Protein engineering of β-1,4-endoglucanase and Chimera construction with β-glucosidase from Clostridium thermocellum for improving ligno-cellulosic biomass saccharification

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Plant cell wall were composed of polymers for example cellulose, various hemicellulose and lignin and can be converted into alcohol that can be served as renewable source of energy. Saccharification of cellulose (major component of plant cell wall) requires three major enzymes i.e. endoglucanases and exoglucanases (cellobiohydolases and β-glucosidase) for its complete hydrolysis. But the use of these enzymes are limited because of their low catalytic efficiency and individual production of each enzyme increases the total cost of bioethanol production. So, it is important to engineer enzymes to i) improve catalytic efficiency and ii) production of chimera having multifunctional activity to reduce the number of enzymes required for complete degradation of lignocellulosic biomass. Site-directed mutagenesis of β-1,4- endoglucanase from family 5 glycoside hydrolase (CtGH5) from Clostridium thermocellum was performed to develop a mutant CtGH5-F194A that gave 40 U/mg specific activity against carboxymethyl cellulose, resulting 2-fold higher activity than wild-type CtGH5. CtGH5-F194A was fused with a β-1,4-glucosidase, CtGH1 from Clostridium thermocellum to develop a chimeric enzyme. The chimera (CtGH1-L1-CtGH5-F194A) expressed as a soluble protein using E. coli BL-21cells displaying 3- to 5-fold higher catalytic efficiency for endoglucanase and β-glucosidase activities. TLC analysis of hydrolysed product of CMC by chimera 1 revealed glucose as final product confirming both β-1,4-endoglucanase and β-1,4-glucosidase activities, while the products of CtGH5-F194A were cellobiose and cellooligosaccharides. Protein melting studies of CtGH5-F194A showed melting temperature (Tm), 68ºC and of CtGH1, 79ºC, whereas, chimera showed 78ºC. The action of Chimera (CtGH1-L1-CtGH5-F194A) was analysed on pretreared Sorghum stalk biomass. The TLC analysis showed Chimera displayed higher accumulation of glucose after 48h than the mixture of CtGH1 and CtGH5-F194A. Therefore, Chimera acts on the pretreated Sorghum stalk biomass and efficiently releases the glucose as final product. The insilico molecular modelling was performed to determine the molecular structure of Chimera. The modelled structure of the Chimera showed a stable modular conformation with 99.9% of residues in allowed region analysed by Ramachandran plot. The molecular dynamics simulation showed stability of the two modules in the chimeric form. Moreover, SAXS analysis of Chimera displayed elongated structure with two modules in fully folded form in the solution and showed an overall shape similar to a bean-bag contour. The enzymatic saccharifcation using cocktail of cellulases comprising of Chimera and cellobiohydrolases (CtCBH5A) on dual alkali pretreated followed by organosolv of sugarcane bagasse gave maximum TRS yield at 30°C for 96h was 230 mg/g and glucose yield of 137 mg/g of pretreated biomass. The efficacy of the recombinant enzymes was obtained on the dual pretreated sugarcane bagasse. The effectiveness of the pretreatment processes was further confirmed using FESEM and FT-IR analysis.
Supervisor: Arun Goyal