Gene Cloning,Heteroexpression And Characterization Of The Lytic Polysaccharide Monooxygenase From Aspergillus Oryzae

According to gene sequence of the LPMOs (GenBank:BAE61530.1), specific primers were designed and amplified by PCR to get lpmo (Lytic polysaccharide monooxygenases gene which is a fragment with length 1266bp). Than the gene was cloned to T vector and sequenced, the results showed that the gene was 100% compared to the reported genes.LPMOs gene Ipmo, vector pET32a and pPIC9K were digested by the same restriction endonuclease and transformed into E.coli DH5a to obtain the recombinant expression vector pET32a-lpmo (ZH-1) and pPIC9K-lpmo (ZH-2).The recombinant vector pET32a-lpmo and pPIC9K-lp/mo were transformed into E.coli BL21 and Pichia pastor is GS115 using chemical transformation and electroporation methods. After induced by methanol for 5 days, the Pichia pastoris GS115/pPIC9K-lpmo showed high activity.The pET32a-lpmo/BL21 could produce 600mg/L LPMOs after induced by IPTG (0.2-lmg/mL) for 16h.The optimum temperature for the expression was 16-28℃.The recombinant protein was purified by Ni affinity chromatography and it was showed that the molecular weight of recombinant protein was amount to 68 kDa after SDS-PAGE analysis.Based on the enzyme characterization study, the optimum reaction temperature of purified LPMOs for chitosan was 50 ℃.The optimum pH was 6.0.The metal ions could influence the LPMOs activity at some extent. The low concentration of Cu2+, Al3+, Fe3+ could promote recombinant LPMOs activity, however, the high concentration of Cu2+, Al3+, Fe3+ could inhibit the enzyme activity. Zn2+ had no influences on enzyme activity.This study indicated that LPMOs expression can be improved by constructing heterologous expression system. This work laid the foundation for our team to carry on the consistent research on industrial ethanol producing from cellulose materials degradation.