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Aminotransferase-Catalyzed Conversion of D-Amino Acids to L-Amino Acids Ian G. Fotheringham, Gene E. Kidman3 Brian S. McArthur,s Larry E. Robinaon,g and Mark P. Scollar'J Department of Process Sciences, The NutraSweet Company, Mt. Prospect, Illinois 60056
A microbial bioconversion process is described for the conversion of D-amino acids or the corresponding racemic mixtures t o L-amino acids. T h e process comprises reacting a D-amino acid or a D,L mixture of an amino acid with glucose and ammonia in the presence of an aminotransferase-containing culture of Escherichia coli. T h e aminotransferase is only moderately stereoselective in converting D- and L-amino acids to the keto acids. T h e aminotransferase-mediated conversion of a-keto acid t o L-amino acid exhibits very high stereospecificity, thus rendering the conversion of D-amino acids irreversible. Conversions of leucine, phenylalanine, and homophenylalanine to the corresponding L isomers are described.
The direct conversion of D-amino acids to L-amino acids eliminates the need for costly derivatization and resolution steps required for the recovery and utilization of D-amino acids produced in side reactions of peptide synthesis (1). A microbial whole-cell bioconversion process has been developed utilizing newly discovered activities of Escherichia coli aminotransferases aspartate aminotransferase (AAT)and tyrosine aminotransferase (TAT) with D-amino acids to convert D,L mixtures to L-amino acids in one step (SchemeI). The first half-reaction proceeds with moderate selectivity to yield the corresponding a-keto acid intermediate, and the second half-reaction comprises the wellknown conversion of a-keto acids to L-amino acids (2), which proceeds with near-absolute stereospecificity (3). The microbial conversion is carried out with inexpensive glucose and inorganic salts. Amino acceptor 1 and amino donor 2 (Scheme I) are generated by the actively metabolizing cells, eliminating the need to add them. E. coli strain HW857 [A(aspC)Kanr tyrB/TnlO] was transformed with plasmid vectors carrying the E. coli aspartate (aspC) and aromatic (tyrB) aminotransferase genes. Plasmid pIFlOO contained a subclone of aspC on pAT153, and plasmid pME64 contained a subclone of tyrB on pHC79. The resultant transformants, HW857(pIF100) and HW857(pME64), exhibited elevated levels of AAT (Ill-fold) and TAT (33-fold) (4), respectively, and were employed to convert racemic phenylalanine to L-phenylalanine (90-95% ee) (Figure 1). The conversion reached a maximum a t 28-30 h, at which time the nutrients became depleted and amino acid degradation occurred. No conversion was observed when the host strain HW857 was employed. HW857(pME64) was also used to demonstrate the conversion of D-leucine to L-leucine, presented in Figure 2. This strain was also employed to convert a racemic mixture of homophenylalanine (0.17 g/L) to L-homophenylalanine with an enantiomeric excess of 63% in 30 h. Although the above experiments with bacterial cells containing elevated levels of AAT and TAT strongly suggest these enzymes are responsible for the bioconvert
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Figure 1. Conversion of D,L-phenykdanine to L-phenylalanine by HW857 aspC(pIF100) (A,0 ) and HW857 tyrB(pME64) (A, 0)as a function of time. Colonies were inoculated into defined medium containing K2HPOd (7.5g/L), (NHd)*HPOd(10.0 g/L), MgSOp7Hz0 (1.5 g/L), ferric ammoniumcitrate (0.24 g/L), yeast extract (0.1 g/L), and glucose (20.0 g L) and grown at 37 "C for 16 h. Cells were harvested (time = 0 and resuspended in fresh defined medium containing D,L-phenylalanine (10.0 g/L) and incubated at 37 "C and pH 7.2. The isolation of enantiomerically enriched phenylalanine obtained through the conversion by HW857 aspC(pIF100) (A,0 ) is carried out as follows: at 31 h of conversion,the pH of the reaction mixture is adjusted to 3.5 with HzSO,. The cells are centrifuged and washed with 3-5 volumes of deionizedH2O. The combined supernatantis adjusted to pH 4.5 with NH4OH and concentrated to a wet cake by rotary evaporation. The wet cake is redissolved in dilute HzS04(1%) at 85-90 "C to a concentration of 200 g/L. Crystallization is carried out in the presence of Tween 80 (0.01% ) as follows: NH4OH is added to adjust the pH to between 4.2 and 4.5 while the mixture is cooled slowly to 30 O C . The yield of total phenylalanine is 59% (5.9 g) with an enantiomeric excess of 90%. The levels of D- and L-phenylalanine were determined by HPLC after derivatizationwith l-fluoro-2,4-dinitrophenyl-5-~-alanine amide (FDAA)according to the method of Marfey (5). HPLC conditions: Spheri-5,RP-18 column (10 X 4.6 cm); UV detection at 340 nm; mobile phase A, 0.05 MTEA-phosphate (pH 3.0); mobile phase B, CHBCN;linear gradient 10% B-40% B in 45 min; flow, 2.0 mL/min at 25 O C . sion, we wanted to confirm this by the use of purified enzymes. The efficiency of AAT- and TAT-catalyzed conversions is determined by the relative activity of D and L isomers in the first half-reaction (Scheme I). The data in line 1of Table I illustrate that the conversion of D-phenylalanine to phenylpyruvic acid proceeds with 6-fold greater catalytic efficiency when catalyzed by homogeneous preparations of TAT as compared to AAT in a buffered reaction mixture. The catalytic efficiency of the conversion
0 199 1 American Chemical Society and Amerlcan Institute of Chemical Engineers
Biotechnol. hog., 1991, Vol. 7, No. 4
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more highly expressed in E. coli (4). This may explain the greater than expected conversion efficiency of whole cells bearing the AAT plasmid.
Scheme I
A *, 2
I
(D,L) RCHNH2COOH
\
a o r b
RCOCOOH
a or
(L) RCHNH2COOH
2 R
PhCHt Phenylalanine ICH,)>CHCH2 Levcine PhChsCht Homophenylalanine
1
2 d
--
1
WKetOqlutarate L-GlUtamdte aspartate aminotransferase IAATi
b * cym?iine aminotransferase (TAT1
Aminotransferase-catalyzedwhole-cell bioconversions of racemic amino acids afford a convenient and coateffective approach to the production of L-amino acids. The conversion does not require the addition of cofactors and cosubstrates, derivatization of substrates, or enzyme immobilization. The described process may lead to increased exploration of substrate and stereoselectivityof E. coli aminotransferases and establish their use as a convenient tool in chiral synthesis.
Acknowledgment We are grateful to Dr. M. Herold and Dr. E. Koehler, Biozentrum,CH-4506 Basel, Switzerland,who generously provided us with homogeneous preparations of AAT and
0 0
lo
Hours
2o
30
Figure2. Conversionof Dleucine (A)to L-leucine ( 0 )by HW857 tyrB(pME64) as a function of time. Conditions and methods were the same as described in Figure 1. Cells were harvested (time = 0) and resuspended in fresh defined medium containing &leucine (10.0g/L). Table I. Stereoselectivity of AAT and TAT in the Conversion of D- and L-Phenylalanine to Phenylpyruvic Acid. k = t / K ~M-' , 8-l substrate AAT TAT 3.8 X 109 D-phenylalanine 5.8 X lo2 3.2 X 106 L-phenylalanine 8.4 X 109 a The activities of AAT and TAT were assayed spectrophotometrically (6). Thereactionmixturecontained,in 3.2mL;0.5MKHzPO, (pH 7.2),0.1-12.0 mM D or L-phenylalanine,18.8 mM a-ketoglutarate, 0.19 mM pyridoxal phosphate,and enzyme. The mixture was incubated at 37 OC for 5 min, after which the reaction was stopped with 0.2mL of 10 N NaOH. The optical density was determined at 320 nm against a control to which NaOH had been added prior to addition of enzyme. A molar extinction coefficient of 17 500 M-l cm-1 was used. The catalytic parameters were determined from double-reciprocalplota (7). The values of kat/KM areapparentvalues at pH 7.2 and were calculated for each variable substrate, while pyridoxal phosphate and a-ketoglutarate were at saturating concentrations. of L-phenylalanine to phenylpyruvic acid shown in line 2 of Table I is 38-fold greater for TAT as compared to AAT. This was expected as TAT exhibits a greater catalytic efficiency with aromatic substrates (8). AAT has been previously shown to be more stable than TAT (9) and is
TAT.
Literature Cited (1) Kemp, D. S.In The Peptides; Gross, E., Meienhofer, J., Eds.; Academic Press: New York, 1979; Vol. 1, Chapter 7. (2)Umbarger,H.E. Amino AcidBioeynthesisandItsRegulation. Annu. Rev. Biochem. 1978,47,581-582. (3) Oishi, M. In The Microbial Production of Amino Acids, Yamada, K., et al., ms.; John Wiley & Sons: New York, 1972; Chapter 16. (4)Fotheringham, I. G.,et al. The Cloning and SequenceAnalysis of the aspC and tyrB Genes from Escherichia coli K12. Biochem. J . 1986,234,593-604. (5) Marfey, P. Determination of D-Amino Acids. Use of a Bifunctional Reagent, 1,5-Difluoro-2,5-Nitrobenzene.Carbberg Res. Commun. 1984,49,591-596. (6) Diamondstone,T. I.Assay of TyrosineTransaminase Activity by Conversion of p-Hydroxyphenylpyruvate to p-Hydroxybenzaldehyde. Anal. Biochem. 1966,16,395-401. (7) Lineweaver, H.;Burk, D. The Determination of Enzyme DissociationConstants. J.Am. Chem. SOC.1934,56,658-666. ( 8 ) Powell,J.T.; Morrison,J. F.The Purification and Properties
of the Aspartate Aminotransferase and Aromatic Amino Acid Aminotransferase from Escherichia coli. Eur. J. Biochem. 1978,87,391-400. (9) Mavrides, C.; Orr, W. Multispecific Aspartate and Aromatic Amino Acid Aminotransferases in Escherichia coli. J. Biol. Chem. 1975,250,4128-4133.
Accepted June 3,1991. Registry No. AAT, 9000-97-9; TAT, 9014-55-5;leucine, 6190-5;phenylalanine, 63-91-2;homophenylalanine, 943-73-7.
