Conclusively, LB wheat bran medium resulted in maximum enzyme production in CF whereas, terrific broth was observed to stimulate maximum production of all three recombinant enzymes as secretory fraction; therefore , this medium was used for further production. activities of CotA, Xyl and Pel were obtained, respectively. The cocktail was also found to MK-3102 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. == Introduction == 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; however , due to complex structure of the biomass, accessibility of cellulose to these enzymes MK-3102 is hampered [2]. Hemicellulases, pectinases and laccases are key enzymes which facilitate the hydrolysis process by loosening lignocellulose structure [2]. In other words, depolymerization of plant biomass is achieved by synergistic action Rabbit Polyclonal to p73 of cellulases, hemicellulases, pectinases and ligninases [3]. To improve the production of lignocellulolytic enzymes, various strategies have been adopted, including heterologous expression, use of strong promoter and manipulation of the metabolism of the organisms [4]. Till date, most of these enzymes have been produced individually by respective microorganisms employed and there are hardly any reports on the efficient production of all these enzymes by a single microorganism, which will also be cost effective for degradation of lignocellulosics. Besides, low level of production of these enzymes from wild type microorganisms also limits their utility for commercial exploitation. Recently, combined expression of two or more enzymes MK-3102 using single or multiple plasmids in single host is generating extensive interest, however the major drawback in multiple plasmids based strategy is their maintenance by the host cells [5]. In single plasmid based approach, co-expression of multiple genes can be derived by cloning more than one gene with its expression assembly in a single plasmid [5]. Although attempts have been made to simultaneously express two lignocellulolytic enzymes [6, 7], however , the possibility of simultaneous expression of more than two lignocellulolytic genes in a single host remain to be explored. Liu and Yu developed the process for fungal endoxylanase and -glucosidase production as cocktail inE. coliby developing bi-cistronic vector based upon pET30 plasmid, which resulted in production of enzyme cocktail with enhanced activities [6]. Similarly, Fonseca-Maldonado et al. reported the production of bacterial xylanase and laccase with 44% enhanced activity as cocktail fromPichia pastorisexpression system following homologous recombination based approach [7]. MK-3102 Co-expression of multiple heterologous proteins in same cell provides alternative way of avoidingin vitroreconstitution, reduction in possibilities of improper folding, ease of simultaneous purification and reduction in production cost [8]. Therefore , we have made an attempt to minimize the enzyme production cost following simultaneous expression of three bacterial enzymes, viz. spore coat A protein (CotA) and endoxylanase (Xyl) fromBacillus pumilusMK001 and pectate lyase (Pel) fromB. subtilisRCK inE. MK-3102 coliBL21(DE3) by modifying pETDuet-1 vector. In addition to intracellular fraction, sub-cellular localization of these enzymes has also been investigated. Further, an attempt has also been made to scale up the recombinant enzyme cocktail.