Catalytic action of L-2-halo acid dehalogenase on long-chain L-2-haloalkanoic acids in organic solvents
A.K.M. Quamrul Hasan* Harumi Takada, Nobuyoshi Esaki, and Kenji Soda† Laboratory of Microbial Biochemistry; Institute for Chemical Research, Kyoto University, Uji, Kyoto-Fu 611, Japan
Received December 4, I990/Accepted April 10, I99f
A Lyophilized preparation of L-2-halo acid dehalogenase was not only stable but also catalytically active in anhy- drous dimethyl sulfoxide, toluene, and other organic sol- vents. 2-Halo acids with long alkyl (C t—C ,t) or aromatic (phenyl and benzyl) side chains were inert in water but de- halogenated effectively in anhydrous dimethyl sulfoxide by the lyophilized enzyme. Long-chain 2-haloalkanoic acids such as 2-bromohexadecanoic acid were better as sub- strates than short-chain halo acids (e.g., 2-chIoropropanoic acid). The dehalogenation proceeded with inversion of C 2 configuration to produce the corresponding (2fl’)-2-hydroxy acids in anhydrous dimethyl sulfoxide in the same way as found in water.
Key words: L-2-halo acid dehalogenase • dehalogenation • in dimethyl sulfoxide • 2-hydroxy acids • stereospecific pro- duction of substrate specificity • change in organic solvent
INTRODUCTION
Optically active 2-hydroxy acids are useful starting ma- terials for the synthesis of various pharmaceuticals and agrochemicals. In contrast to amino acids, 2-hydroxy acids are rarely produced by fermentation except lactate. Chiral 2-hydroxy acids can be resolved by chromatogra- phy after their conversion to diastereomeric compounds with optically active amines.7 However, the methods require expensive reagents and tedious procedures. Mi- crobial resolution of racemic 2-hydroxy acids based on enantiospecific dehydrogenation has been reported.” Enantiospecific NADH-dependent 2-hydroxy acid dehy- drogenases have been used to produce optically active 2-hydroxy acids from their corresponding 2-oxo acids.4 However, these methods are only applicable to the pro- duction of a limited number of 2-hydroxy acids that are substrates.
L-2-Halo acid dehalogenase of Pseudomonas putida
dehalogenates u-2-halo acids to produce the correspond- ing 2-hydroxy acids with inversion of C2 configuration and is applicable to the enantioselective synthesis of 2-hydroxy acids.’ However, 2-halo acids, in particular 2-bromo acids, are generally unstable in aqueous solu- tions and give undesirable racemic 2-hydroxy acids
* On study leave from University of Dhaka, Bangladesh. ’ To whom all correspondence should be addressed.
Biotechnology and Bioengineering, Vol. 38, Pp. 1114—1117 (1991) @ 1991 John Wiley & Sons, Inc.
spontaneously. Therefore, their enzymatic dehalogena- tion should be preferably performed in organic solvents in order to obtain 2-hydroxy acids with high optical pu- rity. We here describe the catalysis of L-2-halo acid de- halogenase in organic solvents on various 2-halo acids with long alkyl chains and aromatic side chains, which do not serve as substrates in aqueous environments.
MATERIALS AND METHODS
Chemicals
Toluene, lithium L-lactate, and DL-2-Chloropropanoic acid were purchased from Nacalai Tesque, Kyoto, Japan; lithium D-lactate, L-2-Ch1oropropanoic, and 2-hydroxy- 3-phenylpropanoic acids were from Sigma; 2-hyroxy- hexanoic acid and dimethyl sulfoxide (DMSO) were from Aldrich; 2-hydroxyoctanoic and 2-hydroxyhexa- decanoic acids were from Aldrich; 2-hydroxyoctanoic and 2-hydroxyhexadecanoic acids were from Tokyo Kasei, Tokyo. Other commercial reagents were of ana- lytical grade. 2-Chloro-3-phenylpropanoic, 2-chloro- 3-hydroxypropanoic, and 2-chloroalkanoic acids with C5—C 7 were prepared from the corresponding racemic 2-amino acids by the method of Fu et al.2 2-Hydroxy- heptanoic acid was prepared from 2-chloroheptanoic acid by the method of Cowdrey et al. I
Enzyme Preparation and Assay
L-2-Halo acid dehalogenase was purified from Pseudo- monas putida according to the method described previ- ously.’ Dehalogenation of 2-halo acids was followed spectrophotometrically by determination of the halogen ions released as described previously. 6 One unit of en- zyme was defined as the amount of enzyme that cata- lyzes the dehalogenation of 1 mmol substrate/min.
Enzymatic Reaction in Organic Solvent
2- Halo acids (25 mM, 0.2 mL) dissolved in appropriate organic solvents were mixed with a lyophilized prepara-
CCC 0006-3592/91/0901114—04$04.00
tion of L-2-halo acid dehalogenase (2.5 pg; 0.3 unit). The reactions were carried out with shaking at 30°C for 30 min. After incubation, 0.15M HCSOs was added to stop the reaction. Control experiments were done with enzyme preparations heat inactivated at 95°C for 20 min.
Reaction Mixture Analyses
After the reaction, a decrease in the substrate and an increase in the product were determined by analyzing their methyl esters by gas chromatography (GC). The GC analysis was performed with a GC instrument (Shimadzu GC 14A) equipped with a flame ionization detector and an integrator (Shimadzu CR 6A chromato- pac). A capillary column (Ulbon HR-20, 0.25 mm i.d. x 50 m) was used with helium as carrier gas at a flow rate of 1.4 mL/min. The column temperature was pro- grammed at 5°C/min, from l00°C to 250°C. The in- jector and detector temperatures were set at 270°C. Lactic acid was determined by high-performance liquid chromatography (HPLC) with a Ultron PS-80H column (0.8 cm i.d. x 30 cm) (Shinwa Kako, Kyoto, Japan): solvent, 0.126% perchloric acid in water; flow rate,
0.8 mL/min; detection, 210 nm.
The stereostructure of 2-hydroxyheptanoic acid was examined by the enzymatic method with D- and L-2- hydroxyisocaproate dehydrogenases described by Hum- mel et al.’ and Schutte et al.,’° respectively, except for using 3-acetylpyridine adenine dinucleotide instead of NAD. Optical purity of 2-hydroxyhexadecanoic acid produced enzymatically was calculated from the following equation: optical purity = [(AD — AL)/ (AD + AL)] X 100(%), where AD denotes HPLC peak area derived from the D-enantiomer and AL for the L-enantiomer.
RESULTS
Stability of L-2-Halo Acid Dehalogenase in Anhydrous DMSO
c-2-Halo acid dehalogenase was lyophilized without sig- nificant loss of activity (residual activity, more than 92%). When the lyophilized enzyme was incubated with L-2-Chloropropanoic acid in anhydrous DMSO, lactic acid and chloride ions were produced at a rate of about 6% of that in the standard aqueous medium. Although the activity was low in DMSO, no reaction occurred in the absence of the enzyme or in the presence of the heat-inactivated enzyme. The activity of the lyophilized enzyme did not decrease in DMSO after 4 h. The en- zyme immobilized in polyurethane matrices also de- halogenated L-2-chloropropanoir acid in anhydrous DMSO at the rate approximately that of the lyophilized enzyme. The dehalogenation in anhydrous DMSO pro-
ceeded with inversion of the C configuration with both enzyme preparations.
Substrate Specificity of L-2-Halo Acid Dehalogenase in Anhydrous DMSO
c-2-Halo acid dehalogenase acts exclusively on i.-2- haloalkanoic acids with carbon chain length of less than 4 in the standard aqueous medium.’ However, the lyophilized enzyme dehalogenated various longer chain 2-haloalkanoic acids as well in anhydrous DMSO (Table I). The configuration of 2-hydroxyheptanoic acid was analyzed with D- and L-2-hydroxy acid dehydroge- nases: the 2ft-isomer was obtained with an optical purity of 78% enantiomeric excess. The 2-hydroxyhexa- decanoic acid formed was shown to possess a 77% diastereoisomeric excess of 2fi-isomer when it was ana- lyzed by HPLC after conversion to the carboxamide with (ft)-1-(1-naphthyl)ethylamine.”
2-Haloalkanoic acids with longer carbon chains, such as 2-bromohexadecanoic acid, were good substrates for the enzyme. 2-Chloroalkanoic acids from C, to C 7 were more reactive substrates than 2-chloropropanoic acid. In addition, 2-halo acids with an aromatic side chain, which are inert in water, were also dehalogenated effi- ciently. Thus, the substrate specificity of the enzyme was changed markedly in nonaqueous media. The ste- reochemistry of the enzymatic dehalogenation was not affected by organic solvents: the dehalogenation pro- ceeds with inversion of the configuration.
Enzymatic Dehalogenation of 2-ChIoroheptanoic
Acid in Various Organic Solvents.
The lyophilized enzyme preparation was suspended in various organic solvents containing 2-chloroheptanoic acid, and the chloride produced was determined. The enzyme was found to be active in both polar and non- polar solvents, although polar solvents were generally more suitable (Table II). The DMSO was the best among the solvents examined.
DISCUSSION
Various enzymes are active in nonaqueous media, as shown by Klibanov and co-workers.’ We have shown that L-2-halo acid dehalogenase is also active in various organic solvents. Although the enzyme was inactivated in a mixture of DMSO and water, it was stable in anhy- drous DMSO. Other anhydrous polar solvents such as N,N-dimethylformamide did not inactivate the enzyme. Chymotr ypsin is not inactivated either in DMSO.‘5 N,N-dimethylformamide is a suitable reaction medium for subtilisin .’0 However, most enzymes are inactivated in these polar solvents.’” Therefore, L-2-halo acid de- halogenase is a new addition to such pro-organic solvent
COMMUNICATIONS TO THE EDITOR 1115
Table I. Substrate spectficity of r-2-halo acid dehalogenase in different reaction media and identification of the enzymatic products in DMSO.
Halo acid‘
Relative
activity (to) Retention water DMSO time (min)
Product‘
Hydroxy acid
2-Bromohexanoic — 100
2-Bromooctanoic — 50
2-Bromotetradecanoic — 130
2-Bromohexadecanoic — 270
13.3d
9.1′
2-Hydroxyoctanoic 2-Hydroxyhexa-
decanoic
Dash indicates that the product analysis was not done. ‘ Racemic mixtures were used un less otherwise stated.
Reaction conditions are the same as described in Materials and Methods except that
3.0 units of the freeze-dried enzyme was used with incubation time for 1.5 h. ‘ HPLC. Conditions described in Materials and Methods section.
GC retention times. Conditions described in Materials and Methods.
2-Hydrox yhexadecanoic acid was converted to the 1-(1-naphthy1)ethylamide as de- scribed in Materials and Methods, and analyzed by HPLC with Ultron SIL column (0. 46 cm i.d. x 25 cm) (Shinwa Kako, Kyoto, Japan) and hexane/2-propanol (10:1, v/v) at 0.8 mL/min by monitoring at 280 nm.
Table II. Reactivity of c-2-halo acid dehalogenase with 2-chIoro- heptanoic acid in various solvents.
genation is not influenced by DMSO: (2-fi)-2-hydroxy- heptanoic acid and (2-fi)-2-hydroxyhexadecanoic acid
Solvent
Relative activity (to)’
were produced from the corresponding 2-halo acids with inversion of configuration. The enzyme shows the strict enantiospecificity but low structural specificity in DMSO. The action of the enzyme on longer chain 2-halo acids is probably due to their high solubility in organic solvents.
We are grateful to Dr. Maria-Regina Kula, Institute for Enzyme Technology, Dusseldorf University, Germany, and Dr. Kenji Mori, the University of Tokyo, Japan, for provid- ing us with n- and L-2-hydroxy acid dehydrogenases and (R)- 2-hydroxyhexadecanoic acid, respectively.
‘ Initial activity for c-2-ch1oropropanoic acid in water is 100Wo.
enzymes. It is probably the first example of an enzyme that is active in anhydrous DMSO.
Substrate specificity of enzymes is often affected by organic solvents.” We studied the reactivity in organic solvents of long-chain 2-haloalkanoic acids that are not substrates in water for L-2-halo acid dehalogenase. 2- Chloroalkanoic acids with C, —C 7 are better as sub- strates than L-2-chloropropanoic acid in an hydrous DMSO. This reaction triedium is also suitable for the enzymatic dehalogenation of 2-halo acids with an aro- matic side chain, which are also unreactive in water. However, the stereochemistry of the enzymatic dehalo-
References
1. Cowdrey, W.A. , Hughes, E. D., Ingold, C.K. 1937. J. Chem. Soc. 1208.
2. Fu, S.-C. J., Birnbaum, S. M., Greenstein, J.P. 1954. J. Am. Chem. Soc. 76: 6054.
3. Hiratake, J., Inagaki, M., Yamamoto, Y. , Oda, J. 1987. J. Chem. Soc. Perkin Trans. 1 1053.
4. Hummel, W., Kula, M.-R. 1989. Eur. J. Biochem. 184: 1.
5. Hummel, W., Schutte, H., Kula, M.-R. 1985. Appl. Microbiol. Biotechnol. 21: 7.
6. Iwasak i, I., Utsumi, S., Hagino, K., Ozawa, T. Bull. Chem. Soc.
7. Karlsson, K.-A. , Pascher, I. 1974. Chem. Phys. Lipids 12: 65.
8. Klibanov, A. M. 1989. Trends Boichem. Sci. 14: 141.
1116 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 38, NO. 9, NOVEMBER, 1991
9. Motosugi, K. , Esaki, N. , Soda, K. 1982. Agric. Biol. Chem. 46: 12. Schutte, H., Hummel, W., Kula, M.-R. 1984. Appl. Microbiol. 837. Biotech noI. 19: 167.
10. Riva, S., Chopineau, J., Kieboom, A.P.G., Klibanov, A. M. 13. Singer, S. J. 1962. Adv. Protein Chem . 17: 1.
1988. J. Am . Chem. Soc. 110: 584. 14. Zaks, A., Klibanov, A.M. 1986. J. Am. Chem. Soc. 108: 2767.
11. Sawada, T. , Ogawa, M., Ninomiya, R., Yokose, K., Fujiu, M. , 15. Zaks, A., Klibanov, A. M. 1988. J. Biol. Chem. 263: 3194. Watanabe, K., Suhara, Y. , Maruyama, H. B. 1983. Appl. Envi-
ron . Microbiol. 45: 884.