Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing oxygen-containing organic compound
Patent
1994-11-21
1997-09-09
Marx, Irene
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing oxygen-containing organic compound
435196, 435161, C12P 722
Patent
active
056655754
DESCRIPTION:
BRIEF SUMMARY
The invention concerns a microbial esterase which enantioselectively cleaves 1-arylalkyl esters, a process for the isolation of such a microbial esterase as well as a process for the enantioselective cleavage of 1-arylalkyl esters using such a microbial esterase.
There is a widespread use of enzymes as catalysts in large scale chemical production processes. Of particular importance in this connection is the enantioselectivity of enzymatically catalysed reactions and the property of enzymes not only to convert the natural substrates but also synthetic compounds in a corresponding manner. Enzymes can therefore also be used in the synthesis of chiral building blocks of natural substances, pharmaceutical agents, agrochemicals and fine chemicals (J. Jones, J. Sih and D. Perlmann, Applications of Biochemical Systems in Organic Chemistry, J. Wiley & Sons, New York 1976, as well as R. Prochter and S. Clark, Enzymes in Organic Synthesis, Pittman, London 1985). 1-Arylalkanols (formula I, see FIG. 1) are in this regard important synthetic building blocks for pharmaceutical agents. Numerous esterases and lipases which convert 1-arylalkyl esters and thus lead to 1-arylalkanols are known and are commercially available. However, these previously known esterases either do not cleave enantioselectively (cholesterol esterase from Candida cylindracea and porcine pancreas, esterases from rabbit and porcine liver, lipases from Candida cylindracea, Geotrichum candidum, Penicillium cleave only the 1-arylalkyl esters of formula II (see FIG. 1; lipoprotein lipase and cholesterol esterase from Pseudomonas spec., lipase from Mucor hiemalis, lipase from Pseudomonas fluorescens, P. cepacia, P. spec. and porcine pancreas; see also K. Laumen et al., J. Chem. Soc. Chem. Commun. (1988), 598-600). The enantioselectivity of a reaction is characterized by the E-value according to C.S. Chen et al. (J. Am. Chem. Soc. 104 (1982), 7294). Reactions with an E-value<20 are to be regarded as being non-enantioselective; reactions with an E-value of>50 are referred to as enantioselective in the following. In order to produce the 1-arylalkanol of formula III (see FIG. 1) it has previously been necessary to use classical chemical methods.
The object of the present invention was therefore to provide an esterase which enantioselectively hydrolyzes 1-arylalkyl esters according to formula IV (see FIG. 1).
This object is achieved by a microbial esterase which enantio-selectively cleaves (S)-1-phenylethyl acetate and is obtainable from Arthrobacter spec. (DSM 7034) or Pseudomonas fluorescens (DSM 7033). The esterase obtainable from Arthrobacter spec. (DSM 7034) is preferred.
This enantioselective cleavage reaction is tested in this case in a known manner e.g. by means of a HPLC on a chiral column material such as e.g. cellulose-coated silica gel esterified with benzoate. Alternatively the enantiomeric purity of the product can also be determined with a polarimeter.
The esterase according to the invention has a molecular weight of ca. 100,000 D when determined by means of native polyacrylamide gel electrophoresis and exhibits an isoelectric point of ca. 4.7. This enzyme hydrolyzes substituted and unsubstituted 1-arylalkyl esters and 1-phenylalkyl esters, preferably esters of short-chained carboxylic acids (chain length 1-7 C atoms) such as the (S)-1-phenylethyl ester of acetic acid, the (S)-1-phenylpropyl ester of acetic acid or the (S)-1-(-4-methylphenyl)-ethyl ester of propionic acid. The enzyme can be stabilized by the addition of glycerol.
In order to isolate the esterase according to the invention, the aforementioned bacteria are lysed by known methods e.g. by treatment with lysozyme and benzonase in hypotonic buffer and the crude extract obtained in this manner is treated with a soluble ion exchanger, preferbly Polymin G35 (BASF, Germany). The enzyme solution obtained in this manner is purified twice by hydrophobic chromatography, preferably over phenylsepharose and finally by ion exchange chromatography, preferably on DEAE-sepharose.
Since previou
REFERENCES:
Cambou et al., J. Am. Chem. Soc., 1984, 106, 2687-2692.
Whitesell et al., CHIMIA, 40, 9(Sep.), 1986.
Santaniello et al., Gazzetta Chimica Italiana, 119, 1989, 581-584.
Laumen et al., J. Chem. Soc. Chem. Commun., 1988, 1459-1461.
Janssen et al., Tetrahedron, vol. 47, No. 36, 7645-7662, 1991.
Mori et al., Tetrahedron, vol. 36, 91-96, 1980.
Oritani et al., Agri. Biol. Chem., 37 (8), 1923-1928, 1973.
Danda et al., Tetrahedron, 47(41), 8701-8716, 1991.
Krell Hans-Willi
Rasor Peter
Boehringer Mannheim GmbH
Marx Irene
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