Cloning And Heterologous Expression Of Xylanase Gene From Lentinula Edodes And Its Effect On Rumen Fermentation In Vitro

Hemicellulose is a polysaccharide which is second only to cellulose in the elevent of plant cell wall and its preponderant constituent is xylan.Xylanase is essential for the degradation of xylan into monosaccharides and oligosaccharides.At present,although profuse xylanase genes had been cloned and expressed,their application was still limited by many factors,such as p H adaptability and temperature stability.Our previous research suggested that Lentinus edodes had a powerful ability to degrade lignocellulose and was an ideal source for studying xylanase.In this research,a novel xylanase gene was cloned from the L.edodes and we were succeed to express it by Pichia pastoris X33.The enzymatic properties of recombinant L.edodes xylanase(rLeXyn11A)and its effect on the hydrolysis and in vitro rumen fermentation of agriclitural straws were studied in present research,which supplied theoretical support for the application of rLeXyn11A in production.Experiment 1:The meaning of this experiment was to clone novel xylanase gene from L.edodes by molecular biological methods.Using the c DNA of L.edodes prepared in laboratory as template,the primers were devised to clone the xylanase gene of L.edodes,and a xylanase gene of L.edodes was obtained.Experiment 2:The meaning of this experiment was to heterologously express rLeXyn11A and to study its enzymatic properties.The p PICZ?A-Xyn was constructed and heterologously expressed by P.pastoris X33.The enzymatic properties of rLeXyn11A were investigated using oat xylan as material.The optimum function temperature and p H of rLeXyn11A were 40?and 4.0,respectively,with high temperature resistance and good temperature stability.The activity of rLeXyn11A was promoted by 5 m M Zn²⁺.Experiment 3:The effect of rLeXyn11A on straw hydrolysis was studied.Rice straw,wheat straw and corn straw were chose for hydrolysis test.8?g purified rLeXyn11A was added in the treatment group,and water of same volume was injected into the control group.The amount of releasing sugar was detected after 24h incubation under the optimal conditions.The results of xylose suggested that rLeXyn11A treatment increased the releasing sugars produced with rice straw,wheat straw,and corn straw by 28.9%,16.2%and 24.3%,respectively,contrasted with the control group,and significantly stimulated the hydrolysis of agriculture straws.Experiment 4:The experiment on the influence of rLeXyn11A on in vitro rumen fermentation with agricultural straw.Rice straw,wheat straw and corn straw were selected for hydrolysis test.200?g purified rLeXyn11A was injected with treatment group,and the control group was added with the same amount of buffer,and collecting samples for parameters'determination after incubation at 39?for 48 h.The results of in vitro rumen fermentation revealed that rLeXyn11A significantly increased IVNDFD,the production of synthetic microbial protein,the concentrations of acetic acid,total VFA,and the NH₃-N in the fermentation system of rice straw,wheat straw,and corn straw.High throughput sequencing and real-time PCR data suggested that fermentation substrates controled the effects of rLeXyn11A on ruminal microbial community.With wheat straw,rLeXyn11A increased the relative abundance of representative fibre-degrading bacteria(F.succinogenes,R.flavefaciens,and R.albus).With rice straw,rLeXyn11A increased the relative abundance of fibrolytic bacteria including Firmicutes,Desulfovibrio,Ruminococcaceae and Fibrobacter succinogenes.With corn straw,the fibrolytic bacteria Firmicutes,Christensenellaceae?R?7?group,Saccharofermentans,and Desulfovibrio were increased by rLeXyn11A.The r Llexyn11A can improve the hydrolysis of agricultural straw and in vitro rumen fermentation by increasing the digestion of hemicellulose,changing the formation of microbial communities,fostering the growth of fiber degradation and the generation of VFA,and promoting the synthesis of FLMCP.In this study,rLeXyn11A obained from L.edodes had excellent enzymological properties and its effects on straw degradation and in vitro rumen fermentation were preliminarily researched.This study enriched xylanase resources and provided theoretical support for the application of rLeXyn11A in production.