Expression And Characterization Of A Novel Lytic Polysaccharide Monooxygenases

Lytic polysaccharide monooxygenase(Bt LPMO10A) is a recently discovered class of functional polysaccharide oxidation hydrolase. Oxidation mainly through the catalytic constitute a two histidine residues and Cu2+ in the active site of lytic polysaccharide monooxygenase, which disconnect the cellulose, chitin, starch and other insoluble polysaccharide substrates and form a number of different degree of polymerization of oligosaccharides aldonic acids.How efficient and environmentally friendly production of oligosaccharide which in medical, agricultural and food industry has important applications is the purpose of this research. Recently, to add lytic polysaccharide monooxygenase in chitinase degradation system is a relatively new method to auxiliary insoluble polysaccharide degradation. Use of lytic polysaccharide monooxygenase oxidation to break the chian of chitin oligosaccharide and easy to chitinase further degradation, improving the efficiency of the oxidation reaction.In this paper, it is the first time cloned a lytic polysaccharide monooxygenase from Bacillus thuringiensis subsp.Kurstaki by the method of molecular biology and it is oxidation hydrolase from AA10 hydrolase family. To make target gene connected with vector p ET22 b using RF-clone technology, and then introduced into E. coli BL21(DE3) for heterologous expression.The molecular weight of the recombinant protein is 20 KDa and purified by affinity chromatography to obtain pure protein. Lytic polysaccharide monooxygenase in stable in pH 8.0 Tris-HCl system. BtLPMO10 A can not inhibit the apples rot, fusarium wilt, corn leaf blight and Aspergillus niger, this is indicating that no fungistasis. BtLPMO10 A has a strong substrate-binding capacity to β-chitin ambient conditions at pH 8.0,on the other hand it is no binding with microcrystalline cellulose and chitosan oligosaccharide. MALDI-TOF showed that, under the conditions of existence of Vc, BtLPMO10 A can degrade chitin into different polymerization degree of 2 to 11 of oligosaccharide aldonic acids. This study examines the synergistic action between BtLPMO10 A and chitinase. Comparative analysis the result is the production of disaccharide has a greatly improvement detect by HPLC. The efficiency of enzymatic degradation of chitin has improved 3-10 fold.