(S)-hydroxynitrile lyases with improved substrate acceptance...

Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing nitrogen-containing organic compound

Reexamination Certificate

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C435S232000

Reexamination Certificate

active

06319697

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to (S)-hydroxynitrile lyases which have an altered substrate Acceptance.
2. Description of the Related Art
The implementation of chemical reactions with the aid of biological catalysts is gaining increasing importance, especially in those areas of application in which use can be made of the property, which is frequently pronounced in enzymes, are preferentially transforming one of the two enantiomers in reactions using chiral or prochiral components.
This group of enzymes includes the hydroxynitrile lyase (HNL) family, inter alia
Hevea brasiliensis
HNL (HbHNL) and
Manihot esculenta
HNL (MeHNL). The two enzymes exhibit a high sequence identity and belong to the proteins of the “&agr;/&bgr;-hydrolase fold” type, which exhibit a characteristic tertiary fold and a so-called catalytic triad containing aspartic acid, serine and histidine as the active center. In this context, the active center is located at the inner end of a hydrophobic channel. In principle, HbHNL and MeHNL are suitable for converting a large number of carbonyl compounds, such as aliphatic, alicyclic, unsaturated, aromatic and heteroaromatic aldehydes and ketones, into the corresponding (S)-cyanohydrins. Since HNLs are gaining ever greater importance as biocatalysts for preparing (S)-cyanohydrins, attempts are constantly being made to improve their catalytic activity and substrate acceptance. The substrate acceptance of the HNLs which have so far been available is not satisfactory, particularly in the case of starting compounds possessing bulky residues, as a result of which the corresponding cyanohydrins are obtained either at a low conversion rate and/or in low enantiomeric excess.
The object of the invention was consequently to provide (S)-hydroxynitrile lyases having an improved substrate acceptance.
SUMMARY OF THE INVENTION
The invention consequently relates to (S)-hydroxynitrile lyases which have an altered substrate acceptance, in particular a substrate acceptance which is improved in the case of defined substrates, and which are derived from the (S)-hydroxynitrile lyases obtained from
Hevea brasiliensis
and
Manihot esculenta,
wherein one or more bulky amino acid residues within the hydrophobic channel leading to the active center are replaced by less bulky amino acid residues.
DESCRIPTION OF THE PREPARED EMBODIMENTS
The HNLs according to the invention are mutants of
Hevea brasiliensis
(HbHNL) or
Manihot esculenta
(MeHNL) (S)-HNLs which can be obtained from recombinantly modified microorganisms such as
Pichia pastoris, Saccharomyces cerevisiae
or
Escherichia coli
(WO 97/03204). In this context, the recombinant HNLs which are to be modified can also possess a truncated sequence, which is obtained, for example, by removing the first amino acid(s) in sequence. The mutants possess an altered sequence of those amino acids which form the hydrophobic channel which leads to the active center.
In this context, individual bulky amino acid residues, or several bulky amino acid residues, are replaced by less bulky amino acid residues. Preference is given to replacing tryptophan, as a bulky amino acid residue, with a less bulky amino acid residue such as alanine, glycine, valine or phenylalanine. Particular preference is given to mutants in which the tryptophan at position 128 of the full-length sequence of HbHNL or MeHNL has been replaced by alanine or phenylalanine.
The HNLs according to the invention are prepared by being functionally overexpressed in recombinantly modified microorganisms such as
Pichia pastoris, Saccharomyces cerevisiae
or
Escherichia coli
, for example in analogy with M. Hasslacher et al., J. Biol. Chem. 1996, 271, 5884 or Wajant, H. and Pfizenmaier, K. 1996, J. Biol. Chem. 25830-24834. The mutation is carried out, for example, using a Quikchange™ Side-Directed Mutagenesis Kit (Stratagene) in accordance with the manufacturer's instructions. The Quikchange™ Side-Directed Mutagenesis Kit is a ready-to-use system for preparing specific mutants and is marketed, for example, by Stratagene Cloning Systems, La Jolla, Calif. (USA).
The resulting HNLs are purified by standard methods, for example in analogy with Wajant, H. Pfizenmaier, K., J. Biol Chem. 1996, 25830-25834.
The HNLs according to the invention are suitable for preparing (S)-cyanohydrins at a superior turnover rate and/or in a higher enantiomeric excess as compared with the state of the art. The HNLs according to the invention are employed, in particular, when aliphatic and aromatic aldehydes and ketones are used as substrates. In this context, aliphatic aldehydes are preferably to be understood as being saturated or unsaturated, branched or cyclic aldehydes having 2 to 20 C atoms.
Particular preference is given to saturated or unsaturated branched aldehydes having 4 to 18 C atoms. The aliphatic and aromatic aldehydes may be unsubstituted or substituted by groups which are inert under the reaction conditions, for example by optionally substituted aryl or heteroaryl groups, such as phenyl or indolyl groups, or by halogen, ether, alcohol, acyl, carboxylic acid, nitro or azido groups.
Examples of suitable aliphatic aldehydes are hexanal, hexenal, heptanal, propanal, octanal, octenal and 2-methylpropanal. Examples of suitable aromatic or heteroaromatic substrates are benzaldehyde or variously substituted benzaldehydes, such as 3-phenoxybenzaldehyde, 4-fluoro-3-phenoxybenzaldehyde, 2-chlorobenzaldehyde, 2-nitrobenzaldehyde, 4-methylbenzaldehyde, etc.
The substrates are reacted with a cyanide group donor in the presence of the HNLs according to the invention.
Suitable cyanide group donors are hydrocyanic acid, alkali metal cyanides or a cyanohydrin of the general formula
R
1
R
2
C(OH)(CN)  formula I.
In formula I, R
1
and R
2
denote, independently of each other, hydrogen or an unsubstituted hydrocarbon group, or R
1
and R
2
together denote an alkylene group having 4 or 5 C atoms, with R
1
and R
2
not simultaneously denoting hydrogen. The hydrocarbon groups are aliphatic or aromatic, preferably aliphatic groups. R
1
and R
2
preferably denote alkyl groups having 1-6 C atoms; the cyanide group donor is very preferably acetone cyanohydrin.
The cyanide group donor can be prepared using known methods. Cyanohydrins, in particular acetone cyanohydrin, can also be obtained commercially. Hydrocyanic acid (HCN), KCN, NaCN or acetone cyanohydrin is preferably used as the cyanide group donor, with hydrocyanic acid being particularly preferably used.
In this context, the hydrocyanic acid can also be released from one of its salts, such as NaCN or KCN, only shortly before the reaction and added to the reaction mixture as the substance itself or dissolved form.
The reaction can be carried out in an organic, aqueous or 2-phase system or in an emulsion. The aqueous system used is an aqueous solution or buffer solution which contains the HNL according to the invention. Examples of these solutions or buffer solutions are Na citrate buffer, phosphate buffer, etc.
The organic diluents employed can be aliphatic or aromatic hydrocarbons which are not miscible, or only slightly miscible, with water and which are optionally halogenated, alcohols, ethers or esters, or mixtures thereof. Methyl tert-butyl ether (MTBE), diisopropyl ether, dibutyl ether or ethyl acetate, or a mixture of these compounds, is preferably employed. In this context, the HNLs according to the invention can be present in the organic diluent either as such or immobilized; however, the reaction can also take place in a two-phase system or in an emulsion using non-immobilized HNL.


REFERENCES:
patent: 5346816 (1994-09-01), Griengl et al.
patent: WO97/03204 (1997-01-01), None
GenBank Accession No. AAC49184 (Mar. 1996).*
GenBank Accession No. S45682 (Dec. 1994).*
Wajant et al. Identification of Potential Active-Site Residues in the Hydroxynitrile Lyase fromManihot esculentaby Site-Directed Mutagenesis. J. Biol. Chem. (1996) 271(42): 25830-25834.*
Hasslacher et al. Molecular CLoning of the Ful

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