Biotechnology Letters, Vol 19, No 3, March 1997, pp. 213–215

1 Continuous production of erythrulose using transketolase in a membrane reactor JŸrgen Bongs, Doris Hahn, Ulrich Schšrken, Georg A. Sprenger, 1 Udo Kragl* and Christian Wandrey Institut fŸr Biotechnologie, Forschungszentrum JŸlich GmbH, D-52425 JŸlich, Germany

Transketolase can be used for synthesis of chiral intermediates and . However the enzyme is strongly deactivated by the educts. This deactivation depends on the reactor employed. An enzyme membrane reactor allows the continuous production of L-erythrulose with high conversion and stable operational points. A productivity (space- time yield) of 45 g LÐ1 dÐ1 was reached.

24 pts min base to base from Key words to line 1 of text 1 Introduction (10 mM), thiaminpyrophosphate (0.5 mM) and DTT (1 Transketolase (E.C. 2.2.1.1.) is used for the asymmetric mM). The substrate concentration of was synthesis of natural substances or their precursors 50 mM, the product concentration of L-erythrulose was such as carbohydrates (Drueckhammer et al., 1991), (+)- 50 mM, each compound dissolved in the liquid buffer exo-brevicomin (Myles et al., 1991), or fagomine system. Other reaction conditions were: pH = 7.0; (Effenberger and Null, 1992). In vivo transketolase cata- T = 25°C. lyses the reversible transfer of a two- ketol moiety from a to an . When ␤-hydroxypyruvate Repetitive batch (HPA) is used as the donor substrate the reaction The reaction was performed in a commercially available becomes irreversible (Bolte et al., 1987) (Figure 1) even stirred ultrafiltration cell (Amicon) equipped with an 1 though there is a wide range of possible acceptor UF-membrane (Amicon) with a cut-off of 10 kDa. The substrates (aldehydes) (Demuynck et al., 1991; pH was measured and maintained at 7.0 using a pH Humphrey et al., 1995). Consequently, transketolase is controller (Methrom). The substrate concentrations a versatile tool for C-C-formation and for the insertion were: HPA (50 mM) and glycolaldehyde (50 mM). The of chirality in the scope of chemo-enzymatic synthesis. other reaction parameters are enumerated above.

Recent papers have focused on the preparation of the Continuous stirred tank reactor (CSTR) products (Hobbs et al., 1993); but in regard to the large The continuous production was carried out in a 10 mL scale utilization of transketolase, the stability of the enzyme membrane reactor (EMR). Transketolase was enzyme and its deactivation play an important role. The separated from the product stream by means of the same 1 aim of this work was to identify limitations in the UF-membrane (Amicon) above. Enforced flow across the process in order to overcome unpleasant side reactions membrane and complete mixing in the reactor with a and to improve the performance of the reaction. magnetic stirrer ensured that the EMR behaved as a continuous stirred tank reactor. The substrate concen- Materials and methods trations at the inlet of the reactor were 50 mM. The Strain pH was adjusted to 7.0 with the buffer system above, Transketolase was purified to apparent homogeneity the temperature was 25°C. from recombinant E. coli K12 cells carrying the homol- ogous cloned tktA gene on a pUC-19 derived plasmid. Analytical methods The concentrations of the substrates and the product 1 Enzyme activity assay were monitored by HPLC (Sykam) using a cation- The deactivation of transketolase was monitored by exchange column HPX-87H (BIORAD), eluent 6 mM photometric assay (Sprenger et al., 1995). The buffer sulphuric acid, 65°C, 0.6 mL/min, photometric detec-

system contains glycyl-glycine (50 mM), MgCl2 tion at 195 nm.

© 1997 Chapman & Hall Biotechnology Letters · Vol 19 · No 3 · 1997 213 J. Bongs et al.

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Figure 1 Reaction scheme of a transketolase-catalyzed interconversion.

Results and discussion Figure 3 Concentration change in a batch reactor The source of transketolase used in this work were compared to a continuous stirred tank reactor (CSTR). recombinant E. coli cells (Sprenger et al., 1995). After fermentation in a 200 L scale the purification of the high conversion the substrate concentration is constant crude extract provided up to 720 000 Units. at a low level. Consequently the rate of deactivation 1 should be very small. The enzyme is retained by an The formation of L-erythrulose by transketolase- ultrafiltration (UF)-membrane. Only the product and catalyzed interconversion of HPA and glycolaldehyde the remaining unreacted substrate can pass the was investigated; in the course of the reaction L-erythru- membrane. lose is formed. The rate of deactivation for transketo- lase caused by the reactive ␣-hydroxy aldehyde is much The experimental verification of this strategy is shown higher than by the synthesized product (Figure 2). in Figures 4 and 5. In Figure 4 the reaction is performed as repetitive batch. After complete conversion (2 h) The typical course of the substrate and product concen- the enzyme is filtered by means of a stirred Amicon® trations in a batch and a continuous process are UF-cell (Kragl et al., 1993). Due to the repeated high 1 compared in Figure 3. In case of the batch process in substrate concentration the strong transketolase-deacti- the beginning of the reaction the substrate concentra- vation extends the reaction time. Therefore in the last tion is high, yielding in a high transketolase-deactiva- batch, 7 h instead of 2 h like in the first batch are tion. Contrary to this reactor type in an enzyme required to reach complete conversion. The overall membrane reactor (EMR), which operates as a contin- space-time yield for L-erythrulose is 28 g L–1 d–1 and

1 uously stirred tank reactor with constant discharge, at the half-life of transketolase is only t ⁄2 = 5.6 h.

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Figure 2 Deactivation of transketolase in dependence of Figure 5 Continuously operated reactor: Conversion of buffer, substrate and product. HPA and glycolaldehyde to L-erythrulose.

214 Biotechnology Letters · Vol 19 · No 3 · 1997 Continuous production of erythrulose using transketolase in a membrane reactor

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Figure 4 Repetitive batch: Conversion of HPA and glycolaldehyde to L-erythrulose and decrease of activity of transketolase.

Contrary to this batch operation a stable conversion of Acknowledgement 1 nearly 90% is reached in a continuously operated reactor Part of this work was financed by Deutsche for a period of more than 20 h (residence time ␶ = 2 h), Forschungsgemeinschaft: SFB 380 ‘Asymmetric Synthesis since the deactivation of transketolase is minimal with Chemical and Biological Methods’. (Figure 5). Under these conditions the space-time yield for L-erythrulose is 45 g L–1 d–1 and the half-life of References

1 Bolte, J., Demuynck, C., Samaki, H. (1987) Tetrahedron Lett. 28, transketolase is drastically increased up to t 2= 106 h. ⁄ 5525–5528. After a reduction of the residence time to 0.25 h Dalmas, V., Demuynck, C. (1993) Tetrahedron: Asymm. 4, the conversion decreases to 38%. Accordingly the 2383–2388. substrate concentration raises and no steady-state condi- Demuynck, C., Bolte, J., Hecquet, L., Dalmas, V. (1991) tions are reached anymore due to the strong continual Tetrahedron Lett. 32, 5085–5088. 1 deactivation of transketolase. After increasing the resi- Drueckhammer, D.G., Hennen, W.J., Pederson, R.L., Barbas III, C.F., Gautheron, C.M., Krach, T., Wong, C.-H. (1991) dence time again a constant conversion is reached but Synthesis, 499–525. now at a lower level caused by the deactivated trans- Effenberger, F., Null, V. (1992) Liebigs Ann. Chem., 1211–1212. ketolase. Hobbs, G.R., Lilly, M.D., Turner, N.J., Ward, J.M., Willets, A.J., Woodley, J.M. (1993) J. Chem. Soc. Perkin Trans. 1, 165–166. Humphrey, A.J., Turner, N.J., McCague, R., Taylor, S.J.C. However, the described process for the use of tran- (1995) J. Chem. Soc.,Chem. Commun. 2475–2476. sketolase provides an efficient tool for the continuous Kragl, U., Gödde, A., Wandrey, C., Kinzy, W., Cappon, J.J., large-scale production of L-erythrulose. Further studies Lugtenburg, J. (1993) Tetrahedron: Asymm. 4 (6), 1193–1202. Myles, D.C., Andrulis III, P.J., Whitesides, G.M. (1991) on various aldehydes ought to open a wide range Tetrahedron Lett. 32, 4835–4838. 1 of other products employing the presented reactor Sprenger, G.A., Schörken, U., Sprenger, G., Sahm, H. (1995) optimization. Eur. J. Biochem. 230, 525–532.

Received as Revised 16 January 1997

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