Cloning and expression of β-glucosidase gene from Bacillus licheniformis into E. coli BL 21 (DE3)

Abstract

A 1.4 Kb fragment of Bacillus licheniformis ATCC 14580 encoding β-glucosidase was cloned and expressed in Escherichia coli. β-Glucosidase expressed by E. coli harboring cloned gene was located entirely in the intracellular fraction. Recombinant β-glucosidase protein was purified to homogeneity level and the molecular weight was found to be 53 kDa using sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis. It gave maximum activity at 50• C and pH 6. Km and Vmax were 0.206 mM and 1.26 U/mg, respectively, with p-nitrophenyl-β-D-glucopyranoside, while activation energy Ea, enthalpy of activation ∆H and entropy of activation ∆S were found to be 66.31 kJ/mol, 64.04 kJ/mol and 48.28 J/mol/K, respectively. The pKa1 and pKa2 of the ionizable groups of active site residues involved in Vmax were found to be 5.5 and 7.0, respectively. When the recombinant β-glucosidase protein was used as a member of consortium with endoglucanase and exoglucanase for the saccharification of wheat straw, 123% increase in saccharification was observed.

Microbial β-Glucosidases: Screening, Characterization, Cloning and Applications

“…Generally fungal β-glucosidase exhibits optimal activity at pH of 4.0-6.0 [59, 69,86,92,93], nevertheless β-glucosidase with optimal pH of 2.4 has been reported from T. cylindrosporum Syzx4 [68] and that with optimal pH of 9 and 10 has been reported from Acremonium murorun LPSC 927 and Chaetomium globosum, respectively [94,95]. Bacterial β-glucosidase exhibits optimal activity at pH of 6-7 [44,87,96,97], although those with pH optima of 5, 8 and 10 has been reported from Caldicellulosiruptor saccharolyticus [98], Bacillus halodurans [99], Klebsiella pneumoniae [100], respectively. Yeast β-glucosidase reported from A. pullulans and Candida peltata exhibited optimal activity at pH 5 [80,101] and that from Kluyveromyces marxianus showed optimal activity at pH 5.5 [102].…”

“…However, expression in E. coli has several drawbacks such as formation of inclusion bodies, low secretion efficiency, absence of splicing machinery and inability to perform post-translational modifications such as glycosylation explaining why it is not successfully being used for expression of fungal β-glucosidase enzymes [196,197,198]. E.coli is usually transformed with self-replicated plasmid that does not integrate to chromosomal DNA and keeps on replicating itself independently from cell divisions [199,200].…”

Catalytic properties, functional attributes and industrial applications of β-glucosidases

“… E. coli 52 450 7.0 45 GH1 Chang et al ( 2011 ) Micrococcus antarcticus E. coli 48 472 6.5 25 GH1 Fan et al ( 2011b ) Sphingomonas sp. E. coli 49.3 447 5.0 37 GH1 Wang et al ( 2011 ) Bacillus licheniformis E. coli 53 466 6 50 GH1 Zahoor et al ( 2011 ) Bacillus licheniformis E. coli 52.2 – 6.0 60 GH16 Aftab et al ( 2012 ) Fervidobacterium islandicum E. coli 53.41 459 6.0–7.0 90 GH1 Jabbour et al ( 2012 ) Yeast Candida wickerhamii E. coli 72 – 7 37–40 GH1 …”

Characterization of a beta-glucosidase from Bacillus licheniformis and its effect on bioflocculant degradation

“…β-Glucosidase is distributed throughout a broad range of organisms, including microbes, plants, and animals (Chang et al 2008; Knight and Dick 2004). Some studies have been conducted to isolate novel β-glucosidases from the genus Bacillus , which represents one of the most important groups of bacteria not only for its production of commercially valuable enzymes but also in the study of the mechanism of extracellular polymer secretion (Chamoli et al 2016; Khan and Husaini 2006; Zahoor et al 2011). …”