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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2016, Vol. 36 Issue (10): 35-44    DOI: 10.13523/j.cb.20161006
研究报告     
新颖微生物低温酯酶EstP8的酶学性质研究与在手性催化中的应用
公颜慧1, 马三梅1, 张云2,3, 王永飞1, 胡云峰2,3
1. 暨南大学生物工程学系 广州 510632;
2. 中国科学院南海海洋研究所 中国科学院热带海洋生物资源与生态重点实验室 广州 510301;
3. 中国科学院南海海洋研究所 广东省海洋药物重点实验室 广州 510301
Functional Characterization of a Novel Microbial Psychrophilic Lipase and Its Utilization in Stereo-Selective Biocatalysis
GONG Yan-hui1, MA San-mei1, ZHANG Yun2,3, WANG Yong-fei1, HU Yun-feng2,3
1. Department of Biotechnology, Jinan University, Guangzhou 510632, China;
2. Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China;
3. Guangdong Key Laboratory of Marine Materia Medica, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China
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摘要：

从假单胞菌Pseudonocardia antitumoralis HUP007基因组中克隆了一个1 041bp的酯酶基因EstP8，编码的蛋白具有377个氨基酸残基。在E.coli BL21（DE3）中实现酯酶EstP8的高效异源表达和纯化。EstP8为脂肪酶家族Ⅳ中的一员，具有HGGG保守序列。EstP8最适底物为对硝基苯酚乙酸酯（p-NPO），最适温度和pH分别为50℃和8.0。EstP8催化p-NPO水解反应的活性、Vmax和Km分别达到105.19U/mg、89.4μM/min、1.144mM。EstP8在pH7.0~8.0范围内具有良好的pH稳定性；在4℃时，酯酶相对活力为41.78%，在10~40℃内具有很好温度稳定性。EstP8对大部分金属离子有很好的耐受性，低浓度的Cu2+、Mn2+、Zn2+对该酶的活性有激活作用。辛烷、庚烷、甲苯、丙酮、DMF等有机溶剂对EstP8的活性同样具有激活作用。酯酶EstP8还可以通过水解拆分高效地制备手性（R）-1-苯基乙醇；添加有机溶剂可以很好地促进该酯酶的光学选择性和产率，在共溶剂甲苯的存在下，所制备的（R）-1-苯基乙醇的e.e.和产率可达91%和18%；在共溶剂DMSO的存在下，所制备的（S）-乙酸苏合香酯的e.e.和产率可达98%和60%。酯酶EstP8在手性生物催化等诸多工业领域有很好的应用潜力。
关键词： 手性拆分;  高对映选择性;  金属离子耐受性;  有机溶剂耐受性;  低温酯酶    
Abstract:

From the genome of Pseudomonas antitumoralis HUP007, an esterase gene harboring 1041 bp and encoding a protein EstP8 of 377 amino acid was cloned. Esterase EstP8 was efficiently expressed in E. coli BL21(DE3) and further purified. Esterase EstP8 belongs to the IV family of lipases and contains conserved HGGG motif. The optimal substrate of esterase EstP8 was p-NPO, the optimal working temperature of esterase EstP8 was 50℃ and the optimal working pH of esterase EstP8 was 8.0. The hydrolysis activity of esterase EstP8 toward p-NPO was 105.19 U/mg, with Vmax and Km being 89.4 μmol/min and 1.144 mM, respectively. Esterase EstP8 remained high activity at pH7.0~8.0. The relative activity of esterase EstP8 was 41.78% at 4℃ and esterase EstP8 behaved very good thermo-stability at 10~40℃. EstP8 exhibited very good resistance to most metal ions tested. The addition of Cu2+, Mn2+ or Zn2+ of low concentrations could even stimulate the activity of EstP8. Organic solvents such as isooctane, methylbenzene, acetone and DMF could stimulate the activity of EstP8. Esterase EstP8 could generate chiral (R)-1-phenylethanol through kinetic resolution. The addition of organic solvents could well stimulate the stereo-selectivity and conversion during kinetic resolution. The e.e. and conversion of generated (R)-1-phenylethanol could reach 91% and 18%, respectively, in the presence of methylbenzene. The e.e. and conversion of generated (S)-styrallyl acetate could reach 98% and 60%, respectively, in the presence of DMSO. Psychrophilic esterase EstP8 possesses good potential in diverse industries such as stereo-selective biocatalysis.
Key words: Resistance to metal ions    Psychrophilic esterase    Kinetic resolution    Resistance to organic solvents    High enantio-selectivity
收稿日期: 2016-03-16 出版日期: 2016-10-25
ZTFLH:  Q819  
基金资助:

国家自然科学基金青年科学基金项目（21302199）、中国科学院战略性先导科技专项（XDA11030404）、中国科学院重点部署项目（KGZD-EW-606）、广州市科技计划（210510010012）资助项目
通讯作者: 马三梅,电子信箱:wyfmsm@163.com;胡云峰,电子信箱:yunfeng.hu@scsio.ac.cn     E-mail: yunfeng.hu@scsio.ac.cn;wyfmsm@163.com
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公颜慧
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引用本文:

公颜慧, 马三梅, 张云, 王永飞, 胡云峰. 新颖微生物低温酯酶EstP8的酶学性质研究与在手性催化中的应用[J]. 中国生物工程杂志, 2016, 36(10): 35-44.

GONG Yan-hui, MA San-mei, ZHANG Yun, WANG Yong-fei, HU Yun-feng. Functional Characterization of a Novel Microbial Psychrophilic Lipase and Its Utilization in Stereo-Selective Biocatalysis. China Biotechnology, 2016, 36(10): 35-44.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20161006    或    https://manu60.magtech.com.cn/biotech/CN/Y2016/V36/I10/35

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