WO2014053558A1 - Variants of the parvibaculum lavamentivorans short-chain alcohol dehydrogenase - Google Patents

Abstract

The present invention provides libraries of polypeptides, polynucleotides, or host cells, wherein said library includes at least one polypeptide variant of the Parvibaculum lavamentivorans short-chain alcohol dehydrogenase, or a polynucleotide comprising a nucleotide sequence encoding said variant, and/or a host cell including said polypeptide variant or the polynucleotide comprising the nucleotide sequence encoding said variant. Also disclosed are variants of the Parvibaculum lavamentivorans short- chain alcohol dehydrogenase, polynucleotides comprising a nucleotide sequence encoding said variants, and host cells including said variant or said polynucleotide. Also provided is a method for stereoselective reduction ketones.

Description

Variants of the Parvibaculum lavamentivorans short-chain alcohol dehydrogenase

Field of the Invention

The invention relates to stereoselective oxidoreductase enzymatic activity, and to the use of stereoselective oxidoreductase activity for stereospecific synthesis of compounds. More specifically, the invention relates to enzymes having stereoselective oxidoreductase activity, to nucleotide sequences encoding said enzymes, and to host cells including said enzymes and/or nucleotide sequences.

Background of the Invention Chiral compounds are those molecules having a non-superimposable mirror image. The two mirror images of a chiral compound are called "enantiomers" or "optical isomers". Many naturally occurring biologically active molecules are chiral compounds. Moreover, numerous drug targets for treating a disease distinguish between the two enantiomers of a chiral substrate. Hence, one enantiomer of a drug often possesses a much better therapeutic effect than the other enantiomer. Thus there is a desire for synthesizing the pharmaceutically active enantiomer of compound only.

One such example wherein synthesizing only one of the two enantiomers of a compound is desired is (R)-3-quinuclidinol which is used as intermediate compound in the synthesis of a variety of drugs for treating various disorders. Examples of such drugs and disorders are solifenacin, a competitive muscarinic acetylcholine receptor antagonist for treating

overactive bladder with or without urge incontinence;

palonosetron, a 5-HT₃ antagonist used in the prevention and treatment of chemotherapy- induced nausea and vomiting;

talsaclidine, a non-selective muscarinic acetylcholine receptor agonist that was developed for treating Alzheimer's disease;

revatropate, a antimuscarinic agent which shows some 50-fold selectivity for M1 and M3 receptors in guinea pig trachea and rabbit vas deferens over the M2 subtype in atria, which is an effective bronchodilator that was well tolerated by COPD patients which inhaled revatropate in early clinical trials. (R)-3-quinuclidinol is obtainable by reduction of 3-quinuclidinone. Several approaches for stereospecific chemical reduction of carbonyl groups to produce chiral alcohols have been employed, but turned out to provide insufficient yields of the desired enantiomer. To overcome the drawbacks of conventional chemical processes in synthesizing chiral alcohols, several processes were described using enzymatic reduction of corresponding ketones. References for several enzymatic methods for producing 3-quinuclidinol are found in

WO 2012/007965 A1.

International Publication WO 2012/007965 A1 provides a process for producing optically pure 3-quinuclidinol, i.e. (R)-3-quinuclidinol, by utilizing an oxidoreductase enzyme derived from Saccharomyces species. A >99% GC purity and >95% ee of (R)-3-quinuclidinol is reported.

International publication WO 2010/123062 A1 discloses a method for producing (R)- quinuclidinol by reducing 3-quinuclidinone or a salt thereof in the presence of a polypeptide derived from bacteria of the genus Burkholderia or a recombinant organism capable of producing said polypeptide. Incubating 3-quindolidinone with an extract from recombinant £. coli expressing the Burkholderia gene encoding the polypeptide referred to as RBS in said document in the presence of glucose and NADP⁺ lead to conversion of said substrate at a rate of not lower than 99%, and the measured optical purity was not lower than 99% ee.

However, there is still a need for alternative alcohol dehydrogenases which are easier and more reliable to obtain from recombinant hosts, which may convert a broader spectrum of substrates, and/or which are more suitable for stereoselective synthesis of compounds in an industrial scale.

Parvibaculum lavamentivorans is a member of the alpha-subclass of the Proteobacteria, and classified as novel genus within the bacterium of the family of Phyllobacteriaceae within the order Rhizobiales. P. lavamentivorans was recognized as heterotrophic organism for its capability to omega-oxigenate linear alkylbenzenesulfonate (LAS), a commercially available surfactant, and to shorten the side chain by beta-oxidation to yield sulfophenylcaboxylat.es. Parvibaculum lavamentivorans expresses a short-chain alcohol dehydrogenase.

The inventors of the present invention have utilized protein engineering of a Parvibaculum lavamentivorans short-chain alcohol dehydrogenase for providing variants of its short-chain alcohol dehydrogenase, polynucleotides comprising a nucleotides sequence encoding one of said variants, and host cells including one of said variants and/or one of said polynucleotides. In the process, the inventors generated libraries of variants of the Parvibaculum lavamentivorans short-chain alcohol dehydrogenase, libraries of polynucleotides comprising a nucleotide sequence encoding at least one of said variants, and libraries of host cells including at least one of said variants and/or at least one polynucleotide comprising a nucleotide sequence encoding one of said variants. Suitability of some of the variants of the Parvibaculum lavamentivorans short-chain alcohol dehydrogenase for stereoselective reduction of a ketone to one enantiomer of the corresponding alcohol was proven by utilizing 3-quinuclidinone as substrate.

Summary of Invention

In a first aspect, the invention provides libraries of elements, said elements being selected from the group consisting of polypeptides, polynucleotides and host cells, wherein said library includes at least one polypeptide variant that is based on the wild-type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase and/or at least one polynucleotide comprising a nucleotide sequence that encodes said at least one polypeptide variant.

In a second aspect, the invention provides polypeptides, wherein said polypeptides are variants of the wild-type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase.

In a third aspect, the invention provides kits comprising a plurality of different alcohol dehydrogenases, wherein at least one of said plurality of alcohol dehydrogenases is a variant of the wild-type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase.

In a fourth aspect, the present invention provides polynucleotides comprising a nucleotide sequence which encodes a variant of the Parvibaculum lavamentivorans short-chain alcohol dehydrogenase. In a fifth aspect, the present invention provides host cells which include at least one variant of the wild-type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase and/or at least one polynucleotide comprising a nucleotide sequence encoding for said variant or one of said variants. In a sixth aspect, the present invention provides the use of the variants of the wild-type

Parvibaculum lavamentivorans short-chain alcohol dehydrogenase for converting a suitable ketone to the R-enantiomer of the corresponding alcohol. In a further aspect, the present invention also comprises the use of the wild-type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase for converting a suitable ketone to the R-enantiomer of the corresponding alcohol.

In another further aspect, the present invention provides a process for preparing the R- enantiomer of an alcohol from a suitable ketone, wherein the suitable ketone is reacted with one of the variants of the wild-type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase, or with the wild-type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase.

Detailed Description of Embodiments According to the first aspect, the present invention provides a library of elements. Said elements being selected from the group consisting of polynucleotides, polypeptides and host cells, wherein said library includes at least one polypeptide variant that is based on the wild- type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase and/or at least one polynucleotide comprising a nucleotide sequence that encodes said at least one polypeptide variant, said at least one variant differs from the wild-type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase in at least one of the amino acid residues of the 2^nd sphere of said wild-type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase.

Hence, in an embodiment of the first aspect, the library of elements is a library of

polypeptides, wherein said library comprises at least one polypeptide which is a variant of the Parvibaculum lavamentivorans short-chain alcohol dehydrogenase. Said at least one variant of the Parvibaculum lavamentivorans short-chain alcohol dehydrogenase differs from the wild-type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase in at least one of the amino acid residues of the 2^nd sphere of said wild-type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase.

The term "library of polypeptides" refers to a collection of a plurality of different polypeptide molecules, wherein the different polypeptide molecules within the library differ from each other with respect to their amino acid sequences. It is to be understood that polypeptides constitute the elements of the library in the embodiment wherein the library of elements is a library of polypeptides. The amino acids of the 2 sphere of the wild-type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase are those amino acid residues within the catalytic center of the enzyme which appear not to be involved in the coordination of the enzyme's substrate and the enzyme's cofactor NADH, but which are present at a distance of about 4 A to about 17A, preferably to about 15 A, from the enzyme's substrate and/or the enzyme's cofactor. The amino acids of the 2^nd sphere of the wild-type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase were identified by utilizing the protein homology/analogy recognition engine PHYRE (http://www.sbg.bio.ic.ac.uk/~phyre/) for protein structure prediction (Kelley LA and Sternberg MJE. (2009). Protein structure prediction on the web: a case study using the Phyre server. Nature Protocols 4, 363 - 371 ). The xylitol dehydrogenase of

Gluconobacter oxydans served as template for the prediction of the structure of the wild-type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase.

The amino acid residues of the 2 sphere of the wild-type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase are the amino acid residues

G at position 92, I at position 93, K at position 103, T at position 1 1 1 ,

I at position 146, S at position 147, G at position 151 , G at position 154,

Q at position 155, A at position 158, D at position 160, N at position 162,

V at position 191 , H at position 192, D at position 197, T at position 198,

P at position 199, V at position 201 , K at position 202, N at position 203,

N at position 206, L at position 224, H at position 228, L at position 233,

G at position 234, V at position 261 and D at position 262, with respect to the wild-type Parvibaculum lavamentivorans short-chain alcohol

dehydrogenase.

Hence, in an embodiment of the second aspect, the variant of the wild-type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase differs in at least one of the amino acid residues from the wild-type Parvibaculum lavamentivorans short-chain alcohol

dehydrogenase which are selected from the group of amino acid residues consisting of

G at position 92, I at position 93, K at position 103, T at position 1 1 1 ,

I at position 146, S at position 147, G at position 151 , G at position 154,

Q at position 155, A at position 158, D at position 160, N at position 162,

V at position 191 , H at position 192, D at position 197, T at position 198,

P at position 199, V at position 201 , K at position 202, N at position 203,

N at position 206, L at position 224, H at position 228, L at position 233,

G at position 234, V at position 261 and D at position 262, with respect to the wild-type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase.

In a further and/or additional embodiment, the at least one variant of the Parvibaculum lavamentivorans short-chain alcohol dehydrogenase is a polypeptide comprising an amino acid sequence which is selected from the group consisting of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34 and 36. The at least one variant of the Parvibaculum lavamentivorans short-chain alcohol dehydrogenase may comprise a tag at its N-terminal and/or C-terminal end which may be used for purifying the variant. The term "tag" refers to a peptide sequence that is genetically grafted onto the variant of the of the Parvibaculum lavamentivorans short-chain alcohol dehydrogenase. The tag may be removable from the variant of the Parvibaculum lavamentivorans short-chain alcohol dehydrogenase by chemical or enzymetic means. Examples of tags include AviTag, calmodulin-tag, chitin binding protein (CBP), maltose binding protein-tag, Nus-tag, Strep-tag, Thioredoxin-tag, S-tag. SBP-tag, Softag 1 , Softag 3, Xpress tag, Isopeptag, SpyTag, glutathione-S-transferase-tag, green fluorescent protein-tag, poly(His) tag, FLAG-tag, V5-tag, c-myc-tag, TC-tag, Ty-tag and HA- tag.

In another embodiment, the at least one variant of the Parvibaculum lavamentivorans short- chain alcohol dehydrogenase is a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34 and 36.

In another and/or alternative embodiment of the first aspect, the library of elements is a library of polynucleotides, wherein at least one polynucleotide comprises a nucleotide sequence which encodes one of said variants of the Parvibaculum lavamentivorans short- chain alcohol dehydrogenase, i.e. one of the variants which differs from the wild-type

Parvibaculum lavamentivorans short-chain alcohol dehydrogenase in at least one of the amino acid residues of the 2^nd sphere of said wild-type Parvibaculum lavamentivorans short- chain alcohol dehydrogenase.

The term "library of polynucleotides" refers to a collection of a multitude of polynucleotide molecules, wherein the polynucleotide molecules within the library differ from each other with respect to at least a portion of their nucleotide sequences. It is to be understood that polynucleotides constitute the elements of the library in the embodiment wherein the library of elements is a library of polynucleotides. The term "polynucleotide" comprises desoxyribonucleic acid molecules and ribonucleic acid molecules.

In an embodiment, the at least one polynucleotide comprises a nucleotide sequence which comprises at least one non-silent mutation in at least one of the triplets encoding the amino acid residues of the 2^nd sphere of the Parvibaculum lavamentivorans short-chain alcohol dehydrogenase.

The term "triplet" refers to a set of three successive nucleotide bases within a polynucleotide. The triplets constitute the codons which specify which amino acid will be added next during protein biosynthesis.

In an embodiment, the at least one polynucleotide comprises a nucleotide sequence which comprises at least one non-silent mutation in at least one of the triplets encoding the amino acid residues of the wild-type Parvibaculum lavamentivorans short-chain alcohol

dehydrogenase which are selected from the group of amino acid residues consisting of

G at position 92, I at position 93, K at position 103, T at position 1 1 1 ,

I at position 146, S at position 147, G at position 151 , G at position 154,

Q at position 155, A at position 158, D at position 160, N at position 162,

V at position 191 , H at position 192, D at position 197, T at position 198,

P at position 199, V at position 201 , K at position 202, N at position 203,

N at position 206, L at position 224, H at position 228, L at position 233,

G at position 234, V at position 261 and D at position 262, with respect to the wild-type Parvibaculum lavamentivorans short-chain alcohol

dehydrogenase.

In an additional and/or alternative embodiment, the library of polynucleotides comprises at least one polynucleotide comprising a nucleotide sequence which encodes a variant of the Parvibaculum lavamentivorans short-chain alcohol dehydrogenase, said variant having an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34 and 36.

In an additional and/or alternative embodiment, the library of polynucleotides comprises at least one polynucleotide molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33 and 35. In an embodiment, the polynucleotides of the library of polynucleotides are linear nucleic acid molecules. Said linear polynucleotide molecules may be selected from the group consisting of artificial chromosomes, virus-based nucleic acid molecules, and in-vitro generated nucleic acid molecules such as in-vitro transcribed RNA, products of polymerase chain reactions or other DNA-amplification processes.

In an alternative embodiment, the polynucleotides of the library of polynucleotides are circular nucleic acid molecules. The circular molecules may be selected from the group consisting of plasmids and cosmids.

In another and/or alternative embodiment of the first aspect, the library of elements is a library of host cells, wherein at least one of said host cells includes at least one variant of the Parvibaculum lavamentivorans short-chain alcohol dehydrogenase and/or at least one polynucleotide comprising a nucleotides sequence which encodes one of the variants of the Parvibaculum lavamentivorans short-chain alcohol dehydrogenase. In embodiments wherein the at least one host cells includes at least one of said host cells includes at least one variant of the Parvibaculum lavamentivorans short-chain alcohol dehydrogenase and at least one polynucleotide comprising a nucleotides sequence which encodes one of the variants of the Parvibaculum lavamentivorans short-chain alcohol dehydrogenase, it is preferred that the polynucleotide comprises the nucleotide sequence which encodes the amino acid sequence of the variant of the Parvibaculum lavamentivorans short-chain alcohol dehydrogenase included in said host cell.

The term "host cell" refers to any suitable host which may include a polypeptide and/or polynucleotide that is not part of the naturally occurring polypeptides and/or polynucleotides in said host. The host cell may be a virus, a prokaryotic cell or a eukaryotic cell. In an embodiment, the host cell is E. coli. The host cells of the library of host cells which include a polynucleotide molecule comprising a nucleotide sequence which encodes for a variant of the Parvibaculum lavamentivorans short-chain alcohol dehydrogenase are obtained by means of molecular biological mechanism, i.e. for example by means of infection, transfection or transformation.

In an embodiment, the at least one host cell includes at least one variant of the wild-type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase, said variant of the wild- type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase differs from the wild- type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase in at least one of the amino acid residues of the 2 sphere of said wild-type Parvibaculum lavamentivorans short- chain alcohol dehydrogenase.

In an embodiment of the library of host cells, nucleotide sequence which encodes for a variant of the Parvibaculum lavamentivorans short-chain alcohol dehydrogenase is integrated into the genome of the host cell, i.e. has been integrated into at least one of the host cells chromosomes, or may be present as part of an extrachromosomal nucleic acid molecule.

In a further and/or additional embodiment, the at least one host cell includes at least one variant of the wild-type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase, wherein said at least one variant of the wild-type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase differs in at least one of the amino acid residues from the wild-type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase which are selected from the group of amino acid residues consisting of

G at position 92, I at position 93, K at position 103, T at position 1 1 1 ,

I at position 146, S at position 147, G at position 151 , G at position 154,

Q at position 155, A at position 158, D at position 160, N at position 162,

V at position 191 , H at position 192, D at position 197, T at position 198,

P at position 199, V at position 201 , K at position 202, N at position 203,

N at position 206, L at position 224, H at position 228, L at position 233,

G at position 234, V at position 261 and D at position 262, with respect to the wild-type Parvibaculum lavamentivorans short-chain alcohol

dehydrogenase.

In a further and/or additional embodiment, the at least one host cell includes at least one variant of the wild-type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase, wherein the at least one variant of the Parvibaculum lavamentivorans short-chain alcohol dehydrogenase is a polypeptide comprising an amino acid sequence which is selected from the group consisting of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34 and 36. In another embodiment, the at least one variant of the Parvibaculum

lavamentivorans short-chain alcohol dehydrogenase is a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34 and 36.

In an additional and/or alternative embodiment, the library of host cells includes at least one host cell which includes at least one polynucleotide, wherein said at least one polynucleotide comprises a nucleotide sequence which encodes one of the polypeptide variants of the wild- type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase which differ from the wild-type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase in at least one amino acid residue of the 2^nd sphere of the wild-type Parvibaculum lavamentivorans short- chain alcohol dehydrogenase.

In a further and/or additional embodiment, the library of host cells includes at least one host cell which includes at least one polynucleotide, wherein said at least one polynucleotide comprises a nucleotide sequence which encodes one of the polypeptide variants of the wild- type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase which differ from the wild-type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase in at least one amino acid residue selected from the group consisting of

G at position 92, I at position 93, K at position 103, T at position 1 1 1 ,

I at position 146, S at position 147, G at position 151 , G at position 154,

Q at position 155, A at position 158, D at position 160, N at position 162,

V at position 191 , H at position 192, D at position 197, T at position 198,

P at position 199, V at position 201 , K at position 202, N at position 203,

N at position 206, L at position 224, H at position 228, L at position 233,

G at position 234, V at position 261 and D at position 262, with respect to the wild-type Parvibaculum lavamentivorans short-chain alcohol

dehydrogenase.

In an additional and/or alternative embodiment, the library of host cells comprises at least one host cell including a polynucleotide molecule comprising a nucleotide sequence encoding a variant of the Parvibaculum lavamentivorans short-chain alcohol dehydrogenase, said variant having an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34 and 36.

In an additional and/or alternative embodiment, the library of host cells comprises at least one host cell including a polynucleotide molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33 and 35.

In a preferred embodiment of the library of host cells, said host cells are capable of expressing the variant of the Parvibaculum lavamentivorans short-chain alcohol

dehydrogenase that is encoded by the nucleotide sequence said host cell includes. In an additional and/or alternative embodiment, the library of polynucleotides comprises at least one polynucleotide molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33 and 35.

In an additional and/or alternative embodiment, the library of polynucleotides comprises at least one polynucleotide comprising a nucleotide sequence which encodes a variant of the Parvibaculum lavamentivorans short-chain alcohol dehydrogenase, said variant having an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34 and 36.

According to the second aspect, the invention provides polypeptides, said polypeptides being variants of the wild-type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase, wherein said variants differs from the wild-type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase in at least one of the amino acid residues of the 2^nd sphere of said wild-type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase.

The amino acids of the 2^nd sphere of the wild-type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase are those amino acid residues within the catalytic center of the enzyme which appear not to be involved in the coordination of the enzyme's substrate and the enzyme's cofactor NADH, but which are present at a distance of about 4 A to about 17A, preferably to about 15 A, from the enzyme's substrate and/or the enzyme's cofactor.

The amino acid residues of the 2^nd sphere of the wild-type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase are the amino acid residues

G at position 92, I at position 93, K at position 103, T at position 1 1 1 ,

I at position 146, S at position 147, G at position 151 , G at position 154,

Q at position 155, A at position 158, D at position 160, N at position 162,

V at position 191 , H at position 192, D at position 197, T at position 198,

P at position 199, V at position 201 , K at position 202, N at position 203,

N at position 206, L at position 224, H at position 228, L at position 233,

G at position 234, V at position 261 and D at position 262, with respect to the wild-type Parvibaculum lavamentivorans short-chain alcohol

dehydrogenase.

Hence, in an embodiment of the second aspect, the variant of the wild-type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase differs in at least one of the amino acid residues from the wild-type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase which are selected from the group of amino acid residues consisting of

G at position 92, I at position 93, K at position 103, T at position 1 1 1 ,

I at position 146, S at position 147, G at position 151 , G at position 154,

Q at position 155, A at position 158, D at position 160, N at position 162,

V at position 191 , H at position 192, D at position 197, T at position 198,

P at position 199, V at position 201 , K at position 202, N at position 203,

N at position 206, L at position 224, H at position 228, L at position 233,

G at position 234, V at position 261 and D at position 262, with respect to the wild-type Parvibaculum lavamentivorans short-chain alcohol

dehydrogenase.

In a further and/or additional embodiment of the second aspect, the variant of the

Parvibaculum lavamentivorans short-chain alcohol dehydrogenase is a polypeptide comprising an amino acid sequence which is selected from the group consisting of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34 and 36. The variant of the

Parvibaculum lavamentivorans short-chain alcohol dehydrogenase may comprise a tag at its N-terminal and/or C-terminal end which may be used for purifying the variant. The term "tag" refers to a peptide sequence that is genetically grafted onto the variant of the of the

Parvibaculum lavamentivorans short-chain alcohol dehydrogenase. The tag may be removable from the variant of the Parvibaculum lavamentivorans short-chain alcohol dehydrogenase by chemical or enzymetic means. Examples of tags include AviTag, calmodulin-tag, chitin binding protein (CBP), maltose binding protein-tag, Nus-tag, Strep-tag, Thioredoxin-tag, S-tag. SBP-tag, Softag 1 , Softag 3, Xpress tag, Isopeptag, SpyTag, glutathione-S-transferase-tag, green fluorescent protein-tag, poly(His) tag, FLAG-tag, V5-tag, c-myc-tag, TC-tag, Ty-tag and HA-tag.

In another embodiment of the second aspect, the variant of the Parvibaculum

lavamentivorans short-chain alcohol dehydrogenase is a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34 and 36.

In an additional and/or alternative embodiment, the enzymatic activity of the variant of the Parvibaculum lavamentivorans short-chain alcohol dehydrogenase is dependent on NAD rather than on NADP. This does not mean that the enzymatic activity of the variant of the Parvibaculum lavamentivorans short-chain alcohol dehydrogenase is abolished in the presence of NADP but the absence of NAD. A secondary activity with respect to NADP of about 1/20 of the activity with respect to NAD may be given.

In an additional and/or alternative embodiment, the variant of the Parvibaculum

lavamentivorans short-chain alcohol dehydrogenase accepts 2-propanol as substrate which permits a substrate-coupled regeneration of the cofactor. The variant of the Parvibaculum lavamentivorans short-chain alcohol dehydrogenase is stable in the presence of up to 23 vol.-% 2-propanol within an aqueous solvent, i.e. remains its enzymatic activity. In an additional and/or alternative embodiment, the variant of the Parvibaculum

lavamentivorans short-chain alcohol dehydrogenase has an optimum temperature for converting 3-quinuclidinone to (R)-3-quinduclidinol of about 40°C, preferably of above 40°C, more preferably of about 50°C, and most preferably of more than 50°C. In an additional and/or alternative embodiment, the variant of the Parvibaculum

lavamentivorans short-chain alcohol dehydrogenase has an optimum pH for the reduction reaction of about 6.

In an additional and/or alternative embodiment the enzymatic activity of the variant of the Parvibaculum lavamentivorans short-chain alcohol dehydrogenase is independent of any metal ions, in particular independent of Mg²⁺.

In an additional and/or alternative embodiment, the variant of the Parvibaculum

lavamentivorans short-chain alcohol dehydrogenase is enzymatically stable in the presence of up to 38.5 vol.-% 2-propanol.

According to the third aspect, the invention provides a kit, wherein said kit comprises a plurality of alcohol dehydrogenases, and wherein at least one of the plurality of alcohol dehydrogenases is a variant of the wild-type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase, said variant differs from the wild-type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase in at least one of the amino acid residues of the 2^nd sphere of said wild-type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase.

The term "kit" refers to a kit of parts, wherein said parts are understood to be polypeptides, in particular polypeptides possessing enzymatic alcohol dehydrogenase activity. The expression "plurality of alcohol dehydrogenases" refers to multiple different polypeptides possessing enzymatic alcohol dehydrogenase activity. The "plurality of alcohol

dehydrogenases" may comprise different polypeptides possessing alcohol dehydrogenase activity, wherein the different polypeptides originate from different organisms. In an additional and/or alternative, the "plurality of alcohol dehydrogenases" may comprise different polypeptides possessing alcohol dehydrogenase activity, wherein said polypeptides originate from the same species but differ in their amino acid sequence.

In an embodiment of the third aspect, the kit comprises at least one variant of the wild-type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase which differs in at least one of the amino acid residues from the wild-type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase which are selected from the group of amino acid residues consisting of

G at position 92, I at position 93, K at position 103, T at position 1 1 1 ,

I at position 146, S at position 147, G at position 151 , G at position 154,

Q at position 155, A at position 158, D at position 160, N at position 162,

V at position 191 , H at position 192, D at position 197, T at position 198,

P at position 199, V at position 201 , K at position 202, N at position 203,

N at position 206, L at position 224, H at position 228, L at position 233,

G at position 234, V at position 261 and D at position 262, with respect to the wild-type Parvibaculum lavamentivorans short-chain alcohol

dehydrogenase.

In a further and/or additional embodiment of the third aspect, the kit comprises at least one variant of the Parvibaculum lavamentivorans short-chain alcohol dehydrogenase which is a polypeptide comprising an amino acid sequence which is selected from the group consisting of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34 and 36. the kit comprises at least one variant of the Parvibaculum lavamentivorans short-chain alcohol dehydrogenase which is a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34 and 36.

In an embodiment of the kit, each alcohol dehydrogenase of the plurality of alcohol dehydrogenases is provided in a separate vial.

The kit provides a collection of different alcohol dehydrogenases which may differ in their features such as - for example - substrate specificity, reaction velocity, efficacy, stability and the like. The kit provides the opportunity to determine the most suitable enzyme for a desired reaction to be catalyzed in a trial. The most suitable alcohol dehydrogenase can then be selected for scaling up the reaction to be catalyzed to an industrial scale. According to the fourth aspect, the invention provides a polynucleotide which comprises a nucleotide sequence which encodes a variant of the Parvibaculum lavamentivorans short- chain alcohol dehydrogenase which differs from the wild-type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase in at least one of the amino acid residues of the 2^nd sphere of said wild-type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase.

In an embodiment, the polynucleotide comprises a nucleotide sequence which comprises at least one non-silent mutation in at least one of the triplets encoding the amino acid residues of the 2^nd sphere of the Parvibaculum lavamentivorans short-chain alcohol dehydrogenase. In an embodiment, the polynucleotide comprises a nucleotide sequence which comprises at least one non-silent mutation in at least one of the triplets encoding the amino acid residues of the wild-type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase which are selected from the group of amino acid residues consisting of

G at position 92, I at position 93, K at position 103, T at position 1 1 1 ,

I at position 146, S at position 147, G at position 151 , G at position 154,

Q at position 155, A at position 158, D at position 160, N at position 162,

V at position 191 , H at position 192, D at position 197, T at position 198,

P at position 199, V at position 201 , K at position 202, N at position 203,

N at position 206, L at position 224, H at position 228, L at position 233,

G at position 234, V at position 261 and D at position 262, with respect to the wild-type Parvibaculum lavamentivorans short-chain alcohol

dehydrogenase.

In an additional and/or alternative embodiment, the polynucleotide comprises a nucleotide sequence which encodes a variant of the Parvibaculum lavamentivorans short-chain alcohol dehydrogenase, said variant having an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34 and 36.

In an additional and/or alternative embodiment, the polynucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33 and 35. In an embodiment, the polynucleotide is a linear nucleic acid molecule. Said linear polynucleotide molecule may be selected from the group consisting of artificial

chromosomes, virus-based nucleic acid molecules, and in-vitro generated nucleic acid molecules such as in-vitro transcribed RNA, products of polymerase chain reactions or other DNA-amplification processes.

In an alternative embodiment, the polynucleotide is a circular nucleic acid molecule. The circular molecule may be selected from the group consisting of plasmids and cosmids. According to the fifth aspect, the invention provides a host cell, wherein said host cells includes at least one variant of the Parvibaculum lavamentivorans short-chain alcohol dehydrogenase and/or at least one polynucleotide comprising a nucleotides sequence which encodes one of the variants of the Parvibaculum lavamentivorans short-chain alcohol dehydrogenase. In embodiments wherein the at least one host cells includes at least one of said host cells includes at least one variant of the Parvibaculum lavamentivorans short-chain alcohol dehydrogenase and at least one polynucleotide comprising a nucleotides sequence which encodes one of the variants of the Parvibaculum lavamentivorans short-chain alcohol dehydrogenase, it is preferred that the polynucleotide comprises the nucleotide sequence which encodes the amino acid sequence of the variant of the Parvibaculum lavamentivorans short-chain alcohol dehydrogenase included in said host cell.

The host cell includes at least one variant of the wild-type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase, said variant of the wild-type Parvibaculum

lavamentivorans short-chain alcohol dehydrogenase differs from the wild-type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase in at least one of the amino acid residues of the 2^nd sphere of said wild-type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase.

In a further and/or additional embodiment, the host cell includes at least one variant of the wild-type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase, wherein said at least one variant of the wild-type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase differs in at least one of the amino acid residues from the wild-type

Parvibaculum lavamentivorans short-chain alcohol dehydrogenase which are selected from the group of amino acid residues consisting of

G at position 92, I at position 93, K at position 103, T at position 1 1 1 ,

I at position 146, S at position 147, G at position 151 , G at position 154,

Q at position 155, A at position 158, D at position 160, N at position 162, V at position 191 , H at position 192, D at position 197, T at position 198,

P at position 199, V at position 201 , K at position 202, N at position 203,

N at position 206, L at position 224, H at position 228, L at position 233,

G at position 234, V at position 261 and D at position 262, with respect to the wild-type Parvibaculum lavamentivorans short-chain alcohol

dehydrogenase.

In a further and/or additional embodiment, the host cell includes at least one variant of the wild-type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase, wherein the at least one variant of the Parvibaculum lavamentivorans short-chain alcohol dehydrogenase is a polypeptide comprising an amino acid sequence which is selected from the group consisting of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34 and 36. In another embodiment, the at least one variant of the Parvibaculum lavamentivorans short- chain alcohol dehydrogenase is a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34 and 36.

In an additional and/or alternative embodiment, the host cell includes at least one

polynucleotide, wherein said at least one polynucleotide comprises a nucleotide sequence which encodes one of the polypeptide variants of the wild-type Parvibaculum

lavamentivorans short-chain alcohol dehydrogenase which differ from the wild-type

Parvibaculum lavamentivorans short-chain alcohol dehydrogenase in at least one amino acid residue of the 2^nd sphere of the wild-type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase.

In a further and/or additional embodiment, the host cell includes at least one polynucleotide, wherein said at least one polynucleotide comprises a nucleotide sequence which encodes one of the polypeptide variants of the wild-type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase which differ from the wild-type Parvibaculum lavamentivorans short- chain alcohol dehydrogenase in at least one amino acid residue selected from the group consisting of

G at position 92, I at position 93, K at position 103, T at position 1 1 1 ,

I at position 146, S at position 147, G at position 151 , G at position 154,

Q at position 155, A at position 158, D at position 160, N at position 162,

V at position 191 , H at position 192, D at position 197, T at position 198,

P at position 199, V at position 201 , K at position 202, N at position 203,

N at position 206, L at position 224, H at position 228, L at position 233, G at position 234, V at position 261 and D at position 262, with respect to the wild-type Parvibaculum lavamentivorans short-chain alcohol

dehydrogenase. In an additional and/or alternative embodiment, the host cell includes a polynucleotide molecule comprising a nucleotide sequence encoding a variant of the Parvibaculum lavamentivorans short-chain alcohol dehydrogenase, said variant having an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34 and 36.

In an additional and/or alternative embodiment, the host cell includes a polynucleotide molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33 and 35. In a preferred embodiment, the host cell is capable of expressing the variant of the

Parvibaculum lavamentivorans short-chain alcohol dehydrogenase that is encoded by the nucleotide sequence said host cell includes.

According to the sixth aspect, the invention provides the use of a variant of the Parvibaculum lavamentivorans short-chain alcohol dehydrogenase, wherein said variant differs from the wild-type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase in at least one of the amino acid residues of the 2^nd sphere of said wild-type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase for converting a suitable ketone to the R-enantiomer of the corresponding alcohol. In the use of said variant of the Parvibaculum lavamentivorans short-chain alcohol dehydrogenase the suitable ketone is reacted with said variant of the Parvibaculum lavamentivorans short-chain alcohol dehydrogenase, whereby the ketone is reduced in a stereospecific manner to essentially one of the enantiomers only, namely the R- enantiomer. According to a further aspect, the invention also provides the use of the wild-type

Parvibaculum lavamentivorans short-chain alcohol dehydrogenase for converting a suitable ketone to the R-enantiomer of the corresponding alcohol.

A "suitable ketone" is a ketone that can be converted by the variant of the Parvibaculum lavamentivorans short-chain alcohol dehydrogenase and/or the wild-type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase. In an embodiment of the sixth aspect and the further aspect, the suitable ketone is a bicyclic ketone. In an additional and/or alternative embodiment, the suitable ketone is 3- quinulidinone. The advantage of using the wild-type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase for converting a suitable ketone to the R-enantiomer of the corresponding alcohol resides within - among other properties is the high thermostability of the enzyme, its stability in the presence of organic solvents and its use of NAD(H) as cofactor instead of NADP(H). The advantages of using a variant of the wild-type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase for converting a suitable ketone to the R-enantiomer of the corresponding alcohol resides within the inferior stereoselectivity of a variant for a given substrate compared to the wild-type enzyme.

According to the another further aspect, the invention provides a method for preparing the R- enantiomer of a chiral alcohol, the process comprising

a. reacting a suitable ketone with a variant of the Parvibaculum lavamentivorans short- chain alcohol dehydrogenase which differs from the wild-type Parvibaculum

lavamentivorans short-chain alcohol dehydrogenase in at least one of the amino acid residues of the 2^nd sphere of said wild-type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase in the presence of suitable cofactors in a suitable solvent or with the wild-type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase; and b. isolating the R enantiomer of the alcohol.

In an embodiment of the further aspect, the ketone is a bicyclic ketone.

In an additional and/or alternative embodiment, the ketone is 3-quinulidinone.

The conversion of the ketone to the R-enantiomer of the corresponding alcohol may be performed at suitable reaction conditions, preferably conditions that are optimal for the enzyme's reactivity. For example the conversion may be carried out at a pH of 7 and/or a temperature of 25 °C. However, it is also possible to perform the conversion of the ketone to the R-enantiomer of its corresponding alcohol at a pH which differs therefrom. The conversion may be performed at a pH of about 6, 6.5, 7.5 or 8. In addition, the conversion may be performed at a temperature below or above said 25 °C, in particular if the

temperature optimum for the enzyme is different, and the components of the reaction mixture are stable at said temperature. For example, the wild-type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase has a temperature optimum of about 50 °C. Hence, the conversion may be performed at a temperature of up to 50°C, preferably at a temperature of 30°C or higher, more preferably at a temperature of 37°C or higher, and even more preferably at a temperature of 40°C or higher. It can even be contemplated that the conversion is performed at a temperature above the temperature optimum for enzymatic activity of a given polypeptide.

The present invention will be described with respect to particular embodiments and with reference to the figures, but the invention is not limited thereto, but only to the claims. The darwings described are only schematic and are non-limiting. In the drawings, the size of some of the elements may be exaggerated and not drawn on scale for illustrative purposes.

Where an indefinite or definite article is used when referring to a singular noun, e.g. "a", "an", "the", this includes a plural of that noun unless something else is specifically stated.

Furthermore, the terms first, second, third and the like in the description and in the claims are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein. Moreover, the terms top, bottom, over, under, beyond and the like in the description and in the claims are used for descriptive purposes and not necessarily for describing relative positions. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other orientations than described or illustrated herein. It is to be noticed that the term "comprising", used in the present description and claims, should not be interpreted as being restricted to the means listed thereafter; it does not exclude other elements or steps. Thus, the scope of the expression "a device comprising means A and B" should not be limited to devices consisting only of components A and B. It means that with respect to the present invention, the only relevant components of the device are A and B.

Examples Example 1

Generating variants of the P. lavamentivorans short-chain alcohol dehydrogenase

A library of P. lavamentivorans short-chain alcohol dehydrogenase variants, wherein the mutants were obtained by semi-rationale protein design in that amino acid residues within the catalytic center were identified which might be involved in substrate specificity concerning bicyclic ketones, but appear not to be involved in the coordination of the coenzyme NADH and are also not directly involved in the catalytic mechanism as such. More specifically, those amino acid residues of the second sphere of the catalytic center were considered which had a distance of about 4 A to about 17A, from the enzyme's substrate and/or the enzyme's cofactor, and which were deemed not to be involved in the coordination of the enzyme's substrate and/or the enzyme's cofactor. A total of six amino acids residues suitable for being subjected to mutagenesis were identified: threonine at position 1 1 1 (T-m), alanine at position 158 (A₁₅₈), asparagine at position 162 (N₁₆2), valine at position 201 (V₂oi), lysine at position 202 (K202), and leucine at position 224 (L₂24)-

Based on the nucleotide sequence of the wild-type P. lavamentivorans alcohol

dehydrogenase gene (SEQ ID NO: 1 ) a variant of the P. lavamentivorans alcohol dehydrogenase gene, wherein the codon usage was adapted to the codon usage of

Escherichia coli, was generated. The nucleotide sequence of this gene variant is shown in SEQ ID NO: 3. Based on said variant of the P. lavamentivorans alcohol dehydrogenase gene a library of approximately 1200 E. coli clones, wherein each of said E. coli clones beared a polynucleotide molecule comprising a nucleotide sequence encoding a variant of the P. lavamentivorans short chain alcohol dehydrogenase which had two of said six amino acid residues altered. The number of clones for each of the libraries that were generated was large enough to comprise all theoretically possible variants.

Quadruple mutants were generated by overlap-extension PCR such that individual nucleotides were changed, and the resulting triplets encoded other amino acids. Table 1 provides a summary of the oligonucleotide primers that were used for overlap-extension PCR reactions:

SEQ nucleotide sequence feature

ID NO:

37 5 ' -TTGTCCAGAGCTCATATGGCACGTATGCAGGG-3 ' ADH-5'-Stop

38 5 ' -ATATAAGCTTTCATTAACGGGTGGTATAACC-3 ' ADH-3'-Stop

39 5 ' -GAGCGGTAAGAGCGATTATATGGCTGCCAAAG-3 ' 158K/162M-up

40 5 ' -CTTTGGCAGCCATATAATCGCTCTTACCGCTC-3 ' 158K/162M-down

41 5 ' -GAGCGGTCACAGCGATTATCCCGCTGCCAAAG-3 ' 158H/162P-up

42 5 ' -CTTTGGCAGCGGGATAATCGCTGTGACCGCTC-3 ' 158H/162P-down

43 5 ' -GAGCGGTGCGAGCGATTATGCCGCTGCCAAAG-3 ' 158A/162A-UP

44 5 ' -CTTTGGCAGCGGCATAATCGCTCGCACCGCTC-3 ' 158A/162A-down 45 5 ' -GAGCGGTGACAGCGATTATTCCGCTGCCAAAG-3 ' 158D/162S-up

46 5 ' -CTTTGGCAGCGGAATAATGCGTGTCACCGCTC-3 ' 158D/162S-down

47 5 ' -GAGCGGTGACAGCGATTATGGCGCTGCCAAAG-3 ' 158D/162G-up

48 5 ' -CTTTGGCAGCGCCATAATCGCTGTCACCGCTC-3 ' 158D/162G-down

49 5 ' -GAGCGGTCACAGCGATTATGGCGCTGCCAAAG-3 ' 158H/162G-up

50 5 ' -CTTTGGCAGCGCCATAATCGCTGTGACCGCTC-3 ' 158H/162G-down

51 5 ' -GAGCGGTGACAGCGATTATTGCGCTGCCAAAG-3 ' 158D/162C-up

52 5 ' -CTTTGGCAGCGCAATAATCGCTGTCACCGCTC-3 ' 158D/162C-down

Table 1 : Oligonucleotide primers that were used for overlap-extension PCR reactions in generating quadruple variants of P. lavamentivorans short-chain alcohol dehydrogenase.

The resulting mutants or variants of P. lavamentivorans short-chain alcohol dehydrogenase that were obtained and subjected to further characterization are summarized in Table 2.

Example 2

Enzyme preparation of variants of P. lavamentivorans short-chain alcohol

dehydrogenase

The recombinant/transformed E. coli containing an expression plasmid (based on pET21 a) comprising the nucleotide sequence which encodes a variant of the P. lavamentivorans short-chain alcohol dehydrogenase were grown over night at 37°C in LB-medium

supplemented with 100 μg ml ampicillin with shaking at 200 rpm. Expression of protein was induced by adding 100 μΜ IPTG (final cone.) to culture that was inoculated with the bacteria from the overnight culture at a density of OD₅₈o = 0.05, when said culture had a density of OD₅₈o = 0.5 to 0.7. Cultures were shaken at 200 rpm for about 18 hours at 30°C. Bacteria were harvested by centrifugation for 20 min at 21.000 rpm at 4°C. The cell pellets were re- suspended in a threefold amount of buffer (100 mM Tris/HCI, pH 7.2; 1 mM MgCI₂). The re- suspended cells were subjected to ultrasonic treatment. A crude protein extract was obtained in that the lysed cells were centrifuged for 20 min at 14,000 g at 4°C, and the supernatant was stored at -20°C for further use.

Example 3

Oxidoreductase activity of variants of the P. lavamentivorans short-chain alcohol dehydrogenase The enzymatic activity of the crude protein extracts comprising a variant of the P. lavamentivorans short-chain alcohol dehydrogenase was assayed spectrophotometrically in an NADH dependent assay at 340 nm. The 1 ml standard assay mixture comprised 870 μΙ TEA-buffer (50 mM Tris/HCI, pH 7.0; 1 .15 mM MgCI₂), 100 μΙ 100 mM ethylacetacetate, 20 μΙ 12.5 mM NADH, and 10 μΙ crude protein extract (diluted in 100 mM Tris/HCI (ph 7.2), 1 mM MgCI₂). The reaction ran for 1 min at 30°C.

For determining the optimum temperature for catalytic activity, multiple reactions were performed for 100 hour at temperatures ranging from room temperature to 60°C. Crude extracts were adjusted to an activity of 8 U/ml, concentration of cofactor NAD+ was 0.2 mM, buffer was potassium phosphate buffer (100 mM) including 2 mM MgCI₂, pH 7.5).

Ethylacetonate was used as substrate. For regeneration of cofactor, each reaction included iPrOH. The total amount of organic phase was 80% at a molar ratio of isopropanol (iPrOH) to ethylacetoacetate (EAA) of 2:1. Samples of the reaction were analysed.

Example 4

Synthesis of (R)-3-quinuclidinol from 3-quinuclidinone using variants of the P.

lavamentivorans short-chain alcohol dehydrogenase

In a reaction vessel, 3-quinuclidinone hydrochloride and glucose monohydrate were dissolved in water. The reaction mixture was stirred at 25°C and 2000 rpm. The pH of the reaction mixture was adjusted to 7.0 by adding NaOH. An aliquot of a variant of the P.

lavamentivorans short-chain alcohol dehydrogenase, NAD in enzyme solution, glucose dehydrogenase of B. subtilis and water were added. The reaction mixture was stirred at 25°C under continuous pH adjustment (pH 7.0) for approximately 12 hours till conversion is >99 %. Then sodium hydroxide was added to shift the pH value to 12.0. Subsequently celite and Na₂S0₄ was added, and the solution was warmed to 80°C for 20 min. After cooling to about 35°C, the solids were filtered off, and the pellet was washed twice with 2-butanol. Both phases of the filtrate were mixed and separated. The aqueous phase was again extracted once with 2-butanol. The organic phases were combined and 2-butanol was removed under reduced pressure. The remaining residue was dissolved in a mixture of ethanol and water (9:1 v/v). The resulting solution was stirred and phosphoric acid was added. The mixture was cooled to 20°C and stirred for another 20 min. The product was filtered off, and dried under reduced pressure at 30°C. The product was analyzed by gas chromatography to measure the chemical purity. Also the optical purity of the product with respect to (R)-3-quinculidinol was measured by HPLC.

For analyzing the chemical purity, 2 to 5 mg of the product was dissolved in 200 μΙ water, 200 ml NaOH (10 M) and 300 μΙ MTBE were added. After mixing and centrifugation, the upper layer was analyzed by gas chromatography:

Column: FS-lnnopeg-2000 (30 m x 0.25 mm x 0.25 μπι)

Carrier gas: Helium at 3 ml/min, split 10

Oven program:

Retention times: 3-quinuclidinol: 7.2 min

3-quinuclidinone: 4.9 min.

For analyzing the optical purity, 5 to 10 mg of the product were dissolved in 200 μΙ water, 300 μΙ NaOH (10 M) was added, and extracted with 1 ml MTBE. The organic layer was separated and 10 to 20 mg benzoic anhydride was added and dissolved. The mixture was concentrated at 70°C in an open reaction vessel, 200 μΙ of 2 N hydrochloric acid and 500 μΙ of MTBE was added. The upper layer was discarded after mixing and centrifugation. The aqueous phase was alkalized with 150 μΙ NaOH (10 M) and extracted with 500 μΙ DCM. The organic phase was analyzed by HPLC:

Column: MultoHigh Chiral AM-HR (25 m x 0.25 mm x 0.25 μπι)

Flow: 1 ml/min

Oven temperature: 30°C

Solvents: A: 2-propanol / triethylamine (99.9% : 0.1 %)

B: n-heptane / 2-propanol / triethylamine (98.9% : 1 % : 0.1 %)

Eluent:

Retention times: (R)-3-quinuclidinol: 50 min

(S)-3-quinuclidinol: 41 min.

The optical purity of the reaction product obtained by using the wild-type P. lavamentivorans short-chain alcohol dehydrogenase (SEQ ID NO: 2) was measured to be 84.3 % ee. The optical purity of the variants of P. lavamentivorans short-chain alcohol dehydrogenase that were obtained and subjected to further analysis are summarized in Table 2.

SEQ ID 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 No:

T

K H D D K D D H D

M P P A Q G M A S G G C

R Q S N R R R R Q Q

I W W Y M W W W W W W

e.e. 97.6 96.8 98.1 93.8 95.8 96.5 94.7 95.5 89.5 89.7 98.8 97.7 97.4 97.7 98.8 94.9 Table 2: Variants of P. lavamentivorans short-chain alcohol dehydrogenase, and their stereospecificity (measured as e.e.) in converting 3-quinuclidinone to (R)-3-quinuclidinol.

For all variants of the P. lavamentivorans short-chain alcohol dehydrogenase set forth in Table 2 the measured optical purity was above optical purity that was measured for the wild- type P. lavamentivorans short-chain alcohol dehydrogenase.

Claims

A library of elements, said elements being selected from the group consisting of polynucleotides, polypeptides and host cells, wherein said library includes at least one polypeptide variant that is based on the wild-type Parvibaculum lavamentivorans short- chain alcohol dehydrogenase and/or at least one polynucleotide comprising a nucleotide sequence that encodes said at least one polypeptide variant, said at least one variant differs from the wild-type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase in at least one of the amino acid residues of the 2^nd sphere of said wild-type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase.

The library according to claim 1 , wherein the at least one amino acid residue of the 2^nd sphere appears not to be involved in the coordination of the enzyme's substrate and the enzyme's cofactor NADH, but which is present at a distance of about 4 A to about 17 A, preferably to about 15 A, from the enzyme's substrate and/or the enzyme's cofactor.

The library according to claim 1 or 2, wherein the at least one amino acid residue of the

2 sphere is selected from the group of amino acid residues consisting of

G at position 92, I at position 93, K at position 103, T at position 1 1 1 , I at position 146, S at position 147, G at position 151 , G at position 154, Q at position 155, A at position 158, D at position 160, N at position 162, V at position 191 , H at position 192, D at position 197, T at position 198, P at position 199, V at position 201 , K at position 202, N at position 203, N at position 206, L at position 224, H at position 228, L at position 233, G at position 234, V at position 261 and D at position 262,

with respect to the wild-type P. lavamentivorans short-chain alcohol dehydrogenase.

A polypeptide, wherein said polypeptide is a variant of the wild-type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase, said variant differs from the wild- type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase in at least one of the amino acid residues of the 2^nd sphere of said wild-type Parvibaculum

lavamentivorans short-chain alcohol dehydrogenase.

The polypeptide according to claim 4, wherein the at least one amino acid residue of the 2^nd sphere appears not to be involved in the coordination of the enzyme's substrate and the enzyme's cofactor NADH, but which is present at a distance of about 4 A to about 17 A, preferably to about 15 A, from the enzyme's substrate and/or the enzyme's cofactor.

The polypeptide according to claim 4 or 5, wherein the at least one amino acid residue of the 2 sphere is selected from the group of amino acid residues consisting of

G at position 92, I at position 93, K at position 103, T at position 1 1 1 , I at position 146, S at position 147, G at position 151 , G at position 154, Q at position 155, A at position 158, D at position 160, N at position 162, V at position 191 , H at position 192, D at position 197, T at position 198, P at position 199, V at position 201 , K at position 202, N at position 203, N at position 206, L at position 224, H at position 228, L at position 233, G at position 234, V at position 261 and D at position 262, with respect to the wild-type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase.

The polypeptide according to any one of claims 4 to 6, wherein said polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34 and 36.

A kit comprising a plurality of alcohol dehydrogenases, wherein at least one of said plurality of alcohol dehydrogenases is a variant of the wild-type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase as defined in any one of claims 4 to 7.

The kit according to claim 8, wherein each alcohol dehydrogenase of the plurality of alcohol dehydrogenases is provided in a separate vial.

A polynucleotide comprising a nucleic acid sequence encoding a variant of the wild type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase as defined any one of claims 4 to 7. The polynucleotide according to claim 10, wherein said nucleic acid sequence comprises at least one non-silent mutation in at least one of the triplets encoding the amino acid residues of the 2^nd sphere of the wild-type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase.

12. The polynucleotide according to claim 10 or 1 1 , wherein the nucleic acid sequence is selected from the group consisting of SEQ ID NO: 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33 and 35. 13. A host cell including at least one molecule selected from the group consisting of the variants of the wild-type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase as defined in any one of claims 4 to 7, and the polynucleotides comprising a nucleotide sequence as defined in any one of claims 10 to 12. 14. A process for preparing the R-enantiomer of a chiral alcohol, the process comprising a. reacting a suitable ketone with the wild-type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase or with a variant of the wild-type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase as defined in any one of claims 4 to 7 in the presence of suitable cofactors in a suitable solvent; and

b. isolating the R enantiomer of the alcohol.

15. The process according to claim 14, wherein said suitable ketone is a bicyclic ketone. 16. The process according to claim 14, wherein said suitable ketone is 3-quinuclidone.

17. The process according to claim 14 or 15, the process comprises

a. reacting 3-quinuclidinone with a variant of the Parvibaculum lavamentivorans short-chain alcohol dehydrogenase as defined in any one of claims 4 to 7 in the presence of suitable cofactors in a suitable solvent; and

b. isolating the resulting (R)-3-quinuclidinol.

18. Use of the wild-type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase or of a variant of the wild-type Parvibaculum lavamentivorans short-chain alcohol dehydrogenase as defined in any one of claims 4 to 7 for converting a suitable ketone to the R-enantiomer of the corresponding alcohol.

19. The use according to claim 17, wherein said suitable ketone is 3-quinuclidinone. 20. The use according to claim 18 or 19, wherein said corresponding alcohol is 3- quinuclidinol.