催化Aldol加成反应的新型枯草芽孢杆菌BS168酰基氨肽酶的表达和应用

doi:10.7503/cjcu20150358

高等学校化学学报 ›› 2015, Vol. 36 ›› Issue (12): 2454.doi: 10.7503/cjcu20150358

催化Aldol加成反应的新型枯草芽孢杆菌BS168酰基氨肽酶的表达和应用

于潇潇, 王琦, 周烨, 高仁钧, 王英武()

吉林大学生命科学学院, 分子酶学工程教育部重点实验室, 长春 130012

收稿日期:2015-05-04 出版日期:2015-12-10 发布日期:2015-10-10
作者简介:联系人简介: 王英武, 男, 博士, 教授, 博士生导师, 主要从事生物化学、药物合成和分析领域的研究. E-mail:wyw@jlu.edu.cn
基金资助:
国家自然科学基金(批准号: 20772046)和吉林省自然科学基金(批准号: 20120943)资助

Cloning and Application of a New Acylaminoacyl Peptidase from Bacillus subtilis 168 for Aldol Reaction^†

YU Xiaoxiao, WANG Qi, ZHOU Ye, GAO Renjun, WANG Yingwu^*()

Key Laboratory for Molecular Enzymology and Engineering, the Ministry of Education,School of Life Science, Jilin University, Changchun 130012, China

Received:2015-05-04 Online:2015-12-10 Published:2015-10-10
Contact: WANG Yingwu E-mail:wyw@jlu.edu.cn
Supported by:
† Supported by the National Natural Science Foundation of China(No.20772046) and the Natural Sciences Foundation of Jilin Province, China(No.20120943)

摘要/Abstract

摘要：

从枯草芽孢杆菌(Bacillus subtilis 168)基因组中的ORF32230出发, 通过氨基酸序列分析推测其可能为酰基氨肽酶基因, 并与典型的脯氨酸寡肽酶家族成员一致, 含有2个独立的结构域, 活性中心由催化三联体丝氨酸-天冬氨酸-组氨酸(Ser-Asp-His)组成. 将BSU32230的基因片段与pET-21a载体相连, 转入BLP(DE3)表达菌中, 在0.5 mmol/L 异丙基-β-D-硫代半乳糖苷(IPTG)存在及20 ℃条件下诱导表达该蛋白. 利用硫酸铵沉淀与Ni亲和层析对BSU32230蛋白进行纯化, 并通过实验证明该蛋白同时具有酯酶和肽酶2种活性. 该酶最佳反应温度为50 ℃, 最佳pH值为8.0, 40 ℃下半衰期约29 h, 在pH=4~10范围内稳定.该酶能够在有机相中催化不对称Aldol加成反应, 且反应产物的立体选择性较好(84.6%).

关键词: 酰基氨肽酶, 枯草芽孢杆菌, Aldol反应

Abstract:

Acylaminoacyl peptidase has been named acyl-peptide releasing enzyme(AARE), catalyses the removal of an N-acylated amino acid from N-α-acylpeptides. In this research, a novel acyl-peptide releasing enzyme gene(BSU32230) from Bacillus subtilis 168 was cloned and expressed in Escherichia coli BLP DE3 codon plus to produce acyl-peptide releasing enzyme. The recombinant enzyme was purified in two steps: ammonium sulfate precipitation and Ni²⁺-column affinity chromatography. The optimum temperature and pH of enzyme were 50 ℃ and 8.0, respectively. The half-life of recombinant enzyme at 40 ℃ was 29 h, the enzyme was stable at pH range from 4 to 10. This report demonstrated the acylaminoacyl peptidase from Bacillus subtilis peptidase can catalyze aldol reaction and showed high enantioselectivity. The reaction provided optically active secondary alcohol with satisfying enantioselectivity(84.6% e.e.).

Key words: Acylaminoacyl peptidase, Bacillus subtili, Aldol reaction

中图分类号:

O629.8

TrendMD:

于潇潇, 王琦, 周烨, 高仁钧, 王英武. 催化Aldol加成反应的新型枯草芽孢杆菌BS168酰基氨肽酶的表达和应用. 高等学校化学学报, 2015, 36(12): 2454.

YU Xiaoxiao, WANG Qi, ZHOU Ye, GAO Renjun, WANG Yingwu. Cloning and Application of a New Acylaminoacyl Peptidase from Bacillus subtilis 168 for Aldol Reaction^†. Chem. J. Chinese Universities, 2015, 36(12): 2454.

图/表 8

Scheme 1 Aldol reaction catalyzed by BSU32230

Fig.1 Sequcence aligment of prolyl oligopeptidases

Fig.2 Substrate specificity of purified BSU32230 against different p-nitrophenyl esters(A) and various short peptides(B) (A) a. pNPC2; b. pNPC3; c. pNPC4; d. pNPC8; e. pNPC10; f. pNPC12; g. pNPC16.(B) a. Ala2; b. (D-Ala)2; c. Ala-phe; d. Asp-Ala; e. Ala3; f. Ac-Ala3.

Table 1 Purification of BSU32230

Sample	Total protein/mg	Total activity/U	Specific activity/(U·mg^-1)	Purification fold	Yield(%)
Crude extract	198.00	84.26	0.43	1	100
Ammonium sulfate	78.00	52.61	0.67	1.59	62.44
Ni²⁺ affinity column	4.48	30.73	6.86	16.14	36.47

Fig.3 Effects of temperature(A) and pH(B) on BSU32230 activity

Fig.4 Thermostability(A) and pH stability(B) of BSU32230 pH=4.0—8.0, 50 mmol/L Na2HPO4-NaH2PO4, ■ 1 h, ◆ 24 h; pH=7.0~10.0, 50 mmol/L Tris-HCl, ● 1 h, ▲ 24 h.

Fig.5 Effect of temperature on the asymmetric aldol addition of Acetone and 4-nitrobenzaldehyde catalyzed by BSU32230

Table 2 Comparison of Acyl-peptide releasing enzyme(BSU32230) and Porcine Pancreatic Lipase(PPL) catalyzed aldol additions between 4-nitrobenzaldehyde and Acetone

Enzyme	Temperature/℃	Time/h	Yield(%)	e.e.(%)
BSU32230	50	24	30.9	84.6
PPL	37	24	24.6	82.1
Denatured BSU32230	50	24	<0.5	—
Denatured PPL	37	24	<0.5	—
Control/No enzyme	50	24	<0.5	—

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催化Aldol加成反应的新型枯草芽孢杆菌BS168酰基氨肽酶的表达和应用

Cloning and Application of a New Acylaminoacyl Peptidase from Bacillus subtilis 168 for Aldol Reaction^†

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催化Aldol加成反应的新型枯草芽孢杆菌BS168酰基氨肽酶的表达和应用

Cloning and Application of a New Acylaminoacyl Peptidase from Bacillus subtilis 168 for Aldol Reaction†

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Cloning and Application of a New Acylaminoacyl Peptidase from Bacillus subtilis 168 for Aldol Reaction^†