紫色色杆菌苯丙氨酸羟化酶的异源表达及重组酶学性质研究

生物技术通报 ›› 2014, Vol. 0 ›› Issue (8): 152-158.

紫色色杆菌苯丙氨酸羟化酶的异源表达及重组酶学性质研究

曹淑慧, 周丽, 崔文璟, 刘中美, 周哲敏

江南大学生物工程学院工业生物技术教育部重点实验室, 无锡 214122

修回日期:2014-01-23 出版日期:2014-08-15 发布日期:2014-08-01
作者简介:作者简介: 曹淑慧，女，硕士研究生，研究方向：发酵工程；E-mail：elizabethcsh@sina.com

Heterologous Expression and Characterization of Phenylalanine Hydroxylase from Chromobacterium violaceum

Cao Shuhui, Zhou Li, Cui Wenjing, Liu Zhongmei, Zhou Zhemin

The Key Laboratory of Industrial Biotechnology of the Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122

Revised:2014-01-23 Published:2014-08-15 Online:2014-08-01
Supported by:
国家自然科学基金项目(31070711,31300087);新世纪优秀人才项目(NCET-10-0461);江苏省自然科学基金项目(BK20130131)

摘要/Abstract

摘要：

来源于紫色色杆菌（Chromobacterium violaceum）的苯丙氨酸羟化酶结构简单，性质更接近于人的苯丙氨酸羟化酶，具有潜在的医药应用价值。从紫色色杆菌基因组中克隆得到苯丙氨酸羟化酶基因pah。构建重组表达载体pET24a-pah，并在Escherichia coli BL21（DE3）中实现高效表达。离子层析纯化后，重组蛋白比酶活高达503.2 U/mg。酶学性质研究显示，该重组酶的最适温度为40℃左右，50℃时PAH的半衰期为15 min；最适pH在7.5左右，在pH6-8范围内较稳定。37℃，pH7.5条件下，K_m值为1.5 mmol/L，V_max为0.5 mmol/min，k_cat为5.05/s，催化效率k_cat/K_m 为3.37 L/mmol·s。

关键词: 苯丙氨酸羟化酶, 紫色色杆菌, 表达, 纯化, 酶学性质

Abstract:

Phenylalanine hydroxylase（PAH）derived from Chromobacterium violaceum has potential pharmaceutical value due to its simple structure and the closer property to human PAH. We cloned the pah gene from C. violaceum and constructed recombinant expression vcectors pET24a-pah, finally, the pah gene was efficiently expressed in Escherichia coli BL21（DE3）. The specific activity of recombination protein was 503.2 U/mg after purification. The studies of enzymatic properties showed that the enzyme displayed maximum activity at 40℃, and its half-life was 15 min at 50℃. The optimal pH was pH7.5, and the enzyme activity was stable at the range of pH6-8. Under the conditions of 37℃ and pH7.5, its K_m was 1.5 mmol/L, V_max was 0.5 mmol/min, k_cat was 5.05/s, and the catalytic efficiency（k_cat /K_m）was 3.37 L/mmol·s.

Key words: Phenylalanine hydroxylase, Chromobacterium violaceum, Expression, Purification, Enzymatic properties

曹淑慧, 周丽, 崔文璟, 刘中美, 周哲敏. 紫色色杆菌苯丙氨酸羟化酶的异源表达及重组酶学性质研究[J]. 生物技术通报, 2014, 0(8): 152-158.

Cao Shuhui, Zhou Li, Cui Wenjing, Liu Zhongmei, Zhou Zhemin. Heterologous Expression and Characterization of Phenylalanine Hydroxylase from Chromobacterium violaceum[J]. Biotechnology Bulletin, 2014, 0(8): 152-158.

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