Cloning And Expression And Enzymatic Properties Of Lytic Polysaccharide Monooxygenase And Optimization Of Fermentation Conditions

Lytic polysaccharide monooxygenases(LPMO)are a class of copper metal-dependent enzymes capable of cleaving crystalline polysaccharides(such as cellulose or chitin)in the presence of electron donors;it could synergistic with cellulase significantly and improve the hydrolysis efficiency of cellulase,with important economic value and application prospects.1.The LPMO gene sequence of Talaromyces atroroseus IBT 11181 strain was screened from the NCBI database,and the company successfully synthesized a gene fragment encoding LPMO.The gene was ligated to the pET-28a(+)vector by using EcoRl and Hind111 as restriction sites to construct a recombinant expression vector.The enzymatic analysis showed that the optimum pH of the LPMO was 5.0 and the optimum reaction temperature was 50?.2.Based on the measured enzymatic conditions,the fermentation conditions of the recombinant bacteria were studied.In terms of optimization of medium conditions,the optimum carbon source and nitrogen source are glucose and peptone,respectively.The optimum concentration is 10 g/L,the optimum phosphate addition is 60 mmol/L,and the optimum Mg2+ concentration is 0.25 g/L;optimization of the culture environment,the optimal inoculum volume of the culture solution is 2%,and the optimum liquid volume in the 250 mL shake flask is 40 mL;the optimum induction temperature for inducing the expression of the LPMO is 16?,The IPTG concentration was 0.08 mmol/L.When the cellulose is degraded by the same amount of cellulase under optimum conditions,the amount of reducing sugar is increased by 60%compared to the hydrolysis of the single cellulase.