有机溶剂/缓冲液双相体系中固定化Acetobacter sp.CCTCC M209061细胞催化1-(4-甲氧基)-苯基乙醇不对称氧化反应

doi:10.7503/cjcu20140058

高等学校化学学报 ›› 2014, Vol. 35 ›› Issue (7): 1529.doi: 10.7503/cjcu20140058

有机溶剂/缓冲液双相体系中固定化Acetobacter sp.CCTCC M209061细胞催化1-(4-甲氧基)-苯基乙醇不对称氧化反应

程景¹, 娄文勇^1,²(), 宗敏华²

1. 华南理工大学轻工与食品学院应用生物催化实验室, 2. 制浆造纸工程国家重点实验室, 广州 510640

收稿日期:2014-01-17 出版日期:2014-07-10 发布日期:2014-06-10
作者简介:联系人简介: 娄文勇, 男, 博士, 教授, 博士生导师, 主要从事生物催化方面的研究. E-mail: wylou@scut.edu.cn
基金资助:
国家自然科学基金(批准号: 21222606, 21376096)、广东省自然科学基金重点项目(批准号: S2013020013049)、华南理工大学中央高校基本科研业务费(批准号: 2013ZG0003)和全国百篇优秀博士论文作者资助项目(批准号: 201504)资助

Biocatalytic Asymmetric Oxidation of Racemic 1-(4-Methoxyphenyl) Ethanol Using Immobilized Acetobacter sp.CCTCC M209061 Cells in Organic Solvent-containing Biphasic System^†

CHENG Jing¹, LOU Wenyong^1,^2,^*(), ZONG Minhua^2,^*

1. Lab of Applied Biocatalysis, College of Light Industry and Food Sciences, 2. State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou 510640, China

Received:2014-01-17 Online:2014-07-10 Published:2014-06-10
Contact: LOU Wenyong,ZONG Minhua E-mail:wylou@scut.edu.cn
Supported by:
Supported by the National Natural Science Foundation of China(Nos.21222606, 21376096), the Key Project of Guangdong Natural Science Foundation, China(No S2013020013049), the Fundamental Research Funds for Central Universities——South China University of Technology(No.2013ZG0003) and the Foundation For the Author of National Excellent Doctoral Dissertation of China(No.201504)

摘要/Abstract

摘要：

在有机溶剂/缓冲液双相体系中, 利用固定化醋酸杆菌Acetobacter sp.CCTCC M209061细胞高对映体选择性地催化1-(4-甲氧基)-苯基乙醇(MOPE)的不对称氧化反应, 成功地拆分外消旋MOPE得到对映体纯(S)-MOPE. 与游离细胞相比, 固定化细胞催化反应速度有所降低, 但其稳定性(包括操作稳定性、热稳定性和储藏稳定性)明显提高. 固定化细胞连续使用10批次(每批次12 h)后, 仍能保留其初始催化活性的58%以上, 而游离细胞仅保留约20%的相对活性. 在所考察的不同有机溶剂中, 正己烷不仅能较好地溶解底物, 而且对细胞的生物相容性相对较好, 因而提高了反应底物浓度、反应初速度、对映体回收率及残留底物e.e.值, 是反应体系中最适宜的有机相. 该反应的最适宜正己烷体积分数为60%, 辅底物为50 mmol/L丙酮, 底物浓度为40 mmol/L, 缓冲液pH=6.5, 反应温度为30 ℃; 在此条件下, 反应初速度为80.4 μmol/min, 反应12 h后, 对映体回收率和残留底物e.e.值分别为51.0%和99.9%, 明显好于水单相反应体系.

关键词: 固定化Acetobacter sp.CCTCC M209061细胞, 1-(4-甲氧基)-苯基乙醇, 4-甲氧基苯乙酮, 有机溶剂/缓冲液双相体系, 不对称氧化

Abstract:

The resolution of racemic 1-(4-methoxyphenyl) ethanol(MOPE) to enantiopure(S)-MOPE through highly enantioselective oxidation of MOPE with immobilized Acetobacter sp.CCTCC M209061cells was successfully conducted in organic solvent-containing biphasic system. In spite of exhibiting relatively slower reaction rate than free cells, immobilized cells of Acetobacter sp.CCTCC M209061 showed much higher stability, including operational stability, thermal stability and storage stability. The immobilized cells still retained more than 58% of its original catalytic activity after being repeatedly used for 10 batches(12 h per batch), and the free cells maintained only around 20% of relative activity. Among various organic solvents examined, use of n-hexane as the second phase in a two-phase system enhanced the concentration of substrate, the initial reaction rate, the enantiomer recovery and the residual substrate e.e., resulting from its excellent ability to dissolve MOPE and good biocompatibility with Acetobacter sp.CCTCC M209061 cells. As a result, n-hexane was selected as the most suitable organic phase for the reaction. For the biocatalytic oxidation of MOPE carried out in n-hexane-based biphasic system, the optimum volume fraction of n-hexane, co-substrate and its concentration, substrate concentration, buffer pH and reaction temperature were 60%, 50 mmol/L acetone, 40 mmol/L, 6.5 and 30 ℃, respectively. Under the above-described optimized reaction conditions, the initial reaction rate was 80.4 μmol/min, the enantiomer recovery and the residual substrate e.e. reached 51.0% and 99.9% at reaction time of 12 h, respectively, which were better than the results obtained in aqueous monophasic system.

Key words: ImmobilizedAcetobacter sp.CCTCC M209061 cell, 1-(4-Methoxyphenyl) ethanol, 4'-Methoxyacetophenone, Organic solvent/buffer biphasic system, Asymmetric oxidation

中图分类号:

O643

TrendMD:

程景, 娄文勇, 宗敏华. 有机溶剂/缓冲液双相体系中固定化Acetobacter sp.CCTCC M209061细胞催化1-(4-甲氧基)-苯基乙醇不对称氧化反应. 高等学校化学学报, 2014, 35(7): 1529.

CHENG Jing, LOU Wenyong, ZONG Minhua. Biocatalytic Asymmetric Oxidation of Racemic 1-(4-Methoxyphenyl) Ethanol Using Immobilized Acetobacter sp.CCTCC M209061 Cells in Organic Solvent-containing Biphasic System^†. Chem. J. Chinese Universities, 2014, 35(7): 1529.

图/表 12

Fig.1 Asymmetric oxidation of MOPE catalyzed by immobilized cetobacter sp.CCTCC M20601 cells

Fig.2 Comparison of asymmetric oxidation of MOPE with free cells(a, c) and immobilized Acetobac-ter sp.CCTCC M209061 cells(b, d) in n-hexane/buffer biphasic system Reaction conditions:n-hexane/TEA-HCl buffer(100 mmol/L, pH=6.5)(the volume fraction of n-hexane was 60%), 1.5 g immobilized cells or 0.24 g free cells, 40.0 mmol/L MOPE, 50.0 mmol/L acetone, 30 ℃, 200 r/min.

Fig.3 Operational stability of free cells and immobilized Acetobacter sp.CCTCC M209061 cells in n-hexane/buffer biphasic system Reaction conditions:n-hexane/TEA-HCl buffer(100 mmol/L, pH=6.5)(the volume fraction of n-hexane was 60%), 1.5 g immobilized cells or 0.24 g free cells, 40.0 mmol/L MOPE, 50.0 mmol/L acetone, 30 ℃, 200 r/min. 12 h per batch, the relative activity of the first batch was defined as 100%.

Table 1 Effect of various organic solvents on asymmetric oxidation of MOPE with immobilized cetobacter sp.CCTCC M209061 cellsa

Organic solvent	lgP^b	Initial reaction rate/(μmol·min^-1)	Reaction time/h	Enantiomer recovery^c(%)	e.e.^d(%)
Ethyl acetate/buffer	0.7	7.2	26	24.1	31.7
Isopropyl ether/buffer	1.9	16.8	24	36.0	56.3
Cyclohexane/buffer	3.0	45.0	13	47.2	89.0
n-Hexane/buffer	3.5	51.3	12	48.0	92.3
Isooctane/buffer	3.9	42.5	15	45.5	83.4
n-Nonane/buffer	4.7	34.9	18	42.7	74.5
Dodecane/buffer	6.6	30.3	20	41.0	69.5

Table 2 Partition coefficients of MOAP and MOPE between two phase systems

Media	Partition coefficients between the two phases		Media	Partition coefficients between the two phases
Media	MOAP		MOPE	MOAP	MOPE
Ethyl acetate/buffer	46.4	30.5	Isooctane/buffer	7.0	0.8
Isopropyl ether/buffer	27.1	10.8	n-Nonane/buffer	6.5	0.6
Cyclohexane/buffer	10.7	1.4	Dodecane/buffer	6.0	0.5
n-Hexane/buffer	9.6	1.1

Fig.4 Effect of various organic solvents on glucose metabolic activity retention of Acetobacter sp.CCTCC M209061 cells a. Buffer; b. ethyl acetate; c. isopropyl ether; d. cyclohe-xane; e. n-hexane; f. isooctane; g. n-nonane; h. dodecane.

Fig.5 Effect of n-hexane concentration on asymme-tric oxidation of MOPE with immobilized Acetobacter sp. CCTCC M209061 cells Reaction conditions: 30 ℃, 200 r/min, 30.0 mmol/L MOPE, 1.5 g immobilized cells, n-hexane/TEA-HCl buffer(100 mmol/L, pH=6.5), 70.0 mmol/L acetone.

Table 3 Effect of co-substrate on asymmetric oxidation of MOPE with immobilized Acetobacter sp. CCTCC M209061 cellsa

Co-substrate	Initial reaction rate/(μmol·min^-1)	Reaction time/h	Enantiomer recovery^b(%)	e.e.^c(%)
No co-substrate	37.5	24	40.8	70.0
Acetone	56.0	12	50.5	99.9
2-Butanone	42.2	20	44.1	75.4
2-Pentanone	39.0	22	42.9	88.6
Cyclohexanone	53.1	14	48.0	94.3
Acetylacetone	49.4	16	45.8	72.4
4-Methyl-2-pentanone	38.7	24	42.0	71.8

Fig.6 Effect of acetone concentration on asymmetric oxidation of MOPE with immobilized Acetobacter sp.CCTCC M209061 cells Reaction conditions:30 ℃, 200 r/min, n-hexane/TEA-HCl buffer(100 mmol/L, pH=6.5)(the volume fraction of n-hexane was 60%), 1.5 g immobilized cells, 30.0 mmol/L MOPE.

Table 4 Effect of substrate concentration on asymmetric oxidation of MOPE with immobilized Acetobacter sp.CCTCC M209061 cellsa

Concentration of substrate/(mmol·L^-1)	Initial reaction rate/(μmol·min^-1)	Reaction time/h	Enantiomer recovery^b(%)	e.e.^c(%)
30.0	69.4	9.0	51.8	99.9
35.0	75.0	10.0	51.5	99.9
40.0	80.4	12.0	51.0	99.9
45.0	69.8	16.0	48.8	96.5
50.0	66.0	17.5	48.0	92.3
55.0	63.2	20.0	47.3	90.1

Fig.7 Effect of buffer pH on asymmetric oxidation of MOPE with immobilized Acetobacter sp.CCTCC M209061 cells Reaction conditions: 30 ℃, 200 r/min, n-hexane/TEA-HCl buffer(100 mmol/L)(the volume fraction of n-hexane was 60%), 1.5 g immobilized cells, 50.0 mmol/L acetone, 40.0 mmol/L MOPE.

Fig.8 Effect of reaction temperature on asymme-tric oxidation of MOPE with immobilized Acetobacter sp.CCTCC M209061 cells Reaction conditions: 200 r/min, n-hexane/TEA-HCl buffer(100 mmol/L, pH=6.5)(the volume fraction of n-hexane was 60%), 1.5 g immobilized cells, 50.0 mmol/L acetone, 40.0 mmol/L MOPE.

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有机溶剂/缓冲液双相体系中固定化Acetobacter sp.CCTCC M209061细胞催化1-(4-甲氧基)-苯基乙醇不对称氧化反应

Biocatalytic Asymmetric Oxidation of Racemic 1-(4-Methoxyphenyl) Ethanol Using Immobilized Acetobacter sp.CCTCC M209061 Cells in Organic Solvent-containing Biphasic System^†

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