The Influence To Enzyme Activity And Biochemical Properties Of Xylanolytic Enzyme By Fusion To Xylan Binding Domain

In nature,hemicellulose is the second largest source of biomass that second only to cellulose,its complete degradation requires the synergisitic action of many enzymes.This article mainly focused on the arabinoxylan.Xylan binding domain(XBD)have specific recognition of xylan and bind xylan as a non-catalytic domain.XBD can bind different enzymes of xylan degrading(xylanase and arabinosidase and so on)closely,thus enhance accessibility of substrate to play a better role in performing hydrolysis activity when degrading xylan.In this study,xylan binding domain from different sources were respectively fused to the xylanase and the a-L-arabinofuranosidase gene to construct recombinant enzyme,then expressed in E.coli.Compared to native enzyme,the catalytic properties of the chimeras were characterized.Finally the recombinant xylanases and a-L-arabinofuranosidase were used to evalutate the effect when hydrolyzing wheat arabinoxylan.The specific research content is list below.1.XBD2b was derived from Cellulomonas fimi xylanase 11A and XBD6 was derived from Clostridium thermocellum xylanase(CtXynGH30)by the method of gene synthesis.The XBD2b and XBD6 were fused to the C-terminus of xylanase gene to obtain fusion gene,then to express them in E.coli.The result was as follows:the enzymatic activity(107 U/mL)and specific activity(48 U/mg)of recombinant strain E.coli BL21(DE3)contain pET-20b-xynA-XBD2b increased 3.3-fold and 1.7-fold compared to Kcoli BL21(DE3)contain pET-20b-xynA(25 U/mL and 17.6 U/mg).The enzymatic activity(0.52 U/mL)and specific activity(0.21 U/mg)of recombinant strain Kcoli TOP 10 contain pHsh-xynB-XBD2b both reduced 99%compared to E.coli TOP10 contain pHsh-xynB(65.31 U/mL ? 28.4 U/mg).The enzymatic activity(7.79 U/mL)and specific activity(4.48 U/mg)of recombinant strain E.coli BL21(DE3)contain pET-28a-xynB-XBD2b decreased by 88%and 84.2%compared to E.coli TOP 10 contain pHsh-xynB.Also the activity(32 U/mL)and specific activity(12 U/mg)of recombinant strain E.coli BL21(DE3)contain pET-28a-xynB-XBD6 reduced by 45%and 49%compared to E.coli TOP 10 contain pHsh-xynB(58 U/mL and 23 U/mg),which indicated the fused of XBD2b had positive effect to the enzymatic activity of E.coli BL21(DE3)contain pET-20b-xynA-XBD2b.2.XBD were fused to the C-terminus of arabinofuranosidase gene to obtain fusion gene,then to express them E.coli.The result was as follows:the enzymatic activity(4.52 U/mL)and specific activity(2.1 U/mg)of recombinant strain E.coli TOP 10 contain pHsh-Tm-ara-XBD2b reduced by 50%and 49%respectively compared to E.coli TOP10 contain pHsh-Tm-ara(2.26 U/mL and 1.07 U/mg).The enzymatic activity(0.22 U/mL)and specific activity(0.12 U/mg)of recombinant strain E.coli TOP10 contain pHsh-BP-ara-XBD2b reduced by 93%and 82%compared to the enzymatic activity(3.29 U/mL)and specific activity(0.66 U/mg)of E.coli TOP10 contain pHsh-BP-ara.And the enzymatic activity(6.8 U/mL)and specific activity(5.1 U/mg)of recombinant strain E.coli BL21(DE3)contain pET-28a-Tm-ara-XBD6 increased 0.8 times and 1.04 times compared to E.coli TOP10 contain pHsh-Tm-ara(3.7 U/mL and 2.5 U/mg)respectively,which indicated the fused of XBD6 had positive effect to the enzymatic activity of E.coli BL21(DE3)contain pET-28a-Tm-ara-XBD6.3.The optimal conditions and stability of XynA-XBD2b and XynB-XBD6 were studied,and the recombinant xylanase that unfused XBD as a control.The results showed that the optimum pH,pH stability and temperature stability of XynA-XBD2b did not significant change compared to XynA,the optimum temperature of XynA-XBD2b reduced 5?.And the optimum pH of XynB-XBD6 dropped by 0.4 units compared to XynB;The optimum reaction temperature decreased by 5?;The pH stability of XynB-XBD6 decreased slightly when the pH was over 6.2;The temperature stability of XynB-XBD6 decreased significantly compared to XynB.4.The optimal conditions and stability of Tm-Ara-XBD2b and Tm-Ara-XBD6 were evaluated,and the recombinant arabinofuranosidase that unfused XBD as a control.The optimum pH,pH stability,the optimum temperature and temperature stability of Tm-Ara-XBD2b did not significantly change compared to Tm-Ara.While The optimal pH of Tm-Ara-XBD6 decreased by 0.4 units compared to Tm-Ara;The optimum reaction temperature was stable at 95? for Tm-Ara-XBD6 and Tm-Ara;The pH stability of Tm-Ara-XBD6 slightly reduced when the pH was alkaline;The temperature stability of Tm-Ara-XBD6 significantly reduced compared to Tm-Ara.5.The binding ability to insoluble arabinoxylan of XBD6 protein and the adsorption properties of fused xylanase and arabinosidase with XBD2b on insoluble arabinoxylan were investigated.The results found that XynA-XBD2b and XynB-XBD2b have certain adsorption capacity on insoluble arabinoxylan compared to XynA and XynB,which indicated that XBD2b played a crucial role in adsorption to insoluble xylan.Also,XBD6 had the ability to bind to insoluble xylan.6.The synergy between XynA-XBD2b and Tm-Ara-XBD2b to hydrolyze wheat bran arabinoxylan were studied.The results showed that the reducing sugar content of the synergy between XynA-XBD2b and Tm-Ara-XBD2b to degrade wheat bran arabinoxylan was higher than the synergy between XynA and Tm-Ara,which indicated that XBD2b played an important role in hydrolysis of wheat bran arabinoxylan.And the reducing sugar content increased with Tm-Ara-XBD2b increasing when hydrolysis of wheat bran arabinoxylan,which showed that Tm-Ara-XBD2b played a role in removal of steric effects by collateral arabinose.And the reducing sugar content of the synergy between Tm-Ara-XBD6 and XynB to degrade wheat bran arabinoxylan was higher than the synergy between Tm-Ara and XynB,the reducing sugar content of the synergy between Tm-Ara-XBD6 and XynA-XBD2b to degrade wheat bran arabinoxylan was higher than the synergy between Tm-Ara and XynA-XBD2b,which also indicated that XBD6 played an important role in hydrolysis of wheat bran arabinoxylan.