트랜스아미나제 커플링 반응을 이용한 광학활성 L-아미노산 및 (R)-아민의 동시합성

Theories and Applications of Chem. Eng., 2002, Vol. 8, No. 1

조병관*, 조호정**, 윤형돈**, 김병기*,**

서울대학교 생물화학공학 협동과정*, 서울대학교 응용화학부**

Simultaneous Synthesis of Enantiomerically Pure L-Amino acids and (R)-Amines using Coupling Reaction of Transaminase

Byung-Kwan Cho*, Ho-Jung Cho**, Hyungdon Yun**, Byung-Gee Kim*,**

Interdisciplinary Program of Biochemical Engineering and Biotechnology*, School of Chemical Engineering**, Seoul National University

Introduction

The biosynthesis of optically active amino acids and amines has been received great attention due to their various usages in chiral building blocks for the synthesis of various pharmaceuticals (Fotheringham et al., 1997; Stiring, 1992). Among several enzymatic methods that have been employed for the synthesis of optically active amino acids and amines, transaminase is a promising one owing to its broad substrate specificity and no requirement for external cofactor regeneration. The chiral amino acids and amines can be produced via two reaction schemes of transaminase. One is asymmetric synthesis starting from keto acids or ketone (i.e. backward reaction form amine transfer point of view), and the other is kinetic resolution starting from racemic amino acids or amines (i.e. forward reaction). Both schemes are well developed for the production of various chiral amino acids using □-transaminase, which includes most common transaminase and D-transaminase (Taylor et al., 1998; Galkin et al., 1997). However, the reversible nature of the □-transaminase reaction and the presence of keto acids and amino acids in the reaction mixture cause the low yield of the reaction and the product recovery, which limit commercial feasibility of transaminase technology. The previous studies described the coupled reactions capable of driving equilibrium toward product formation.

As another means to alleviate limitations of □-aminotransferase, and to produce chiral amines at the same time, coupling of □-aminotransferase with □-transaminase reaction is suggested here. It was possible to shuttle the pyruvate and alanine in the reaction as amino acceptor and amino donor for □-transaminase and □-transaminase, respectively. Therefore, if amino donors and acceptors are well designed to utilize the properties of □- and □-transaminase, very efficient coupled transaminase reaction can be devised, so that both chiral amino acid and amine compounds can be enriched at the same time. Using this coupling system, simultaneous production system of L-amino acids and (R)-amines without the need for any extra amino donor and acceptor, can be easily and efficiently developed.

Result and Discussion

화학공학의 이론과 응용 제8권 제1호 2002년

□/□-Transaminase coupling reaction

For the co-synthesis of L-amino acids and (R)-amines, one of □-transaminases was coupled with an □-transaminase showing high enantioselectivity for (S)-enantiomer of amines which we have screened from soil (Shin and Kim, 1997). In reaction (A), aliphatic L-amino acids were produced from the corresponding □-keto acids by AlaAT with the consumption of L-alanine. The L-alanine is continuously regenerated by the coupled reaction of □-transaminase using racemic amines as an amino donor. In reaction (B), aromatic L-amino acids were produced from the corresponding □-keto acids by TyrAT or AspAT using L-aspartic acid as an amino donor. The resulting oxaloacetate is easily decomposed to pyruvate and carbon dioxide, spontaneously or enzymatically (Rozzell, 1985). Similarly, racemic amines are resolved via the coupled reaction of □-transaminase using pyruvate as an amino acceptor (Figure 1).

(A)

(B)

Figure 1. Schematic diagram of co-synthesis of L-amino acids and (R)-amines with transaminase coupled reaction. (A) AlaAT/□AT system, (B) TyrAT/□AT system.

Simultaneous production of L-amino acids and (R)-amines using coupling reaction

As depicted in Figure 2, 10 mM of 2-oxo-4-phenylbutyrate and 10 mM of racemic □-methylbenzylamine was converted to L-homophenylalanine and (R)-□-methylbenzylamine with 83% (>99% ee) and 48% (93% ee) yield, respectively. When 10mM of racemic 1-aminoindan was used, L-homophenylalanine and (R)-1-aminoindan were achieved with 78% (>99% ee) and 43% (76% ee) yields, respectively. Although 1-aminoindan had showed higher substrate specificity than that of □-methylbenzylamine (Shin and Kim, 2001), somewhat low yield of L-homophenylalanine caused to decrease the yield of (R)-1-aminoindan. Judging from the yield of L-homophenylalanine and (R)-□-methylbenzylamine, oxaloacetate decarboxylation seems to smoothly proceed and shift the equilibrium of □-transaminase reaction to the product formation, and the resulting pyruvate is subsequently used as an amino acceptor for □-AT. Aromatic amino acids such as L-homophenylalanine and L-tyrosine are easily precipitated in the reaction mixtures due to their low solubility as the reactions proceed, and the precipitation drives the reaction equilibrium to the product formation (Galkin et al., 1997; Cho et al., submitted).

Similarly using AlaAT/□AT coupling reaction, aliphatic L-amino acids and (R)-amines were synthesized at high optical purities and high conversion rates. Figure 3 shows the co-synthesis of L-2-aminobutyrate and (R)-amines using AlaAT/□AT coupling reaction. L-2-aminobutyrate was produced from 2-oxobutyrate with yields of 81% and 90% with simultaneous production of (R)-□-methylbenzylamine and (R)-1-aminoindan, respectively. The ee values of L-2-aminobutyrate were much more than 99 %. The yield of (R)-amines increased as the yields of L-amino acids increased. As 1-aminoindan is better substrate than □-methylbenzylamine, the somewhat higher yield of (R)-1-aminoindan is the cause of the 90% yield of L-aminobutyrate. In AlaAT/□AT coupling reaction, L-alanine and pyruvate were regenerated via transamination. In the point of AlaAT, the reaction equilibrium was shifted to product formation via removing pyruvate by □-AT reaction. □-Transamination reaction rate was dependent on □-transamination. Because AlaAT/□AT coupling reaction showed higher □-transamination reaction rate than TyrAT/□AT coupling reaction, AlaAT/□AT coupling reaction showed higher □-TA reaction rate than TyrAT/□AT.

Figure 2. Co-synthesis of L-homophenylalanine and (R)-amines. (A), L-homophenylalanine(□); acetophenone(□). (B), L-homophenylalanine(□); 1-indanone(□).

Figure 3. Co-synthesis of L-aminobutyrate and (R)-amines. (A), L-aminobutyrate(□); acetophenone(□). (B), L-aminobutyrate(□); 1-indanone(□).

Two-Liquid-Phase reaction

The □-transaminase showed severe product inhibition by acetophenone in □/□ transaminase coupling reaction. So, an aqueous/organic two-phase system was applied to overcome this problem. Through solvent screening, iso-octane was selected as the best organic phase. Acetophenone extracting capacity and the biocompatibility of the solvent to the cloned cells were important factors in the reaction rate at high concentration of □-methylbenzylamine. Using the optimal volume ratio in the biphasic system with iso-octane, 100 mM □-methylbenzylamine could be resolved successfully with asymmetric synthesis of L-unnatural amino acids using coupling reaction.

Conclusion

In this report, removal of the byproduct or regeneration of the substrate was attempted by coupling of □-transaminase reaction which exhibits high equilibrium constant. This coupling not only enhances the substrate conversion yield of □-transaminase reaction hampered by the equilibrium constant but also resolves racemic amines simultaneously, so that another chiral compound can be produced during the reaction. Using broad substrate specificity of transaminse, many different special chiral amino acids as well as chiral amines can be produced in this coupling reaction. For the purpose, three pairs of □- and □-transaminase are examined. All performed well and achieved high yields of corresponding L-amino acids and (R)-amines with high ee values. Variation of L-amino acids produced by the coupling reactions is determined by the substrate specificity of □-transaminase. The substrate specificity of TyrAT is aromatic and dicarboxy L-amino acids, such as L-phenylalanine, L-homophenylalanine, and L-aspartate and that of AlaAT is aliphatic L-amino acids, such as L-valine, L-leucine and L-2-aminobutyric acid. As AlaAT and □AT can shuttle alanine successfully, provided with □-transaminases which can preferably use □-ketoglutarate and oxalacetate as an amine acceptor rather than pyruvate, shuttling of glutamate and aspartate would be also possible with right combination of □-transaminases, such as TyrAT. Racemic amines can be also varied, so that interesting chiral amines are produced.

Co-expression of the both transaminases in one cell has some limitations in controlling the ratio of the two enzymes and supplying scarce substrates. However, it can achieve efficient coupling of the reaction system by readily providing necessary substrates. The control of different enzyme ratios can be manipulated by using appropriate promoters to change their expression levels. In this system, only the product inhibition of the ketone produced from the □-transaminase reaction is severe problem. However, the unfavorable role of the ketone product was successfully overcome by liquid/liquid biphasic reaction system. Besides, in order to enhance the reaction rate of decarboxylation of oxalacetate by temperature, thermo-stable transaminases can be also used in this reaction system. The application of this coupling reaction for the synthesis of other diverse substrates should be further carried out to confirm the efficiency and versatility of this reaction, and it will expedite to prove the concept of using recombinant cell as a cell factory for synthesis of fine chemicals.

References

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2. Stirling, D. I. : in Collins A. N., Sheldrake G. N. and Crosby J.(Eds), "The use of aminotransferases for the production of chiral amino acids and amines", In Chirality in Industry, John Wiley & Sons, 209(1992).

3. Taylor, P. P., Pantaleone, D. P., Senkpeil, R. F. and Fortheringham, I. G. : Trends Biotechnol., 16, 412(1997).

4. Galkin, A., Kulakova, L., Yoshimura, T., Soda, K. and Esaki, N. : Appl. Environ. Microbiol., 63, 4651(1997).

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7. Cho B. K. and Kim B. G., Biotechnol. Bioeng., submitted, (2002).