Preparation,charaterization And Catalytic Mechanism Analysis Of Phenylalanine Aminomutase

Phenylalanine aminomutase(PAM)is a member of the MIO(4-methylene-imidazol-5-one)family.MIO,as a new cofactor,is different from endogenous cofactors,for example PLP,NADH and so on,it is formed post-translationally and autocatalytically via the condensation of an amino acid triad(Ala-Ser-Gly).MIO is believed to function as an electrophile to attack the NH2 of a-phenylalanine to shift from the C?site to the Cp site to produce ?-phenylalanine.So PAM can be used to synthesize high value-added ?-phenylalanine.In order to establish enzymatic synthesis process of ?-phenylalanine,the phenylalanine aminomutase gene(pam.)from Pantoea agglomerans was cloned.The recombinant PaPAM was constructed to achieve high expression in E.coli and purified to prepare high-purity recombinant enzyme.The fermentation,induction expression conditions and enzymatic properties of PaPAM were explored.On the basis of this,the catalysis mechanism of PaPAM was analyzed by NMR and MS.The solving the bottleneck problem that restricts(3-phenylalanine production,and lay a foundation for further designing the prexperimental results obtained in this paper have great significance for oduction process of?-phenylalanine.The main research contents are as follows:(1)The phenylalanine aminomutase gene(pam)from Pantoea agglomerans was cloned,the expression vector was constructed for heterologous expression.The results showed that the gene pam with 1626 bp encoding 541 amino acids of PaPAM was successfully cloned.The expression vector pET28a-pam was constructed and transferred into E.coli BL21 for induced expression using isopropy-?-D-thiogalactoside(IPTG).The electrophoretically pure recombinant phenylalanine aminomutase(PaPAM)was produced by affinity chromatograph.The enzyme activity reached 2.5 U/mg under the optimum conditions of 30 ?,pH 9.0 and 1.5 mol/L NH4+,The enzyme exhibited high stability at 30 ??50 ? and pH 8?10.Metal ions and surfactants have different effects on enzyme activity.Na+,Mg2+,Ca2+,and Fe3+ have little effect on PaPAM activity,while SDS and Triton 100 can strongly inhibit activity.MS and NMR results indicated that the PaPAM can isomerisation a-phenylalanine to form P-phenylalanine.(2)The catalysis mechanism of PaPAM was analyzed by NMR and MS.The NMR and MS analytical results showed that the Ca-NH2 and pro-3R hydrogen are intramolecularly exchange,which revealed that the PaPAM shuttles the a-NH2 of the substrate to the ? site to replace the pro-3R hydrogen.Simultaneously,the pro-3R hydrogen is shifted to the a site to produce ?-phenylalanine.(3)The medium composition and the induced expression conditions of the PaPAM were optimized.The results showed that the best carbon and nitrogen sources were glycerol and tryptone,and the most suitable concentration was 1.5%.The addition of different metal ions in the medium had different effects on recombinant growth and enzyme production.Mg2+,Ca2+and K+ had little effect on PaPAM growth and enzyme production,while Zn2+ had an inhibitory effect.The cell concentration OD600=0.6 was the best time to add the IPTG with the final concentration was 0.4 mmol/L.And the optimal temperature for induction was 26 ?,time was 14 h.