Cloneing, Expression And Chracterizaton Of Lytic Polysaccharide Monooxygenases In AA10 Family

Cellulose and chitin are two of the most abundant renewable resources on the earth, which have promising and wide application prospects at the aspect of being developed and transformed into biofuels. Meanwhile, it is significantly meaningful for solving the resource, environment and energy issues. Classical enzymatic degradation of cellulose and chitin are the use of glycoside hydrolase systems to catalyze reaction, which break the glycosidic bonds of polysaccharides by hydrolysis reaction. However, the catalytic efficiency of glycoside hydrolase targeted on substrate crystalline regions is very low, thus limiting the biomass degradation.In recent years, the enzymatic degradation of crystalline polysaccharides had been reconsidered after the recent landmark discovery of a new class of oxidases termed lytic polysaccharide monooxygenases（LPMO）. LPMO is a new type of oxidase that acts on crystalline polysaccharides, which can cleave glycoside bonds of chitin（or cellulose） by an oxidative function, generating oxidized and non-oxidized chain ends. This reaction leads to the structures of substrate becoming loose that are benefits for further degradation by glycoside hydrolase system. LPMOs are mainly distributed in two families: AA9 family（formerly GH61） and AA10 family（formerly CBM33）. Currently, there are many unsolved issues in the study of LPMOs, especially for the LPMOs’ substrate selectivity and functional differentiation in AA10 family. Therefore, to explore and study more LPMOs from different sources are not only important for deep exploration of LPMO, but also significant for effective degradation of crystalline polysaccharides.Actinosynnema mirum DSM 43827 strain and Thermobifida fusca YX strain are study object in this project. According to the comparison of gene sequences, we chose three LPMO genes from AA10 family for researching which were Amir₁822（am1）, Amir₅334（am5） gene originated from Actinosynnema mirum DSM 43827 strain and Tfu₁268（tf1） gene from Thermobifida fusca YX strain. These LPMO genes had been cloned successfully by PCR and transformed into E. coli for co-expression with maltose binding protein（MBP） tag. The fusion protein was purified by Ni-column affinity chromatography and cleavaged by Factor Xa to obtain the mature LPMOs. The analysis of enzymatic characterization show Am5 was a LPMO that could act on chitin but not cellulose, but Am1 could not act on either cellulose or chitin. Mass spectrometry results indicated Am5 break glycosidic bonds of chitin by oxidation and generated a series of oxidized chito-oligosaccharides. Reductant could impact seriously on the activity of Am5, which was necessary to the reaction catalyzed by Am5. The results of chitin degradation by chitinase with Am5 suggested that in the presence of a reductant such as ascorbic acid, Am5 can significantly boost chitinase efficiency to much higher levels than before. The affinity of Am5 demonstrated Am5 combined with chitin more strongly than cellulose. Moreover, we used the homology modeling method to calculate the tertiary structure of Am5 protein based on its amino acid sequences, and predict that the active sites of Am5 were N-terminal conserved His 31 and His 106.A novel lytic polysaccharide monooxygenase with high selectivity acted on chitin was identified through cloning, co-expressing and characterizing three LPMO genes from AA10 family in this project. It not only pointed out a new effective method for the degradation of chitin, but also provided new resource and reference for further research and application of LPMO.