Production and Characterization of Phenylalanine Aminomutase from Streptomyces Maritimus and Synthesis of β-Arylalanine†

doi:10.7503/cjcu20160624

Abstract

Abstract:

The gene of phenylalanine aminomutase was cloned from Streptomyces maritimus. The corresponding enzyme(SmPAM) was expressed to synthesize the β-arylalanine in E. coli. A pair of primers was designed to amplify the gene pam of SmPAM, and the pamof 1569 bp was cloned into pET28a to construct pET28a-pam which was transferred into E. coli BL21 to express the recombinant SmPAM. The pam of SmPAM was cloned and expressed in E. coli. The activity was 2.5 U/mg at optimum condition of 30 ℃ and pH=9. Moreover, the enzyme exhibited excellent thermostablity and pH stability, no activity decrease was observed at 60—70 ℃ even incubated for 3 h, and about 98% of activity remained after incubation at pH=9—11 for 24 h. The enzyme displayed broad substrate spectrum, and could be used to synthesize the β-arylalanine with different groups at benzene ring, among these β-arylalanines, the yield of 2-nitro-β-phenylalanine was the maximum and reached 93%. The SmPAM displayed excellent stability and broad substrate spectrum with potential prospectin industrial application.

Key words: Phenylalanine aminomutase, β-Arylalanine, Gene cloning, Heterologous expression

CLC Number:

O629

TrendMD:

ZHU Longbao, TAO Yugui, GE Fei, LI Wanzhen, LIU Yi, DU Guocheng. Production and Characterization of Phenylalanine Aminomutase from Streptomyces Maritimus and Synthesis of β-Arylalanine^†[J]. Chem. J. Chinese Universities, 2017, 38(2): 206.

Figures/Tables 7

Fig.1 Agarose gel electrophoresis of gene DNA, PCR products and the digested pET28a-pamM: marker; lane 1: product of PCR; lane 2: genomeDNA; lane 3: the pET28a-pam was digested by EcoRⅠ and NdeⅠ.

Fig.2 SDS-PAGE of recombinant enzyme expressed in E.coli BL21(DE3) and purification of the recombinant SmPAM using HisTrapTM/FFM: marker; lane 1: E.coli BL21; lane 2: cell extracts of E.coli BL21/pET28a-pam without induction by IPTG; lanes 3 and 5: the supernatant fraction after sonication of E.coli BL21/pET28a-pam induced by IPTG(the arrow in lane 3 represents the expressed SmPAM); lane 4: the precipitated fraction after sonication of E.coli BL21/pET28a-pam induced by IPTG; lane 6: the purified SmPAM by HisTrap TM/Faffinity chromatography column.

Fig.3 Determination of molecular mass of natural SmPAM on Sephadex G-75

Fig.4 Effects of temperature(A) and temperature stability(B) on the activity of enzyme

Fig.5 Effects of pH(A) and pH stability(B) on SmPAM activity

Fig.6 Effects of metal ions(A) and surfactants(B) on the activity of recombinant SmPAM(A) a. Na+; b. Mg2+; c. Ca2+; d. Fe2+; e. Cu2+; f. Zn2+; g. Mn2+; h. Co3+; (B) a. CTAB; b. Triton X-100; c. Tween 80; d. SDS.

Table 1 Synthesis of β-arylalanine by SmPAM*

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