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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.

journal_name

Protein Expr Purif

journal_title

Protein expression and purification

authors

Kumar S,Jain KK,Singh A,Panda AK,Kuhad RC

doi

10.1016/j.pep.2014.12.003

subject

Has Abstract

pub_date

2015-06-01 00:00:00

pages

43-51

eissn

1046-5928

issn

1096-0279

pii

S1046-5928(14)00288-5

journal_volume

110

pub_type

杂志文章

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