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

doi:10.7503/cjcu20140058

Abstract

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

CLC Number:

O643

TrendMD:

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^†[J]. Chem. J. Chinese Universities, 2014, 35(7): 1529.

Figures/Tables 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.

References 18

[1]	Zilbeyaz K., Kurbanoglu E. B., Bioresour. Technol., 2008, 99(6), 1549—1552
[2]	Goldberg K., Schroer K., Lutz S., Appl. Microbiol. Biotechnol., 2007, 76(2), 249—255
[3]	Hillier M. C., Marcoux J. F., Zhao D. L., Grabowski E. J., J. Org. Chem., 2005, 70(4), 8385—8394
[4]	Lee J. H., Han K., Kim M. J., Park J., Eur. J. Org. Chem., 2010, 2010(6), 999—1015
[5]	Wang W., Lou W. Y., Zong M. H., Chin. J. Catal., 2011, 32(6), 1003—1010
	(汪薇, 娄文勇, 宗敏华. 催化学报, 2011, 32(6), 1003—1010)
[6]	Gong G. H., Hou Y., Zhao Q., Process Biochem., 2010, 45(9), 1445—1449
[7]	Hatzakis N. S., Smonou L., Bioorgan. Chem., 2005, 33(4), 325—337
[8]	Egi M., Sugiyama K., Saneto M., Angew. Chem., 2013, 125(13), 3742—3746
[9]	Sonnenberg J. F., Pichugin D., Top. Catal., 2013, 56(14), 1199—1207
[10]	Wang X. T., Chen X. H., Xu Y., Lou W. Y., J. Biotechnol., 2013, 163(10), 292—300
[11]	Chen X. H., Lou W. Y., Zong M H., BMC Biotechnol., 2011, 11(1), 110—118
[12]	Cao H., Sun Q., Li C., Li K. X., Chem. J. Chinese Universities, 2013, 34(8), 1873—1879
	(曹红, 孙倩, 李春, 李开雄. 高等学校化学学报, 2013, 34(8), 1873—1879)
[13]	Chang M. Y., Juang R. S., Enzym. Microb. Technol., 2005, 36(1), 75—82
[14]	Guisan J.M., Immobilization of Enzymes and Cells, Humana Press, Totowa, 2006, 160—175
[15]	Castro G. R., Knubovets T.,Crit. Rev. Biotechnol., 2003, 23(3), 195—231
[16]	Klibanov A. M., Nature, 2001, 409(6817), 241—246
[17]	Goretti M., Ponzoni C., Caselli E., Bioresour. Technol., 2011, 102(5), 3993—3998
[18]	Li H. D., Sun Z. C., Ni Y., Chem. Res. Chinese Universities, 2013, 29(6), 1140—1148
	(Ed.: V, Z)