Expression,Characterization And Chiral Synthesis Of Low-Specificity L-Threonine Aldolase From Pseudomonas Putida KT2440

Threonine aldolase is a kind of pyridoxal-5'-phosphate(PLP)-dependent enzymes,which catalyzes the reversible aldol reaction of glycine with various aromatic and aliphatic aldehydes to form ?-hydroxy-a-amino acids with two chiral stereocenters.?-Hydroxy-a-amino acids are an important precursor of the active ingredients of many drugs.It is not only widely used in the pharmaceutical and fine chemical industries,but also has a good industrial application prospect.In this study,the low specificity L-threonine aldolase gene(ItaE)was cloned using PCR amplification technique from the genome of Pseudomonas putida KT2440.The recombinant expression plasmid pET28a-KT2440 was constructed and transformed into Escherichia coli BL21(DE3)to obtain recombinant E.coli BL21(DE3)/pET-KT2440.The enzymatic properties was characterized.The key amino acids Thr206 and lys207 were mutated by site-directed mutagenesis.The reaction conditions of LTA biosynthesis for 3-(4-methylsulfonylphenyl)serine were studied.The main results are as follows:(1)The ItaE gene was amplified by PCR technique using the genome of P.putida KT2440 as template.The recombinant plasmid pET28a-KT2440 was constructed and transformed into E.coli BL21(DE3)to constructe the recombinant expression strain E.coli BL21(DE3)/pET-KT2440.SDS-PAGE analysis showed that LTA was highly expressed in E.coli BL21(DE3)and the molecular weight was about 40 kDa,which was consistent with the theoretical value.The temperature induced by IPTG was optimized.The recombinant protein was highly expressed at 250C with the induction of 0.5 mmol·L-1 IPTG.It was purified by Ni2+affinity chromatography and a single band was observed in SDS-PAGE gels.(2)The enzyme activity of LTA was determined.The results of enzymatic properties indicated that the optimal reaction temperature of LTA was 50?.After the incubation at less than 45? for 1 h,the residual enzyme activity could still be retained above 80%.Its optimal pH was 8.0 and it was stable below 45? and pH between 5.0 and 9.0.And LTA exhibited Km and kcat values of 23.95 mmol·L-1 and 19216.6 s-1 respectively under the optimal conditions.One mmol·L-1 Mg2+ and Ca2+ obviously stimulated enzyme activity,and 1 mmol·L-1 Mn2+slightly promoted the enzyme activity.Ni2+,Cu2+,Zn2+and Fe2+of 1 mmol·L-1 obviously inhibited it,and Co2+had a slight inhibitory effects.The organic solvent resistance results showed that the LTA presented good resistance in tert-butyl methyl ether(TBME).The residual activity was retained over 90%after incubation of the enzyme in TBME for 1 h,whereas it retained less than 20%in dimethyl sulfoxide(DMSO),absolute ethanol(EtOH),tetrahydrofuran(TF),acetonitrile(CH3CN).(3)The mutant strain was constructed and the enzymatic activity of the mutant enzyme was determined.Five site-directed mutants were constructed using the ItaE gene as a template.All the mutants were highly expressed in E.coli BL21(DE3).The purified enzyme with a single band was obtained through Ni2+affinity chromatography.The enzyme activity of the mutants was determined.The mutants of K207 lost most the L-threonine cleavage activity,and only retained 0.3-4.8%of enzyme activity compared with the wild type,indicating that the Lys207 is essential for enzyme catalytic function.However,T206S had little effect on the enzyme activity The results of circular dichroism thermal denaturation experiment showed that the denaturation temperature(Tm)of wild type was 49.2?,while the denaturation temperature of mutant T206S was 53.7?.The denaturation temperature was increased by 4.5? compared with the wild-type LTA,suggesting that T206S is closely related to the thermal stability.(4)Reaction conditions for the synthesis of 3-(4-Methylsulfonylphenyl)serine were determined using wild type LTA and T206S as catalyst.The optimal temperature and pH were 50? and 8.0 of 3-(4-Methylsulfonylphenyl)serine biosynthesis by wild type LTA and reached the highest value of 41%in 3 h,while The mutant enzyme T206S optimal temperature and pH were 558 and 8.0 and reached the highest value of 43%in 3 h,The optical purity was higher at temperature of 30? and pH 8.0 of wild type LTA,while the T206S was higher at temperature of 35? and pH 8.0.And with the reaction time increased,the optical purity continued to be decreased.