Expression,Purification And Characterization Of Insect LPMO1

Chitin is the second-largest polysaccharide in nature besides cellulose.It is a linear polymer of?-1,4 linked N-acetylglucosamine.It is widely found in the epidermis,trachea and midgut of insects,and can protect insects from external aggressions.Enzymes involved in the metabolism of chitin play an important role in the development of insects.Therefore,they have received extensive attention and are important potential targets for pesticides design.The lytic polysaccharide monooxygenase(LPMO)of the AA15 family is a type of copper ion-dependent enzyme discovered that participates in the degradation of insect chitin in recent years.It can break chitin through oxidative cleavage,thereby significantly improving the degradation efficiency of chitinase.However,the current understanding of the LPMO activity of this family is very limited.Revealing its catalytic activity will further clarify the mechanism of insect chitin degradation and contribute to the utilization of chitin as biomass resources.In this thesis,the AA15 family of cleavage polysaccharide monooxygenase(OfLPMO1)from Ostrinia furnacalis was selected as the research object,and the following researches were perfomed:(1)Cloning and recombinent expression of OfLPMO1.The catalytic domain and full-length yeast expression vector of the cleavage polysaccharide monooxygenase(OfLPMO1)of AA15 family of Ostrinia furnacalis were successfully constructed(OfLPMO1-CD/OfLPMO1-FL)and transferred into Pichia pastoris to achieve soluble expression.The recominant proteins OfLPMO1-CD and OfLPMO1-FL were then purified to homogeneous through chitin affinity chromatography.The yields were 7 mg/L and 13.4 mg/L,respectively.(2)An evaluation system for measuring LPMO activity was established using 2,6-dimethoxyphenol(2,6-DMP)and H₂O₂as substrates,and BtLPMO10A from microorganisms(Bacillus thuringiensis)as a model.According to this method,the chitin substrate was found to improve the stability of BtLPMO10A.(3)The enzymatic properties of OfLPMO1.The comparative analysis showed that OfLPMO1-FL had a higher catalytic and binding activity than OfLPMO1-CD,and the binding ability of OfLPMO1s toward?-chitin was much stronger than that toward?-chitin.Both the oxidative and binding activities of OfLPMO1 were dependent on Cu²⁺.(4)The application of OfLPMO1 to degrade chitin.The chitin degradation ability of OfLPMO1-FL is stronger than that of BtLPMO10A.The electron microscope showed that it could destroy the compacted structure of chitin,resulting in a fluffy and porous surface.OfLPMO1 was proved to be able to synergize with the chitinases Of Chi-h,SmChiA,SmChiB and SmChiC during the degradation of chitin.