# Patent

## Title
Lipase variants

## Abstract

The invention relates to a lipase variant which can be prepared by carrying out, on the amino acid sequence of a starting lipase selected from the lipase homologous family I.1 or I.2, at least one amino acid substitution in those positions which correspond to the positions 17, 29, 30, 52, 86, 117, 122, 160, 163, 167, 265, 266, 286, 289 in the prototype lipase sequence SEQ ID NO: 1.

## Background
RELATED APPLICATIONS
This application is a national stage application (under 35 U.S.C. 371) of PCT/EP02/11620 filed Oct. 17, 2002, which claims benefit of German application 101 51 292.9 filed Oct. 22, 2001 and German application 102 05 444.4 filed Feb. 8, 2002.
FIELD OF THE INVENTION
The present invention relates to lipase variants having increased specific activity, processes for the preparation of these lipase variants, and the use of these lipase variants as catalysts in chemical reactions, in particular for the preparation of optically active compounds.
DESCRIPTION OF THE BACKGROUND
The use of the naturally occurring biocatalysts in chemical reactions is limited by numerous factors such as, for example, lack of activity, stability and enantioselectivity. It is therefore desirable to have available novel biocatalysts which have been optimized with respect to these properties.
An industrially important application of lipases is the preparation of optically active amines, alcohols, carboxylic acids and carboxylic acid esters.
EP 0 548 228 discloses optimized lipase variants for use as detergent additives.
SUMMARY OF THE INVENTION
The present invention overcomes the problems and disadvantages associated with current strategies and designs and provides new lipase variants and methods.
One embodiment of the invention is directed to lipase variants comprising an amino acid sequence of a staffing lipase wherein the starting lipase is selected from the group consisting of a of lipases of lipase homologous families I.1 and I.2, wherein the amino acid sequence of said lipase variant contains at least one amino acid substitution at a position that corresponds to position 17, 29, 30, 52, 86, 117, 122, 160, 163, 167, 265, 266, 286, or 289 of SEQ ID NO: 1.
Another embodiment of the invention is directed to formulations containing lipase variants of the invention. Lipase variants of these formulations may be immobilized on a solid carrier material or in cells.
Another embodiment of the invention is directed to nucleic acid sequences and constructs of nucleic acids that encode lipase variants of the invention.
Another embodiment of the invention is directed to host organisms that contain nucleic acids of the invention.
Another embodiment of the invention is directed to processes for production of lipase comprising culturing an organism that expresses a lipase variant of the invention in a culture medium and, if desired, purifying the lipase variant from the culture mixture.
Another embodiment of the invention is directed to processes for enzyme-catalytic conversion or enantioselective conversion of substrates, which comprises reacting the substrates in the presence of the lipase variants of the invention. Preferred substrates are alcohols, amines, amino esters and carboxylic acid esters or racemic alcohols, amines, amino esters and carboxylic acid esters.


DESCRIPTION OF THE INVENTION
The invention is based on the object of making available novel lipase variants having improved properties, e.g. increased specific activity.
Lipase variants have been found which can be prepared by carrying out in the amino acid sequence of a starting lipase, selected from the lipase homologous family I.1 or I.2, at least one amino acid substitution in those positions which correspond to the positions 17, 29, 30, 52, 86, 117, 122, 160, 163, 167, 265, 266, 286, 289 in the sequence of the enzymatically active prototype lipase SEQ ID NO: 1.
The lipase having the amino acid sequence SEQ ID NO. 1 is designated below as a prototype lipase sequence. It is in this case the lipase of the strain Pseudomonas spec. DSM 8246.
This strain Pseudomonas spec. DSM 8246 is also designated as Burkholderia plantarii. The lipases from Burkholderia plantarii and Pseudomonas glumae have the same sequence (Frenken et al., Cloning of the Pseudomonas glumae lipase gene and determination of the active site residues, Appl. Environ. Microbiol. 1992, 58, 3787-3791).
Starting lipases of the lipase homologous families I.1 and I.2 are understood according to the invention as meaning the lipases which can be classified in the homologous classes (table 1) described by Arpigny and Jäger in Biochem. J. 199, 343, 177. Reference is expressly made hereby to this classification. Preferred starting lipases are the lipases from Pseudomonas aeruginosa, Vibrio cholerae, Pseudomonas fragi, Acinetobacter calcoaceticus, Pseudomonas wisconsinensis, Pseudomonas fluorescens, Pseudomonas vulgaris, Burkholderia cepacia, Burkholderia glumae, Pseudomonas spec. DSM 8246, Burkholderia plantarii, Chromobacterium viscosum and Pseudomonas luteola. 
A particularly preferred starting lipase is the lipase from Pseudomonas spec. DSM 8246, whose amino acid sequence is shown in SEQ ID NO: 1.
The amino acid sequences of these preferred lipases are described in Nardini et al., J. Biol. Chem. 2000, 275, 40, page 31223.
It is also possible, however, additionally to use other lipases as starting lipases of the lipase homologous families I.1 and I.2, as long as they satisfy the classification criteria of Arpigny and Jäger.
Lipases and lipase variants are understood according to the invention as meaning lipases, i.e. enzymes which have the enzymatic activity of a lipase. As a rule, such lipase variants are prepared by specific genetic modification of starting lipases. “Evolutive processes”, in which a starting molecule is optimized by undirected mutation and selection, are a further possibility of preparing the lipase variants according to the invention.
Amino acid substitution is understood according to the invention as meaning the replacement of an amino acid in the amino acid sequence of the starting lipase by another amino acid, preferably a natural amino acid. Natural amino acids are understood as meaning Ala, Asp, Asn, Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp and Tyr.
Since the amino acid sequences of the starting lipases as a rule do not have the same length as the prototype lipase sequence, the positions of the prototype lipase sequence cannot be transferred directly to the starting lipase, but the corresponding homologous positions must be identified. In order to determine these positions, a sequence alignment with the prototype lipase sequence is carried out, such as is described in Nardini et al., J. Biol. Chem. 2000, 275, 40, page 31223 (FIG. 3). Reference is hereby expressly made to this document and the further documents mentioned therein with respect to the sequence alignment and the homologous positions.
As is inferred, for example, from the homology comparison in Nardini et al., J. Biol. Chem. 2000, 275, 40, page 31223, the position Met16 of the lipase from Pseudomonas aeruginosa, corresponds to the position Leu44 in the lipase from Vibrio cholerae, to the position Leu17 in the lipase from Pseudomonas Fragi, to the position Leu49 in the lipase from Acinetobacter calcoaceticus, to the position Val37 in the lipase from Pseudomonas wisconsinensis, to the position Met17 in the lipase from Pseudomonas fluorescens, to the position Leu16 in the lipase from Pseudomonas vulgaris, to the position Leu17 in the lipase from Burkholderia cepacia, to the position Leu17 in the lipase from Burkholderia glumae (identical to SEQ ID NO: 1), to the position Leu17 in the lipase from Chromobacterium viscosum, and to the position Leu57 in the lipase from Pseudomonas luteola. 
A preferred embodiment of the invention is lipase variants which can be prepared from starting lipases which are selected from the following group:

    
    
        A lipase from Pseudomonas aeruginosa, 
        B lipase from Vibrio cholerae, 
        C lipase from Pseudomonas fragi, 
        D lipase from Acinetobacter calcoaceticus, 
        E lipase from Pseudomonas wisconsinensis, 
        F lipase from Pseudomonas fluorescens, 
        G lipase from Pseudomonas vulgaris, 
        H lipase from Burkholderia cepacia, 
        I lipase from Burkholderia glumae, 
        J lipase from Burkholderia plantarii, 
        K lipase from Chromobacterium viscosum, 
        L lipase from Pseudomonas luteola, 
        M lipase from Pseudomonas spec. DSM 8246
    
    


Further preferred embodiments are those lipase variants which are mutated in the following positions compared with the starting lipase:

    A lipase from Pseudomonas aeruginosa, mutated in position Met17,
    B lipase from Vibrio cholerae, mutated in position Leu44,
    C lipase from Pseudomonas Fragi, mutated in position Leu17,
    D lipase from Acinetobacter calcoaceticus, mutated in position Leu49,
    E lipase from Pseudomonas wisconsinensis, mutated in position Val37,
    F lipase from Pseudomonas fluorescens, mutated in position Met17,
    G lipase from Pseudomonas vulgaris, mutated in position Leu16,
    H lipase from Burkholderia cepacia, mutated in position Leu17,
    I lipase from Burkholderia glumae, mutated in position Leu17,
    J lipase from Burkholderia plantarii, mutated in position Leu17,
    K lipase from Chromobacterium viscosum, mutated in position Leu17,
    L lipase from Pseudomonas luteola, mutated in position Leu57,
    M lipase from Pseudomonas spec. DSM 8246, mutated in position Leu17.


Further preferred embodiments are those lipase variants which carry the following substitutions compared with the starting lipase:

    A lipase from Pseudomonas aeruginosa, where Met16 is replaced by Ala16, Thr16 or Phe16,
    B lipase from Vibrio cholerae, where Leu44 is replaced by Ala44, Thr44 or Phe44,
    C lipase from Pseudomonas Fragi, where Leu17 is replaced by Ala17, Thr17 or Phe17,
    D lipase from Acinetobacter calcoaceticus, where Leu49 is replaced by Ala49, Thr49 or Phe49,
    E lipase from Pseudomonas wisconsinensis, where Val37 is replaced by Ala37, Thr37 or Phe37,
    F lipase from Pseudomonas fluorescens, where Met17 is replaced by Ala17, Thr17 or Phe17,
    G lipase from Pseudomonas vulgaris, where Leu16 is replaced by Ala16, Thr16 or Phe16,
    H lipase from Burkholderia cepacia, where Leu17 is replaced by Ala17, Thr17 or Phe17,
    I lipase from Burkholderia glumae, where Leu17 is replaced by Ala17, Thr17 or Phe17,
    J lipase from Burkholderia plantarii, where Leu17 is replaced by Ala17, Thr17 or Phe17,
    K lipase from Chromobacterium viscosum, where Leu17 is replaced by Ala17, Thr17 or Phe17,
    L lipase from Pseudomonas luteola, where Leu57 is replaced by Ala57, Thr57 or Phe57,
    M lipase from Pseudomonas spec. DSM 8246, where Leu17 is replaced by Ala17, Thr17 or Phe17.


Further preferred embodiments are those lipase variants which carry at least one of the following amino acid substitutions compared with the starting lipase from Pseudomonas spec DSM 8246:

    Leu17 replaced by Ala17, Thr17 or Phe17, Met17 
    Tyr29 replaced by Ser29, Thr29, Phe29, Glu29 
    Trp30 replaced by His30, Phe30 
    Phe52 replaced by Ser52, Thr52 or Tyr52, Leu52 
    His86 replaced by Trp86, Thr86, Ser86 
    Ser117 replaced by Ala117, Thr117, Met117 
    Phe122 replaced by Leu122 
    Ala160 replaced by Phe160, Leu160, Ile160 
    Ala163-replaced by Phe163, Leu163, Ile163 
    Leu167 replaced by Ala167, Val167, Ser167, Thr167 
    Leu265 replaced by Ala265, Val267 or Met265, Ser265, Thr265 
    Val266 replaced by Ala266, Leu266, Met266, Ser266, Lys266 
    Leu286 replaced by Ala286, Met286, Val286 or Ile286, Ser286 
    Ile289 replaced by Ala289, Val289 or Leu289.


A particularly preferred embodiment is a lipase variant which carries two of the following amino acid substitutions compared with the starting lipase from Pseudomonas spec DSM 8246:

    
    
        Leu17 replaced by Ala17, Thr17, Phe17, Met17 
        Phe52 replaced by Leu52, Ser52, Thr52 or Tyr52.
    
    


A further subject of the invention are nucleic acid sequences which encode the lipase variants according to the invention described above. Suitable nucleic acid sequences are obtainable by back-translation of the polypeptide sequence according to the genetic code.
Preferably, the codons used for this are those frequently used in accordance with the codon usage specific to the organism. The codon usage can easily be determined with the aid of computer analysis of other, known genes of the organism concerned.
Should the lipase variant be expressed, for example, in a bacterium, it is frequently advantageous to use the codon usage of the bacterium in the back-translation.
A further subject of the invention is nucleic acid constructs which contain the nucleic acid sequences according to the invention. These nucleic acid constructs preferably carry, in addition to the nucleic acid sequences, also regulatory sequences which are advantageous, for example, for expression, replication or recombination.
A further subject of the invention are host organisms which can be transformed using these nucleic acid constructs. Suitable host organisms are unicellular or multicellular organisms, microorganisms being preferred as host organisms.
A further subject of the invention is processes for the preparation of lipase variants by culturing host organisms, which have been transformed using a nucleic acid-sequence coding for the lipase variants, under those conditions which allow the production of the lipase variants in these host organisms. Subsequently, when the concentration of the lipase variant in the culture medium has reached the desired level, the lipase variant can be isolated from the culture medium. Depending on the desired intended use of the lipase, this can also be purified using customary processes of protein chemistry such as precipitation and chromatography. For some intended uses, however, the entire host organism can also be used, if appropriate after prior destruction.
The invention furthermore relates to lipase formulations comprising at least one of the lipase variants according to the invention.
The lipase variants can be used in this formulation in isolated form, immobilized on a solid carrier material or in cells. Such lipase formulations can contain, in addition to the lipase variant, also further stabilizers, detergents and enzyme substrates.
Methods for the immobilization of lipases are described, for example, in WO 00/05354, EP 1069183.
Preferably, the lipase variants are used in immobilized form.
The invention furthermore relates to a process for the enzyme-catalytic conversion or enantioselective conversion of substrates by reacting the substrates in the presence of the lipase according to the invention.
The lipase variants according to the invention can accordingly be used as catalysts in chemical reactions. The lipase variants according to the invention can be employed in a multiplicity of chemical reactions.
The catalytic properties of the lipase variants according to the invention are measured using reference reactions. Thus, good higher specific activities compared with the starting lipases can also be determined:
The determination of the specific activity of a lipase variant is carried out according to the invention in the following reference reaction, in which the lipase or lipase variant catalyzes the reaction of racemic trans-methoxycyclohexanol with vinyl laurate to give lauric acid 1R,2R-2-methoxycyclohexyl ester and 1S,2S-methoxycyclohexanol.
The specific activity is determined in this reference reaction under the following conditions:
The reference reaction is carried out in microtiter plates having 96 cavities. The microtiter plate contains, per cavity

    A the lipase-containing lyophyllisate of 150 μl of culture supernatant of cultures of natural or recombinant organisms which express a lipase or
    B the corresponding, firmly prespecified amount of isolated lipase.


For separation of the racemates, racemic trans-methoxycyclohexanol and vinyl laurate are employed in the molar ratio 1:0.65 [i.e. per plate 10 g of methoxycyclohexanol and 11.3 g of vinyl laurate]. 200 μl of this mixture are pipetted into each well.
Each plate is sealed using a ‘plate sealer’, closed with a lid and incubated for 96 h at room temperature in a revolving shaker at 150 rpm. 100 μl per cavity in each case are then removed, treated with ethyl acetate and analyzed by gas chromatography.
The amount of methoxycyclohexyl laurate formed is measured relative to the amount of the lipase employed (case B) or relative to the optical density of the cell suspensions in the cavities of the microtiter plates (case A).
In case A, it must be guaranteed that all further measurement parameters and other parameters, such as, for example, induced and constitutive expression, are identical in the organisms which express the respective lipase.
A larger amount of methoxycyclohexyl laurate measured according to this method per amount of lipase means a higher specific activity.
Enzyme-catalytic conversions are understood as meaning chemical reactions of substrates which lipases can catalyze. The following reactions may be mentioned by way of example:

    acylation or enantioselective acylation of alcohols,
    acylation or enantioselective acylation of amines,
    acylation or enantioselective acylation of amino esters, such as, for example, amino acid esters,
    hydrolysis or enantioselective hydrolysis of carboxylic acid esters,
    acylation or enantioselective acylation of cyanohydrins,
    hydrolysis or enantioselective hydrolysis of cyanohydrin esters,
    asymmetrisization of meso-diols or
    asymmetrisization of meso-diesters by hydrolysis.


Preferred processes are processes for the

    acylation or enantioselective acylation of alcohols,
    acylation or enantioselective acylation of amines,
    acylation or enantioselective acylation of amino esters, such as, for example, amino acid esters or a process for the hydrolysis or enantioselective hydrolysis of carboxylic acid esters.


The process for the enzyme-catalytic conversion or enantioselective conversion of substrates comprises reacting the substrates in the presence of the lipase variants according to the invention.
Depending on whether the type of reaction necessitates it, further reagents are preferably added here. Thus, an acylation, for example, necessitates the addition of an acylating agent, while hydrolysis, for example, needs no further addition of reagents.
Substrate is understood as meaning a chemical compound which can be reacted by enzyme catalysis, i.e. chemically modified, by lipases. In enantioselective conversions, stereoisomer mixtures of which only one stereoisomer is reacted are likewise substrates.
By way of example, alcohols, amines, amino esters, amides, carboxylic acid esters, thioesters, thiols, cyanohydrins, cyanohydrin esters and meso-diols and their stereoisomer mixtures may be mentioned as substrates. Preferred substrates are alcohols, amines, amino esters and carboxylic acid esters or racemic alcohols, amines, amino esters and carboxylic acid esters.
The process is preferably carried out in solution, in the case of liquid substrates with or without solvent. Solvents which can be used are, for example, water, organic solvents or alternatively aqueous/organic two-phase mixtures.
Preferably, the organic solvents used are dioxane, THF, diethyl ether, methyl t-butyl ether (MTBE), toluene or heptane. The aqueous/organic two-phase mixture employed is preferably a water/MTBE mixture in any desired ratio.
When carrying out the process in solution, the substrate concentration is not critical, but is preferably between 0.5% by weight and 50% by weight based on the solution; 20 to 30% by weight is particularly preferred. The temperature when carrying out the process is likewise not critical, but is restricted upwardly by the temperature stability of the enzyme. Preferably, the process is carried out at 0° C. to 60° C.; 15° C. to 40° C. is particularly preferred.
The process can be carried out continuously or batchwise. For carrying out the process continuously, a liquid mobile phase is passed in a manner known per se, for example, into a reactor through a packed bed of immobilized lipase variant. The mobile phase can be either a solution of substrate (and reagents) or the liquid substrate (and reagents) without solvent. The flow rate is not critical and depends on process technology viewpoints such as the height, diameter and particle size of the packed bed, and on the design of the reactor.
Reactors used for the continuous process are preferably the reactors customary for continuous, heterogeneous catalytic processes (fluid/solid reactions) (J. Hagen, Chemische Reaktionstechnik, VCH, Weinheim 1992, pp. 165-169). By way of example, fluidized bed reactors and fixed bed reactors, such as a tubular reactor, column reactor, chamber reactor, shelf reactor, tube bundle reactor and catalytic furnace, may be mentioned.
In the batchwise process procedure, the immobilized lipase variants are suspended in a solution of substrate (and reagents) or in liquid substrates (and reagents) with or without solvent in a reactor in a manner known per se and the suspension is thoroughly mixed. Reactors used for the batchwise process are preferably the reactors having a shaking, mixing or stirring device customary for batchwise, heterogeneous catalytic processes (fluid/solid reactions). By way of example, the stirred vessel and designs derived therefrom and also reaction vessels having a shaking device may be mentioned.
After completion of the reaction (achievement of the thermodynamic equilibrium), the immobilized lipase variant is isolated, for example by decanting, centrifuging off or filtering off and washing, and used in further reactions.
In a preferred embodiment of the process, substrates which contain acylatable functional groups such as, for example, hydroxyl or amino groups, such as alcohols, amines or amino acid esters, are acylated or enantioselectively acylated in the presence of the immobilized lipase as catalyst and an acylating agent.
This enzyme-catalytic conversion is preferably carried out in an organic solvent such as, for example, dioxane, THF, diethyl ether, methyl t-butyl ether (MTBE), toluene or heptane.
A process for the acylation or enantioselective acylation of alcohols, amines or amino acid esters or racemic alcohols, amines or amino acid esters in the presence of an acylating agent and the lipase variant of SEQ. ID. NO.1 is particularly preferred.
There is virtually no restriction with respect to the alcohols, amines and amino acid esters. Thus it is possible to use mono- and polyhydric alcohols, such as, for example

    1-phenylethanol,
    optionally substituted 2-chloro-1-phenylethanol,
    pent-3-yn-2-ol,
    1-butyn-3-ol,
    2-hydroxy-4-phenylbutyric acid esters,
    a-methyl-(1,3)-benzodioxole-5-ethanol,
    2-propanol-1-(1,3-benzodioxol-4-yl),
    trans-2-methoxycyclohexanol,
    2-methoxy-2-phenylethanol,
    cis-2-methyl-4-hydroxypyran or
    trans-2-methyl-4-hydroxypyran
    or their stereoisomer mixtures,
    mono- and polyhydric amines or their stereoisomer mixtures or
    α, β or γ-amino acid esters such as, for example, the optionally halogen-substituted C1-C4-alkyl, alkylaryl, aryl, C2-C6-alkenyl or C2-C6-alkynyl esters of the natural amino acids or their stereoisomer mixtures.


Optionally substituted 2-chloro-1-phenylethanol is understood as meaning in particular 2-chloro-1-phenylethanol and substituted 2-chloro-1-phenylethanol. Preferred substituted 2-chloro-1-phenylethanols are alcohols of the formula III




where

    n is 1 to 3 and
    R, independently of one another, are halogen, in particular F or Cl, C1-C4-alkoxy, in particular methoxy, and NO2.


A particularly preferred substituted 2-chloro-1-phenylethanol is 2-chloro-1-(m-chlorophenyl)ethanol.
The substrate preferably used is racemic trans-2-methoxycyclohexanol.
Acylating agents are understood as meaning organic compounds which can function as acyl transfer agents in the presence of lipases in solution. The following may be mentioned by way of example:

    aliphatic, araliphatic or aromatic carboxylic acids which are optionally substituted by halogen, such as Cl, Br, I, F (acylation), such as
    C1-C6-alkanecarboxylic acids, e.g. formic acid, acetic acid, propionic acid, butyric acid or
    such as araliphatic or aromatic carboxylic acids, e.g. benzoic acid, 3-phenylpropionic acid or
    the corresponding carboxylic acid esters (transesterification), such as, for example,
    3-phenylpropionic acid esters or alkyl acetates, such as, for example, ethyl acetate.


Preferred carboxylic acid esters as acylating agents are vinyl esters of the formula I




in which

    R1 is hydrogen or a C1-C4-alkyl group, preferably a methyl group and
    R2 is hydrogen, C1-C18-alkyl optionally substituted by halogen, phenyl or (C1-C3-)alkoxy-(C1-C4)-alkyl.


Preferred vinyl esters of the formula I are vinyl formate, vinyl acetate, vinyl propionate, vinyl butyrate and vinyl laurate.
Acylating agents are furthermore aliphatic, cycloaliphatic, araliphatic or aromatic carboxylic anhydrides and mixed carboxylic anhydrides (acylation) such as acetic anhydride, succinic anhydride (SA), butyric anhydride, 2-ethylhexanoic anhydride or methylsuccinic anhydride. In the case of the use of succinic anhydride (SA) or other poorly soluble anhydrides as acylating agents, propylene carbonate can be particularly advantageously admixed in order to bring the SA into solution. This is especially of importance with respect to a continuous process.
A particularly preferred acylating agent is vinyl laurate.
In a further preferred embodiment of the process, the carboxylic acid esters are hydrolyzed or enantioselectively hydrolyzed in the presence of the lipase variants or lipase formulations according to the invention.
In this case, no further reagents have to be added; nevertheless, the presence of water is necessary. Preferably, the hydrolysis of carboxylic acid esters is carried out by addition of water using a preferably two-phase system such as, for example, water/MTBE in the presence of the lipase variants or lipase formulations according to the invention.
There is virtually no restriction with respect to the carboxylic acid esters. Thus it is possible, for example, compounds of the formula II or their stereoisomer mixtures




where

    R3, R4 and R5 independently of one another are hydrogen,
    halogen, such as, for example, F, Cl, Br or I,
    a branched or unbranched, optionally substituted
    C1-C8-alkyl radical, such as, for example, optionally substituted methyl, ethyl, propyl, 1-methylethyl, butyl, 1-methylpropyl, 2-methylpropyl, 1,1-dimethylethyl, pentyl, 1-methylbutyl, 2-methylbutyl, 1,2-dimethylpropyl, 1,1-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, hexyl, 1-methylpentyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl, 1-ethylbutyl, 2-ethylbutyl, 1-ethyl-2-methylpropyl, heptyl or octyl,
    C6-alkenyl radical, such as, for example, optionally substituted 2-propenyl, 2-butenyl, 3-butenyl, 1-methyl-2-propenyl, 2-methyl-2-propenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-methyl-2-butenyl, 2-methyl-2-butenyl, 3-methyl-2-butenyl, 1-methyl-3-butenyl, 2-methyl-3-butenyl, 3-methyl-3-butenyl, 1,1-dimethyl-2-propenyl, 1,2-dimethyl-2-propenyl, 1-ethyl-2-propenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-methyl-2-pentenyl, 2-methyl-2-pentenyl, 3-methyl-2-pentenyl, 4-methyl-2-pentenyl, 3-methyl-3-pentenyl, 4-methyl-3-pentenyl, 1-methyl-4-pentenyl, 2-methyl-4-pentenyl, 3-methyl-4-pentenyl, 4-methyl-4-pentenyl, 1,1-dimethyl-2-butenyl, 1,1-dimethyl-3-butenyl, 1,2-dimethyl-2-butenyl, 1,2-dimethyl-3-butenyl, 1,3-dimethyl-2-butenyl, 1,3-dimethyl-3-butenyl, 2,2-dimethyl-3-butenyl, 2,3-dimethyl-2-butenyl, 2,3-dimethyl-3-butenyl, 1-ethyl-2-butenyl, 1-ethyl-3-butenyl, 2-ethyl-2-butenyl, 2-ethyl-3-butenyl, 1,1,2-trimethyl-2-propenyl, 1-ethyl-1-methyl-2-propenyl or 1-ethyl-2-methyl-2-propenyl,
    C3-C6-alkynyl radical, such as, for example, optionally substituted 2-propynyl, 2-butynyl, 3-butynyl, 1-methyl-2-propynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-methyl-3-butynyl, 2-methyl-3-butynyl, 1-methyl-2-butynyl, 1,1-dimethyl-2-propynyl, 1-ethyl-2-propynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl, 1-methyl-2-pentynyl, 1-methyl-2-pentynyl, 1-methyl-3-pentynyl, 1-methyl-4-pentynyl, 2-methyl-3-pentynyl, 2-methyl-4-pentynyl, 3-methyl-4-pentynyl, 4-methyl-2-pentynyl, 1,1-dimethyl-2-butynyl, 1,1-dimethyl-3-butynyl, 1,2-dimethyl-3-butynyl, 2,2-dimethyl-3-butynyl, 1-ethyl-2-butynyl, 1-ethyl-3-butynyl, 2-ethyl-3-butynyl or 1-ethyl-1-methyl-2-propynyl
    or C3-C8-cycloalkyl radical, such as, for example, optionally substituted cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl, cyclooctyl,
    an optionally substituted
    aryl radical, such as, for example, optionally substituted phenyl, 1-naphthyl or 2-naphthyl,
    arylalkyl radical, such as, for example, optionally substituted benzyl,
    heteroaryl radical, such as, for example, optionally substituted 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-furyl, 3-furyl, 2-pyrrolyl, 3-pyrrolyl, 2-thienyl, 3-thienyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl, 2-pyrimidyl, 4-pyrimidyl, 5-pyrimidyl, 6-pyrimidyl, 3-pyrazolyl, 4-pyrazolyl, 5-pyrazolyl, 3-isothiazolyl, 4-isothiazolyl, 5-isothiazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl, 3-pyridazinyl, 4-pyridazinyl, 5-pyridazinyl or 6-pyridazinyl, preferably 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-thiazolyl, 4-thiazolyl or 5-thiazolyl,
    or heterocycloalkyl or -alkenyl radical.


Possible mono to triple substituents of the C1-C8-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C3-C8-cycloalkyl, aryl, arylalkyl, heteroaryl, heterocycloalkyl or -alkenyl radicals are, for example, halogen, nitro, amino, hydroxyl or cyano groups, C1-C4-alkyl, C1-C4-haloalkyl, C1-C4-alkoxy, C1-C4-haloalkoxy, C1-C4-alkylthio, hetaryl, aryl radicals or the radical —O—CO—C1-C4-alkyl.
Preferred carboxylic acid esters are, for example,

    1-butyn-3-ol acetate,
    1-butyn-3-ol butyrate,
    1-phenylethyl acetate or
    2-acetoxy-4-phenylbutyric acid esters.


The process for the enantioselective enzyme-catalytic conversion of substrates using the lipase variants or lipase formulations according to the invention can be used for the separation of stereoisomers and in particular for the separation of enantiomers or diastereomers from a stereoisomer mixture of the substrate. Particularly preferably, it is used for the separation of enantiomers or diastereomers from racemic substrates and thus for the preparation of optically active compounds from the respective racemic mixtures.
Owing to the enantioselective substrate specificity of the lipase variants or lipase formulations according to the invention, only one enantiomer, for example, of the racemic substrate is converted; the other enantiomer does not react. The resulting products can be easily separated in a manner known per se by chemical, physical and mechanical separation methods.
Crystallization, precipitation, extraction in two-phase solvent systems, chromatographic separation processes, such as HPLC, GC or column chromatography on silica gel or thermal separation processes such as distillation may be mentioned by way of example.
Accordingly, the present invention furthermore relates to a process for the preparation of optically active compounds, which comprises reacting stereoisomer mixtures or racemates of substrates, which can be reacted by means of enzyme catalysis by lipases, enantioselectively in the presence of the lipase variants according to the invention and then resolving the mixtures.
Using the process according to the invention, preferably the optically active compounds can be prepared which can be reacted as a stereoisomer mixture as substrates of lipases, or at least one stereoisomer of their stereoisomer mixture can be reacted as a substrate of lipases.
A process for the enantioselective acylation of alcohols, amines or amino acid esters is preferably used for the separation of racemic alcohols, amines or amino acid esters and thus for the preparation of optically active alcohols, amines or amino acid esters.
A process for the enantioselective hydrolysis of carboxylic acid esters is preferably used for the separation of racemic carboxylic acid esters and thus for the preparation of optically active carboxylic acid esters.
The lipase variants or lipase formulations according to the invention have the advantage that they have an increased specific activity together with constant enantioselectivity.
The examples below illustrate the invention:
General Experimental Conditions
In all cases in which no detailed description of the experiments takes place, molecular biology methods were used which are known and are described, for example, in Current Protocols of Molecular Biology, 1999, John Wiley & Sons.
Bacterial strains used:
Escherichia coli XL1-Blue (Stratagene)

    Escherichia coli XJS5037 (pRK2013) (Proc. Natl. Acad. Sci., 1979, 1648-1652) Pseudomonas spec. DSM 8246


Plasmids used:

    pBluescriptIIKS (Stratagene),
    pBP1500: Derivative of pML131 (Gene, 1990, 89:37-46) with a 2.6 kBp DNA fragment which comprises the lipase operon from Pseudomonas spec. DSM 8246. pBP1500 codes for an enzymatically inactive lipase (lipA−),
    pBP1520: Derivative of pML131 (Gene, 1990, 89:37-46) with a 2.6 kBp DNA fragment which comprises the lipase operon from Pseudomonas spec. DSM 8246. pBP1520 codes for an enzymatically active lipase (lipA),
    pBP2112: Derivative of pML131 (Gene, 1990, 89:37-46) with a 2.6 kBp DNA fragment which-comprises the lipase operon from Pseudomonas spec. DSM 8246 and which comprises the mutation for the production of the lipase variant LipA L17A.


EXAMPLE 1
Preparation of a Lipase Variant Having Increased Specific Enzyme Activity
A) Culturing of the Bacteria
The recombinant strains of Escherichia coli were cultured either on solid LB agar or in LB medium at 37° C. in a rotary shaker at 200 rpm for 16 h. The medium contained the antibiotics necessary for election Pseudomonas spec. DSM 8246 and the recombinant derivatives of this strain were either incubated on solid FG agar or in FP medium at 30° C. in a rotary shaker at 200 rpm for 24 h or in (NH4)2SO4 6 g/l, CaCl2 0.02 g/l, MgSO4×7H2I 1 g/l, KH2PO4 3.5 g/l, K2HPO4 3.5 g/l, yeast extract 5 g/l, glucose 5 g/l, trace salt solution 10 ml/l at 30° C. in a rotary shaker at 150 rpm for 24 h. The medium contained the antibiotics necessary for selection. As incubation vessels, petri dishes, culture tubes or microtiter plates were used.
The following antibiotics (selection) were used: tetracycline 10 μl/ml (XL1-Blue), ampicillin 100 μg/ml (pBluescriptIIKS), gentamicin 10 μg/ml (pBP plasmids in E. coli), gentamicin 45 μg/ml (pBP plasmids in Pseudomonas spec. DSM 8246), chloramphenicol 20 μg/ml (Pseudomonas spec. DSM 8246), kanamycin 25 μg/ml (Escherichia coli XJS5037 (pRK2013) or Pseudomonas spec. DSM 8246)
B) Mutagenesis
The mutant L17A of lipA from Pseudomonas spec. DSM 8246 was produced by site-directed mutagenesis using the overlap extension PCR method (Recombinant PCR, 1990, 177-183, Academic Press, San Diego).
Chromosomal DNA from Pseudomanas spec. DSM 8246 was used as matrix DNA. The primers employed were:








(SEQ ID NO: 2)










MAT16
5′-GATCGACGTAAGCTTTAACGATGGAGAT-3′



 








(SEQ ID NO: 3)










MAT109
5′-CGGTGCCCGCGGCGCCGTGGACGAGGATCACCG-3′



 








(SEQ ID NO: 4)










MAT108
5′-CGTCCACGGCGCCGCGGGCACCGACAAGTTCGC-3′



 








(SEQ ID NO: 5)










MAT19
5′-CATCGGGCGAGCTCCCAGCCCGCCGCG-3′







In the first PCR step, a subfragment of lipA was produced using MAT16 and MAT109 and a further subfragment of lipA was produced using MAT108 and MAT19. The subfragments were separated in an agarose gel and purified using the GFX kit (Pharmacia). The fragment lipA* which codes for the lipase variant L17A was produced from the purified fragments in the second PCR step using MAT16 and MAT19.
The PCR was carried out according to the procedure of the manufacturer using the GC-rich kit (Pharmacia) with the following temperature profile: (1) 95° C.-3 min, (2) 95° C.-30 s, (3) 40° C.-30 s, (4) 72° C.-30 s, (5) 95° C.-30 s, (6) 60° C.-30 s, (7) 72° C. 30 s, (8) 72° C.-10 min; steps (2)-(4) with 5 cycles; then steps (5)-(7) 15 cycles.
The fragment lipA* and the vector pBluescriptIIKS were cleaved using HindIII and SacI, separated in an agarose gel, purified using the GFX kit and ligated. The batch was then transformed into E. coli. The recombinant plasmid was purified and sequenced in order to detect the mutation. Except for the mutation L17A, no further mutations were detected in the DNA.
C) Cloning
The vector pBP1500 and the recombinant pBluescript derivative with lipA* was cleaved using HindIII and SacI and ligated. The resultant recombinant plasmid was designated pBP2112. pBP2112 was transformed into E. coli XL1-Blue. pBP2112 was then transferred to Pseudomonas spec. DSM 8246 by conjugation of E. coli XL1-Blue with the aid of the helper strain E. coli XJS5037 (pRK2013). The recombinant strain was designated Pseudomonas spec. DSM 8246 (pBP2112).
D) Enzyme Production and Work-up
Pseudomonas spec. DSM 8246 (pBP2112) was incubated at 30° C. for 24 h in 96-cavity microtiter plates each containing 250 μl of medium per cavity on a rotary shaker at 150 rpm. As a control, Pseudomonas spec. DSM 8246 (pBP1520) was cultured. This strain carries the lipA gene of Pseudomonas spec. DSH 8246 in plasmid-coded form (comparison example). The following medium was employed for the culture of the bacteria: (NH4)2SO4 6 g/l, CaCl2 0.02 g/l, MgSO4×7H2O 1 g/l, KH2PO4 3.5 g/l, K2HPO4 3.5 g/l, yeast extract 5 g/l, glucose 5 g/l, trace salt solution 10 ml/l, kanamycin 25 μl/ml, gentamicin 45 μg/ml. After the culture, the microtiter plates were centrifuged. The culture supernatants were then transferred to a polypropylene microtiter plate, frozen at −80° C. for 16 h and then freeze-dried for 48 h.
The chromosomal copy of the lipase gene is not expressed under the culturing conditions. The comparison example therefore also carries the natural lipase in plasmid-coded form.
E) Resolution and Analysis
The resolution of rac-trans-2-methoxycyclohexanol was carried out in the microtiter plates containing the freeze-dried supernatants. For the resolution, methoxycyclohexanol and vinyl laurate were employed in a molar ratio of 1:0.65 [i.e. 10 g of methoxycyclohexanol and 11.3 g of vinyl laurate per plate]. 200 μl of this mixture were pipetted into each cavity. Each plate was sealed using a ‘plate sealer’, closed with a cover and incubated for 96 h at room temperature in a rotary shaker at 150 rpm.
100 ml per cavity were then removed, treated with ethyl acetate and analyzed by gas chromatography.
Table 1 contains the measured amounts of methoxycyclohexanyl laurate formed in % by weight.









TABLE 1





Example 1E




Amount of methoxy-
Comparison example


cyclohexanyl laurate
Amount of methoxy-


formed in [% by
cyclohexanyl laurate


weight] using the
formed in [% by


lipase variant LipA
weight] using the


L17A from
lipase from


Pseudomonas spec.
Pseudomonas spec.


DSM 8246 (pBP2112)
DSM 8246 (pBP1520)
Media blank







14.9
2.6
0.02


18.6
1.0
0.02


21.2
2.5
0.02


18.1
2.7
0.02


16.8
2.2
0.03


14.0
1.7
0.02


15.1
1.8
0.03


10.3
0.8
0.02


Mean = 16.1
Mean = 1.9
Mean = 0.02









In comparison with the natural enzyme, the lipase variant LipA L17A showed an activity which was higher by the factor 8.5 in the resolution of methoxycyclohexanol.
The specific enzyme activity is in this case defined as the amount of methoxycyclohexanyl laurate formed in [% by weight] divided by optical density of the bacterial suspension at OD660 nm. The amount of active enzyme formed correlates in a range of an optical density of OD660 nm≈0.0 to 0.7+/−0.2 linearly with the turbidity of the bacterial suspension.
The following values resulted here for the specific activity:









TABLE 2





Specific activity of
Comparison example:



the lipase variant
Specific activity of


LipA L17A from
the lipase from


Pseudomonas spec.
Pseudomonas spec.


DSM 8246 (pBP2112)
DSM 8246 (pBP1520)


in the reaction
in the reaction


according to example
according to example


1E [in % by weight]
1E [in % by weight]
Media blank







16.4
3.1
0.5


20.2
1.2
0.5


22.1
3.0
0.5


20.3
3.4
0.5


19.1
2.8
0.8


14.4
2.2
0.5


17.4
2.4
0.8


10.8
1.0
0.5


Mean = 17.6
Mean = 2.4
Mean = 0.6









In comparison with the natural enzyme, the lipase variant LipA L17A showed a specific enzyme activity which was higher by the factor 7.3 in the resolution of methoxycyclohexanol.

## Claims

1. A lipase variant comprising an amino acid sequence of a starting lipase that has been substituted, wherein the starting lipase is selected from the group consisting of lipases of homologous families I.1 and I.2, wherein the substitution is at least one amino acid substitution at a position that corresponds to position 17, 29, 30, 52, 86, 117, 122, 160, 163, 167, 265, 266, 286, or 289 of SEQ ID NO: 1, and wherein the variant has lipase activity.

2. The lipase variant of claim 1, wherein the starting lipase is a lipase from an organism selected from the group of organisms consisting of Pseudomonas aeruginosa, Vibrio cholerae, Pseudomonas fragi, Acinetobacter calcoaceticus, Pseudomonas wisconsinensis, Pseudomonas fluorescens, Pseudomonas vulgaris, Burkholderia cepacia, Burkholderia glumae, Burkholderia plantarii, Chromobacterium viscosum, Pseudomonas luteola, and Pseudomonas spec. DSM 8246.

3. The lipase variant of claim 1 selected from the group consisting of:
a lipase from Pseudomonas aeruginosa, with a substitution at position Met16;
a lipase from Vibrio cholerae, with a substitution at position Leu44;
a lipase from Pseudomonas Fragi, with a substitution at position Leu7;
a lipase from Acinetobacter calcoaceticus, with a substitution at position Leu49;
a lipase from Pseudomonas wisconsinensis, with a substitution at position Val37;
a lipase from Pseudomonas fluorescens, with a substitution at position Met17;
a lipase from Pseudomonas vulgaris, with a substitution at position Leu16;
a lipase from Burkholderia cepacia, with a substitution at position Leu17;
a lipase from Burkholderia glumae, with a substitution at position Leu17;
a lipase from Burkholderia plantarii, with a substitution at position Leu17;
a lipase from Chromobacterium viscosum, with a substitution at position Leu17;
a lipase from Pseudomonas luteola, with a substitution at position Leu57; and
a lipase from Pseudomonas spec. DSM 8246, with a substitution at position Leu17.

4. The lipase variant of claim 1 selected from the group consisting of:
a lipase from Pseudomonas aeruginosa, wherein Met16 is substituted with Ala16, Thr16 or Phe16;
a lipase from Vibrio cholerae, wherein Leu44 is substituted with Ala44, Thr44 or Phe44;
a lipase from Pseudomonas Fragi, wherein Leu17 is substituted with Ala17, Thr17 or Phe17;
a lipase from Acinetobacter calcoaceticus, wherein Leu49 is substituted with Ala49, Thr49 or Phe49;
a lipase from Pseudomonas wisconsinensis, wherein Val37 is substituted with Ala37, Thr37 or Phe37;
a lipase from Pseudomonas fluorescens, wherein Met17 is substituted with Ala17, Thr17 or Phe17;
a lipase from Pseudomonas vulgaris, wherein Leu16 is substituted with Ala16, Thr16 or Phe16;
a lipase from Burkholderia cepacia, wherein Leu17 is substituted with Ala17, Thr17 or Phe17;
a lipase from Burkholderia glumae, wherein Leu17 is substituted with Ala17, Thr17 or Phe17;
a lipase from Burkholderia plantarii, wherein Leu17 is substituted with Ala17, Thr17 or Phe17;
a lipase from Chromobacterium viscosum, wherein Leu17 is substituted with Ala17, Thr17 or Phe17;
a lipase from Pseudomonas luteola, wherein Leu57 is substituted with Ala57, Thr57 or Phe57;
a lipase from Pseudomonas spec. DSM8246, wherein Leu17 is substituted with Ala17, Thr17 or Phe17.

5. The lipase variant of claim 1, wherein the at least one amino acid substitution comprises two substitutions that correspond to the positions 17 and 52 of SEQ ID NO: 1.

6. The lipase variant of claim 1, wherein the starting lipase is from Pseudomonas spec. DSM 8246 and the at least one substitution is selected from the group of substitutions consisting of:
Leu17 replaced by Ala17, Thr17, Phe17, or Met17;
Tyr29 replaced by Ser29, Thr29, Phe29, or Glu29;
Trp30 replaced by His30 or Phe30;
Phe52 replaced by Ser52, Thr52, Tyr52 or Leu52;
His86 replaced by Trp86, Thr86 or Ser86;
Ser117 replaced by Ala117, Thr117 or Met117;
Phe122 replaced by Leu122;
Ala160 replaced by Phe160, Leu160 or Ile160;
Ala163 replaced by Phe163, Leu163 or Ile163;
Leu167 replaced by Ala167, Val167, Ser167 or Thr167;
Leu265 replaced by Ala265, Val267, Met265, Ser265 or Thr265;
Val266 replaced by Ala266, Leu266, Met266, Ser266 or Lys266;
Leu286 replaced by Ala286, Met286, Val286, Ile286 or Ser286; and
Ile289 replaced by Ala289, Val289 or Leu289.

7. A nucleic acid sequence that encodes the lipase variant of claim 1.

8. A nucleic acid construct comprising the nucleic acid sequence of claim 7.

9. An isolated host cell transformed with the nucleic acid construct of claim 8.

10. A process for production of lipase comprising culturing an organism that expresses the lipase variant of claim 1 in a culture medium.

11. A lipase formulation comprising the lipase variant of claim 1 and a stabilizer, detergent, enzyme substrate, or a combination thereof.

12. A lipase formulation comprising the lipase variant of claim 1 that is immobilized on a solid carrier material or is in cells.

13. A process for enzyme-catalytic conversion or enantioselective conversion of substrates, which comprises reacting the substrates in the presence of the lipase variant of claim 1.

14. The process of claim 13, wherein the substrates comprise alcohols, or amine or amino acid esters that are acylated in the presence of an acylating agent.

15. The process of claim 13, wherein the substrates comprise carboxylic acid esters that are hydrolyzed.

16. A process for preparation of optically active compounds, which comprises reacting stereo isomer mixtures or racemates of substrates in an enzyme-catalyzed manner enantioselectively in the presence of the lipase variant of claim 1; and then resolving the mixtures.

17. The process of claim 16, wherein the substrates comprise alcohols, amines or amino acid esters that are enantioselectively acylated in the presence of an acylating agent.

18. The process of claim 10, further comprising recovering said lipase variant from the culture medium.

19. A lipase variant consisting of an amino acid sequence of a starting lipase that has been substituted, wherein the starting lipase is selected from the group consisting of lipases of homologous families I.1 and I.2, wherein the substitution is at least one amino acid substitution at a position that corresponds to position 17, 29, 30, 52, 86, 117, 122, 160, 163, 167, 265, 266, 286, or 289 of SEQ ID NO: 1, and wherein the variant has lipase activity.