Engineering a bifunctional laccase xylanase chimera for application in pulp an paper industry

Abstract

An enzyme cocktail (xylanase, lipase, protease, pectinase and and#945; -amylase) from Bacillus halodurans was employed for upscaled biobleaching of untreated and ODL treated agro based pulp at Shreyans Paper Limited, Ahmedgarh Ludhiana and 50 and 40% respectively, reduction in chemical usage were achieved. A very simple and economical medium of soybean meal medium was optimized for cost-effective and sustainable production of laccase from Rheinheimera sp. In addition, it is focusing on the development of a novel chimeric protein of industrially relevant enzymes. As the efficient usage of enzymes in large scale industrial applications demands progression in multi-enzyme systems by tailoring and optimizing catalytic sites through enzyme engineering. Efforts have been carried out to engineer a functional laccase-xylanase chimera for the eco-friendly recycling of old newsprint pulp. Restriction digestion-ligation and fusion using linker and overlap approaches have been used to construct a chimera of truncated xylanase from B. halodurans and truncated laccase from Rheinheimera sp. possessing two catalytic properties and produced from a single host with improved efficiency. Although PCR showed genes fusion but the expressed xylanase-laccase chimera cleaved proteolytically to yield separate xylanase and laccase polypeptides in E. coli BL21 (DE3) and B. subtilis KO7. Chimera showed xylanase activity only in culture supernatant because of its extracellular secretion using its own native signal peptide and laccase activity was only observed in sonicated supernatant of culture, indicating its intracellular localization due to absence of signal peptide in laccase gene. This enzyme cocktail improved brightness of deinked paper by 8.07 and 27.7% in comparison to 60% chemical treated and untreated pulp, respectively, thus achieving 40% reduction in