The Cloning, Expression Of Xylanase Gene From Aspergillus Niger F19 In Escherichia Coli And Improvement Of Its Thermostability

Xylan is the most abundant renewable polysaccharide fibers in nature secondary only to cellulose,which is mainly found in the plant cell walls.Different xylans have different origins,branches and there are many substitute groups on the backbone and side chains. For the complexity of the structure of the xylan,the degradation of xylan needs many kinds of hydrolases.Xylanases(EC 3.2.1.18) can catalyze the hydrolysis of internalβ-1,4 bonds of xylan into xylooligosaccharides and D-xylose,therefore,xylanase is the key hydrolases for the degradation of xylan.In this present dissertation,xylanase gene xynA and xynB were cloned and expressed in E.coli,and the purification and characterization of recombinant XynB were investigated,using Aspergillus niger F19 as the experimental material.After this,we tried to improve the thermal stability of the recombinant XynB,the results showed that:1.The cloning,expression of xynA and xynB gene in E.coliThe xynA and xynB gene were isolated from the genomic DNA of A.niger F19, cloned and expressed in E.coll.Two pairs of primers were designed according to the reported full sequences of xynA and xynB gene from A.niger in GenBank.xynA and xynB gene fragments were successfully amplified by PCR reactions under the suitable conditions.The xynA and xynB gene without origin signal peptide sequences were fused into the E.coli expression vector pET-28a(+) with correct open reading frame,then transfered into E.coli BL21,finally the recombinant strain BLX1 and BLX2 were obtained.After induced by IPTG,the xynA gene was successfully expressed in the recombinant strain BLX1;the expression product existed as inclusion bodies and intracellular soluble form,and xynB gene in BLX2 as intracellular soluble form.2.Purification of XynBMost of the product of recombinant XynB existed as the intracellular soluble form, and the xynB gene was expressed in pET-28a(+),therefore the C-terminal of the XynB has 6×His protein tag,thus we purified the XynB with the Ni-NTA affinity column.The purity of recombinant XynB was fine and a single band was founded in the SDS-PAGE analysis.3.The characterization of XynBThe purified XynB had a K_m of 12.5±0.08 mg/mL,a V_max of 289±10.8μmol/min/mg and a specific activity of 13500±128.4 IU/mg,using oat spelt xylan as a substrate.The optimal pH of XynB was 5.0,it sustained above 75%of the enzyme activity of XynB among the pH from 4.0 to 7.0 using the purified XynB for assay.The XynB showed high pH stability in room temperature for 6h among the pH from 3.0 to 8.0,more than 68%of the enzyme activity remained.The optimal temperature of XynB was 50℃,the recombinant XynB showed low enzyme activity at the temperature above 60℃or below 30℃.The thermal stability of XynB is low,and the residual activity of the XynB after incubated at 60℃for 30 min is 20%.It was stable at the temperature below 40℃.The effect of 10 mM/L metallic ions on XynB showed that Zn²⁺ and Co²⁺ activated the recombinant xylanase by 8%and 15%,respectively.The Mn²⁺ restrained the XynB by 21%,and other metallic ions showed no effect on it.4.The improvement of thermo-stability on XynBXynB had a high specific activity but low thermo-stability,so it's difficult to put it into industrial application.To improve the thermal stability of XynB,two site-directed mutagenesis strategies were applied.The first one was to substitute the surface serine and threonine into argnine.The second one was introduce the disulfide bridge into discatalytic domain of XynB.Then the mutated gene constructed to pET-28a(+) and transformed into BL21 for the expression of mutated XynB.However,inclusion bodies formed during the process of expression and no enzyme activity detected on the supernatant of cell disruption liquid.To determine the effect of the site-directed mutagenesis on the thermal stability of XynB,high excretive eukaryotic expression system of wild type and mutated type of XynB is constructing.Still,large-scale industrial application research of this enzyme is in progress.