Journal of Bioscience and Bioengineering VOL. 119 No. 6, 648e651, 2015 www.elsevier.com/locate/jbiosc
Purification and characterization of a novel NADPH-dependent 2-aminoacetophenone reductase from Arthrobacter sulfureus Guogang Zhao,1, 2 Wanru Sun,1 and Jianjun Wang1, * State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, PR China1 and Department of Internal Medicine, University of Kentucky School of Medicine, Lexington, KY 40536, USA2 Received 8 June 2014; accepted 5 November 2014 Available online 6 December 2014
A novel 2-aminoacetophenone reductase was purified to homogeneity from Arthrobacter sulfureus BW1010. The enzyme is a monomer with a molecular weight of approximately 60 kDa. Using NADPH as coenzyme, it catalyzes the reduction of ketones, especially amine phenyl ketones, and stereospecifically reduces 2-aminoacetophenone to (S)-2amino-1-phenylethanol (e.e > 99.8%) with the optimal pH at 7.5. Ó 2014, The Society for Biotechnology, Japan. All rights reserved. [Key words: 2-Aminoacetophenone reductase; (S)-2-Amino-1-phenylethanol; Arthrobacter sulfureus; Reduction; NADPH-dependent]
There is a group of reductases that catalyze the reduction of various aldehydes and ketones. These reductases exist widely in living organisms, and participate in the degradation of biogenic and xenobiotic carbonyl compounds as their physiological functions (1). Some members of them have been characterized and shown the ability of reducing aldehydes or ketones to optically pure alcohols, which are important intermediates for synthesis of various chiral pharmaceuticals (2e6). Optically active 2-amino-1-phenylethanol (PEA) is a useful building block for adrenergic drugs, which are utilized in clinical treatment of asthma, glaucoma and cardiovascular diseases (7). It is a simple and direct way to obtain desired optically pure intermediates through stereo-selective bio-reduction of the prochiral a-amino phenylketones using enzymes or microbial cells. However, this approach has been little utilized in the synthetic organic chemistry. Baker’s yeast (Saccharomyces cerevisiae) has been used to reduce corresponding amine-phenylketones to (R)-PEA derivatives with low yield (8), and a 2-aminoacephenone reductase (2-AAPR) from Burkholderia sp. YT that catalyzes the reduction of 2aminoacephone (2-AAP) to (R)-PEA has been purified and characterized (9). It is of great interest to mine more novel enzymes with favorable properties for the reduction. Arthrobacter sulfureus BW1010 is a 2-AAPR producer. Under aerobic conditions and with racemic PEA as an inducer, the yield of whole cells produced (S)-PEA (e.e. > 99%) reached 75% within 6 h (10). In this communication, we report on the purification and characterization of the novel NADPH-dependent 2-AAPR from A. sulfureus BW1010 cells.
* Corresponding author. Tel./fax: þ86 10 64807707. E-mail address: [email protected] (J. Wang).
MATERIALS AND METHODS Chemicals 2-AAP, racemic PEA, and (R)- and (S)-PEA (e.e. 97%) were purchased from Acros Co. (Belgium). Hydroxypropyl-b-cyclodextrin was purchased from Beckman Coulter Ltd. (USA). HiPrep16/10 Phenyl (high sub), Mono Q HR5/5 and Superdex 200 HR10/30 were obtained from GE Amersham Biosciences (USA). All other chemicals were analytical grade. Growth conditions A. sulfureus BW1010 was pre-cultivated in 250 ml flasks containing 50 ml of basal medium broth (pH 7.0) consisting of 1% glucose, 1% Bacto Tryptone, 0.5% yeast extract, 0.3% (NH4)2SO4 and 0.3% KH2PO4 (w/v) with constant agitation at 220 rpm at 30 C for 12 h. To induce the production of 2-AAPR, the cultures were inoculated in 500 ml flasks containing 100 ml of basal medium supplemented with 0.1% racemic PEA (w/v) as inducer. Incubation was performed aerobically for another 48 h as described above. Cells were harvested by centrifugation at 4 C, washed with ice-cold phosphate buffer (50 mM, pH 7.0) twice, and processed further for enzyme purification. Enzyme and protein assays The standard assay mixture (1.0 ml) for reduction contained 20 mM potassium phosphate buffer (pH 7.0), 5 mM 2-AAP and 0.1 mM NADPH and appropriate amount of enzyme. The reaction was conducted at 25 C, and measured by the increase in absorbance at 340 nm. One unit of enzyme activity is defined as the amount of enzyme that catalyzed the oxidation of 1.0 mmol NADPH per min at 25 C. Protein content was determined by the method of Bradford (11) with bovine serum albumin as the standard. Enzyme purification All purification procedures were performed at 0e4 C in buffer A (20 mM TriseHCl, 1 mM EDTA, 1 mM DTT, pH 8.0), unless other specified. All the columns used were equipped on an ÄKTA FPLC system (GE Amersham Biosciences). The cells (wet weight,150 g) were resuspended in 300 ml of buffer A, and disrupted with a sonic disintegrator at 0 C. After centrifugation at 15,000 g for 30 min, the supernatant was fractionated with solid ammonium sulfate. The precipitates obtained with 30e60% saturation were collected, dissolved in 25 ml of buffer A and dialyzed for 24 h against buffer A containing 500 mM (NH4)2SO4. The active solution was applied to a HiPrep16/10 Phenyl (high sub) column (1.6 mm 10 cm) pre-equilibrated with buffer A containing 500 mM (NH4)2SO4. After the column was washed with the pre-equilibrated buffer, the enzyme was eluted with a linear gradient of 500 to 0 mM (NH4)2SO4. Active fractions were pooled and dialyzed against buffer A overnight. The dialysate was loaded on a Mono Q HR5/5 column (0.5 mm 5 cm) pre-equilibrated with buffer A and subsequently eluted with a linear gradient of 250e500 mM KCl. The fractions showing enzymatic activities were pooled, concentrated and loaded onto a Superdex 200 HR10/ 30 column (1 mm 30 cm) pre-equilibrated with buffer A containing 100 mM KCl. The
1389-1723/$ e see front matter Ó 2014, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2014.11.004
VOL. 119, 2015
PURIFICATION AND CHARACTERIZATION OF 2-AAP REDUCTASE
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TABLE 1. Purification of 2-AAPR from A. sulfureus BW1010. Purification step Crude extract Ammonium sulfate Phenyl-Sepharose Mono Q Superdex 200
Total activity (U) 96.5 96.5 40.8 32.5 20.2
Total protein (mg) 1900 456 45 2.6 0.9
Specific activity (U/mg) 0.05 0.21 0.91 12.5 22.4
Fold 1 4.2 18 246 441
Yield (100%) 100 100 42 34 21
enzyme was eluted with the equilibration buffer. The active fraction was collected, added 20% (v/v) glycol, and stored at 20 C. SDS-PAGE and determination of molecular mass of 2-AAPR Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 10% acrylamide gel) was carried out as described by Laemmli (12). Gels were stained with Coomassie Brilliant Blue R-250. The molecular mass of the native purified 2-AAPR was estimated by gel filtration. Gel filtration was performed on a Superdex 200 HR column with 100 mM phosphate buffer and 100 mM KCl (pH 7.0) at a flow rate of 0.4 ml/min. The calibration proteins (GE Amersham Biosciences) with indicated molecular masses were used as references: ferritin (450 kDa), aldolase (158 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), and chymotrypsinogen A (25 kDa). N-terminal amino acid sequence Purified 2-AAPR was blotted from 10% SDS-PAGE gel to a polyvinylidene difluoride membrane, and was stained with Coomassie Brilliant Blue R-250. The protein band was cut out, and the N-terminal sequence analysis was carried out using an Applied Biosystems 477A protein sequencer (Life Technologies, USA).
FIG. 2. Effect of pH on the activity of 2-AAPR from A. sulfureus BW1010. The enzymatic activity was measured in 50 mM of citrate-K2HPO4 (pH 4.0e5.5, squares), KH2PO4eK2HPO4 (pH 5.5e8.0, circles), and TriseHCl (pH 8.0e10.0, triangles). Enzymatic reduction of 2-AAP A reaction mixture (2.5 ml) containing 50 mmol of KH2PO4eK2HPO4 (pH 7.0), 10 mM of 2-AAP and 2 mM of NADPH, 20 mM glucose and 0.1 U of glucose dehydrogenase were incubated at 30 C, followed by addition of 5 U of 2-AAPR. After 2 h incubation, the reaction mixture was passed through a 0.22 mm filter, and then analyzed by capillary electrophoresis. Analytical method of 2-AAP and PEA by capillary electrophoresis The analytical assay by capillary electrophoresis was performed as described (10). The retention times were 15.5 min for 2-AAP, 16.9 min for (R)-PEA, and 17.3 min for (S)-PEA, respectively.
RESULTS Purification of AAPR 2-AAPR was purified 441-fold with a recovery of 21% from the cell-free extract of A. sulfureus BW1010 (Table 1), and showed a single band on SDS-PAGE (Fig. 1). The specific activity of purified 2-AAPR was 22.4 U/mg using 2-AAP as the substrate. Subunit structure The molecular mass of native 2-AAPR was calculated to be 58 kDa based on the result of gel filtration (Fig. 1A). The subunit molecular mass of purified 2-AAPR was estimated to be approximately 60 kDa by SDS-PAGE (Fig. 1B). The results indicated that 2-AAPR was a monomer. The N-terminal amino acid sequence of 2-AAPR was TEFPHAGLIV. The amino acid sequence revealed no significant similarity to those of any other proteins deposited in the BLAST database. TABLE 2. Effect of various metal ions and chemical reagents on the activity of 2-AAPR from A. sulfureus BW1010. Chemicals
FIG. 1. Analysis of molecular weight and purity of 2-AAPR from A. sulfureus BW1010. (A) Determination of molecular weight by gel filtration chromatography. The molecular mass of the purified enzyme was calculated based on the following equation: lgMr ¼ 3.48Kav þ 2.90 (R2 ¼ 0.9935). Kav, the effective distribution coefficient; Mr, the molecular mass. Open circle, purified enzyme; closed circles, marker proteins: 1, ferritin (450 kDa); 2, aldolase (158 kDa); 3, bovine serum albumin (66 kDa); 4, ovalbumin (45 kDa); and 5, chymotrypsinogen A (25 kDa). (B) SDS-PAGE analysis of 2AAPR. Lane 1, the crude extract; lane 2, the fraction from ammonium sulfate precipitation; lane 3, the Phenyl-sepharose fraction; lane 4, the Mono Q fraction; and lane 5, the Superdex 200 fraction; lane 6, the molecular mass makers.
None FeCl3 MgCl2 CaCl2 FeCl2 ZnSO4 CuCl2 CoCl2 MnSO4 HgCl2 AgNO3 EDTA PCMB 2-Mercaptoethanol Dithiothreitol PMSF NaN3
Concentration (mM)
Relative activity (%)
1 1 1 1 1 1 1 1 0.1 0.1 1 1 1 1 1 1
100 78 140 98 46 0 0 97 100 0 0 100 25 100 100 98 100
Enzyme activity was measured as described in method section. The enzyme activity without addition of any chemicals was taken as 100%. 0.2 U of enzyme was used in each assay.
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ZHAO ET AL.
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TABLE 3. Substrate specificity of 2-AAPR from A. sulfureus BW1010. Substrate Acetone 2-Butanone 2-Pentanone 2-Hexanone 2-Heptanone 2-Octanone Acetophenone 2-Aminoacetophenone 2-Amnio-40 -chloroacetophenone 2-Amnio-40 -amnioacetophenone 2-Amnio-40 -hydoxyacetophenone
Relative activity (%) 99.8%) (Fig. 3). The hydride anion from NADPH is probably transferred to the re-face of the carbonyl, suggesting that the stereochemistry of 2-AAPR follows Prelog’s rule (17). Thus, 2-AAPR from A. sulfureus BW1010, in combination with the NADPH regeneration system, would be efficient in bio-preparation of optically active (S)-PEA drugs, such as (S)-Sotalol against arrhythmias. References 1. Shimizu, S., Ogawa, J., Kataoka, M., and Kobayashi, M.: Screening of novel microbial enzymes for the production of biologically and chemically useful compounds, Adv. Biochim. Eng. Biotechnol., 58, 45e87 (1997).
651
2. Hata, H., Shimizu, S., Hattori, S., and Yamada, H.: Ketopantoyl-lactone reductase from Candida parapsilosis: purification and characterization as a conjugated polyketone reductase, Biochim. Biophys. Acta, 990, 175e181 (1989). 3. Yamada, H., Shimizu, S., Kataoka, M., Sakai, H., and Miyoshi, T.: A novel NADPH-dependent aldehyde reductase, catalyzing asymmetric reduction of bketo acid esters, from Sporobolomyces salmonicolor: purification and characterization, FEMS Microbiol. Lett., 70, 45e48 (1990). 4. Katoka, M., Doi, Y., Sim, T. S., Shimizu, S., and Yamada, H.: A novel NADPHdependent carbonyl reductase of Candida macedoniensis: purification and characterization, Arch. Biochem. Biophys., 294, 469e474 (1992a). 5. Katoka, M., Sakai, H., Morikawa, T., Katoh, M., Miyoshi, T., Shimizu, S., and Yamada, H.: Characterization of aldehyde reductase from Sporobolomyces salmonicolor, Biochim. Biophys. Acta, 1122, 57e62 (1992b). 6. Itoh, N., Morihama, R., Wang, J. C., Okada, K., and Mizuguchi, N.: Purification and characterization of phenylacetaldehyde reductase from a styrene-assimilating Corynebacterium strain, ST-10, Appl. Environ. Microbiol., 63, 3783e3788 (1997). 7. Hong, Y. P., Gao, Y., Nie, X. Y., and Zepp, C. M.: Asymmetric reduction of aketoimines with oxazaborolidine catalysts: a novel, practical approach to chiral arylethanolamines, Tetrahedron Lett., 35, 5551e5554 (1994). 8. Izumi, T. and Fukaya, K.: Bake’s yeast reduction of a-(acylamino) acetopenones and lipase catalyzed resolution of 2-acylamino-1-arylethanols, Bull. Chem. Soc. Jpn., 66, 1216e1221 (1993). 9. Yamada-Onodera, K., Takase, Y., and Tani, Y.: Purification and characterization of 2-aminoacetophenone reductase of newly isolated Burkholderia sp. YT, J. Biosci. Bioeng., 104, 416e419 (2007). 10. Zhao, G., Wang, J., Ma, K., Wu, S., Liu, L., Yang, L., and Sun, W.: Transform 2aminoacetophenone to (S)-2-amino-1-phenylethanol by Arthrobacter sulfureus, Biotechnol. Lett., 26, 1255e1259 (2004). 11. Laemmli, U. K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature, 227, 680e685 (1970). 12. Bradford, M. M.: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem., 72, 248e254 (1976). 13. Kuhn, A., van Zyl, C., van Tonder, A., and Prior, B. A.: Purification and partial characterization of an aldo-keto reductase from Saccharomyces cerevisiae, Appl. Environ. Microbiol., 61, 1580e1585 (1995). 14. Forrest, G. L. and Gonzalez, B.: Carbonyl reductase, Chem. Biol. Interact., 129, 21e40 (2000). 15. Muheim, A., Waldner, R., Sanglard, D., Reiser, J., Schoemaker, H. E., and Leisola, M. S. A.: Purification and properties of alcohol-dehydrogenase from the white-rot fungus Phanerochaete chrysosporium, Eur. J. Biochem., 195, 369e375 (1991). 16. Shaw, J. P., Rekik, M., Schwager, F., and Harayama, S.: Kinetic studies on benzyl alcohol dehydrogenase encoded by TOL plasmid pWWO, J. Biol. Chem., 268, 10842e10850 (1993). 17. Prelog, V.: Specification of the stereospecificity of some oxidoreductases by diamond lattice sections, Pure Appl. Chem., 9, 119e130 (1964).
Purification and characterization of a novel NADPH-dependent 2-aminoacetophenone reductase from Arthrobacter sulfureus Guogang Zhao,1, 2 Wanru Sun,1 and Jianjun Wang1, * State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, PR China1 and Department of Internal Medicine, University of Kentucky School of Medicine, Lexington, KY 40536, USA2 Received 8 June 2014; accepted 5 November 2014 Available online 6 December 2014
A novel 2-aminoacetophenone reductase was purified to homogeneity from Arthrobacter sulfureus BW1010. The enzyme is a monomer with a molecular weight of approximately 60 kDa. Using NADPH as coenzyme, it catalyzes the reduction of ketones, especially amine phenyl ketones, and stereospecifically reduces 2-aminoacetophenone to (S)-2amino-1-phenylethanol (e.e > 99.8%) with the optimal pH at 7.5. Ó 2014, The Society for Biotechnology, Japan. All rights reserved. [Key words: 2-Aminoacetophenone reductase; (S)-2-Amino-1-phenylethanol; Arthrobacter sulfureus; Reduction; NADPH-dependent]
There is a group of reductases that catalyze the reduction of various aldehydes and ketones. These reductases exist widely in living organisms, and participate in the degradation of biogenic and xenobiotic carbonyl compounds as their physiological functions (1). Some members of them have been characterized and shown the ability of reducing aldehydes or ketones to optically pure alcohols, which are important intermediates for synthesis of various chiral pharmaceuticals (2e6). Optically active 2-amino-1-phenylethanol (PEA) is a useful building block for adrenergic drugs, which are utilized in clinical treatment of asthma, glaucoma and cardiovascular diseases (7). It is a simple and direct way to obtain desired optically pure intermediates through stereo-selective bio-reduction of the prochiral a-amino phenylketones using enzymes or microbial cells. However, this approach has been little utilized in the synthetic organic chemistry. Baker’s yeast (Saccharomyces cerevisiae) has been used to reduce corresponding amine-phenylketones to (R)-PEA derivatives with low yield (8), and a 2-aminoacephenone reductase (2-AAPR) from Burkholderia sp. YT that catalyzes the reduction of 2aminoacephone (2-AAP) to (R)-PEA has been purified and characterized (9). It is of great interest to mine more novel enzymes with favorable properties for the reduction. Arthrobacter sulfureus BW1010 is a 2-AAPR producer. Under aerobic conditions and with racemic PEA as an inducer, the yield of whole cells produced (S)-PEA (e.e. > 99%) reached 75% within 6 h (10). In this communication, we report on the purification and characterization of the novel NADPH-dependent 2-AAPR from A. sulfureus BW1010 cells.
* Corresponding author. Tel./fax: þ86 10 64807707. E-mail address: [email protected] (J. Wang).
MATERIALS AND METHODS Chemicals 2-AAP, racemic PEA, and (R)- and (S)-PEA (e.e. 97%) were purchased from Acros Co. (Belgium). Hydroxypropyl-b-cyclodextrin was purchased from Beckman Coulter Ltd. (USA). HiPrep16/10 Phenyl (high sub), Mono Q HR5/5 and Superdex 200 HR10/30 were obtained from GE Amersham Biosciences (USA). All other chemicals were analytical grade. Growth conditions A. sulfureus BW1010 was pre-cultivated in 250 ml flasks containing 50 ml of basal medium broth (pH 7.0) consisting of 1% glucose, 1% Bacto Tryptone, 0.5% yeast extract, 0.3% (NH4)2SO4 and 0.3% KH2PO4 (w/v) with constant agitation at 220 rpm at 30 C for 12 h. To induce the production of 2-AAPR, the cultures were inoculated in 500 ml flasks containing 100 ml of basal medium supplemented with 0.1% racemic PEA (w/v) as inducer. Incubation was performed aerobically for another 48 h as described above. Cells were harvested by centrifugation at 4 C, washed with ice-cold phosphate buffer (50 mM, pH 7.0) twice, and processed further for enzyme purification. Enzyme and protein assays The standard assay mixture (1.0 ml) for reduction contained 20 mM potassium phosphate buffer (pH 7.0), 5 mM 2-AAP and 0.1 mM NADPH and appropriate amount of enzyme. The reaction was conducted at 25 C, and measured by the increase in absorbance at 340 nm. One unit of enzyme activity is defined as the amount of enzyme that catalyzed the oxidation of 1.0 mmol NADPH per min at 25 C. Protein content was determined by the method of Bradford (11) with bovine serum albumin as the standard. Enzyme purification All purification procedures were performed at 0e4 C in buffer A (20 mM TriseHCl, 1 mM EDTA, 1 mM DTT, pH 8.0), unless other specified. All the columns used were equipped on an ÄKTA FPLC system (GE Amersham Biosciences). The cells (wet weight,150 g) were resuspended in 300 ml of buffer A, and disrupted with a sonic disintegrator at 0 C. After centrifugation at 15,000 g for 30 min, the supernatant was fractionated with solid ammonium sulfate. The precipitates obtained with 30e60% saturation were collected, dissolved in 25 ml of buffer A and dialyzed for 24 h against buffer A containing 500 mM (NH4)2SO4. The active solution was applied to a HiPrep16/10 Phenyl (high sub) column (1.6 mm 10 cm) pre-equilibrated with buffer A containing 500 mM (NH4)2SO4. After the column was washed with the pre-equilibrated buffer, the enzyme was eluted with a linear gradient of 500 to 0 mM (NH4)2SO4. Active fractions were pooled and dialyzed against buffer A overnight. The dialysate was loaded on a Mono Q HR5/5 column (0.5 mm 5 cm) pre-equilibrated with buffer A and subsequently eluted with a linear gradient of 250e500 mM KCl. The fractions showing enzymatic activities were pooled, concentrated and loaded onto a Superdex 200 HR10/ 30 column (1 mm 30 cm) pre-equilibrated with buffer A containing 100 mM KCl. The
1389-1723/$ e see front matter Ó 2014, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2014.11.004
VOL. 119, 2015
PURIFICATION AND CHARACTERIZATION OF 2-AAP REDUCTASE
649
TABLE 1. Purification of 2-AAPR from A. sulfureus BW1010. Purification step Crude extract Ammonium sulfate Phenyl-Sepharose Mono Q Superdex 200
Total activity (U) 96.5 96.5 40.8 32.5 20.2
Total protein (mg) 1900 456 45 2.6 0.9
Specific activity (U/mg) 0.05 0.21 0.91 12.5 22.4
Fold 1 4.2 18 246 441
Yield (100%) 100 100 42 34 21
enzyme was eluted with the equilibration buffer. The active fraction was collected, added 20% (v/v) glycol, and stored at 20 C. SDS-PAGE and determination of molecular mass of 2-AAPR Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 10% acrylamide gel) was carried out as described by Laemmli (12). Gels were stained with Coomassie Brilliant Blue R-250. The molecular mass of the native purified 2-AAPR was estimated by gel filtration. Gel filtration was performed on a Superdex 200 HR column with 100 mM phosphate buffer and 100 mM KCl (pH 7.0) at a flow rate of 0.4 ml/min. The calibration proteins (GE Amersham Biosciences) with indicated molecular masses were used as references: ferritin (450 kDa), aldolase (158 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), and chymotrypsinogen A (25 kDa). N-terminal amino acid sequence Purified 2-AAPR was blotted from 10% SDS-PAGE gel to a polyvinylidene difluoride membrane, and was stained with Coomassie Brilliant Blue R-250. The protein band was cut out, and the N-terminal sequence analysis was carried out using an Applied Biosystems 477A protein sequencer (Life Technologies, USA).
FIG. 2. Effect of pH on the activity of 2-AAPR from A. sulfureus BW1010. The enzymatic activity was measured in 50 mM of citrate-K2HPO4 (pH 4.0e5.5, squares), KH2PO4eK2HPO4 (pH 5.5e8.0, circles), and TriseHCl (pH 8.0e10.0, triangles). Enzymatic reduction of 2-AAP A reaction mixture (2.5 ml) containing 50 mmol of KH2PO4eK2HPO4 (pH 7.0), 10 mM of 2-AAP and 2 mM of NADPH, 20 mM glucose and 0.1 U of glucose dehydrogenase were incubated at 30 C, followed by addition of 5 U of 2-AAPR. After 2 h incubation, the reaction mixture was passed through a 0.22 mm filter, and then analyzed by capillary electrophoresis. Analytical method of 2-AAP and PEA by capillary electrophoresis The analytical assay by capillary electrophoresis was performed as described (10). The retention times were 15.5 min for 2-AAP, 16.9 min for (R)-PEA, and 17.3 min for (S)-PEA, respectively.
RESULTS Purification of AAPR 2-AAPR was purified 441-fold with a recovery of 21% from the cell-free extract of A. sulfureus BW1010 (Table 1), and showed a single band on SDS-PAGE (Fig. 1). The specific activity of purified 2-AAPR was 22.4 U/mg using 2-AAP as the substrate. Subunit structure The molecular mass of native 2-AAPR was calculated to be 58 kDa based on the result of gel filtration (Fig. 1A). The subunit molecular mass of purified 2-AAPR was estimated to be approximately 60 kDa by SDS-PAGE (Fig. 1B). The results indicated that 2-AAPR was a monomer. The N-terminal amino acid sequence of 2-AAPR was TEFPHAGLIV. The amino acid sequence revealed no significant similarity to those of any other proteins deposited in the BLAST database. TABLE 2. Effect of various metal ions and chemical reagents on the activity of 2-AAPR from A. sulfureus BW1010. Chemicals
FIG. 1. Analysis of molecular weight and purity of 2-AAPR from A. sulfureus BW1010. (A) Determination of molecular weight by gel filtration chromatography. The molecular mass of the purified enzyme was calculated based on the following equation: lgMr ¼ 3.48Kav þ 2.90 (R2 ¼ 0.9935). Kav, the effective distribution coefficient; Mr, the molecular mass. Open circle, purified enzyme; closed circles, marker proteins: 1, ferritin (450 kDa); 2, aldolase (158 kDa); 3, bovine serum albumin (66 kDa); 4, ovalbumin (45 kDa); and 5, chymotrypsinogen A (25 kDa). (B) SDS-PAGE analysis of 2AAPR. Lane 1, the crude extract; lane 2, the fraction from ammonium sulfate precipitation; lane 3, the Phenyl-sepharose fraction; lane 4, the Mono Q fraction; and lane 5, the Superdex 200 fraction; lane 6, the molecular mass makers.
None FeCl3 MgCl2 CaCl2 FeCl2 ZnSO4 CuCl2 CoCl2 MnSO4 HgCl2 AgNO3 EDTA PCMB 2-Mercaptoethanol Dithiothreitol PMSF NaN3
Concentration (mM)
Relative activity (%)
1 1 1 1 1 1 1 1 0.1 0.1 1 1 1 1 1 1
100 78 140 98 46 0 0 97 100 0 0 100 25 100 100 98 100
Enzyme activity was measured as described in method section. The enzyme activity without addition of any chemicals was taken as 100%. 0.2 U of enzyme was used in each assay.
650
ZHAO ET AL.
J. BIOSCI. BIOENG.,
TABLE 3. Substrate specificity of 2-AAPR from A. sulfureus BW1010. Substrate Acetone 2-Butanone 2-Pentanone 2-Hexanone 2-Heptanone 2-Octanone Acetophenone 2-Aminoacetophenone 2-Amnio-40 -chloroacetophenone 2-Amnio-40 -amnioacetophenone 2-Amnio-40 -hydoxyacetophenone
Relative activity (%) 99.8%) (Fig. 3). The hydride anion from NADPH is probably transferred to the re-face of the carbonyl, suggesting that the stereochemistry of 2-AAPR follows Prelog’s rule (17). Thus, 2-AAPR from A. sulfureus BW1010, in combination with the NADPH regeneration system, would be efficient in bio-preparation of optically active (S)-PEA drugs, such as (S)-Sotalol against arrhythmias. References 1. Shimizu, S., Ogawa, J., Kataoka, M., and Kobayashi, M.: Screening of novel microbial enzymes for the production of biologically and chemically useful compounds, Adv. Biochim. Eng. Biotechnol., 58, 45e87 (1997).
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