信号肽对亮氨酸脱氢酶在<i>Bacillus subtilis</i>中分泌表达的影响及酶学性质研究

doi:10.13523/j.cb.20180407

中国生物工程杂志

2018, Vol. 38

Issue (4): 46-53 DOI: 10.13523/j.cb.20180407

研究报告

信号肽对亮氨酸脱氢酶在Bacillus subtilis中分泌表达的影响及酶学性质研究

王男,金吕华,张玲,林荣,杨海麟(

)

江南大学生物发酵与分离实验室无锡 214122

The Effect of Signal Peptides on the Expression of Leucine Dehydrogenase and Enzymatic Properties in Bacillus subtilis

Nan WANG,Lv-hua JIN,Ling ZHANG,Rong LIN,Hai-lin YANG(

)

Jiangnan University, Laboratory of Biological Fermentation and Separation, Wuxi 214122, China

全文: PDF(937 KB) HTML

摘要：

根据信号肽N端电荷数,选择Sec及Tat两种途径的信号肽构建枯草芽孢杆菌穿梭质粒,首次实现Bacillus cereus源亮氨酸脱氢酶基因在Bacillus subtilis中的分泌表达。Tat途径信号肽PhoD促进蛋白质分泌的效果最好,胞外酶活力达20.25U/ml,为不添加信号肽的2.2倍,信号肽N端较多的电荷数,可能有利于多聚体蛋白的分泌。对表达产物进行纯化和酶学性质测定。结果表明,纯酶比酶活为13U/mg;L-Leucine为底物时酶的K_m为6.17mmol/L,V_max为14.49μmol/(L·min);底物特异性研究发现,酶与天然底物L-Leucine的亲和性最好,对一些脂肪族氨基酸也有活性,对芳香族氨基酸L-Phenylalanine无活性;酶的最适pH为10.5~12.0,pH稳定范围为5.0~11.0;最适反应温度为55℃;圆二色谱变温扫描酶二级结构变化,α螺旋含量随温度升高逐渐降低;差示扫描微量热技术(DSC)测定酶的解折叠温度(T_m值)为64.13℃,表明该酶具有较好耐热性。

关键词： 信号肽; 亮氨酸脱氢酶; 枯草芽孢杆菌; 酶学性质

Abstract:

According to the different N-terminal positive charges, Sec and Tat passway signal peptides were selected to construct the shuttle plasmid with pMA5, which was the first time that the leucine dehydrogenase gene from Bacillus cereus was efficiently secreted in Bacillus sutilis. The results showed that PhoD signal peptide had the highest exogenous activity, reaching 20.25U/ml, which was 2.2 times higher than that without signal peptide. The enzymatic properties of the purified leucine dehydrogenase were investigated. The purified enzyme expression level was 13U/mg; the Michaelis constants K_m and V_max were 6.17mmol/L and 14.49μmol/(L·min) for L-Leucine, separately. The enzyme had the best affinity with the natural substrate L-Leucine, and showed activity toward some the aliphatic amino acids; no activity was observed for the Aromatic amino acids L-Phenylalanine; the optimum pH was 10.5-12.0 and the stability was maintained at pH 5.0-11.0; the optimum reaction temperaturewas 55℃, The changed of the secondary structure at different temperatures by circular dichroism proved that the α-helical t α-helix content gradually decreased with increasing temperature; DSC measured T_m value of 64.13℃,indicating that the enzyme had high thermal stability.

Key words: Signal peptides Leucine dehydrogenase Bacillus subtilis Enzymatic properties

收稿日期: 2017-11-23 出版日期: 2018-05-08

ZTFLH:

Q816

基金资助: * 江苏省产学研资助项目(BY2016022-40)

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引用本文:

王男,金吕华,张玲,林荣,杨海麟. 信号肽对亮氨酸脱氢酶在Bacillus subtilis中分泌表达的影响及酶学性质研究[J]. 中国生物工程杂志, 2018, 38(4): 46-53.

Nan WANG,Lv-hua JIN,Ling ZHANG,Rong LIN,Hai-lin YANG. The Effect of Signal Peptides on the Expression of Leucine Dehydrogenase and Enzymatic Properties in Bacillus subtilis. China Biotechnology, 2018, 38(4): 46-53.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20180407 或 https://manu60.magtech.com.cn/biotech/CN/Y2018/V38/I4/46

表1 引物序列表

图1 重组质粒pMA5-leudh的PCR (a)与双酶切(b)验证

图2 重组菌SDS-PAGE分析

图3 信号肽构建示意图

表2 不同信号肽引导的LeuDH活性比较

图4 不同信号肽介导的LeuDH发酵上清SDS-PAGE分析

图5 重组亮氨酸脱氢酶纯化UV峰图(a)及SDS-PAGE鉴定(b)

图6 LeuDH对底物L-Leucine的动力学曲线

表3 LeuDH氧化反应底物特异性

图7 LeuDH最适pH(a)及pH稳定性(b)

图8 LeuDH最适温度(a)及温度稳定性(b)

表4 不同温度下LeuDH二级结构分析

图9 不同温度下LeuDH圆二色谱分析

图10 重组LeuDH Tm值

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