复合交联酶聚集体的制备及催化羰基不对称还原合成手性醇

doi:10.7503/cjcu20170509

高等学校化学学报 ›› 2018, Vol. 39 ›› Issue (1): 54.doi: 10.7503/cjcu20170509

复合交联酶聚集体的制备及催化羰基不对称还原合成手性醇

杨猛¹, 江惠娟¹, 宁晨曦², 魏东芝², 苏二正^1,²()

1.南京林业大学轻工与食品学院, 南京210037
2.华东理工大学生物反应器工程国家重点实验室, 上海200237

收稿日期:2017-07-26 出版日期:2018-01-10 发布日期:2017-11-28
作者简介:联系人简介: 苏二正, 男, 博士, 教授, 主要从事酶与生物催化方面的研究. E-mail:ezhsu@njfu.edu.cn
基金资助:
国家自然科学基金(批准号: 31401679)、江苏省“六大人才高峰”高层次人才项目(批准号: 2015-JY-016)、中国博士后科学基金(批准号: 2016M600417, 2017T100373)、江苏省第五期“333工程”培养资金资助项目(批准号: BRA2017458)和生物反应器工程国家重点实验室开放基金资助

Preparation of Combined Cross-linked Enzyme Aggregates and Its Application in Synthesis of Chiral Alcohols by the Asymmetric Reduction of Carbanyl Group^†

YANG Meng¹, JIANG Huijuan¹, NING Chenxi², WEI Dongzhi², SU Erzheng^1,^2,^*()

1. College of Light Industry and Food Engineering, Nanjing Forestry University, Nanjing 210037, China
2. State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China

Received:2017-07-26 Online:2018-01-10 Published:2017-11-28
Contact: SU Erzheng E-mail:ezhsu@njfu.edu.cn
Supported by:
† Supported by the National Natural Science Foundation of China(No.31401679), the Six Talent Peaks Project in Jiangsu Province, China(No.2015-JY-016), the China Postdoctoral Science Foundation(Nos.2016M600417, 2017T100373), the 333 Project of Jiangsu Province, China(No.BRA2017458) and the Open Funding Project of the State Key Laboratory of Bioreactor Engineering, China

摘要/Abstract

摘要：

尝试建立一种复合交联酶聚集体辅酶再生方法. 以Corynebacterium sp. 酮还原酶KRED 30和Bacillus subtilis D-葡萄糖脱氢酶GDH 1为模式酶, 通过对沉淀剂类型、戊二醛浓度、交联温度和时间的优化, 制备了高活力复合交联酶聚集体. 酶学性质研究结果表明, 与自由酶混合物相比, 复合交联酶聚集体的稳定性和底物耐受性得到明显提升. 催化性能研究结果表明, 仅添加少量辅酶启动反应, 在水相体系中复合交联酶聚集体可有效催化4-氯乙酰乙酸乙酯(COBE)和间氯苯乙酮(CPO)还原合成手性醇, 连续催化10次, 残余活性仍保留70%以上; 在双相体系中, 复合交联酶聚集体的催化性能更加优越, 催化COBE的总转化数(TTN)达到6595, 催化CPO的TTN达到7500. 结果表明, 复合交联酶聚集体是一种可行的辅酶再生方法.

关键词: 酮还原酶, D-葡萄糖脱氢酶, 辅酶再生, 复合交联酶聚集体, 手性醇

Abstract:

Using ketoreductase KRED 30 from Corynebacterium sp. and the D-glucose dehydrogenase from Bacillus subtilis as the model enzymes, a combined cross-linked enzyme aggregates(combi-CLEAs) with high activity was prepared by optimization of the precipitant type, glutaraldehyde concentration, cross-linking temperature and time. Characterization of the combi-CLEAs showed that the stability and substrate tolerance of free enzymes mixture were significantly improved after combined cross-linking. Investigation of catalytical performance showed that combi-CLEAs could effectively catalyze the reduction of ethyl 4-chloroacetoacetate(COBE) and 3'-chloroacetophenone(CPO) for the synthesis of corresponding chiral alcohols in aqueous system. Only a little cofactor was needed to start the reactions. The catalytical activity could retain more than 70% after continuously catalyzing for 10 batches. The result showed that catalytical performance of combi-CLEAs in the biphasic system was much better than that in aqueous system. The total turnover number(TTN) for catalyzing COBE could attend 6595, and TTN for catalyzing CPO could attend 7500. Thus, cofactor regeneration by combi-CLEAs is feasible.

Key words: Ketoreductase, D-Glucose dehydrogenase, Cofactor regeneration, Combined cross-linked enzyme aggregates(Combi-CLEAs), Chiral alcohol

TrendMD:

杨猛, 江惠娟, 宁晨曦, 魏东芝, 苏二正. 复合交联酶聚集体的制备及催化羰基不对称还原合成手性醇. 高等学校化学学报, 2018, 39(1): 54.

YANG Meng, JIANG Huijuan, NING Chenxi, WEI Dongzhi, SU Erzheng. Preparation of Combined Cross-linked Enzyme Aggregates and Its Application in Synthesis of Chiral Alcohols by the Asymmetric Reduction of Carbanyl Group^†. Chem. J. Chinese Universities, 2018, 39(1): 54.

图/表 7

Scheme 1 Schematic view for the preparation of combined cross-linked enzyme aggregates and application in cofactor regeneration

Fig.1 Effect of glutaraldehyde content on the catalytical activity of the prepared combi-CLEAs during the cross-linking process

Fig.2 Effects of cross-linking time and temperature on the catalytical activity of the prepared combi-CLEAs during the cross-linking process

Fig.3 SEM images of combi-CLEAs in a less-structured form with different scale bar

Fig.4 Effects of substrate concentration on the catalytic activity of combi-CLEAs(a) and free enzymes mixture(b) in aqueous system(A, C) and biphasic system(B, D)

Fig.5 Reusability of combi-CLEAs catalyzed the reduction of ethyl 4-chloro-3-oxobutanoate(A) and 3'-chloroacetophenone(B) in aqueous system

Fig.6 Time courses for free enzymes mixture(A, C), combi-CLEAs(B, D) catalyzed the reduction of ethyl 4-chloro-3-oxobutanoate(A, B), 3'-chloroacetophenone(C, D) in biphasic system

参考文献 21

[1]	Chittamuru S., Reddy B. V. S., Rao A. B., J. Appl. Pharm., 2016, 8(3), 1—4
[2]	Ramesh N. P., Biomolecules, 2013, 3(4), 741—777
[3]	Liu C., Zhou Y., Yang L., Yang N., Zhang A., Chem. Res. Chinese Universities, 2015, 31(4), 669—673
[4]	Li H. D., Sun Z. H., Ni Y., Chem. Res. Chinese Universities, 2013, 29(6), 1140—1148
[5]	Jaeger K. E., Eggert T., Curr. Opin. Biotechnol., 2002, 13, 390—397
[6]	Detry J., Rosenbaum T., Lütz S., Hahn D., Jaeger K. E., Müller M., Eggert T., Appl. Microb. Biotech., 2006, 72, 1107—1116
[7]	Demir A. S., Şeşenoglu Ö., Eren E., Hosrik B., Pohl M., Janzen E., Kolter D., Feldmann R., Dünkelmann P., Müller M., Adv. Synth. Catal., 2002, 344, 96—103
[8]	Honda K., Inoue M., Ono T., Okano K., Dekishima Y., Kawabata H., J. Biosci. Bioeng., 2017, 123(6), 673—678
[9]	Hummel W., Gröger H., J. Biotech., 2014, 191, 22—31
[10]	Uppada V., Bhaduri S., Noronha S. B., Curr. Sci., 2014, 106, 946—957
[11]	Wu H., Tian C., Song X., Liu C., Yang D., Jiang Z., Green Chem., 2013, 15(7), 1773—1789
[12]	Wang M. L., Du G., Liu D. S., Chem. J. Chinese Universities, 2006, 27(9), 1686—1688
	(王梦亮, 杜刚, 刘滇生.高等学校化学学报, 2006, 27(9), 1686—1688)
[13]	Bastos F. M., França T. K., Machado G. D., Pinto G. F., Oestreicher E. G., Paiva L. M., J. Mol. Catal. B: Enzym., 2002, 19, 459—465
[14]	El-Zahab B., Jia H., Wang P., Biotechnol. Bioeng., 2004, 87, 178—183
[15]	Cao L., van Rantwijk F., Sheldon R. A., Org. Lett., 2000, 2, 1361—1364
[16]	Mu Y. Y., Zhen Q. N., Wang M. F., Qi W., Su R. X., He Z. M., Chem. J. Chinese Universities, 2014, 35(6), 1212—1218
	(慕洋洋, 甄倩楠, 王梦凡, 齐崴, 苏荣欣, 何志敏.高等学校化学学报, 2014, 35(6), 1212—1218)
[17]	Talekar S., Desai S., Pillai M., Nagavekar N., Ambarkar S., Surnis S., Ladole M., Nadar S., Mulla M., RSC. Adv., 2013, 3(7), 2265—2271
[18]	Jung D. H., Jung J. H., Seo D. H., Ha S. J., Kweon D. K., Park C. S., Bioresour. Technol., 2013, 130, 801—804
[19]	Bradford M. M., Anal. Biochem., 1976, 72, 248—254
[20]	Shimizu S., Kataoka M., Katoh M., Morikawa T., Miyoshi T., Yamada H., Appl. Environ. Microb., 1990, 56, 2374—2377
[21]	Schoevaart R., Wolbers M. W., Golubovic M., Ottens M., Kieboom A. P. G., van Rantwijk F., van der Wielen L. A. M., Sheldon R. A., Biotechnol. Bioeng., 2004, 87(6), 754—762

复合交联酶聚集体的制备及催化羰基不对称还原合成手性醇

Preparation of Combined Cross-linked Enzyme Aggregates and Its Application in Synthesis of Chiral Alcohols by the Asymmetric Reduction of Carbanyl Group^†

RichHTML

PDF (PC)

可视化

被引次数

摘要/Abstract

引用本文

使用本文

图/表 7

参考文献 21

相关文章 2

编辑推荐

Metrics

本文评价

[1]	丰险, 穆晓清, 聂尧, 徐岩. 亮氨酸脱氢酶催化底物偶联法合成α-酮异己酸[J]. 高等学校化学学报, 2019, 40(4): 698.
[2]	王梦亮, 杜刚, 刘滇生 . 光控生物不对称还原苯乙酮的研究[J]. 高等学校化学学报, 2006, 27(9): 1686.

复合交联酶聚集体的制备及催化羰基不对称还原合成手性醇

Preparation of Combined Cross-linked Enzyme Aggregates and Its Application in Synthesis of Chiral Alcohols by the Asymmetric Reduction of Carbanyl Group†

RichHTML

PDF (PC)

可视化

被引次数

摘要/Abstract

引用本文

使用本文

图/表 7

参考文献 21

相关文章 2

编辑推荐

Metrics

本文评价

Preparation of Combined Cross-linked Enzyme Aggregates and Its Application in Synthesis of Chiral Alcohols by the Asymmetric Reduction of Carbanyl Group^†