Cloning And Expression Of Straw Degradation Strains Trichoderma Aureoviride Xylanase Gene

Xylan is the major component of hemicellulose,and hemicellulose is the second most abundant resources in nature in addition to cellulose.Xylanase can degrade the Xylan that abound in nature. It has important application prospect and commercial value in papermaking、textile、food 、 feed 、 energy industry and so on,and the different sources of xylanase have different degradation characteristics,therefore,it is necessary to study on the different sources of xylanase.In this study, use the genetic engineering to clone a unknow Trichoderma aureoviride xylanase gene,and construction of engineering strain with high yield xylanase,and to study the physicochemical properties,enzymatic properties,kinetic constant and so on of the xylanase.The main results of this research are as follows:1.According to the information of conservative sequence on the Gen Bank of Trichoderma viride 、 Trichoderma reesei and Trichoderma asperellum xylanase gene,design Trichoderma aureoviride xylanase gene cloning primers P1 and P2 by using the Primer5.0 software,and successful clone a coding sequence of an unknown Trichoderma aureoviride xylanase gene,and submitted to the NCBI database,named as TAX1 gene.Accession No.KM076643,and filled the blank of International.2.Using RT-PCR method,successful clone a c DNA sequence of Trichoderma aureoviride xylanase gene,through the comparison of c DNA sequence and DNA sequence,determine that TAX1 gene has a intron the size of a 114 bp.3.Analyzed the sequence of xylanase protein,and predicted the protein physicochemical properties and signal peptide and conserved regions of the protein,the number of this protein is:AIG72020.1,and speculated the molecular weight of this protein is 24.2044 KD,the oretical isoelectric point is 6.82,the molecular formula is C1086H1602N292O339S1,unstable coefficient is 17.81,this protein is a kind of stable protein.And it is carried on the SDS-PAGE analysis and enzyme activity electrophoresis.Get a specific protein bands about the size of 24 KD,and the time of the highest enzyme activities is the fifth day.4.Create a expression vector TAX1-p PIC9 K by TAX1 gene in Pichia pastoris,and the recombinant vector was transformed into Pichia pastoris strain of GS115,the recombinant pichia pastoris was induced and expressed,by using the real-time fluorescence quantitative PCR to analysis the relative quantitative of expression,when 4 days,the relative expression quantity reached the highest medium by methanol induction.5.The recombinant xylanase was make to the enzymatic characteristics analysis,and the optimal reaction temperature of the xylanase is 50℃,it has a better stability in the 40 ℃ to 60 ℃ of temperature range.The optimal p H of the xylanase is 6.0,the xylanase has good stability between p H4.0-6.0,show that the xylanase is an acidic protein.The optimum substrate is xylan,while Xylanase also has a better degradation ability pretreatment to the straw.When xylan as substrate,the xylanase:Vmax=44.25mg/ml · min,Km=0.36mg/ml,Kcat=Vmax/E=0.204S-1,Kcat/K m=0.57ml/S·mg.6.The xylanase enzyme activity of the recombinant pichia pastoris was measured,and to know that the optimum enzyme production time is 5 days,the optimal p H is 6.0.At this point,the enzyme activity of the recombinant pichia pastoris reached 215.28IU/ml.In the same conditions,to compare the xylanase activity with the original strain, xylanase activity of recombinant pichia pastoris was 3.49 times of that of the original strain.