Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Reexamination Certificate
2002-01-23
2004-12-07
Patterson, Jr., Charles L. (Department: 1652)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
C435S145000, C435S123000, C435S158000, C435S280000
Reexamination Certificate
active
06828115
ABSTRACT:
The invention relates to improved epoxide hydrolases which can be isolated from bacteria of the genus
Streptomyces
, a novel process for the enzymatic separation of epoxide enantiomer mixtures, a novel detection method for epoxide hydrolase activity, a screening method for detecting epoxide hydrolase activity and the use of bacteria of the genus
Streptomyces
and the resultant epoxide hydrolases for enantioselective epoxide hydrolysis.
The increasing importance of enantiomerically pure compounds, especially in the pharmaceutical and agrochemical industries, requires reliable and economic access to optically active substances. To prepare enantiomerically pure diols and epoxides, a number of methods are available.
In asymmetric chemical synthesis of epoxides, synthesis starts from a prochiral compound. By using a chiral reagent, for example a chiral peracid, a chiral dioxirane or oxaziridine or a chiral borate, a chiral auxiliary or a chiral, metallic or nonmetallic catalyst, a chiral epoxide is formed. The best known pathway for synthesizing chiral epoxides is the Sharpless epoxidation of alkenols, for example allyl alcohols, with hydroperoxides in the presence of transition metal catalysts.
Preparation of enantiomerically pure diols by a biochemical pathway is also known. Microorganisms having epoxide hydrolase activity catalyze the regiospecific and enantiospecific hydrolysis of epoxides. They cleave the ether bond in epoxides, forming diols. Some bacterial strains have already been described which enable a broad selection of racemic epoxides to be hydrolyzed enantioselectively. However, the number of known epoxide hydrolases and their application to organic synthesis has been restricted to date. Known strains having epoxide hydrolase activity are, for example,
Aspergillus niger
LCP521
, Bacillus sulfurescent
ATCC 7159
, Rhodococcus
species NCIMB 11216 and others.
However, the known strains having epoxide hydrolase activity often have a restricted substrate spectrum and low reaction rates. Also, the enantioselectivities which can be achieved using these strains are frequently too low [Grogan, G., et al., FEMS Microbiology Lett. 141 (1996), 239-243; Kroutil, W., et al., Tetrahedron Lett. (1996), 8379-83821. The known strains are difficult to manipulate genetically and some are difficult to culture. Therefore, to date, only two epoxide hydrolases are available in recombinant form in
E. coli [Corynebacterium sp
. C12, (Misawa, E., et al., Eur. J. Biochem. 253 (1998), 173-183) and
Agrobacterium radiobacter
AD1 (Rink, R., et al., J. Biol. Chem. 272 (1997), 14650-14657)]. Even purification of the epoxide hydrolases obtained from the microorganisms has to date only been described for
Rhodococcus species
NCIMB 11216 [Faber, K., et al., Biotechnology Lett. 17 (1995), 893-898] and
Norcardia
EH 1 [Kroutil, W., et al J. Biotechnol. 61 (1998), 143-150]. This was highly complex in both cases. The enrichment of epoxide hydrolases from
Corynebacterium sp
. C12 is described by Misawa, E., et al., Eur. J. Biochem. 253, (1998) 173-183.
In addition, the search for novel epoxide hydrolase-containing microorganisms is made difficult owing to the fact that screening for novel epoxide-hydrolase-producing microorganisms, for example in collections of microbiological strains, has hitherto been highly time-consuming. This is due to the fact that, for screening, usually methods are used in which the individual batches must be worked up and analyzed individually by gas chromatography or liquid chromatography.
It is a first object of the invention, therefore, to provide novel epoxide hydrolases having an expanded substrate spectrum and/or improved reactivity and/or improved enantioselectivity. In addition, the novel epoxide hydrolases should be more readily accessible, in particular, because they can be isolated from nonpathogenic organisms which can be readily cultured, and, in addition, if appropriate are readily accessible to methods of molecular biology.
It is a second object of the invention to provide a method for the more rapid and simpler detection of epoxide hydrolase, which should also allow improved screening for epoxide-hydrolase-producing micro-organisms.
It is a third object of the invention to provide an improved biochemical process for separating epoxide enantiomer mixtures and thus an improved process for the enantioselective reaction of epoxides which permits a simpler route to the enantiomerically pure diols and/or epoxides.
It is a fourth object of the invention to provide novel epoxide-hydrolase-producing microorganisms.
We have found that the above first object is achieved, surprisingly, by providing epoxide hydrolases (E.C. 3.3.2.3) from microorganisms of the genus
Streptomyces
. Epoxide hydrolase activity has not previously been described in microorganisms of this genus.
The inventive epoxide hydrolases have at least one of the following advantageous properties, compared with previously known epoxide hydrolases:
improved enantioselectivity in the resolution of enantiomeric epoxides;
improved (expanded) substrate spectrum;
improved reactivity;
enhanced accessibility to methods of molecular biology;
improved biochemical accessibility because the microorganisms are easier to culture.
For the purposes of the invention, “improved enantio-selectivity” is when, for substantially the same conversion rate, a higher enantiomeric excess is achievable.
For the purposes of the invention, an “expanded substrate spectrum” is that racemic mixtures of a plurality of epoxides are converted.
For the purposes of the invention, an “improved reactivity” is that the reaction takes place with a higher space-time yield.
The invention relates in particular to those epoxide hydrolases from
Streptomyces
that have at least one of the following properties:
a) hydrolytic epoxide cleavage of a styrene oxide, for example styrene oxide or a derivative thereof which is monosubstituted or polysubstituted on the phenyl ring or epoxide ring, such as in particular a styrene oxide which is monosubstituted in the meta or para position by nitro or halogen, in particular chlorine or bromine, and at least one further compound selected from the group consisting of ethyl 3-phenylglycidate, n-hexane-1,2-oxide, n-decane-1,2-oxide and indene oxide, which can be unsubstituted or monosubstituted or polysubstituted by substituents preferably in accordance with the above definition;
b) conversion of a racemate of styrene oxide with an enantioselectivity E>2, for example ≧10, for instance from 10 to 100, to give (S)-phenyl-1,2-ethanol according to the reaction equation (A) given below, this conversion being able to be carried out using whole cells or a cell homogenate or an enriched or purified enzyme preparation, and preferably taking place in the presence of a cosolvent, for example from 5 to 10% (v/v) DMSO.
According to a preferred embodiment, the epoxide hydrolases provided are those which can be isolated from bacteria of the genus
Streptomyces
, in particular from the species
S. griseus, S. thermovulgaris, S. antibioticus, S. arenae
and
S. fradiae
, preferably from the strains
Streptomyces griseus
(DSM 40236 and DSM 13447),
Streptomyces thermovulgaris
(DSM 4044 and DSM 13448),
Streptomyces arenae
Tü (DSM 40737 and DSM 12134)
Streptomyces antibioticus
Tü4 (DSM 12925) or
Streptomyces fradiae
Tü27 (DSM 12131).
Particular preference is given to the epoxide hydrolase which can be isolated from
Streptomyces antibioticus
Tü4 (DSM 12925). This enzyme is characterized by its pronounced enantioselectivity and the conversion of (R/S)-styrene oxide (I) according to the following reaction equation (A)
Reaction equation (A):
to (S)-phenyl-1,2-ethanediol (III), with the non-hydrolysis of (R)-styrene oxide (II). Thus this enzyme in a reaction medium containing 10% (v/v) DMSO as solubilizer, catalyzes the above conversion at an enantioselectivity of E=13 and an enantiomeric excess ee[%]=99 for (II) and ee[%]=14 for (III).
Isolation
Enzelberger Markus
Hauer Bernhard
Schmid Rolf D.
Wohlleben Wolfgang
Zocher Frank
BASF - Aktiengesellschaft
Keil & Weinkauf
Patterson Jr. Charles L.
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