Kinetic resolution of aminoacid ester with immobilized ...

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2Istituto di Biochimica delle Macromolecole, UniversitA, Napoli, Italy. SUMMARY. Cells of Sulfolobus solfataricus trapped in sodium alginate have been used to.
Biotechnology Letters Vol 12 No i0 Received as revised 2nd August

717-720

(1990)

KINETIC RESOLUTION OF AMINOACID ESTER WITH IMMOBILIZED CELLS OF

SULFOLOBUS SOLFATARICUS A. Trincone 1, G. Palmieri 1, L. Lama 1, B. Nicolaus 1, M. De Rosa 2 and A. Gambacorta I

lIstituto per la Chimica di Molecole di Interesse Biologico del C.N.R. Via Toiano n. 6, 80072, Arco Felice, Napoli, Italy 2Istituto di Biochimica delle Macromolecole, UniversitA, Napoli, Italy SUMMARY. Cells of Sulfolobus solfataricus trapped in sodium alginate have been used to hydrolyze a range of aminoacid esters with useful stereoselectivity for L-enantiomer at different temperatures.

INTRODUCTION

Enantioselective production of chemicals is an important aim in organic synthesis and in this respect interest in biological methods in the asymmetric synthesis (Whitesides and Wong, 1985) has recently increased. The use of oxidoreductases, from various sources, including archaeobacteria, in the production of chiral alcohols is a well documented and convenient technique allowing usually high optical purity of the products (Keinan and Hafeli, 1986; Trincone et aI., 1990). Commercially pure hydrolytic enzymes and whole cells have been extensively used for the hydrolysis of racemic esters of N-acetylaminoacid esters (Glanzer et al., 1987a), 1alkyn-3-ols (Glanzer et al., 1987b), meso-substrates (Whitesides and Wong, 1985) etc. obtaining chiral products. Enantiomerically specific transesterification is also a useful procedure in the organic synthesis. However, the wide biotechnotogical applications in stereospecific organic synthesis of high value added products has been limited by the low stability of different mesophilic enzymes to organic solvent and to heat. Our attention was focused on the possible applications of thermophilic microorganisms. Many enzymes from these microorganisms have been found to be thermostable, capable of acting at high temperature and in addition showing a general stability to organic solvent and to immobilization procedures. In this paper we report preliminary results on the hydrolytic potentialities of

Sulfolobus sotfataricus a thermoacidophilic archaeobacterium growing at 87~ C and pH 3,5. MATERIALS AND METHODS Microorganism and culture conditions. Sulfolobus

solfataricus strain MT4 (ATCC n. 49155), was isolated from an acid hot spring in Agnano, Naples (De Rosa et al., 1980). The organism was grown at 88 ~ C in a 90 1 fermenter, with low mechanical agitation and aeration flux of 30 ml min -1 1-1 of broth. The microorganism was grown in the standard saline

717

solution using glucose as sole carbon source, the pH of the culture medium was adjusted to 3.5 with 0.1 M H2SO 4 (De Rosa et al., 1980). Cells were harvested in stationary phase of growth by continuous flow centrifuge. The pellet was washed twice with an iso-osmotic saline solution, p H 6, and collected by centrifugation at 3000 g for 30 min and lyophilized. Cell i m m o b i l i z a t i o n . Wet cells (10 g) were mixed thoroughly with 3% sterile sodium alginate solution (Vorlop and Klein, 1983). The resulting homogeneous mixture was extrupted drop by drop through a syringe into 0.2 M CaCI2 solution in 10 mM acetate buffer p H 5.5. The bead diameter ranged from 3 to 4 m m and their wet weight was ca. 44 g. Kinetic resolution of D.L a m i n o a c i d esters. In a typical experiment 2.25 g of wet beads were added to 6 ml of 10 mM Tris-HC1 buffer pH 7.5 containing 46 ~rnoles of aminoacid ester and 100 ~1 of methanol as cosolvent. The mixture was incubated at different temperatures and appropriate control experiments were run in parallel. Samples were withdrawn at intervals and the remaining substrate was monitored as below described. Optical purities both of the resulting aminoacid and of the remaining ester were detected by chiral plate analysis. Analytical methods. Aminoacid ester was assayed by hydroxamate reaction (Pesez, M. and Bartos J., 1974). Purification of unreacted aminoacid ester was achieved by preparative TLC in the appropiate solv'~nt and its hydrolysis was performed by 1N HC1 at 70 ~ C for 5 hours. Chiral vlate analysis. Chiral plate, a glass plate TLC coated with chiral selector and copper ions (Mackerey-Nagel), was eluted using as solvent system C H 3 O H / H 2 0 / C H 3 C N (1: 1: 4, by vol.). Compounds were detected by spraying with ninhydrin reagent. RESULTS AND DISCUSSION A kinetic resolution of D, L aminoacid esters is obtained with immobilized cells of

Sulfolobus solfataricus in alginate beads. With this method racemic phenylalanine, methionine, serine, valine and alanine methyl esters have been hydrolyzed (Fig. 1). At 50 % of conversion of the ester, the remaining substrate is shown to be the D-enantiomer with high optical purity as judged by chiral plate analysis. These preliminary data indicate the possibility to perform the hydrolysis on different aminoacid esters. A more efficient hydrolysis was observed with methionine methyl ester, and similar efficiency for L-alanine and L-valine methyl ester. In each case the rate of hydrolysis of the D-enantiomer was significantly slower. However Sulfolobus solfataricus alginate beads are able to hydrolyze also the D-enantiomer, reaching 100% of conversion in each case within 24 h at 40 ~ C. Results of the investigation on the hydrolysis of different alkyl esters of alanine are presented in Table 1. These results indicate that an increase of the alkyl group of the aminoacid esters gives rise a more efficient hydrolysis. After three hours all the compounds are ca. 80% hydrolyzed. Chiral plate analysis of the hydrolyzed product and of the remaining ester at different times does not show differences in stereospecificity with respect to the variation of alkyl chain tested; from methyl and ethyl esters of D, L-alanine, pure Laminoacid is obtained in 30 min. Since a more efficient hydrolysis of the butyl derivative occurs, D-aminoacid is also present at 30 min and its concentration increases with reaction

time. To analyse the dependence of the stereoselectivity of the reaction upon the temperature, experiments using phenylalanine methyl ester were performed at 20, 40 and 60 ~ C (Fig. 2).

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7t~

ro-.---o---o40 80

10

20

90

40

SO

60~gO

120 ~SO 1~ ~ ( r ~ )

Fig. 1: Kinetic resolution of different aminoacid methyl esters at 25~ C ( r alanine methyl ester; x, methionine; O, serine; @, phenylalanine; A, valine). The high selectivity of the hydrolysis for the natural enantiomer is apparent at different temperatures and an increase in the hydrolysis is observed on increasing the temperature of the reaction. In these conditions 0,7 mmoles of substrate are converted with 1 g of immobilized wet cells in 1 h at 60~ C obtaining a chemical yield of 50% of L-aminoacid. For comparison 1,9 ~moles h -1 of N-acetyl phenylalanine ethyl ester is converted with 1 g of fermenting

Saccharomyces

cerevisiae

obtaining the ester

with ee of 97% as judged by

Table 1: Hydrolysis of different alkyl ester of D, L alanine at 40~ C.

T (min)

Me

Ethyl

Butyl

30

52.2

53.2

70.6

60

68.9

70.0

78.8

90

70.7

71.2

80.0

120

82.2

70.4

81.2

180

84.0

82.6

88.8

measurement of optical rotation (Glanzer et al., 1987a). In the experiments performed at 40 ~ C, doubling the amount of the cells gave a three-fold lowering of the time needed to achieve 50% of conversion, while the system is still active at 20 ~ C being thus suitable for biotrasformations involving thermally unstable compounds. Recently, different hydrolytic activities such as aminopepfidases, serineproteinases, a thiol proteinase and a carboxypeptidase have been described in Sulfolobus solfataricus

719

(Fusi et al.,

9o 80 70 GO

5O

4o 3O

,o /

,/

lo

n

~

i

!

i

i

t

4

i

t

J

i

~

J

i

i

10 20 30 40 50 GO 70 80 90 100 110 120 130 140 150 160 170 I ~

~(min)

Fig. 2: Enantio selective hydrolysis of phenylalanine methyl ester at different temperatures ( @, 20~C; A, 40~ C; O, 60~ C; n, 40~ C doubling the amount of catalyst, see text).

1990; Hanner et al., 1990). All these enzymes were stable up to temperature of 80-90 ~ C and could be involved in the hydrolysis of aminoacid esters. The optimization of this procedure such as the possibility to use organic solvent and other immobilization techniques to increase stereoselection for

chiral synthon production in organic synthesis is currently under

investigation. A C K N O W L E D G E M E N T The authors should like to acknowledge the able technical assistance of Mr. E. Pagnotta, Mrs. I. Romano, Mrs. M. Della Volpe and wish thank Mr. R. Turco for art work. This work was supported in part by the CNR target project on Biotechnology and Bioinstrumentation. REFERENCES Whitesides, G. M. and Wong, C. H. (1985) Angew. Chem. Int. ed. engl. 24, 8, 617-718. Keinan, E., Hafeli, K. E., Kamal, K. S. and Lamed, R. (1986). J. Am. Chem. Soc. 108, 162. Trincone, A., Lama, L., Lanzotti, V., Nicolaus, B., De Rosa, M., Rossi, M. and Gambacorta, A. (1990). Biotechnology and Bioengineering 35, 559-564. Glanzer, B. I., Faber, K. and Griengl, H. (1987a). Tetrahedron 43, 24, 771-778. Glanzer, B. I., Faber, K. and Griengl, H. (1987b). Tetrahedron 43, 24, 5791-5796. De Rosa, M., Gambacorta, A., Nicolaus, B., Buonocore, V. and Poerio, E. (1980). Biotechnol. Lett. 2, 29-34. Pesez, M. and Bartos J. (1974). In: Colorimetric and fluorimetric analysis of organic compounds and drugs. Schawrtz, M. K. ed. pp 291-325. New York. Fusi, P., Burlini, N., Villa, M., Tortora, P. and Guerritore, A. (1990). In: Abstract Book of "Thermophily Today Meeting" held in Viterbo May 7-9, 1990. Hanner, M., Redl, B. and Stoffler, G. (1990); Biochim. Biophys Acta 2, 148-153.

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