department of Microbiology, University of Delhi South Campus, New Delhi 110021, India. Electronic address: kuhad85@gmail.com.
authors.forename: Ramesh Chander
authors.initials: RC
authors.lastname: Kuhad
mesh_terms: D001412:Bacillus subtilis / Q000737:chemistry* / Q000201:enzymology; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D002118:Calcium / Q000737:chemistry; D020134:Catalytic Domain; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D002479:Inclusion Bodies / Q000737:chemistry*; D007700:Kinetics; D010368:Pectins / Q000737:chemistry*; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D011133:Polysaccharide-Lyases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011485:Protein Binding; D011487:Protein Conformation; D058849:Protein Refolding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012995:Solubility; D013696:Temperature; D014508:Urea / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D010368:Pectins; D014508:Urea; D011133:Polysaccharide-Lyases; C023911:pectate lyase; D002118:Calcium; C003181:polygalacturonic acid
keywords: CD spectra; Inclusion bodies; Recombinant pectate lyase; Refolding