Expressing LPMOs Proteins Of Neurospora Crassa In Pichia Pastoris And The Application In The Degradation Of Lignocellulose

Lytic Polysaccharide Monooxygenase(LPMOs)are a class of cellulose degrading auxiliary enzymes that can break the polysaccharide chain and improve the hydrolysis performance of typical cellulase with the aid of electron donors such as cellobiose dehydrogenase(CDH).In this study two LPMO genes and one CDH gene from Neurospora crassa were transformed into Pichia pastoris.The enzymatic properties of recombinant LPMO-00760,LPMO-00836 and CDH were characterized,as well as their effect on the hydrolysis capacity of commercial cellulase cocktails.The LPMO-00760,LPMO-00836 and CDH genes of Neurospora crassa were confirmed successful expression in Pichia pastoris by gene sequencing,Western blot and specific substrate activity electrophoresis.The specific enzyme activity of recombinant LPMO-07760 and LPMO-00836 were 13.74 U/g and 7.85 U/g,respectively,and have optimum reaction temperatures at 35 ? and 40 ? respectively,and the optimum reaction pH was 7.5 and 8.0,respectively.Manganese ions and copper ions can increase their enzymatic activity.The recombinant CDH has an enzyme activity of 250 U/L,and has the best thermal stability between 30 and 40 ?,with an optimum pH of 5.5.Recombinant LPMO can effectively boost the property of the commercial cellulase cocktail Celluclast 1.5L.When microcrystalline cellulose was used as the substrate,the glucose yield increased by 5.58 and 5.83 times,respectively,after the addition of recombinant LPMO-07760 or LPMO-00836,compared to Celluclast 1.5L alone.When ascorbic acid or recombinant CDH was used as the electron donor,the hydrolysis conversion rate of the Zizania latifolia straw increased by 19% and 19.8%,respectively,after adding LPMO-07760 and increased by 15.6% and 20%,respectively,after the adding of LPMO-00836.Recombinant CDH is better as an electron donor than ascorbic acid.