WO2012156151A1

WO2012156151A1 - METHOD FOR ω-AMINOCARBOXYLIC ACID CONDENSATION

Info

Publication number: WO2012156151A1
Application number: PCT/EP2012/056251
Authority: WO
Inventors: Andreas Schmid; Bruno BÜHLER; Nadine LADKAU; Inna HERMANN
Original assignee: Evonik Degussa GmbH
Current assignee: Evonik Operations GmbH
Priority date: 2011-05-16
Filing date: 2012-04-05
Publication date: 2012-11-22
Anticipated expiration: 2013-11-16

Abstract

A method for converting an ω-aminocarboxylic acid or ester thereof into the corresponding lactam, comprising the steps (a) providing the ω-aminocarboxylic acid or ester thereof and (b) contacting the ω-aminocarboxylic acid or ester thereof with an ω-lactam hydrolase in an aqueous solution.

Description

Method for ω-aminocarboxylic acid condensation

The present invention relates to a method for converting an ω-aminocarboxylic acid or ester thereof into the corresponding lactam, comprising the steps (a) providing the o-aminocarboxylic acid or ester thereof and (b) contacting the ω-aminocarboxylic acid or ester thereof with an o- lactam hydrolase in an aqueous solution, and a use of an o-lactam hydrolase for converting an o-aminocarboxylic acid or ester thereof into a lactam. Polyamides are polymers comprising repeating amide groups. The term "polyamides" usually designates synthetic, commercially available thermoplastics and is distinct from the chemically related proteins. Polyamides are derived from primary amines comprising the functional group -CO-NH- or secondary amines (-CO-NR-, wherein R is an organic residue). However, derivatives, more specifically aminocarboxylic acids, lactams and diamines, may also be used to make polyamides.

Many commercially sought-after polyamides are produced starting from lactams. For example, "polyamide 6" is often obtained by way of polymerisation of ε-caprolactam, and "polyamide 12" may be made by polymerising laurolactam. Other commercially relevant products include copolymers of lactams, for instance copolymers of ε-caprolactam and laurolactam.

Laurinlactam is conventionally synthesized using an inefficient multi-step-procedure, which starts with the trimerisation of butadiene. The trimerisation product cyclododecatriene is subsequently hydrogenated and the resulting cyclododecane is oxidized to yield cyclododecanone. The latter is then reacted with hydroxylamine to cyclododecane oxime, which is finally, in a reaction involving a Beckmann rearrangement, converted to laurolactam.

The shortcomings of such methods include the accumulation of large quantities of salts, in particular sodium sulphate, which have to be disposed of. Some of the steps have yields of no more than 20 %, making the overall method rather inefficient. Finally, some of the reactants are obtained by cracking fossil materials rather than renewable raw materials, which is undesirable in terms of sustainability. PCT/EP 2008/067447 describes a biological system that may be used to produce chemically related products, more specifically ω-aminocarboxylic acids, more specifically a cell that comprises a range of enzymatic activities and so is capable of converting carboxylic acids to the corresponding ω-aminocarboxylic acids. Nonwithstanding the advantages of the system, it may not, as such, be used to produce the corresponding lactams, as it lacks an enzymatic activity capable of catalysing the condensation of the ω-aminocarboxylic acid produced, and the final condensation has to be accomplished by other means. Therefore, the problem underlying the present invention is to provide a method that may be used to catalyse the conversion of an aminocarboxylic acid or ester thereof to the corresponding lactam and is acceptable in terms of sustainability, /^'. e. independent from reactants derived from fossil fuels. Another problem underlying the present invention is to provide a biological system capable of carrying out the complete synthesis of lactams starting from the respective carboxylic acid or ester thereof. Another problem underlying the present invention is to provide a reaction system that allows for the removal of the lactam from an aqueous phase upon condensation of the corresponding carboxylic acid or ester thereof.

The problem underlying the present invention is solved by the subject matter of the attached independent and dependent claims.

In a first aspect, the problem underlying the present invention is solved by a method for converting an ω-aminocarboxylic acid or ester thereof to the corresponding lactam, comprising the steps

(a) providing the ω-aminocarboxylic acid or ester thereof and

(b) contacting the ω-aminocarboxylic acid or ester thereof with an o-lactam hydrolase in an aqueous solution.

In an embodiment of the first aspect of the present invention, the aqueous solution in step b) is, initially, essentially free of the corresponding lactam. In an embodiment of the first aspect of the present invention, the ratio of the o-aminocarboxylic acid or ester thereof, respectively, to the corresponding lactam is initially at least 20 : 1 , preferably 50 : 1. In an embodiment of the first aspect of the present invention, the method further comprises step

(c) contacting the aqueous solution with an organic solution comprising at least one organic solvent. In an embodiment of the first aspect of the present invention, the at least one organic solvent is selected from the group of alkyi benzenes, wherein the alkyi substituent has one to four carbon atoms, preferably one carbon atom.

In an embodiment of the first aspect of the present invention, the at least one organic solvent is selected from the group comprising fatty acids and fatty acid esters.

In an embodiment of the first aspect of the present invention, the ω-lactam hydrolase is an ω- laurolactam hydrolase. In an embodiment of the first aspect of the present invention, the ω-lactam hydrolase is selected from the group comprising the laurolactam hydrolases from Acidovorax sp. T31 , Rhodococcus sp. U224, Cupriavidus sp T7, Sphingomonas sp. U298 and Cupriavidus sp. U124 and homologues thereof. In an embodiment of the first aspect of the present invention, the ω-aminocarboxylic acid or ω- aminocarboxylic acid ester is selected from the group of compounds represented by formula (I)

wherein x is 6 to 20, preferably 9 to 15, and wherein R³ is selected from the group comprising H and alkyi, which alkyi comprises 1 to 4 carbon atoms.

In an embodiment of the first aspect of the present invention, the ω-aminocarboxylic acid or ester thereof is 12-aminolaureate or 12-aminolaureate ester, respectively, and is preferably 12- aminolaureate methyl ester. In an embodiment of the first aspect of the present invention, the provision of the ω- aminocarboxylic acid in step (a) is accomplished by way of an in situ reaction. In an embodiment of the first aspect of the present invention, the ω-lactam hydrolase is associated with a viable cell.

In an embodiment of the first aspect of the present invention, the ω-lactam hydrolase is a preparation of a cell expressing said ω-lactam hydrolase.

In a second aspect, the problem underlying the present invention is solved by a use of an ω- lactam hydrolase for converting an ω-aminocarboxylic acid or ester thereof into a lactam.

In an embodiment of the second aspect of the present invention, the ω-lactam hydrolase is selected from the group comprising the ω-lactam hydrolases from Acidovorax sp. T31 , Rhodococcus sp. U224, Cupriavidus sp T7, Sphingomonas sp. U298 and Cupriavidus sp. U124 and homologues thereof.

In a third aspect, the problem underlying the present invention is solved by a recombinant cell comprising an enzyme capable of catalysing the conversion of carboxylic acids or esters thereof to the corresponding ω-hydroxycarboxylic acids or esters thereof, an enzyme capable of catalysing the conversion of the corresponding ω-hydroxycarboxylic acids or esters thereof to the corresponding ω-oxocarboxylic acids or esters thereof, an enzyme capable of catalysing the conversion of ω-oxocarboxylic acids or esters thereof to ω-aminocarboxylic acids or esters thereof and an ω-lactam hydrolase.

In an embodiment of the third aspect, the ω-lactam hydrolase is an ω-laurolactam hydrolase.

In an embodiment of the third aspect, the problem underlying the present invention the enzyme capable of catalysing the conversion of carboxylic acids or carboxylic esters to the corresponding ω-hydroxycarboxylic acids or esters is selected from the group comprising enzymes of the AlkB-type, AlkB from Pseudomonas putida and homologues thereof, wherein the enzyme capable of catalysing the conversion of the corresponding o-hydroxycarboxylic acids or esters to the corresponding ω-oxocarboxylic acids or esters is selected from the group comprising enzymes of the AlkB-type, AlkB from Pseudomonas putida, preferably P. putida GP01, and homologues thereof, wherein the enzyme capable of catalysing the conversion of ω- oxocarboxylic acids or esters to ω-aminocarboxylic acids or esters is selected from the group comprising ω-transaminase CV2025 from Chromobacterium violaceum DSM30191 and homologues thereof, and wherein the ω-lactam hydrolase is selected from the group comprising the o-lactam hydrolases from Acidovorax sp. T31 , Rhodococcus sp. U224, Cupriavidus sp T7, Sphingomonas sp. U298 and Cupriavidus sp. U124 and homologues thereof.

In a fourth aspect, the problem underlying the present invention is solved by a bioreactor comprising an organic phase and an aqueous phase, wherein the aqueous phase comprises the cell according to the third aspect or a recombinant cell expressing an ω-lactam hydrolase and the organic phase comprises at least one organic solvent.

In an embodiment of the fourth aspect, the problem is solved by a bioreactor, wherein the ω- lactam hydrolase is an ω-laurolactam hydrolase, and wherein the aqueous phase comprises ω-aminocarboxylic acid or ω-aminocarboxylic acid ester is selected from the group of compounds represented by formula (I)

wherein x is 6 to 20, preferably 9 to 15, and wherein R³ is selected from the group comprising H and alkyl, which alkyl comprises 1 to 4 carbon atoms.

Further embodiments of the third and fourth aspects include the embodiments of the first aspect of the present invention.

The present invention is based on the surprising finding that ω-lactam hydrolases, a class of enzymes previously known solely for their ability to catalyse the hydrolysis of lactams, may actually be used to bring about the reverse reaction, /^'. e. the condensation of o-aminocarboxylic acids or esters thereof to the corresponding lactam. In a preferred embodiment, the term "converting an ω-aminocarboxylic acid or ester thereof into the corresponding lactam", as used herein, means that an ω-aminocarboxylic acid or ester thereof condenses, forming a cyclic lactam, with a molecule of water being released. For example, the lactam corresponding to 12-aminolaureate is laurolactam.

The co-aminocarboxylic acid or ester thereof in contacted with the ω-lactam hydrolase in an environment compatible with hydrolase activity. The person skilled in the art is familiar with standard parameters, such as ionic strength, temperature and the composition of suitable aqueous buffers that need to be considered with respect to enzyme activity and is capable of determining suitable conditions within the scope of routine experimentation.

In a preferred embodiment, the lactam hydrolase is a recombinant lactam hydrolase, preferably on a plasmid. In a preferred embodiment, the term "recombinant" hydrolase, as used herein, means that the nucleic acid encoding the hydrolase is not an endogenous nucleic acid of the organism used to express it, but has been made using genetic engineering. The person skilled in the art is familiar with suitable plasmids, nucleic acid elements, for example promotor sequences and methods that may be used to make such plasmids.

The present inventors have found that the ratio of ω-aminocarboxylic acid or ester thereof to the corresponding lactam in the aqueous solution may affect the efficiency of the reaction. In a preferred embodiment, the aqueous solution is, initially, essentially free of the corresponding lactam. In a preferred embodiment, the term "essentially free of the corresponding lactam", as referred herein means that a fresh solution of the acid or ester is used that has not previously been in contact with any lactam hydrolase activity. In a preferred embodiment, the aqueous solution comprises only traces of a corresponding lactam. In a more preferred embodiment the ratio of the ω-aminocarboxylic acid or ester thereof, respectively, to the corresponding lactam is, initially, at least 5 : 1 , more preferred 10 : 1 , 20 : 1 , 25 : 1 , 50 : 1 , 100 : 1 , 200 : 1 , 500 : 1 , most preferred, 1000 : 1. Following or simultaneously with step b), the aqueous solution comprising the lactam product may be contacted with an organic solution comprising at least one organic solvent. In a preferred embodiment, the reaction is carried out in a biphasic system comprising an aqueous phase comprising hydrolase activity and an organic phase. The person skilled in the art is aware that the presence of organic solvents may be detrimental to the activity and/or stability of polypeptide enzymes such as hydrolases, but is able, within the scope of routine experimentation, to identify organic solvents and/or conditions compatible with hydrolase activity.

In a preferred embodiment, the term "ω-aminocarboxylic acid", as used herein, refers to a molecule selected from the group represented by formula (I)

wherein x is 5 or more, preferably 5 to 19, most preferably 11. In another preferred embodiment, said term comprises any molecule comprising a hydrocarbon chain substituted at one terminal C atom with an amine group and substituted with a carboxyl group at its other terminal C atom. In a preferred embodiment, the term "laurolactam," as used herein and used interchangeably with the term "laurinlactam", refers to the condensed compound specified by x being 1 1. In a preferred embodiment, any dissociable chemical compound or function, as used herein, refers both to the various undissociated and dissociated states of said compound or function, including the various salts and complexes of the compound or function. For example, the term "R-COOH" is to encompass, amongst others, R-COO^", R-COOH and R-COO^"Na⁺. In a preferred embodiment of the present invention, the term "ω-lactam", as used herein, refers to the molecule formed by condensation of an ω-aminocarboxylic acid. In other words, the ω- lactam is a cyclic molecule that comprises an alkylene group, and the two terminal C atoms of the alkylene group are connected via a peptide bond. In a preferred of the present invention, the o-lactam is o-aminolactam.

In a preferred embodiment, the organic solvent comprised by the organic solution of step c) is a monosubstituted benzene, more preferably an alkyl benzene, more preferably an alkyl benzene, wherein the alkyl substituent comprises one to four carbon atoms. In a most preferred embodiment, the organic solvent is methyl benzene or ethyl benzene.

The teachings of the present invention may not only be carried out using the exact amino acid or nucleic acid sequences of biological macromolecules described herein, but the present invention also comprises the use of homologues of such macromolecules obtained by deleting, adding or substituting single or multiple amino acids or nucleic acids, respectively. In a preferred embodiment, the term "homologue" of a nucleic acid sequence or amino acid sequence, as used herein, refers to another nucleic acid or amino acid sequence, respectively, that has, with respect to the aforementioned nucleic acid or amino acid sequence, a homology of 70, 75, 80, 85, 90, 92, 94, 96, 98, 99 % or more. The prior art describes algorithms that may be used to calculate the degree of homology of two sequences, for example. In a more preferred embodiment of the present invention, the homologue has, preferably in addition to the sequence identity specified before, the biological activity of the original molecule. For example, an amino acid homologue of a polypeptide enzyme has the same or essentially the same enzymatic activity as the polypeptide enzyme. In a preferred embodiment, the term "essentially the same enzymatic activity" refers to a level of activity with respect of the original substrates well beyond the background activity and/or within 3, preferably 2, more preferably 1 orders of magnitude of the K_m and/or k_cat values displayed by the original molecule with respect to said substrates. In a preferred embodiment, the term "homologue" of an amino acid sequence or nucleic acid sequence comprises one or more active portions and/or fragments of the amino acid sequence or nucleic acid sequence, respectively. In a preferred embodiment, the term "active portion", as used herein, refers to an amino acid sequence or a nucleic acid sequence, which is less than the full length amino acid sequence or codes for less than the full length amino acid sequence, respectively, wherein the amino acid sequence or the amino acid sequence encoded, respectively "retains at least some of its essential biological activity", e. g. as a lactam hydrolase. In a preferred embodiment, the term "retains at least some of its essential biological activity", as used herein, means that the amino acid sequence in question has a biological activity exceeding and distinct from the background activity. In a preferred embodiment, the term "homologue" of a nucleic acid comprises nucleic acids the complementary strand of which hybridises, preferably under stringent conditions, to the reference nucleic acid. Stringency of hybridisation reactions is readily determinable by one of ordinary skilled in the art, and in generally is an empirical calculation dependent on probe length, washing temperature and salt concentration. In general longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures. Hybridisation generally depends on the ability of denatured DNA to reanneal to complementary strands are present in an environment below their melting temperature. The higher the degree of desired homology between the probe and hybridisable sequence, the higher the relative temperature, which may be used. As a result it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperature less so. For additional explanations of stringency of hybridisation reactions, see Ausubel et al. (1995). In another preferred embodiment, the term "homologue" of a nucleic acid or amino acid refers to a nucleic acid or amino acid, respectively, that has, at least to some degree the same biological activity and/or function as the reference nucleic acid or amino acid, respectively. In a preferred embodiment, the term "homologue" of a nucleic acid sequence, as used herein, refers to any nucleic acid sequence that encodes the same amino acid sequence as the reference amino acid sequence, in line with the degeneracy of the genetic code.

In a preferred embodiment, the term "the provision of an ω-aminocarboxylic acid in step a) is accomplished by way of an in situ reaction", as used herein, means that the o-aminocarboxylic acid is not added as a ready-made reactant to the reaction mixture, but is instead produced in situ by one ore more reactions, preferably enzymatically catalysed reactions, that produce ω- aminocarboxylic acid. The person skilled in the art is familiar with techniques that allow for the in situ production of molecules in a cell or in a reaction mixture, in particular using enzymatic approaches. In the case of ω-aminocarboxylic acid, a suitable suite of enzymatic reactions is outlined in PCT/EP 2008/067447, which is incorporated in its entirety by reference. The teachings of the present invention may be carried out using a multitude of different cells. The cell may be a prokaryotic cell, preferably a bacterial cell. In a preferred embodiment of the present invention, the cell is from the group of bacteria comprising Escherichia, Corynebacterium and Pseudomonas, preferably E. coli. In another preferred embodiment of the present invention, the cell is a eukaryotic cell, more preferably a lower eukaryotic cell, for example a fungal cell. In a more preferred embodiment of the present invention, the lower eukaryotic cell is selected from the group comprising cells from Saccharomyces, Candida and Pichia. In a most preferred embodiment, the eukaryotic cell is Saccharomyces cerevisiae or Candida tropicalis. In a preferred embodiment, the term "viable cell", refers to a cell that is metabolically active and has intact biological outer membranes. If a viable cell is used, the ω-lactam hydrolase activity may be associated with the membrane, for example as a fusion polypeptide comprising a hydrolase sequence fused to a protein associated with said membrane, or it may be located inside the cell, for example as a protein expressed in the cytosol of a cell. In a preferred embodiment, the term "ω-hydrolase that is a preparation of a cell", as used herein, refers to the various preparations obtainable from a cell associated with ω-hydrolase activity, from a crude lysate of a cell expressing ω-hydrolase activity to a partially or fully purified ω-lactam hydrolase. The person skilled in the art is familiar with techniques that may be used to lyse cells and purify biologically active enzymes from the resulting lysate, for example by subjecting the cell to a freeze-thaw protocol followed by affinity chromatography. In a preferred embodiment, the term "bioreactor", as used herein, refers to a reaction vessel that allows for the use of biological cells, under carefully controllable conditions such as oxygen concentration, nutrients, pH and temperature values, for carrying out biotechnological processes in a directed manner. In a preferred embodiment, the bioreactor is a fermentation reactor. The person skilled in the art is familiar with various kinds of bioreactors well suited to carrying out such biological processes at various scales.

In a preferred embodiment, the "AlkB-type" of polypeptides, as used herein, refers to the group of oxidoreductases comprising AlkB from the Pseudomonas putida AlkBGT system (data base accession number: CAB54050.1) and homologues thereof. AlkB depends on two other polypeptides, AlkG and AlkT, the latter an FAD-dependent rubredoxin reductase transferring electrons to AlkG. AlkG is a rubredoxin, an iron redox protein that functions as AlkB's direct electron donor. In another preferred embodiment, the term "AlkB-type", as used herein, refers to cytochrome-independent monoxygenase using at least one of rubredoxin or a homologue thereof as an electron donor.

The present invention is further illustrated by the following figures and non-limiting examples from which further features, embodiments, aspects and advantages of the present invention may be taken.

Fig. 1 summarises the suite of reactions used to produce ω-lactam hydrolase substrates ALSME (o-amino lauric acid methyl ester) and ALS (ω-amino lauric acid) in situ.

Fig. 2 shows data demonstrating conversion of ALSME and ALS produced in situ by transamination of OLSME (ω-οχο lauric methyl ester) using E. coli cells expressing the CV2025 o-transaminase and the Acidovorax sp. T31 hydrolase. Fig. 2A and Fig. 2B show the concentrations over time of ALSME, ALS, OLSME and HSL (co-hydroxy lauric acid), HLSME (o- hydroxy lauric acid methyl ester), LL and CLS (ω-carboxy lauric acid), respectively.

Fig. 3 shows the concentrations of ALSME, ALS and laurolactam over time in a reaction sample comprising 1 mM ALSME and E. coli BL21 (DE3) (pCOM10_acido) at 1.1 1 g/L at pH 10. Fig. 4 shows the results of gas chromatographic analysis of a sample taken after biotransformation of ALSME using E. coli BL21 (DE3) (pCOM 10_acido) at pH 10.

Example 1 : Cloning of hydrolase genes

The ω-laurolactam hydrolase gene from Acidovorax sp. T31 was cloned into the vector plasmid pCOMIO via Sail and Ndel restriction sites. The ω-laurolactam hydrolase genes used were synthesized by ATG:biosynthetics GmbH (Merzhausen, (G)) and delivered in a Blue Screen pBSK-vector. The ω-laurolactam hydrolase containing pBSK plasmids were then digested by restriction enzymes Sail and Ndel according to manufacturer's instructions. The large fragment comprising approximately 1500 bp was isolated from the agarose gel after electrophoresis. The vector pCOMIO was digested using restriction enzymes Sail and Ndel as well and dephosphorylated and ligated with the hydrolase DNA insert using T4 ligase (Fermentas, Burlington (CA)) according to manufacturer's instructions to yield plasmid pCOM10_acido.

Example 2: Transformation of cells with plasmids

Electroporation was used to introduce the isolated plasmid DNA into the cell. For preparation of electrocompetent cells, 3 ml_ LB culture was inoculated with a single colony of the strain required from the agar plate and incubated over night at 37 °C and 200 rpm. 20 ml_ fresh LB medium were then diluted using 200 of the preculture and incubated at 37 °C and 200 rpm until a specified final optical density of 0.6 at 600 nm was reached. The cells were harvested in the early exponential growth phase by centrifugation (4700 rpm; 4 °C; 15 min) and washed twice with 50 mL cold 10 % glycerol solution. After each washing step, the cells were spun down (4700 rpm, 4 °C, 15 min). Finally, the cell pellet was resuspended in 0.8 mL ice-cold 10 % glycerol solution and aliquots of 100 were transferred into Eppendorf cups for storage at -80°C or were directly used for transformation. For electroporation, the electrocompetent cells were thawed on ice and transferred to pre-cooled 2 mm electroporation cuvettes (PEQLAB Biotechnologie GmbH (Erlangen (G))). After addition of 2 to 10 isolated plasmid DNA, the cuvette was placed in the electroporator (EquiBio, Easyject PRIMA) and a voltage of 2.5 kV was applied. Subsequently, 900 of pre warmed LB medium was added and the mixture was incubated in a thermoshaker (Eppendorf, Hamburg (G)) for 45 min at 37 °C and 700 rpm. Transformed cells were plated as described above.

Example 3: Analysis of samples Samples were analysed using gas chromatography (GC), High Performance Liquid Chromatography (HPCL) and/or Gas chromatography-mass spectrometry. The approach of gas chromatography was applied for determination and quantification of laurolactam, 12-hydroxy lauric acid methyl ester (HLSME) and 12-oxolauric acid methyl ester (OLSME). UltraTM gas chromatograph (Thermo Fisher Scientific Inc., Waltham (USA)), equipped 30 m FactorFour capillary column (VF-5ms, Varian, Middelburg, NL) and a flame ionization detector. Nitrogen was used as carrier gas at a flow rate of 1.5 mL/min. 1 of the sample volume was injected for analysis. For determination of small amounts of substrate and product concentrations, the GC started with an initial oven temperature of 70 °C followed by an increase to 120 °C with a rate of 15 °C/min. The temperature was increased to 170 °C with a rate of 15 °C/min. Subsequently, the temperature was elevated to 180 °C at a rate of 0.3 °C/min followed by a temperature increase to 250 °C at a rate of 20 °C/min. Finally, the temperature was increased to 300°C with a rate of 100°C/min and kept for 2.5 min.. This splitless method was chosen in case the product concentration expected in a sample was less than 0.1 mM. Otherwise, GC analysis was performed with split injection as described as follows: for analysis of laurolactam, 12-hydroxy lauric acid methyl ester and 12-oxo lauric acid methyl ester in higher concentrations, a Focus gas chromatograph (Thermo Fisher Scientific Inc., Waltham (USA)) equipped with OPTIMA delta 3 capillary column (Macherey-Nagel GmbH & Co. KG, Duren (G)) was used. Here, the initial temperature of 80 °C was increased at a rate of 15 °C/min to 180 °C and then from 180 °C to 195 °C by 3 °C/min. Finally, the temperature was increased to 300°C with a rate of 100°C/min and kept for 2.5 min.. A split ratio of 23: 1 was selected. HPCL analyses were performed using a LaChrome HPLC system (VWR Hitachi, Darmstadt (G)) with an integrated Phenomenex Luna C8 silica based column (4.6 x 150 mm, 5 μ, 100 A). The injection volume was set to 20 and the temperature of the column was adjusted to 40 °C. The flow rate of the mobile phase composing mobile phase A, B and C was 0.8 mL/min. Mobile phase A consists of filtrated water completed with 0.4% trifluoracetic acid (TFA) while mobile phase B contains methanol (HPLC grade) completed with 0.2% TFA. Mobile phase C consists of pure acetonitrile (HPLC grade). Detection was carried out by a Corona charged aerosol detector (ESA Biosciences Inc., MA, (USA)).

For evidence of laurolactam, a CP-3800 gas chromatograph linked with a 1200 quadrupole mass spectrometer (Varian, Inc. (Palo Alto (USA)) was applied. The integrated capillary column was a 30 m FactorFour capillary column (VF-5ms, Varian, Middelburg, NL). In the scan mode, mass fragments were monitored from m/z 40 to m/z 650. Therefore, the initial temperature of the column of 80 °C was raised to 160°C at a ratio of 15 °C/min. Then, the temperature of 160 °C was further increase to 250 °C at a ratio of 10 °C/min. The final temperature of 300 °C which is reached at a ratio of 100 °C/min was then maintained for 2 min. The injector was adjusted to the splitless mode and the initial temperature of the injector of 90 °C was increased to 250 °C/min at a rate of 200 °C/min. Subsequently, the temperature of 250 °C was raised to 280 °C with a ratio of 30 °C/min and then cooled down to 90°C at a ratio of 70 °C/min.The sim mode was used to scan the specific mass fragments of m/z 30, m/z 41 , m/z 55, m/z 86, m/z 98, m/z 100, m/z 1 12, m/z 126, m/z 140, m/z 154, m/z 168 and m/z 197. The column as well as the injector was heated as described above.

Example 4: Production of laurolactam starting with ALSME and ALS generated in situ and using viable cells expressing Acidovorax sp. T31 hydrolase

In order to produce lactam hydrolase substrates ALSME and/or ALS in situ, the condensation reaction performed by ω-laurolactam hydrolase was coupled with a transamination reaction catalyzed by a ω-transaminase starting from 12-oxolauric acid methyl ester (OLSME). In the penultimate step of the biosynthesis route for laurolactam synthesis (Fig. 1) the ω-transaminase from Chromobacterium violaceum 2025 catalyzes the transamination reaction of 12-oxolauric acid methyl ester to 12-aminolauric acid methyl ester (ALSME) which is subsequently hydrolyzed by host intrinsic esterases to 12-aminolauric acid (ALS). From there, 12-aminolauric acid is available for the ω-laurolactam hydrolase catalyzed condensation reaction. For that intension, E. coli BL21 (DE3) (paCYCDuet::TA) harboring the ω-transaminase gene was transformed with the pCOM10_ac/^'cfo plasmid according to standard protocols.

E. coli strains transformed using plasmid pCOM 10_acido, see Examples 1 and 2, were plated out on LB agar, containing appropriate antibiotics, and were incubated over night at 37 °C. Afterwards, a single picked colony was used to inoculate 3 mL LB comprising antibiotics and the culture was then incubated (37 °C and 200 rpm) for 8 h. For adaption to mineral medium, 0.5 mL of the preculture was transferred to 100 mL M9* medium supplemented with glucose and antibiotics. The M9* culture was then incubated at 30 °C and 200 rpm over night. Finally, the overnight culture was inoculated with the required amount of M9* medium to a starting optical density (OD450) of approximately 0.2. The culture was incubated at 30 °C and 200 rpm. All shaking flask experiments were performed in 500 mL flasks comprising 100 mL of medium prepared using fresh glucose and antibiotics.

For expression of the ω-laurolactam hydrolase and the CV2025 ω-transaminase genes, respectively, LB and M9* cultures of recombinant E. coli BL21 (DE3) were prepared as described in Example 2, except for the fact that E. coli BL21 (DE3) (paCYCDuet::TA, pCom10_acido) harboring the ω-transaminase gene (PCT/EP2008/067447) and the ω- laurolacatam hydrolase from Acidovorax sp. T31 was used. When the optical density reached 0.5, cultures were induced using 0.025 % dicyclo propyl ketone (DCPK) and 1 mM isopropyl^-D-thiogalactopyranoside (IPTG).. The cells were harvested 5 h after induction (4 °C, 10 min, 4700 rpm).

After 5 h incubation after induction, the cells were centrifuged in 50 mL falcon tubes for 10 min at 4700 rpm and 4 °C. Immediately after centrifugation, the cells were resuspended in nitrogen- free 50 mM potassium phosphate buffer containing 1 % (w/v) glucose at a final cell concentration of approximately 1 gcDw L. For the preparation of 50 mM potassium phosphate buffer pH 7.4, solutions of 50 mM K₂HP0₄ and 50 mM KH₂P0₄were mixed separately and were then titrated until the pH reached a value of 7.4.. The cell suspension was transferred in 1 mL aliquots into sterile Pyrex tubes and incubated in a shaking water bath for 5 min at 30 °C and 350 rpm. After 5 min adaptation, the biotransformation was started by addition of the substrate. As substrate, 12-oxo lauric acid methyl ester (applied concentration in assay: 2,5 mM) were used. If not stated otherwise, biotransformation was stopped after 0, 5, 15, 30, 60, 120 min by adding 1 mL ice-cold diethyl ether containing 0.2 mM dodecane as internal standard for GC analysis or by adding 500 ice-cold acetonitrile containing 0.75 mM tetradecanedioic acid as internal standard.

Biotransformation was started by addition of 2.5 mM 12-oxolauric acid methyl ester dissolved in ethanol. Fig. 2 illustrates substrate depletion as well as product formation observed during the biotransformation of 12-oxolauric acid methyl ester (OLSME).

Example 5: Production of laurolactam starting with ready-made ALSME using viable cells expressing Acidovorax sp. T31 hydrolase E. coli BL21 (DE3) (pCom10_acido) was grown, induced and harvested as described in Example 4. The cell pellet was resuspendend to a cell concentration of 1 gCDW/L in 100 mM sodium carbonate buffer (pH 10) containing 1 % (w/v) glucose. Therefore, 100 mM NaHC0₃ and 100 mM NaHC0₃ solutions were prepared and titrated until pH 10 was reached. The cell suspension was transferred in 1 ml_ aliquots into sterile Pyrex tubes and incubated in a shaking water bath for 5 min at 30 °C and 350 rpm. After 5 min adaptation, the biotransformation was started by addition of ALSME (applied concentration in assay: 1 ,0 mM) The biotransformation was stopped and the samples were prepared for analysis as described in Example 4.

Products formed include the desirable condensation product laurolactam (step 4 or/and step 4'), the latter at concentrations of up to 0.14 mM after 60 min biotransformation. To confirm the formation of laurolactam, the biotransformation samples were additionally analyzed by GC-MS which confirmed that laurolactam was the formed product. At this point, it has to be noted that the concentration of laurolactam might be underestimated, since ALSME and laurolactam have nearly the same retention time on the GC column, as shown in Fig. 4.

References

To the extent it is referred herein to various documents of the prior art, such documents the complete bibliographic data of which read as follows, are incorporated herein in their entirety by reference.

F. M. Ausubel (1995), Current Protocols in Molecular Biology. John Wiley & Sons, Inc. A. M. Lesk (2008), Introduction to Bioinformatics, 3^rd Edition

PCT/EP 2008/067447: ω-aminocarboxylic acids, ω-aminocarboxylic acid esters, or recombinant cells which produce lactams thereof

The features of the present invention disclosed in the specification, the sequence listing, the claims and/or the drawings may both separately and in any combination thereof be material for realising the invention in various forms thereof.

Claims

1. A method for converting an ω-aminocarboxylic acid or ester thereof into the corresponding lactam, comprising the steps

(a) providing the ω-aminocarboxylic acid or ester thereof and

(b) contacting the ω-aminocarboxylic acid or ester thereof with an ω-lactam hydrolase in an aqueous solution, wherein the ω-lactam hydrolase is an ω-laurolactam hydrolase, and wherein the ω-aminocarboxylic acid or ω-aminocarboxylic acid ester is selected from the group of compounds represented by formula (I)

2. The method according to claim 1 , wherein the aqueous solution in step b) is, initially, essentially free of the corresponding lactam.

3. The method according to any of claims 1 to 2, wherein the ratio of the ω-aminocarboxylic acid or ester thereof, respectively, to the corresponding lactam is initially at least 20: 1 , preferably 50: 1.

4. The method according to any of claims 1 to 3, further comprising

(c) contacting the aqueous solution with an organic solution comprising at least one organic solvent.

5. The method according to claim 4, wherein the at least one organic solvent is selected from the group of alkyl benzenes, wherein the alkyl substituent has one to four carbon atoms, preferably one carbon atom.

6. The method according to claim 4, wherein the at least one organic solvent is selected from the group comprising fatty acids and fatty acid esters.

7. The method according to claim 6, wherein the ω-lactam hydrolase is selected from the group comprising the laurolactam hydrolases from Acidovorax sp. T31 , Rhodococcus sp. U224, Cupriavidus sp T7, Sphingomonas sp. U298 and Cupriavidus sp. U124 and homologues thereof.

8. The method according to claim 7, wherein the ω-aminocarboxylic acid or ester thereof is 12-aminolaureate or 12-aminolaureate ester, respectively, and is preferably 12- aminolaureate methyl ester.

9. The method according to any of claims 1 to 8, wherein the provision of the ω- aminocarboxylic acid in step (a) is accomplished by way of an in situ reaction.

10. The method according to any of claims 1 to 9, wherein the ω-lactam hydrolase is associated with a viable cell.

1 1. The method according to any of claims 1 to 10, wherein the ω-lactam hydrolase is a preparation of a cell expressing said ω-lactam hydrolase.

12. A use of an ω-lactam hydrolase for converting an ω-aminocarboxylic acid or ester thereof into a lactam, wherein the ω-lactam hydrolase is an ω-laurolactam hydrolase, and wherein the ω-aminocarboxylic acid or ω-aminocarboxylic acid ester is selected from the group of compounds represented by formula (I)

(CH₂)_x - COOR³ (l), wherein x is 6 to 20, preferably 9 to 15, and wherein R³ is selected from the group comprising H and alkyl, which alkyl comprises 1 to 4 carbon atoms.

13. The use according to claim 12, wherein the ω-lactam hydrolase is selected from the group comprising the ω-lactam hydrolases from Acidovorax sp. T31 , Rhodococcus sp.

U224, Cupriavidus sp. T7, Sphingomonas sp. U298 and Cupriavidus sp. U124 and homologues thereof.

14. A recombinant cell comprising an enzyme capable of catalysing the conversion of carboxylic acids or esters thereof to the corresponding ω-hydroxycarboxylic acids or esters thereof, an enzyme capable of catalysing the conversion of the corresponding ω- hydroxycarboxylic acids or esters thereof to the corresponding ω-oxocarboxylic acids or esters thereof, an enzyme capable of catalysing the conversion of ω-oxocarboxylic acids or esters thereof to ω-aminocarboxylic acids or esters thereof and a ω-lactam hydrolase, wherein the ω-lactam hydrolase is an ω-laurolactam hydrolase.

15. The cell according to claim 14, wherein the enzyme capable of catalysing the conversion of carboxylic acids or carboxylic esters to the corresponding ω-hydroxycarboxylic acids or esters is selected from the group comprising enzymes of the AlkB-type, AlkB from Pseudomonas putida and homologues thereof, wherein the enzyme capable of catalysing the conversion of the corresponding ω- hydroxycarboxylic acids or esters to the corresponding ω-oxocarboxylic acids or esters is selected from the group comprising enzymes of the AlkB-type, AlkB from Pseudomonas putida and homologues thereof, wherein the enzyme capable of catalysing the conversion of ω-oxocarboxylic acids or esters to ω-aminocarboxylic acids or esters is selected from the group comprising ω- transaminase CV2025 from Chromobacterium violaceum DSM30191 and homologues thereof, and wherein the recombinant ω-lactam hydrolase is selected from the group comprising the ω-lactam hydrolases from Acidovorax sp. T31 , Rhodococcus sp. U224, Cupriavidus sp T7, Sphingomonas sp. U298 and Cupriavidus sp. U124 and homologues thereof.

16. A bioreactor comprising an organic phase and an aqueous phase, wherein the aqueous phase comprises the cell according to claim 16 or 17 or a recombinant cell expressing an ω-lactam hydrolase and the organic phase comprises at least one organic solvent, wherein the ω-lactam hydrolase is an ω-laurolactam hydrolase, and wherein the aqueous phase comprises ω-aminocarboxylic acid or ω- aminocarboxylic acid ester is selected from the group of compounds represented by formula (I)