Cloning,Expression And Characterization Of Lytic Polysaccharide Monooxygenase Gene PdLPMO9A From Pleurotus Djamor

In recent years,enzyme conversion of biomass rich in crystalline carbohydrates such as cellulose in straw as energy and other uses has been a research hotspot.However,it has proven difficult to establish an enzyme system with satisfactory efficiency.The newly discovered Lytic Polysaccharide Monooxygenase（LPMO）can oxidatively cleave crystalline polysaccharides and enhance the activity of traditional hydrolases,opening up a new way for enzymes to convert biomass.In this study,cloning and se-quence analysis of Pd LPMO9A,a cleavage polysaccharide monooxygenase gene of Pleurotus djamor,constructed the mature peptide sequence of Pd LPMO9A into an ex-pression vector,and transformation by electroporation into Pichia pastoris.The Pd LPMO9A recombinase was induced and expressed by methanol,and the enzyme was preliminarily purified by means of ultrafiltration,salting out,and dialysis.Character-ize the enzymatic properties and study its synergistic effect with cellulase.The main results are as follows:1.The c DNA sequence of Pd LPMO9A gene was cloned,the total length is 966 bp.Bioinformatics analysis of its sequence revealed that its molecular weight is 33.24 k Da,isoelectric point is 5.06,contains signal peptide sequence,CBM domain and GH61glycoside hydrolase domain,and the protein sequence is related to the related sequence of Pleurotus ostreatus recent.2.The mature peptide recombinant expression vector was constructed and trans-ferred to the Pichia pastoris GS115 strain to obtain the genetically engineered strain.Through methanol-induced expression,the optimal expression conditions were deter-mined:the amount of methanol added was 1.5%,the p H was 6.0,and the time was 120h.At this time,the enzyme activity reached 10.857 U/L,and the molecular weight of the enzyme protein was 43 k Da.3.The enzymatic properties of recombinant Pd LPMO9A were characterized,and its optimal temperature and p H value were determined,which were 60℃and 6.0,re-spectively,and had good stability at a temperature of 50℃and a p H of 6.0.It shows enzymatic activity on the substrates CMC-Na,Avicel and filter paper,and has the best degradation effect on Avicel.A certain concentration of metal ions such as Cu²⁺can promote enzyme activity,and 0.1 m M Cu²⁺has the best effect on promoting enzyme activity.4.Using Avicel and corn stover as substrates,it was found that recombinant Pd LPMO9A can promote the degradation of the substrate by cellulase and has a syner-gistic effect.The addition of ascorbic acid will promote the positive synergistic effect of Pd LPMO9A and cellulase on Avicel.The addition of Cu²⁺can significantly promote the positive synergistic effect of Pd LPMO9A and cellulase in the degradation of corn stover.