Recombinant Expression Of Rice Xylanase Inhibitor(RIXI)and Its Inhibitory Activity On Family 11 Xylanases

?-D-1,4-xylanase?EC 3.2.1.8?is one of important enzymes in xylan degradation,applied widely in industry.Recently,studies found that feed cereals were proved to contain proteinaceous xylanase inhibitors,which decrease the practical effect of xylanases,and application effect.This study cloned rice xylanase Inhibitor gene,rixi,and expressed in Escherichia coli BL21?DE3?and Pichia pastoris GS115.The inhibitory activity of recombinant inhibitor on different sources xylanases was assayed.The main results are as follows:1.The rixi gene was cloned and expressed in E.coli BL21?DE3?.The rixi gene was amplified by PCR using rice genome as template.The open reading frame of rixi?GenBank Accession number:KY807992?was 915 bp,which encoded 304 amino acids.The predicted molecular weight of the deduced protein was 33.9 kDa.The rixi gene was inserted into pCold TF vector and transformed into E.coli BL21?DE3?.After induction with IPTG at 15�C for 24 h,recombinant protein reERIXI,both exist in the cell and the medium.The reERIXI was purified by high affinity Ni-charged resin.Under the control of promoter lac of pCold TF,the recombinant protein was bound with the Trigger Factor,whose molecular mass was 54 kDa.The molecular mass of reERIXI was about 89.8 kDa based on the results of SDS-PAGE and Western Blot.Inhibitory activity test by DNS method indicated that reERIXI could inhibit catalytic activity of many family 11 endoxylanases.The inhibitory rate of TfxA_CD214 and reBaxA50 by reERIXI was approximately 35.13%,61.98%,respectively.When the interaction time was over 30 min,the inhibitory rate of reERIXI on TfxA_CD214 and reBaxA50 were the best with the mass ratio at 1:1and 2:3,respectively.The optimal temperature of reERIXI inhibitory activity on reBaxA50 and TfxA-CD214 were 60�C,and 50�C,respectively.ReERIXI showed high thermostability.2.The rixi was inserted into pPICZ?A vector and expressed in Pichia pastoris GS115.The recombinant pPICZ?A-rixi plasmid was transformed into P.pastoris GS115 cells.The positive transformant PPrixi9 was induced by methanol at 30�C for 72 h.The recombinant inhibitor,rePPRIXI,was purified by high affinity Ni-charged resin.The SDS-PAGE and Western Blot indicated that the molecular mass of rePPRIXI was 47.0 kDa.Inhibitory activity test by DNS method indicated that the inhibitory rate of TfxA-CD214 and reBaxA50 was 30.09%,55.54%,respectively.After reaction for40 min,mass ratio of rePPRIXI with TfxA_CD214 and reBaxA50 at 1:3 and 1:2,the inhibitory activity was high.3.The fluorescence spectroscopy and circular dichroism spectrum were used to study spectral characteristics of interaction between recombinant inhibitor and xylanases.The results showed that inhibitor quenched the fluorescence of xylanases?TfxA_CD214 and reBaxA50?through a static process.For inhibitor reERIXI5,the binding constant K_A of TfxA_CD214 and reBaxA50 were 8.081�10^-3(L�?mol^-1)and 1.833�10^-1(L�?mol^-1),and the binding sites were 0.70649 and1.35748,respectively.For inhibitor rePPRIXI9,the binding constant K_A of TfxA_CD214 and reBaxA50 were 1.485�10^-1(L�?mol^-1),1.846�10^-1(L�?mol^-1),and the binding sites were 1.05085 and 0.86621 respectively.The results demonstrated that there was weak interaction between inhibitor and xylanase.CD results suggestion that the secondary conformational changes of reBaxA50 and TfxA_CD214 with recombinant inhibitor,the secondary structure of xylanases were loosened,which indicated that the main driving force of the xylanase protein binding with reERIXI may be hydrogen bond.4.The effect of reERIXI on the hydrolysis product of xylan was studied by high performance liquid chromatography-evaporative light scattering detector?HPLC-ELSD?.The hydrolysis products of beechwood xylan by TfxA_CD214were X2-X5.After interaction with reERIXI,the concentration of hydrolysis products decreased without modifying the type of hydrolysis products.Hydrolysis products of beechwood xylan by reBaxA50 were X2-X6.After interaction with reERIXI,the concentration of hydrolysis products decreased without modifying the kinds of hydrolysis products..5.Yeast two-hybrid system was used to explore the interactivity level between the inhibitory protein and the enzyme.The fusion yeast was constructed and hybridized,then culture and observation of colony production in defect medium.The interaction level was measured by detecting the activity of beta galactosidase.The results showed that there was a interaction between RIXI inhibitor and xylanases.The strength was reBaxA50>reBaxA>TfxA_CD214>TfxA_CD in turn,which coincide exactly with the results of DNS assay.It can be concluded that the inhibitory activity of RIXI on Bacillus amylosus xylanase was higher than that of truncated Thermomonospora fusca xylanases.Sensitivity of mutant xylanases?TfxA_CD214 and reBaxA50?to recombinant RIXI was higher than that of wild type.