| Cellulose is a macromolecular polysaccharide with basic structure of glucose,which widely exists in nature.However,the traditional method of degradation of cellulose is to hydrolyze it by glycoside hydrolase system.Because of the crystal structure of cellulose,it is difficult to degrade cellulose,which limits the catalytic efficiency of glycoside hydrolase.So that the biological energy in nature can not be used to the maximum extent.Therefore,the development of new biofuels is of great practical significance and can help to solve the practical problems such as resources and environment in nature at present.Recent studies have shown that lyase(Lytic Polysaccharide Monooxygenases,LPMO can contribute to the degradation of crystalline polysaccharides more efficiently and rapidly.Lyase is a kind of enzyme which can break cellulose glycoside bond by oxidation in the presence of bivalent metal ions and reductants,thus loosening the structure of substrate.It helps to hydrolyze more thoroughly in the next step.LPMO mainly includes auxiliary activity 9 familyand auxiliary activity 10 family.Among them,all the LPMO of AA9 family acted on cellulose substrate,while most of the LPMO of AA10 family acted on chitin substrate,and only a few of them acted on cellulose substrate.At present,there are still many problems in the study of LPMO,such as how to improve the expression of LPMO,how to be more efficient,and how to produce oligosaccharide substances which have application value in medical treatment,food and so on.Therefore,how to use bioenergy efficiently is the first problem that we need to solve,and it plays a positive role in solving the problems of resource shortage and environmental pollution in the world at present.In this study,the expression vector p PICZ? A-Pclpmo was constructed by synthesizing the(Phanerochaete chrysosporium)lpmo gene.The recombinant protein was expressed in Pichia pastoris GS115,and the expression of the recombinant protein was 450 mg/l.Using microcrystalline cellulose as substrate,the optimum reaction temperature of recombinant Pc LPMO and p H,were 60 ? and 6.0,respectively.The activity of recombinant Pc PLMO on straw fiber treated by alkali pretreatment was higher,and its thermal stability was 23.3 U/ml.Pc PLMO.The residual activity at 80 ?,90 ? and 100 ? for 60 min,was 80%,62% and 48%,respectively,and the residual activity was 80%,62% and 48% at 80 ?,90 ? and 100 ?,respectively.Through Endo H deglycosylation analysis,Pc LPMO was highly glycosylated,which promoted the thermal stability of the enzyme at high temperature.Pc LPMO and cellulase co-hydrolyzed straw substrates showed a certain synergistic effect.When single cellulase and Pc LPMO(20U/g)were used to hydrolyze corn straw,wheat straw and soybean straw,the enzyme activities were 20.3 U / ml and 21.7 U / ml,respectively,and the enzyme activities of corn straw,wheat straw and soybean straw were 20.3 U / ml and 21.7 U / ml,respectively.20.8 U/ml and 24.9 U / ml,25.5 U / ml,26.4 U / ml.When,Pc LPMO(20U/g and cellulase were added into the whole reaction system,the enzyme activities of corn straw,wheat straw and soybean straw were 32.7 U / ml,34.2 U / ml and 34.3U / ml,respectively,when the enzyme activity was 32.7 U / ml,34.2 U / ml and 34.3 U / ml,respectively.The results show that there are copper binding sites in its structure,which can improve its enzyme activity. |
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