Study On The Mining And Synergy Of Hemicellulose-degrading Enzyme System And Its Application In Improving Feed Utilization

Hemicellulose is the main component of plant cell walls and is the second most abundant biomass in nature.It is widely used in food,feed,bioenergy,pulp and paper industries.Its efficient degradation and utilization has always been the main focus of the industry.Due to the complex structure of hemicellulose,it could be only partially degraded by ?-xylanase and ?-mannanase,its complete degradation required a series of carbohydrate hydrolases.It is necessary to deeply study the enzymes that degrade the products produced by ?-xylanase and ?-mannanase and degrade the side chain of hemicellulose,including ?-L-arabinofuranosidase,feruloyl esterase,acetylxylan esterase and ?-galactosidase,?-xylosidase and ?-mannosidase.The purpose of this study is to explore the hemicellulose-degrading exo-glycosidase and side-chain degrading enzymes that have the potential to be used in the feed industry based on the complex chemical structure of hemicellulose in feed materials.A hemicellulose-degrading enzyme system that efficiently degrades hemicellulose in feed would be selected by studying the synergy between hemicellulose degrading enzymes.The multi-enzymes system will use to improve the utilization of feed ingredients and alleviate the shortage of feed ingredients.The main research contents and conclusions are as follows:(1)Without further degradation by xylosidase,xylan can only be degraded into oligosaccharide products by ?-xylanase.In order to degrade xylan into monosaccharides,six ?-xylosidases from thermophilic microorganism were recombinantly expressed in Pichia pastoris in this study.Among them,?-xylosidases(Hixyl43A,Tlxyl43 A,Ptxyl43A and An Xln D)from H.insolens,T.lanuginosus,P.thermophila and A.niger were high expression with the levels of 537.4 U/m L,354.7 U/m L and 445.2 U/m L and 900.9 U/m L,respectively.Recombinant Hixyl43 A,Ptxyl43A,Tlxyl43 A and An Xln D and ?-xylanase Xyn11A(from N.patriciarum)have a synergistic effect on the hydrolysis of wheat bran.Compared with the hydrolysis by Xyn11 A lonely,the release of xylose during the hydrolysis process was increased by 101%,98%,97%,and 18%,respectively.Comprehensively comparing recombinant expression,enzymatic properties and synergistic effects,Hixyl43 A has the most potential for application in the feed industry.(2)The arabinofuran side chain of xylan hinders the degradation of hemicellulose by ?-xylanase.In this study,six ?-L-arabinofuranosidases were codon-optimized and recombinantly expressed in P.pastoris and ?-L-arabinofuranosidases with high activity and excellent stability were screened for hydrolysis of substituents of ?-L-arabinofuranoside residues in hemicellulose.Among them,the ?-L-arabinofuranases from H.insolens(Hiabf43B)and B.adolescentis(de Ax)were gained good yields of 3.69 g/L and 3.89 g/L,and displayed the maximum activties of 73.1 U/m L and 38.7 U/m L in 7 L fermentor,respectively.The wheat bran was hydrolyzed by the combination of Hiabf43 B,de Ax,Xyn11 A and Hixyl43 A,showing a significant synergistic effect,and the relaese of xylose were increased by 56.2% compared with the absence of ?-L-arabinofuranoside.(3)In order to further improve the degradation efficiency of xylan,this study excavated feruloyl esterase and acetyl xylan esterase to remove ferulic acid substitution and acetyl substituents from the xylan.Among them,the feruloyl esterase from A.niger(An Fae A)and acetyl xylan esterase from T.purpureogenus(Tp AXEI)were codon-optimized and expressed in P.pastoris with 12.63 g/L and 10.70 g/L,and displayed the maximum activities of 190 U/m L and 450 U/m L in 7 L fermentor,respectively.The wheat bran was hydrolyzed by the combination of An Fae A,Tp AXEI,Hiabf43 B,de Ax,Hixyl43 A and Xyn11 A,showing a significant synergistic effect,and the release of xylose was increased by 9.8% and the reducing sugar increased by 10.8% compared with the absence of these two enzymes.(4)In order to improve the degradation efficiency of mannan in hemicellulose,a novel ?-mannosidase(Lrman5A),?-mannanase(Lrman5B)and ?-galactosidase(Lr Agal36A)were identified from L.ramosa in this study.Lrman5 A,Lr Man5 B and Lr Agal36 A were highly expressed in P.pastoris with the level of 4.30 g/L and 27.9 U/m L,8.36 g/L and 8228 U/m L,and 4.36 g/L and 953.6 U/m L in 7 L fermentator,respectively.The recombinant enzymes displayed excellent thermal stability and had broad-range p H stability.The synergistic hydrolysis galaco-mannan by Lr Man5 B,Lrman5A and Lr Agal36 A can significantly increase the release of mannose and reducing sugars,which improve the degradation efficiency of hemicellulose.(5)Adding the hemicellulose degrading enzyme system selected in this study to the feeding diet of weaned piglets,on the basis of conventional hemicellulase,the DMD of feed increased by 6.16%,and the ADG of piglets increased 4.6%,the FCR to meat decreased by 4.2%,and the diarrhea rate decreased by 61.7%.The results demonstrated that the addition of hemicellulose side chain degrading enzymes and exo-glycosidases to the feed of weaned piglets can increase feed utilization,improve animal growth performance.It provides new ideas for the development of the next generation of feed enzymes.