| Article ID | Journal | Published Year | Pages | File Type |
|---|---|---|---|---|
| 2020363 | Protein Expression and Purification | 2015 | 9 Pages |
•The recombinant active pectate lyase has been generated in E. coli BL21(DE3).•The conditions for refolding pectate lyase from inclusion bodies have been optimized.•Change in protein structure after ligand interaction was revealed by CD spectroscopy.•Enzyme was observed to be capable of degrading methylated and unmethylated pectin.
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 ∼3194 IU/mg and ∼1493 IU/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 60 min 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 Ca2+ 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.
