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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. [ABSTRACT FROM AUTHOR]

Pretreatment of rice straw was optimized by physical and chemical methods. Among all the pretreatment methods followed by saccharification, autoclaved ammonia pretreatment significantly enhanced the liberation of reducing sugars i.e. 233.76 ± 1.23 mg/g substrate as compared to other methods. Further statistical optimization by response surface methodology (RSM) including ammonia concentration, substrate concentration and autoclaving time, enhanced the saccharification rate by 1.9-fold i.e. 451.96 mg/g substrate at 5 % rice straw, 12 % ammonia and autoclave time of 30 min after saccharification of 6 h. Maximum liberation of reducing sugars (635.37 mg/g substrate) was attained under these optimized conditions at 60 °C after 48 h. Fourier-transform infrared spectroscopy, X-ray diffraction and scanning electron microscope analysis clearly confirmed the removal of lignin in pretreated biomass. Ammonia pretreated enzymatic hydrolysate was fermented by Saccharomyces cerevisiae at 30 °C, pH 7.0, 150 rpm and 20 % hydrolysate, resulted in 24.37 g/L bioethanol after 72 h. Image 1 • Various pretreatment strategies (physical and chemical) were employed for pretreatment of rice straw. • Ammonia pretreatment significantly enhanced the enzymatic accessibility and liberation of reducing sugars. • Fermentation of enzymatic hydrolysate (20%) resulted in 24.37 g/L ethanol using S.cerevisiae. [ABSTRACT FROM AUTHOR]

Lignocellulosic biomass has been considered as an important and sustainable source of renewable energy. Cellulose constitutes the major component of the lignocellulosic biomass and also offers maximum recalcitrance towards its fullest utilization. The enzymatic breakdown of cellulose is achieved through cellulases. Diverse forms of microbes including fungi, bacteria, actinomycetes and yeast are known to produce cellulases that have found extensive application in various industries. Due to the current global political unrest over oil prices and the threat of global warming following combustion of fossil fuels, the paradigm of research is now focused on biofuel production from plant biomass. Conventional approaches have not been economically feasible for meeting the demands of the industry. This review provides an update regarding the status of present microbial cellulase production technologies and research with special reference to solid state fermentation and different molecular techniques such as mutagenesis, metabolic engineering and heterologous gene expression of cellulases from different microbial domains with improved catalytic and stability properties. Metagenomic and genomic studies for mining of novel cellulase genes in addition to screening of culturable strains using conventional methods have been advanced. In addition the bottlenecks associated with cellulase production and how the future research needs to be directed to provide a comprehensive technology for the production of cellulases with novel traits for application at an industrial level without economic constraints are discussed. [ABSTRACT FROM AUTHOR]