一种新型亮氨酸5-羟化酶NmLEH的异源表达、纯化及酶学性质分析 <sup>*</sup>

doi:10.13523/j.cb.20191204

中国生物工程杂志

2019, Vol. 39

Issue (12): 24-34 DOI: 10.13523/j.cb.20191204

研究报告

一种新型亮氨酸5-羟化酶NmLEH的异源表达、纯化及酶学性质分析 ^*

朱梦露^1,^2,³,王雪雨¹,刘鑫¹,路福平^1,^2,³,孙登岳^1,^2,^3,^**(

),秦慧民^1,^2,^3,^**(

)

1 １天津科技大学生物工程学院天津 300457
2 天津科技大学工业发酵微生物教育部重点实验室天津 300457
3 工业酶国家工程实验室天津 30045

Heterologous Expression, Purification and Enzymatic Properties of a Novel Leucine 5-Hydroxylase

ZHU Meng-lu^1,^2,³,WANG Xue-yu¹,LIU Xin¹,LU Fu-ping^1,^2,³,SUN Deng-yue^1,^2,^3,^**(

),QIN Hui-min^1,^2,^3,^**(

)

1 College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China
2 Tianjin Key Laboratory of Industrial Microbiology, Tianjin 300457, China
3 National Engineering Laboratory for Industrial Enzymes, Tianjin 300457, China

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摘要：

羟基化氨基酸是一种新型氨基酸衍生物,可广泛用作化工材料的前体物及医药合成的中间体。将来源于Nostoc minutum的新型L-亮氨酸5-羟化酶 (NmLEH) 通过重组质粒在大肠杆菌中异源表达。结果表明,在BL21(DE3) 宿主细胞中,诱导温度为25℃,IPTG诱导浓度为0.5mmol/L,诱导10h时,蛋白质表达量最高 (0.45mg/ml);通过Ni-亲和层析和凝胶过滤层析两步分离纯化获得了高度纯化的重组NmLEH蛋白;对NmLEH的酶学性质进行了表征,该酶的最适反应温度为25℃,最适pH 为7.5,在pH 7.0~9.0较为稳定,最适底物为亮氨酸和甲硫氨酸;同源序列分析表明NmLEH属于亚铁和α-酮戊二酸依赖性双加氧酶家族[Fe(II)/αKG-Dos],并预测了该酶的保守催化活性位点(H150、D152、H236);通过同源建模得到了该蛋白质的模拟结构,分析了该蛋白质催化活性中心的形成机制。

关键词： 亮氨酸5-羟化酶; 表达纯化; 酶学性质; 底物特异性; 催化活性中心

Abstract:

Hydroxyl amino acids are a novel kind of amino acid derivatives, which are widely used as a chemically synthetic intermediate.A novel L-leucine-5-hydroxylase (NmLEH) from Nostoc minutum was cloned and inserted into recombinant plasmid, and its expression conditions were optimized. The results showed that when the enzyme was transferred into the BL21 (DE3) host, the induction temperature was 25℃ and the IPTG induction concentration was 0.5mmol/L, the expression levels of NmLEH was highest after induction of 10 (0.45 mg/ml). Using purification process of Ni-affinity chromatography and gel filtration, highly purified recombinant NmLEH was obtained. The enzymatic properties of NmLEH were characterized, and the optimum reaction temperature of the enzyme was 25℃, and the optimum pH was 7.5; the NmLEH enzyme was active at pH 7.0-9.0, and the optimum substrate for the NmLEH were the leucine and methionine. Sequence alignment and phylogenesis analysis implied that residues of H150, H236 and D152 constitute the catalytic triad of NmLEH, which is completely conserved in the Fe(II)/αKG-dependent dioxygenase [Fe(II)/αKG-Dos] superfamily. The formation mechanism of catalytic active site was analyzed based on NmLEH structural model analysis.

Key words: Leucine 5-hydroxylase Expression purification Enzymatic properties Substrate specificity Catalytic active site

收稿日期: 2019-04-24 出版日期: 2020-01-15

ZTFLH:

Q814

基金资助: * 国家自然科学基金(31771911);天津市自然科学基金(16JCQNJC09200);天津市大学生创新创业训练计划项目(1000204)

通讯作者: 孙登岳,秦慧民 E-mail: dysun09@163.com;huiminqin@tust.edu.cn

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引用本文:

朱梦露,王雪雨,刘鑫,路福平,孙登岳,秦慧民. 一种新型亮氨酸5-羟化酶NmLEH的异源表达、纯化及酶学性质分析 ^*[J]. 中国生物工程杂志, 2019, 39(12): 24-34.

ZHU Meng-lu,WANG Xue-yu,LIU Xin,LU Fu-ping,SUN Deng-yue,QIN Hui-min. Heterologous Expression, Purification and Enzymatic Properties of a Novel Leucine 5-Hydroxylase. China Biotechnology, 2019, 39(12): 24-34.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20191204 或 https://manu60.magtech.com.cn/biotech/CN/Y2019/V39/I12/24

图1 L-亮氨酸和L-甲硫氨酸催化反应示意图

表1 梯度洗脱程序

图2 NmLEH菌落PCR和酶切验证

图3 不同诱导条件下可溶性蛋白的浓度

图4 NmLEH蛋白纯化示意图

图5 温度对NmLEH酶活和热稳定性的影响

图6 pH对NmLEH酶活和催化稳定性的影响

表2 NmLEH酶动力学参数

表3 NmLEH的底物选择性

图7 酶辅因子对NmLEH活性的影响

图8 HPLC检测亮氨酸催化产物

图9 NmLEH氨基酸序列比对

图10 NmLEH系统树分析

图11 NmLEH模拟结构及活性中心示意图

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