Streptomyces maritimus苯丙氨酸变位酶的制备、表征及催化合成β-芳香丙氨酸

doi:10.7503/cjcu20160624

高等学校化学学报 ›› 2017, Vol. 38 ›› Issue (2): 206.doi: 10.7503/cjcu20160624

Streptomyces maritimus苯丙氨酸变位酶的制备、表征及催化合成β-芳香丙氨酸

朱龙宝¹, 陶玉贵¹, 葛飞¹, 李婉珍¹, 刘义²(), 堵国成³

1. 安徽工程大学生物与化学工程学院, 芜湖 241000
2. 西华大学食品与生物工程学院, 成都 610039
3. 江南大学工业生物技术教育部重点实验室, 无锡 214122

收稿日期:2016-09-02 出版日期:2017-02-10 发布日期:2016-12-19
作者简介:联系人简介: 刘义, 男, 博士, 副教授, 主要从事酶工程方面的研究. E-mail: myputer@163.com
基金资助:
国家自然科学基金(批准号: 31671797, 21506172)和安徽省高校自然科学基金(批准号: KJ2016A801)资助

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

ZHU Longbao¹, TAO Yugui¹, GE Fei¹, LI Wanzhen¹, LIU Yi^2,^*(), DU Guocheng³

1. School of Biological and Chemical Engineering, Anhui Polytechnic University, Wuhu 24100, China
2. School of Food and Biotechnology, Xihua University, Chengdu 610039, China
3. Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology,Jiangnan University, Wuxi 214122, China

Received:2016-09-02 Online:2017-02-10 Published:2016-12-19
Contact: LIU Yi E-mail:myputer@163.com
Supported by:
† Supported by the National Natural Science Foundation of China(Nos.31671797, 21506172) and the Natural Science Fund for Colleges and Universities in Anhui Province, China(No.KJ2016A801)

摘要/Abstract

摘要：

克隆Streptomyces maritimus来源的苯丙氨酸变位酶(SmPAM)基因, 并进行异源表达, 制备了重组SmPAM, 用于一步合成高附加值的β-芳香丙氨酸. 提取S. maritimus的基因组, 设计1对特异性引物, 采用PCR扩增编码SmPAM的结构基因pam, 与表达载体pET28a连接, 构建重组表达质粒pET28a-pam, 转入E.coli BL21中表达, 采用亲和层析柱分离纯化重组酶SmPAM. 结果表明, 克隆得到编码523个氨基酸长度的SmPAM基因pam, 并在大肠杆菌中实现了高效表达, 制得电泳纯的重组SmPAM. 该酶在最适温度30 ℃, pH=9.0条件下活力达到2.5 U/mg, 具有较高的热稳定性和pH稳定性, 在60~70 ℃下保持3 h未见活性下降, 在pH=9~11保持24 h, 残余酶活力达到98%. SmPAM具有较宽的底物谱, 可催化苯环上携带不同基团的3-芳香丙烯酸合成β-芳香丙氨酸, 当苯环上携带吸电子基团时催化反应更易完成, 其中2-硝基-β-苯丙氨酸的产率最高, 达到93%.

关键词: 苯丙氨酸变位酶, β-芳香丙氨酸, 基因克隆, 异源表达

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

中图分类号:

O629

TrendMD:

朱龙宝, 陶玉贵, 葛飞, 李婉珍, 刘义, 堵国成. Streptomyces maritimus苯丙氨酸变位酶的制备、表征及催化合成β-芳香丙氨酸. 高等学校化学学报, 2017, 38(2): 206.

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^†. Chem. J. Chinese Universities, 2017, 38(2): 206.

图/表 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*

参考文献 22

[1]	Kudo F., Miyanaga A ., Eguchi T., Nat. Prod. Rep., 2014, 31(8),1056—1073
[2]	Grayson J. I., Roos J., Osswald S., Org. Process. Res. Dev., 2011, 15(5), 1201—1206
[3]	Zhang X. H., Ye W. J., Wang K. L., TianY. S., Xiao X., Chem. Res. Chinese Universities,2015, 31(2), 203—207
[4]	Ratnayake N. D., Theisen C., Walter T., Walker K. D., J. Biotechnol., 2016, 10(217), 12—21
[5]	Liu J. Z., Xiong J. B., Zhao G. H., Liu Q., Jiao Q. C., Chem. J. Chinese Universities,2010, 31(11), 2234—2238
	(刘均忠, 熊吉滨, 赵根海, 刘茜, 焦庆才. 高等学校化学学报, 2010, 31(11), 2234—2238)
[6]	Li D. C., Jia L., Wang X. F., Wei D. Z., Prep. Biochem. Biotechnol,2013, 43(2), 207—216
[7]	Mathew S., Bea H., Nadarajan S. P., Chung T., Yun H., J. Biotechnol., 2015, 20(196—197), 1—8
[8]	Mathew S., Jeong S. S., Chung T., Lee S. H., Yun H., Biotechnol. J., 2016, 11(1),185—190
[9]	Zhang K., Liang X., He M., Wu J., Zhang Y., Xue W., Jin L., Yang S., Hu D., Molecules,2013, 18(6), 6142—6152
[10]	Heberling M. M., Wu B., Bartsch S., Janssen D. B., Curr. Opin. Chem. Biol., 2013, 17(2), 250—260
[11]	Lohman J. R., Shen B., Methods Enzymol., 2012, 516, 299—319
[12]	Cooke H. A., Christianson C. V., Bruner S. D., Curr. Opin. Chem. Biol., 2009, 13(4), 460—468
[13]	Walter T., King Z., Walker K. D.,Biochemistry,2016, 55(1), 1—4
[14]	Weise N. J., Parmeggiani F., Ahmed S. T., Turner N. J., J. Am. Chem. Soc., 2015, 137(40), 12977—12983
[15]	Lovelock S. L., Lloyd R. C., Turner N. J., Angew. Chem. Int. Ed. Engl., 2014, 53, 4652—4656
[16]	Heberling M. M., Masman M. F., Bartsch S., Wybenga G. G., Dijkstra B. W., Marrink S. J., Janssen D. B., ACS Chem. Biol., 2015, 10(4), 989—997
[17]	Zhu L. B., Cui W., Fang Y. Q., Liu Y., Gao X. X., Zhou Z. M., Biotechnol. Lett., 2013, 35, 751—756
[18]	Parmeggiani F., Lovelock S. L., Weise N. J., Ahmed S. T., Turner N. J., Angew. Chem. Int. Ed. Engl., 2015, 54(15), 4608—4611
[19]	Wu B., Szymanski W., de Wildeman S., Poelarends G. J., Feringa B. L., Janssen D. B., Adv. Synth. Catal., 2010, 352(9), 1409—1412
[20]	Wu B., Szymanski W., Wybenga G. G., Heberling M. M., Stefaan de Wildeman S. B., Poelarends G. J., Dijkstra B. L., Bauke W., Janssen D. B., Angew. Chem. Int. Ed. Engl., 2012, 51(2), 482—486
[21]	Feng L., Wanninayake U., Strom S., Geiger J., Walker K. D., Biochemistry,2011, 50(14), 2919—2930
[22]	Wu B., Szymanski W., Wietzes P., de Wildeman S., Poelarends G. J., Feringa B. L., Janssen D. B., ChemBioChem,2009, 10(2), 338—344

Streptomyces maritimus苯丙氨酸变位酶的制备、表征及催化合成β-芳香丙氨酸

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

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