华根霉脂肪酶有机相酯合成活性的重塑

doi:10.7503/cjcu20180437

高等学校化学学报 ›› 2019, Vol. 40 ›› Issue (4): 747.doi: 10.7503/cjcu20180437

华根霉脂肪酶有机相酯合成活性的重塑

张璋¹, 王栋^1,³(), 王晓雷², 徐岩^1,³()

1. 工业生物技术教育部重点实验室, 江南大学生物工程学院酿酒科学与酶技术中心, 生物工程学院, 无锡 214122
2. 微生物代谢国家重点实验室, 代谢与发育科学国际合作联合实验室, 上海交通大学生命科学技术学院, 上海 200240
3. 江南大学产业技术研究院, 宿迁 223814

收稿日期:2018-06-12 出版日期:2019-04-03 发布日期:2018-10-22
作者简介:
联系人简介: 王栋, 男, 博士, 教授, 主要从事酶工程及发酵方面的研究. E-mail: dwang@jiangnan.edu.cn;徐岩, 男, 博士, 教授, 博士生导师, 主要从事发酵工程及酶工程方面的研究. E-mail: yxu@jiangnan.edu.cn
基金资助:
国家自然科学基金(批准号: 31271920)、国家重点研究发展计划项目(批准号: 2016YFD0400503)、江苏高校品牌专业建设工程资助项目(TAPP)和江苏省高校品牌特色专业建设工程(批准号: PPZY2015A056)资助.

Regulation of Ester Synthesis Activity of Rhizopus chinensis Lipase^†

ZHANG Zhang¹, WANG Dong^1,^3,^*(), WANG Xiaolei², XU Yan^1,^3,^*()

1. Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, Center for Brewing Science and Enzyme Technology, Wuxi 214122, China
2. State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic & Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China;
3. Suqian Industrial Technology Research Institute of Jiangnan University, Suqian 223814, China

Received:2018-06-12 Online:2019-04-03 Published:2018-10-22
Contact: WANG Dong,XU Yan E-mail:dwang@jiangnan.edu.cn;yxu@jiangnan.edu.cn
Supported by:
† Supported by the National Natural Science Foundation of China(No.31271920), the National Key Research and Development Program of China(No.2016YFD0400503), Top-notch Academic Programs Project of Jiangsu Higher Education Institutions(TAPP), and the Priority Academic Program Development of Jiangsu Higher Education Institutions, China(No.PPZY2015A056).

摘要/Abstract

摘要：

研究了具有较高酯合成活性的华根霉膜相关脂肪酶及其沉淀酶蛋白的重折叠处理过程, 发现膜成分及表面活性剂可能是影响其活性的关键因素. 进一步考察了影响异源表达的可溶性华根霉脂肪酶r27RCL酯合成活性重塑的关键因子及作用阶段. 研究结果表明, 表面活性剂对脂肪酶的酯合成活性具有关键影响, 直接添加表面活性剂可使酯合成活性显著提高. 在7种不同表面活性剂中, 两性离子表面活性剂LPC14将r27RCL的酯合成活性提高了5.75倍. 分子动力学模拟结果表明, 在有机相反应中, 表面活性剂的添加使脂肪酶催化三联体之间的氢键作用力得到加强, 从而提高了脂肪酶的有机相酯合成能力.

关键词: 表面活性剂, 脂肪酶, 非水相催化, 酯合成活性重塑, 分子动力学模拟

Abstract:

The regaining of ester synthesis catalytic ability of extracted and expressed Rhizopus chinensis lipase was investigated in this work. After de novo folding in a mimicking membrane environment, the maximum ester synthesis specific activity of extracted lipase precipitated protein(eRCL-P) was re-obtained and even increased for over 5 times. The refolding treatment of r27RCL, a Pichia pastoris expressed lipase with a high hydrolysis activity but low ester synthesis activity, with different membrane mimicking factors also increased the organic catalysis ability of the protein. The best enhancement of ester synthesis activity was shown when r27RCL was directly mixed with detergent, which indicated the interaction between lipase and regulation factor was established during mixing stage in water solution. Moreover, seven different detergents of three diversity types were introduced to confirm the result, and the zwitterionic detergent LPC14 increased the ester synthesis activity of r27RCL for the best effect as 5.75 times. Although the circular dichroism spectrum showed that the secondary structure of r27RCL had no obviously changes, the molecular dynamics simulation demonstrated that, in organic reaction, the hydrogen bonds between the catalytic triad of r27RCL have been enhanced after mixing with detergent, which may therefore stimulate the ester synthesis activity of lipase. In addition, these results confirmed the regulation of lipase ester synthesis activity, which will surely establish the basics for further research and production.

Key words: Detergent, Lipase, Non-aqueous catalysis, Ester synthesis activity regulation, Molecular dynamics simulation

中图分类号:

O621.25

TrendMD:

张璋, 王栋, 王晓雷, 徐岩. 华根霉脂肪酶有机相酯合成活性的重塑. 高等学校化学学报, 2019, 40(4): 747.

ZHANG Zhang,WANG Dong,WANG Xiaolei,XU Yan. Regulation of Ester Synthesis Activity of Rhizopus chinensis Lipase^†. Chem. J. Chinese Universities, 2019, 40(4): 747.

图/表 8

Fig.1 Extraction and isolation of R. chinensis lipase1. The extracted lipase from R. chinensis, eRCL; 2. the supernatant of the extract, eRCL-S; 3. the precipitate of the extract, eRCL-P; M. low molecular protein marker .

Table 1 Activity assay of R. chinensis lipase

Sample	Concentration/(mg·mL^-1)	Hydrolysis activity/(U·mg^-1)	Ester synthesis activity /(U·mg^-1)
eRCL	0.75±0.03	11.4±0.6	10.4±0.6
eRCL-S	0.35±0.02	9.8±0.08	8.1±0.04
eRCL-P	0.98±0.03	3.6±0.05	1.8±0.11
eRCL-P refolding	0.22±0.01	1.1±0.01	0.8±0.01
eRCL-P refolding with eRCL-S	0.65±0.02	8.8±0.06	9.1±0.03

Fig.2 Effect of additives on the activities of R. chinensis lipase r27RCL in refoldingr27RCL, the commercial R. chinensis lipase; MC, membrane component; TX, detergent Triton X-100; with MC, r27RCL refolding with MC; with TX, r27RCL refolding with Triton X-100.

Fig.3 Effect of adding different stages of MC and Triton X-100 on activity of lipase r27RCLMC, membrane component; TX, detergent Triton X-100; with MC in re-, adding MC in renaturation during refolding process; with TX in re-, adding Triton X-100 in renaturation during refolding process; +MC, r27RCL mixed with membrane component directly; +TX, r27RCL mixed with Triton X-100 directly.

Fig.4 Effects of different detergents on the activity of r27RCLa. r27RCL; b. CTAB; c. Façade-EM; d. C12E8; e. Trion X-100; f. DiC6PC; g. LPC14; h. CHAPS.

Fig.5 Far-UV circular dichroism spectra of lipase r27RCL(a) and r27RCL with detergent LPC14(b)

Fig.6 RMSD(A, B) and RMSF(C, D) value of r27RCL(A, C) and r27RCL with LPC14(B, D) in heptane(a) and water(b)

Fig.7 Simulated 3D structure of catalytic triad of r27RCL in different systemsCrystal structure of r27RCL was downloaded from PDB(ID 4L3W) and was showed in green. The simulated 3D structure of r27RCL in water and heptane were showed in cyan and magenta, respectively. The amino acids of catalytic triad of r27RCL and LPC14 were showed as stick.

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华根霉脂肪酶有机相酯合成活性的重塑

Regulation of Ester Synthesis Activity of Rhizopus chinensis Lipase^†

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华根霉脂肪酶有机相酯合成活性的重塑

Regulation of Ester Synthesis Activity of Rhizopus chinensis Lipase†

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Regulation of Ester Synthesis Activity of Rhizopus chinensis Lipase^†