Cloning, expression, purification, biochemical, functional and structure characterization of pectin methylesterase (CtPME) from family 8 carbohydrate Esterase (CE8) from clostridium thermocellum ATCC 27405 and its applications in textile and food industry

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Pectin is a hetero polysaccharide, present in middle lamella of plant cell wall. Pectin degrading enzymes are pectinases, pectin methylesterase (PME), polysaccharide lyases (PL) and polygalacturonase (PG). Pectinases are widely used in food, beverage, juice clarification and textile industry processing applications. PME hydrolyzes the ester decorations from pectin and releases pectate and methanol. Further, pectate can be degraded by PL and PG to produce pectic oligosaccharides. Pectin methylesterase (CtPME) from Clostridium thermocellum ATCC 27405 was cloned, expressed and biochemically characterized. The modeled structure of CtPME showed the right handed parallel β helices. Molecular Dynamic (MD) simulation confirmed the CtPME stability. Small angle X-ray scattering analysis showed the globular shape of CtPME similar to the modeled structure of CtPME. CtPME or CtPL1B or mixture of both enzymes were explored for degumming of jute fiber and bioscouring of cotton fabric in textile industry. FESEM images revealed that the mixed enzymes treated jute fibers were having smooth surface against the rough surface of the control. Smooth surface of jute fibers facilitates their conversion into yarn. Enzyme treated cotton fabric showed enhanced water absorption capacity, which is highly desirable in textile industry. The enzymes treated jute fiber and cotton fabric gave higher Young’s Modulus and ultimate tensile strength than the control. Enzymatic degumming and bioscouring are environment friendly alternatives to chemicals being utilized by textile industry in green process. Pectin was isolated from sweet orange peels by ultrasound-assisted method. Orange peels are waste and suitable source for extraction of pectin for its use in pharmaceutical industry. FESEM and AFM analysis of Extracted Orange Pectin (EOP) showed heterogeneous surface and wrinkled fiber net structure. The DLS analysis of EOP showed polydispersity. HPSEC analysis of EOP gave the molecular weights, 90 kDa and 1.3 kDa. DSC-TGA analyses of EOP showed thermal degradation at 225°C. XRD analysis showed semi-crystalline nature. FTIR and NMR analyses showed 68% esterification by galacturonic residues. EOP hydrolyzed by both pectinases (CtPME or CtPL1B) produced pectic oligosaccharides (POS) of mainly degree of polymerization (DP), DP2 and DP3. POS displayed anti-cancer activity.
Supervisor: Arun Goyal