Heterologous Expression Of Arginine Deiminase And Its Application

Citrulline is a non-protein amino acid.It can't involve in the protein synthesis although it has the abilty to form the peptide bonds.In recent years,many researches showed that it has good physiological function in many fields,such as pharmacy,food and health care industries.Arginine deiminase(ADI,EC 3.5.3.6)can hydrolyzed L-arginine into L-citrulline and ammonia.Therefore,L-citrulline produced by enzymatic synthesis with ADI has board application prospect.In this study,the arginine deiminase gene of Enterococcus faecalis(E.faecalis)SK32.001 was cloned and expressed in Escherichia coli(E.coli).Then the enzymeatic properties of ADI were studied.Moreover,immobilized ADI which provided support in theory for the application of producing industrial citrulline continuously was also studied.In order to obtain higher enzyme activity and more stable ADI,enzymeatic properties of ADI was changed by molecular modification technology.Methods and experiment results were introduced here,briefly.(1)The heterologous expression of ADI gene and purification of the recombinant enzyme were studied.Using E.faecalis SK32.001 genome as the template,the ADI gene(arcA)whose length is 1227 bp was gained by PCR amplication.Then the target gene fragment was connected with the expression vector pET-28a-c(+).The recombinant plasmid was expressed into the host E.coli BL21(DE).Finally,the genetic engineering bacteria E.coli BL21(DE)/pET-28a-ADI which can overexpress the gene of ADI was gained.The plasmid was overexpressed by IPTG in E.coli,and the crude enzyme was loaded onto a column packed with Ni2+-chelating Sepharose Fast Flow resin for purification.Then purified enzyme was analyzed by SDS-PAGE and 49,100 exogenous proteins were observed.(2)The induced expression condition of the recombinant enzyme was studied.The optimal induced conditions were IPTG concentration of 0.4 mmol/L when the bacteria cincentation OD600 value was 0.5~0.6,and the induced condition was 28°C,9 h.Under the optimal condition,the fermentation enzyme activity increased by 41.5%.(3)Characterization of the recombinat enzyme.It was stable within pH range of 5.0 to 5.5 and within temperature range of 20°C to 25°C.The optimum pH and temperature of the enzyme was 5.5 and 55°C,respectively.Fe3+ enhanced its enzymatic activity.The chemical modifiers and the three-dimensional structural model of the recombinant enzyme indicated that Lys,Trp and Cys were the important amino acid residues for the enzyme.The Km and Vmax of L-arginine were measured to be 10.1 mmol/L and 378.1 ?mol/min/mg,respectively.The bioproductivity of L-citrulline in the resting recombinant cells was 48.5 g/L/h,and the molar yield was 96.9%.(4)The immobilization of the recombinant enzyme was studied.ADI was immobilized on 7 kinds of ion-exchange resins,in which D301-G was the best material for the immobilization.And enzyme was immobilized by cross-linkage of glutaraldehyde.According to single-factor experiments,the optimal immobilized conditions were enzyme load of 156 U/g resin,4-hour absorption at 28°C,pH 6.0;the glutaraldehyde concentration of 0.07% and 4-hour cross-linking action at 4°C.The optimal temperature and pH of the immobilized enzyme was 50°C and 5.0,respectively,and it presented higher thermostability and higher Km than those of the free enzyme.The immobilized enzyme retained 57.7% of its initial enzyme activity after having been reused for eight times.(5)The molecular modification of the recombinant enzyme was studied.Error-prone PCR technique was used to construct mutant library,a mutation M3-34(R15K)showed higher enzyme activity.Then,software B-FITTER was combined to identify the amino acid residues which had an adverse effect on the thermal stability.On the basis of the mutant M3-34,the single-site variants,G264 F,G264P,G264 S,F269A,F269 P,F269D,F269 Y,G292P,G292 D and G292 C were constructed,and the enzyme activity of the mutants were 9.3%,102.6%,6.9%,8.6%,32.9%,96.4%,21.5%,253.6%,13.6%,9.5% that of the wild-type,respectively.The mutants,G264 Y,F269D and G292 P whose enzyme activity didn't reduce,showed different properties.The thermostability of mutants F297 D and G292 P under 40°C was increased by 3.2 and 5.5 times than that of the wild-type,respectively.And the composite mutant F269Y/G292 P showed higher catalytic efficiency and theromstability.In addition,at pH 7.0,G264 Y,F269D,G292 P and composite mutant F269Y/G292 P exhibited higher conversion ratio than wild-type and M3-34,and the optimum temperatures were 55°C.