Recombinant Expression Of Xylanase Inhibitor RiceXIP And Its Competitive Inhibition On Catalytic Activity Of GH 11 Endo-xylanase

Rice xylanase inhibiting protein?riceXIP?,is one of the XIP-type xylanase inhibiting proteins in rice,which has been discovered in recent years.This xylanase inhibiting protein,riceXIP,expressed by the gene of ricexip have different inhibiting effect on GH 11 family endo-xylanases.In this paper,ricexip,which was cloned from Oryza sativa,was successfully expressed in Escherichia coli BL21?DE3?and Pichia pastor GS115,respectively.The inhibitory effect of the recombinant protein on the catalytic activity of GH 11 endo-xylanase was systematic researched and the interaction mechanism between them was preliminarily discussed using the fluorescence spectrum,circular dichroism and molecular docking simulation.In addition,the interaction effect of riceXIP and xylanases was detected by yeast hybrid system.1.The gene,ricexip,was amplified by PCR using the gene of Oryza sativa as template.It's size was 873 bp,containing 290 amino acid residues,and the N-terminal of the sequence contains a signal states coded by 28 amino acid.The protein molecular weight was 32.0 kDa.The ricexip was ligated to the pCold TF vector,transformed into E.coli BL21?DE3?and formed the E.coli engineering bacteria,ColdXIP-5.ColdXIP-5 was induced by IPTG at 15°C for 24 h and then the reEriceXIP was secreted into the culture medium and retained in the cell cytoplasm.The purification of reEriceXIP was using Ni²⁺affinity chromatography.The SDS-PAGE analysis of reEriceXIP showed the size was 87.8 kDa.reEriceXIP has inhibitory effect on 11 kinds of endo-xylanases and the inhibitory effect was better when reEriceXIP interacted with TfxA_CD214,reBaxA454 and TfxA_CD526.The optimum reaction conditions of reEriceXIP on the TfxA_CD214,reBaxA454 and TfxA_CD526 occurred at 40°C for 30 min.The results of inhibitory kinetics of reEriceXIP showed that the K_m of TfxA_CD increased gradually,while V_maxax remained unchanged with the increase of inhibitory protein dose.So the inhibition between inhibitor and xylanase was competitive and K_i was 7.0 nM.HPLC-ELSD assay showed that the hydrolysis of beechwood xylan reduced when reEriceXIP incubated with xylanases?TfxA_CD214,reBaxA454 and TfxA_CD526?.But the type of hydrolysis product was not changed.2.The ricexip was ligated to the pPICZ?A vector,transformed into P.pastoris GS115 and formed the P.pastoris engineering bacteria,PPricexip1.PPricexip1 was induced by MeOH at 30°C and the concentration of supernatant and the concentration of bacterial OD_600nm reached the maximum after inducted 144 h.rePriceXIP was secreted into the culture medium and it was purified using Ni²⁺affinity chromatography.The SDS-PAGE analysis of rePriceXIP showed the size was 44.0kDa.rePriceXIP has inhibitory effect on 11 kind of endo-xylanases and the inhibitory effect was better when rePriceXIP interacted with TfxA_CD214,reBaxA454 and TfxA_CD526.And the optimum reaction conditions of rePriceXIP on TfxA_CD214,reBaxA454 and TfxA_CD526 occurred at 40°C for 30 min.The results of inhibitory kinetics of rePriceXIP showed that K_m increased gradually,while V_max remained unchanged with the increase of inhibitory protein dose.So the inhibition between inhibitor and xylanase was competitive and the K_i was 12.3 nM.3.The interaction mechanism between riceXIP and xylanases?TfxA_CD214,reBaxA454 and TfxA_CD526?were analyzed by fluorescence spectrum and circular dichroism,respectively.The fluorescence spectrum showed that it is static quenching that exsist in the inhibitor-xylanase.It is speculated that there is one binding site between inhibitory proteins and those three xylanases,and the binding effect works through hydrogen bond.Structures of riceXIP and BaxA were determined by a molecular replacement method with the program SWISS-MODEL.The simulation result of molecular docking between riceXIP and BaxA by ZDOCK SERVE showed that the?47?-shaped loop?L?₄?₅?of riceXIP inserted into the groove of xylanase and there are hydrogen bonds between R148 of riceXIP and the sites?E78,E172,N35 and Y80?of BaxA.4.Rice xylyase inhibitor gene,ricexip,ligated to pGBKT7 vector and transformed into AH109.The xylanase gene?tfxa_cd,tfxa_cd214,tfxa_cd526,baxa and baxa454?were ligated to the pGADT7 vector and transformed into Y187.Then the two yeast cells were hybridized to observe the colony growth.The results showed that there are interaction between inhibiting protein expressed by ricexip and xylanases.The interaction strength order was:riceXIP/TfxA_CD526>riceXIP/TfxA_CD214>riceXIP/TfxA_CD and riceXIP/reBaxA454>riceXIP/reBaxA respectively.