Mining Of New Mannanase Gene And Construction Of Efficient Expression System For Marine Microorganisms

As an important hemicellulase,mannanase can hydrolyze mannan into mannose and mannan oligosaccharide.In recent years,people have gradually known and developed mannose enzyme,mannanase now has important value in the food,feed,pharmaceutical,oil,textile,paper processing industries,so its application potential.The main purpose of this study is to explore some new mannitases with promising applications.Mannan is an important component of hemicellulase present in plant cell walls.Mannan biodegradation is mainly executed by mannanase.In this study,a new type of mannanase from Alteromonadaceae Bs31,named ManBs31,was recombinantly expressed and biochemically characterized.Amino acid sequence analysis showed that ManBs31 consists of multiple functional domains,among which ManBs31-GH5,which belongs to the 5 family of glycoside hydrolases,is responsible for substrate hydrolysis.ManBs31-GH5 was successfully produced in an Escherichia coli system and purified by Ni-chromatography.Enzyme activity assay showed that ManBs31-GH5 exhibited maximum catalytic activity at p H 6 and 70 °C.Among the common types of mannan substrates,ManBs31-GH5 had the highest hydrolysis activity toward konjac glucomannan,with a specific enzyme activity of 1070.84U/mg.The hydrolysis products of mannans by ManBs31-GH5 were mannan-oligosaccharides of various lengths but without monosaccharides.Therefore,the enzyme is suitable for preparing functional mannan-oligosaccharides added to food.At the same time,in this study,the successful production of ManBs31-GH5 by Pichia pastoris system provided a solid foundation for the industrial application of the enzyme.Mannanase catalyzes the fracture of ?-1,4-mannosidic linkages in mannans,and has various functions in different biotechnology industries.In the above study,a high temperature mannanase plant was obtained from the marine microbial environment,further excavation of mannanase genes with different properties in this environment.Therefore,a series of experimental designs of ?-mannanase from Verrucomicrobiae DG1235(Man DG1235),revealing the enzymatic properties of the enzyme.The amino acid arrangement of the mannanase gene indicates that Man DG1235 belongs to the glycoside hydrolyase 26 family and has very few amino acid sequence homologies than those reported ?-mannanase.Man DG1235 was successfully expressed and purified in the Escherichia coli expression system,Man DG1235 has typical properties of cold active enzymes including high activity and thermal instability at low temperature,Man DG1235 shows maximum activity at p H 8.0 and 20 ?,indicating that it is a slightly alkaline ?-mannitase.Man DG1235 is capable of hydrolysis of multiple Mannan substrates and is active for certain types of glucans.Structural models established by homologous modeling suggest that Man DG1235 has four mannose binding subbits which are symmetrically arranged in activated cracks,long rings connecting ?2 and ?2,such as ?2 and ?2 in Cj Man26 C produce space-boundary active site cracks in glycine,which may lead to extra-acting features of Man DG1235,especially cutting mannose from the non-reduced end of the substrate.By further studying the p ET28a-Man DG1235 of recombinant mannanase,Five pairs of primers were designed,Man DG1235-W101 A,Man DG1235-D103 A,Man DG1235-D232 N,Man DG1235-Y142 W,Man DG1235-Y261 W,are the five point mutations aims to change p H preferences through point mutations.The results were as follows: the optimum p H for Man DG1235-W101 A was 7.0,Man DG1235-D103 A optimal p H is 9.0,Man DG1235-D232 N protein is insoluble,Man DG1235-Y142 W protein is insoluble,Man DG1235-Y261 W no enzyme activity.