Expression Of Ferulic Acid Esterase,Xylanase And Cellulase From Anaerobic Fungus And Study On Their Enzymatic Hydrolysis Interactions

The effective utilization of agricultural by-products such as straw can supplement the demand of animal husbandry feed,but the utilization efficiency of straw is not high enough.As one of the ways to degrade straw,enzymatic hydrolysis can make the best use of straw,but its low efficiency and high cost make it unable to be widely used in production.High efficient crude fiber degrading enzymes have always been a research hotspot,most of which come from fungi.After previous studies,it was found that when anaerobic fungi degraded crude fiber and secreted a large amount of cellulase and xylanase,ferulic acid esterase also had high expression.So how does ferulic acid esterase affect the enzymatic hydrolysis efficiency of cellulase and xylanase?In this study,ferulic acid esterase,cellulase and xylanase from anaerobic fungi were selected and expressed in prokaryotic and eukaryotic cells,respectively.Purify and explore their enzymatic properties,and one of the three enzymes with the best activity was selected.To explore the effect of ferulic acid esterase on the enzymatic hydrolysis efficiency of xylanase,and the effect of ferulic acid esterase on the degradation efficiency of xylanase and cellulase composite enzyme,so as to provide reference for the development of auxiliary enzyme to improve the degradation efficiency of crude wood fiber.1 Expression and screening of ferulic acid esterase genes from anaerobic fungiSix ferulic acid esterases from anaerobic fungi were named ferulic acid esterase N.1,N.3,N.6,N.7,N.8 and N.9.The gene sequence was synthesized according to the preference of the expression system.Prokaryotic E.coli BL21（DE3）used p ET-28a vector,eukaryotic P.pastoris GS115 used p HBM905BDM vector,purified protein,determined the enzyme properties.The results show that:ferulic acid esterase N.1,N.7 and N.9 were successfully expressed and active in E.coli,ferulic acid esterase N.6 expressed in E.coli has no activity,ferulic acid esterase N.3 and N.8 expressed in E.coli as inclusion bodies.P.pastoris successfully expressed ferulic acid esterase N.1,N.7 and N.9 with activity,no P.pastori was screened to express ferulic esterase N.3,N.6 and N.8.The optimal p H of ferulic acid esterases were 7;the optimal temperature of ferulic acid esterase N.1,7 and 9 expressed in E.coli were 37℃,45℃and 37℃,respectively,and the optimal temperature of ferulic acid esterase N.1,7 and 9 expressed in P.pastoris were 45℃.The p H stability and temperature stability of ferulic acid esterases expressed by P.pastoris were better than those expressed by E.coli.Ferulic acid esterase N.1 has the best p H stability,and the temperature stability of N.1 was slightly lower than ferulic acid esterase N.7.Ca²⁺significantly increased the activity of ferulic acid esterase N.1 and N.7 expressed in P.pastoris（P<0.05）,while Ca²⁺had no significant effect on the activities of ferulic acid esterase N.1 and N.7 expressed in E.coli（P>0.05）.The specific activity of ferulic acid esterases expressed by P.pastoris was higher than that expressed by E.coli,and the highest V_max of N.1 was 96.26 U/mg,which indicated that ferulic acid esterase N.1 expressed by P.pastoris was the best enzyme.2 Expression and screening of xylanase genes from anaerobic fungiXylanase C,D and F from rumen anaerobic fungi synthesize gene sequences according to the preference of the expression system.Prokaryotic E.coli BL21（DE3）used p ET-28a vector,eukaryotic P.pastoris GS115 used p HBM905BDM vector.The proteins were expressed,purified and the enzymatic properties were determined.Then a xylanase with the highest activity was obtained.Xylanase C,D and F were successfully expressed in the two systems.The results showed that:the optimal temperature of xylanase C,D and F were 45℃,37℃and 45℃,respectively;the optimal p H of xylanase C,D and F expressed in E.coli were 7,7 and 6,respectively;the optimal p H of xylanase C,D and F expressed in P.pastori were 6,7 and 6,respectively.The p H stability and temperature stability of xylanase C,D and F expressed in the two expression systems were similar,and the p H stability and temperature stability of xylanase C was the highest,maintained more than 80%activity after 24 h incubation at 37℃.Ca²⁺and Mg²⁺promoted the activity of xylanases in varying degrees（P<0.05）,while Cu²⁺inhibited the activity of xylanases in varying degrees（P<0.05）.Three kinds of xylanases had high degradation ability to beech xylan.Compared with E.coli,P.pastoris increased the activity of xylanases.The results showed that the activity of xylanase F expressed by P.pastoris was the best.3 Expression and screening of cellulase genes from anaerobic fungiTwo cellulase 8 and 9 from rumen anaerobic fungi synthesize gene sequences according to the codon preference of the expression system.Prokaryotic E.coli BL21（DE3）used p ET-28a vector,eukaryotic P.pastoris GS115 used p HBM905BDM vector,purified protein,determined the enzyme properties and then obtained a cellulase with the highest activity.The results showed that the cellulases expressed in E.coli had no activity,while the cellulases 8 and 9 with biological activity were successfully expressed in P.pastoris.The optimal temperature of cellulase 8 and 9 were 37℃and 45℃,respectively,and the optimal p H was 7.The two cellulases had high stability at low temperature such as 28℃and 37℃,and retained more than 80%activity after 24 h incubation;cellulase 8 had better p H stability than 9,and retained more than 80%activity after 24 h incubation at p H=6.Ca²⁺,K²⁺and Zn²⁺promoted the activity of cellulase 8,Mg²⁺,Mn²⁺and Cu²⁺significantly inhibited the activity of cellulase 8（P<0.05）.Na⁺,K²⁺and Mg²⁺significantly increased the activity of cellulase 9,and Zn²⁺,Cu²⁺and Fe²⁺significantly inhibited the activity of cellulase 9（P<0.05）.Cellulase 8 has higher degradation ability to CMC-Na and fucoidan,cellulase 9 has more extensive degradation ability to CMC-Na,fucoidan,soluble starch and PNPG.The V_max of cellulase 8 and 9 were 23.23 U/g and 31.33 U/g,respectively.These results indicated that the activity of cellulase 9 expressed by P.pastoris was the best.4 Effect of ferulic acid esterases on enzymatic hydrolysis efficiency of cellulases and xylanasesIn this study,the best activity of ferulic acid esterase,xylanase and cellulase were selected in the early stage.Different proportions of ferulic acid esterases were added to xylanases and cellulases to explore the changes of substrate degradation efficiency.To explore the effect of different proportions of cellulase and xylanase on straw degradation efficiency.To add 10%ferulic acid esterases to the compound enzyme with the highest enzymatic hydrolysis efficiency,to explore the effect of ferulic acid esterase on straw degradation efficiency of compound enzyme.The results showed that:compared with xylanase without ferulic acid esterase,only 10%ferulic acid esterases could significantly improve the degradation efficiency of xylan（P<0.05）,and the other proportions（20%,30%,40%）could significantly reduce the degradation efficiency of xylan（P<0.05）.The addition of ferulic acid esterases in cellulases could not improve the degradation efficiency of cellulose,and with the increasing of ferulic acid esterases addition ratio,the degradation efficiency of cellulose gradually decreased.When 5%and 10%xylanases were contained in cellulases,the degradation efficiency of rice straw and corn stover was significantly improved（P<0.05）.When the substrate was corn stalk,only 5%xylanases in cellulases could significantly improve the degradation efficiency of substrate（P<0.05）.Adding 10%ferulic acid esterases in composite enzyme could significantly improve the degradation efficiency of rice straw and corn straw（P<0.05）.The results showed that ferulic acid esterase could significantly improve the degradation efficiency of xylanase and cellulase.In conclusion,compared with E.coli,P.pastoris can improve the activity and stability of FAE,xylanase and cellulase.Adding 10%ferulic acid esterases to xylanase can significantly promote the degradation efficiency of xylanase to xylan;When the ratio of xylanase and cellulase is compound enzyme,the degradation efficiency of compound enzyme to different types of straw is different,and the optimal ratio is also different;Adding 10%ferulic acid esterases to the complex enzyme can significantly improve the straw degradation efficiency of the complex enzyme.