Abstract
This study attempted to reduce the enzyme production cost for exploiting lignocellulosic materials by expression of multiple genes in a single host. Genes for bacterial laccase (CotA), pectate lyase (Pel) and endoxylanase (Xyl), which hold significance in lignocellulose degradation, were cloned in pETDuet-1 vector containing two independent cloning sites (MCS). CotA and xyl genes were cloned in MCS1 and MCS 2, respectively. Pel gene was cloned by inserting complete cassette (T7 promoter, ribosome binding site, pel gene, His tag and complete gene ORF) preceded by cotA open reading frame in the MCS1. IPTG induction of CPXpDuet-1 construct in E. coli BL21(DE3) resulted in expression of all three heterologous proteins of ~65 kDa (CotA), ~45 kDa (Pel) and ~25 kDa (Xyl), confirmed by SDS-PAGE and western blotting. Significant portions of the enzymes were also found in culture supernatant (~16, ~720 and ~370 IU/ml activities of CotA, Pel and Xyl, respectively). Culture media optimization resulted in 2, 3 and 7 fold increased secretion of recombinant CotA, Pel and Xyl, respectively. Bioreactor level optimization of the recombinant cocktail expression resulted in production of 19 g/L dry cell biomass at OD600nm 74 from 1 L induced culture after 15 h of cultivation, from which 9, 627 and 1090 IU/ml secretory enzyme activities of CotA, Xyl and Pel were obtained, respectively. The cocktail was also found to increase the saccharification of orange peel in comparison to the xylanase alone. Thus, simultaneous expression as well as extra cellular secretion of these enzymes as cocktail can reduce the enzyme production cost which increases their applicability specially for exploiting lignocellulosic materials for their conversion to value added products like alcohol and animal feed.
Highlights
Rising cost of fossil fuels, their dwindling resources and increasing environmental concerns have motivated researchers to look for alternate liquid transportation fuels especially biofuels from plant biomass [1]
B. pumilus MK001, B. subtilis RCK and different E. coli strains, BL21(DE3), BL21(DE3) containing pTUM4 plasmid and E. coli BL21(DE3) arctic expression host, were obtained from Lignocellulose Biotechnology Laboratory, Department of Microbiology, University of Delhi South Campus, New Delhi
Nucleotide sequencing and restriction map analysis of each gene revealed the presence of two BamHI recognition sites in pel ORF and one HindIII recognition site in xyl ORF which resulted in visualization of five bands after digestion with BamHI/HindIII restriction enzymes
Summary
Rising cost of fossil fuels, their dwindling resources and increasing environmental concerns have motivated researchers to look for alternate liquid transportation fuels especially biofuels from plant biomass [1]. Degradation of the plant biomass using chemicals for this purpose is considered as uneconomic and unfriendly to the environment [2]. Use of biocatalyst such as cellulases, hemicellulases, pectinases and ligninases on the other hand is preferred due to cost effective production and less impact on the environment [1]. Hydrolysis of the lignocellulosic biomass for the production of fermentable sugars is catalyzed mainly by cellulases; due to complex structure of the biomass, accessibility of cellulose to these enzymes is hampered [2]. Hemicellulases, pectinases and laccases are key enzymes which facilitate the hydrolysis process by loosening lignocellulose structure [2]. Low level of production of these enzymes from wild type microorganisms limits their utility for commercial exploitation
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