Recombinant Expression Of Bacillus Amyloliquefaciens Xylanase A Gene In Pichia Pastoris And Its Directed Evolution

The ?-1,4-endo-xylanase(hereinafter referred as xylanase)is one of key enzymes for xylan hydrolysis and can be widely used in food,feed and paper industries.Most of coventional wild-type xylanases or recombinant xylanases showed low catalytic activities and poor stabilities,which were hard to meet industrial needs.This study cloned Bacillus amyloliquefaciens xylanase A gene(baxA)and got secretory expression in E.coli BL21(DE3)and P.pastoris GS115;in order to obtain mutants with more ideal properties,directed evolution techniques(error-prone PCR and DNA shuffling)was used for enzymatic modification;meanwhile,some key amino acid sites or domains related to catalytic activity and thermostability of B.amyloliquefaciens xylanase A were revealed.The main results are as follows:1.The baxA gene was cloned,and engineered E.coli strain pCbaxA15 which could achieve secretory expression was constructed.Take B.amyloliquefaciens which was isolated earlier in the laboratory as the original strain,specific primers were designed and baxA gene was cloned.The baxA gene(GenBank Accession number: KM 624029)encoded an open reading frame with 213 amino acid residuces,including 6 potential glycosylation sites.The theoretical molecular mass of the deduced protein was 23.3 kDa.The baxA gene was inserted into novel expression vector pCold TF and transformed into E.coli BL21(DE3)competent cells.The recombinant xylanase(reBaxA)was secreted into medium during inducible expression at 15°C.The reBax A was purified via high affinity Ni-charged resin,and the specific activity of purified re Bax A was 2.63 U/mg.The SDS-PAGE and Western Blot indicated that the molecular mass of this recombinant xylanase was about 77.3 kDa.Characterization results showed that the optimum temperature and pH of reBaxA were 55° C,pH 6.0;Km,Vmax were 16.05 mg/m L,45.66 ?mol/min/m L,respectively.The oat spelt,beechwood,birchwood xylans were hydrolyzed by reBaxA successively.The hydrolysis products were xylobiose to xylopentaose(X2-X5),with xylotetraose(X4),xylotriose(X3),xylopentaose(X5)as main product,respectively.The baxA gene was ligated with pET30a(+),transformed into E.coli BL21(DE3)and induced by IPTG.The SDS-PAGE,Western Blot and activity determination indicated that baxA gene got recombinant expression.Specific bands were obvious,while expression product did not show any xylanase activity,suggesting that recombinant protein formed inclusion body.2.The engineered P.pastoris strain PPbax A10 which could achieve secretory expression was constrcted.The baxA gene was inserted into shuttle vector pPICZ?A.The recombinant plasmid pPICZ?A-baxA was linearized and electroporated into P.pastoris GS115 competent cells.The engineered P.pastoris strain PPbax A10 was obtained after Zeocin resistance test,PCR identification and activity measurement.This strain was then inoculated to 50 m L YPD medium and added methanol to induce expression after 24 h cultivation.120 h later,the specific activity of PPbax A10's fementation supernatant was 8.18 U/mg and Km,Vmax were 5.41 mg/mL,22.42 ?mol/min/m L,respectively.RePPBaxA10 showed highest activity under the condition of 50°C and pH 5.0.3.The error-prone PCR was applied to the construction of mutant library.The mutant pCbax A50 which showed higher enzymatic activity was screened.In the PCR amplification system of baxA gene,the concentrations of Mg2+ and dTTP,dCTP were increased,the Mn2+ was added.Three protective bases were introduced into restriction sites of both primers,thus gel-purified PCR product could directly used for double digestion.The digested product was inserted into pCold TF and transformed into E.coli BL21(DE3)to construct mutant library.1463 clones were obtained;pCbax A50 with higher extracellular recombinant xylanase activity was screened via 96-well plate high-throughout screening strategy.The sequencing results showed one amino acid mutation,S138 T.The specific activity of reBax A50 was 9.38 U/mg,and optimum occurred at 50°C and pH 5.0.The half-life of mutant xylanase at 60°C was 9.74 min.According to TGA-DSC analysis,the Tm values of reBaxA50 and its parent were 89.15 and 88.95°C,respectively.4.The Thermomonospora fusca xylanase gene was used to ameliorate B.amyloliquefaciens xylanase A by DNA shuffling.Both the sequence of catalytic domain in T.fusca xylanase gene(tfxCD)and the sequence which encoded mature peptide of B.amyloliquefaciens xylanase A(mbaxA)were cloned by a pair of universal primers according to the vectors' sequences.The PCR products of two genes were mixed,randomly fragmentated by DNase?,did PCR with no primers and then did PCR again with specific primers.The product was double enzyme digested,ligated with expression vector pCold TF and transformed into E.coli BL21(DE3)to construct mutant library,2250 clones were obtained.The mutants with higher activity,named pCbaxA153,pCbaxA241,pCbaxA428 were screened by 96-well plate high throughout strategy.Besides,pCbax A199 was picked as it showed decreasment in the enzymatic activity.The amino acid mutations,N29 S,T33I,S31 R,and I51 V were appeared in the mutant xylanases,reBax A153,reBaxA199,reBaxA241,reBax A428,respectively.Their specific activities were 11.95,2.17,11.45,10.26 U/mg in turn.Among these mutant xylanases,re Bax A199 showed best stability in alkaline condition,while reBax A241 had best thermostability.Furthermore,the most stbable mutant xylanase in acid was re BaxA428.The reBaxA153 and reBax A428 had similar hydrolysis patterns towards different xylans.However,reBax A241 could hydrolyze all three xylans thoroughly.Different amino acid mutations might be the cause of variations in enzymatic activities,properties and hydrolysis characteristics.