Studies On Expression And Function Of Two Novel β-mannanases From Flavobacterium Saccharophilum

β-mannanase is an enzyme that hydrolyzes the β-1,4 glycosidic bond in β-mannan,which is widely used in feed,food and other industries.Mannan-oligosaccharides have important physiological activities,such as regulating intestinal microbial environment,inhibiting pathogen growth and inhibiting enteritis.β-mannanase is a good tool for the preparation of mannan-oligosaccharides.It has the advantages of mild reaction conditions and low cost.It is of great significance to develop and prepare highly active β-mannanase.In this paper,two newβ-mannanases were found and characterized,and the prebiotic activity of oligosaccharides was studied.Two β-mannanases Fs2621 and Fs2625 of glycoside hydrolase family 26 from Flavobacterium saccharophilum were prepared by molecular cloning technology.The molecular weights of the proteins were 42.6 k Da and 45.8 k Da,respectively.The enzymatic properties of recombinant enzymes Fs2621 and Fs2625 were studied with locust bean gum as substrate.The specific enzyme activity of Fs2621 was 261.6 U / mg and that of Fs2625 was1546.2 U / mg.The optimum p H of both enzymes was 6.0,and the optimum reaction temperature was 45 o C.Neither of them could hydrolyze mannose disaccharide,but Fs2621 could completely hydrolyze linear mannose oligosaccharides with a degree of polymerization of 3-6,and Fs2625 could only completely hydrolyze mannose hexasaccharide with a higher degree of polymerization.The analysis of the products of Fs2621 and Fs2625 enzymatic hydrolysis of galactomannan showed that the hydrolysis products of both enzymes included mannose and mannose oligosaccharides with a degree of polymerization of 2-11.The difference was that the enzymatic hydrolysis product of Fs2621 was mainly mannose disaccharide,and the enzymatic hydrolysis product of Fs2625 was mannose,mannose disaccharide and mannose oligosaccharides with higher degree of polymerization.The enzymatic hydrolysates of the two enzymes were used as the sole carbon source to cultivate five kinds of Lactobacillus.The results showed that these manno-oligosaccharides could promote the growth of five kinds of Lactobacillus in different degrees.The growth of Lactobacillus casei was the most obvious,and the promotion of Lactobacillus paracasei was the weakest.The manno-oligosaccharides produced by the hydrolysis of gelatin promoted the growth of Lactobacillus most obviously,and the manno-oligosaccharides produced by the hydrolysis of galactomannan by the two enzymes were beneficial to the growth of Lactobacillus.In summary,two new β-mannanases,Fs2621 and Fs2625,were successfully obtained in this paper.The properties and functions of the two enzymes were clarified.The research results lay a foundation for the industrial preparation of mannan oligosaccharides and provide a meaningful reference for its application in functional food or nutrition industry.