Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
  • C12P17/10—Nitrogen as only ring hetero atom
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
  • C12N9/86—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in cyclic amides, e.g. penicillinase (3.5.2)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
  • C12Y305/02—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in cyclic amides (3.5.2)
  • C12Y305/02012—6-Aminohexanoate-cyclic-dimer hydrolase (3.5.2.12)

Definitions

• the present invention relates to a method for converting an ⁇ -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 ⁇ -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.
• polyamide 6 is often obtained by way of polymerisation of ⁇ -caprolactam
• polyamide 12 may be made by polymerising laurolactam.
• copolymers of lactams for instance copolymers of ⁇ -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 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 a method for converting an ⁇ -aminocarboxylic acid or ester thereof to the corresponding lactam, comprising the steps
• the aqueous solution in step b) is, initially, essentially free of the corresponding lactam.
• the ratio of the o-aminocarboxylic acid or ester thereof, respectively, to the corresponding lactam is initially at least 20 : 1 , preferably 50 : 1.
• the method further comprises step
• 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.
• the at least one organic solvent is selected from the group comprising fatty acids and fatty acid esters.
• the ⁇ -lactam hydrolase is an ⁇ - laurolactam hydrolase.
• the ⁇ -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.
• the ⁇ -aminocarboxylic acid or ⁇ - 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 R 3 is selected from the group comprising H and alkyi, which alkyi comprises 1 to 4 carbon atoms.
• the ⁇ -aminocarboxylic acid or ester thereof is 12-aminolaureate or 12-aminolaureate ester, respectively, and is preferably 12- aminolaureate methyl ester.
• the provision of the ⁇ - aminocarboxylic acid in step (a) is accomplished by way of an in situ reaction.
• the ⁇ -lactam hydrolase is associated with a viable cell.
• the ⁇ -lactam hydrolase is a preparation of a cell expressing said ⁇ -lactam hydrolase.
• the problem underlying the present invention is solved by a use of an ⁇ - lactam hydrolase for converting an ⁇ -aminocarboxylic acid or ester thereof into a lactam.
• the ⁇ -lactam hydrolase is selected from the group comprising the ⁇ -lactam hydrolases from Acidovorax sp. T31 , Rhodococcus sp. U224, Cupriavidus sp T7, Sphingomonas sp. U298 and Cupriavidus sp. U124 and homologues thereof.
• a recombinant cell comprising an enzyme capable of catalysing the conversion of carboxylic acids or esters thereof to the corresponding ⁇ -hydroxycarboxylic acids or esters thereof, an enzyme capable of catalysing the conversion of the corresponding ⁇ -hydroxycarboxylic acids or esters thereof to the corresponding ⁇ -oxocarboxylic acids or esters thereof, an enzyme capable of catalysing the conversion of ⁇ -oxocarboxylic acids or esters thereof to ⁇ -aminocarboxylic acids or esters thereof and an ⁇ -lactam hydrolase.
• the ⁇ -lactam hydrolase is an ⁇ -laurolactam hydrolase.
• the enzyme capable of catalysing the conversion of carboxylic acids or carboxylic esters to the corresponding ⁇ -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 ⁇ -oxocarboxylic acids or esters is selected from the group comprising enzymes of the AlkB-type, AlkB from Pseudomonas putida, preferably P.
• the enzyme capable of catalysing the conversion of ⁇ - oxocarboxylic acids or esters to ⁇ -aminocarboxylic acids or esters is selected from the group comprising ⁇ -transaminase CV2025 from Chromobacterium violaceum DSM30191 and homologues thereof, and wherein the ⁇ -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.
• 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 ⁇ -lactam hydrolase and the organic phase comprises at least one organic solvent.
• the problem is solved by a bioreactor, wherein the ⁇ - lactam hydrolase is an ⁇ -laurolactam hydrolase, and wherein the aqueous phase comprises ⁇ -aminocarboxylic acid or ⁇ -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 R 3 is selected from the group comprising H and alkyl, which alkyl comprises 1 to 4 carbon atoms.
• formula (I) wherein x is 6 to 20, preferably 9 to 15, and wherein R 3 is selected from the group comprising H and alkyl, which alkyl comprises 1 to 4 carbon atoms.
• the present invention is based on the surprising finding that ⁇ -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.
• the lactam corresponding to 12-aminolaureate is laurolactam.
• the co-aminocarboxylic acid or ester thereof in contacted with the ⁇ -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.
• the lactam hydrolase is a recombinant lactam hydrolase, preferably on a plasmid.
• the term "recombinant" hydrolase 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 ratio of ⁇ -aminocarboxylic acid or ester thereof to the corresponding lactam in the aqueous solution may affect the efficiency of the reaction.
• the aqueous solution is, initially, essentially free of the corresponding lactam.
• 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.
• the aqueous solution comprises only traces of a corresponding lactam.
• the ratio of the ⁇ -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.
• the aqueous solution comprising the lactam product may be contacted with an organic solution comprising at least one organic solvent.
• the reaction is carried out in a biphasic system comprising an aqueous phase comprising hydrolase activity and an organic phase.
• 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.
• ⁇ -aminocarboxylic acid 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.
• 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.
• the term “laurolactam,” as used herein and used interchangeably with the term “laurinlactam” refers to the condensed compound specified by x being 1 1.
• any dissociable chemical compound or function 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.
• R-COOH is to encompass, amongst others, R-COO " , R-COOH and R-COO " Na + .
• ⁇ -lactam refers to the molecule formed by condensation of an ⁇ -aminocarboxylic acid.
• the ⁇ - 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.
• the o-lactam is o-aminolactam.
• 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.
• 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.
• 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 homologue has, preferably in addition to the sequence identity specified before, the biological activity of the original molecule.
• an amino acid homologue of a polypeptide enzyme has the same or essentially the same enzymatic activity as the polypeptide enzyme.
• 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 ca t values displayed by the original molecule with respect to said substrates.
• 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.
• 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.
• 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.
• 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 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.
• the term "homologue" of a nucleic acid sequence 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.
• the term "the provision of an ⁇ -aminocarboxylic acid in step a) is accomplished by way of an in situ reaction" 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 ⁇ - aminocarboxylic acid.
• an in situ reaction 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 ⁇ - 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.
• the cell may be a prokaryotic cell, preferably a bacterial cell.
• the cell is from the group of bacteria comprising Escherichia, Corynebacterium and Pseudomonas, preferably E. coli.
• the cell is a eukaryotic cell, more preferably a lower eukaryotic cell, for example a fungal cell.
• the lower eukaryotic cell is selected from the group comprising cells from Saccharomyces, Candida and Pichia.
• the eukaryotic cell is Saccharomyces cerevisiae or Candida tropicalis.
• 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 ⁇ -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.
• ⁇ -hydrolase that is a preparation of a cell refers to the various preparations obtainable from a cell associated with ⁇ -hydrolase activity, from a crude lysate of a cell expressing ⁇ -hydrolase activity to a partially or fully purified ⁇ -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.
• 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.
• 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.
• the "AlkB-type" of polypeptides 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.
• the term "AlkB-type" refers to cytochrome-independent monoxygenase using at least one of rubredoxin or a homologue thereof as an electron donor.
• Fig. 1 summarises the suite of reactions used to produce ⁇ -lactam hydrolase substrates ALSME (o-amino lauric acid methyl ester) and ALS ( ⁇ -amino lauric acid) in situ.
• ALSME o-amino lauric acid methyl ester
• ALS ⁇ -amino lauric acid
• Fig. 2 shows data demonstrating conversion of ALSME and ALS produced in situ by transamination of OLSME ( ⁇ - ⁇ 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 ( ⁇ -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.
• the ⁇ -laurolactam hydrolase gene from Acidovorax sp. T31 was cloned into the vector plasmid pCOMIO via Sail and Ndel restriction sites.
• the ⁇ -laurolactam hydrolase genes used were synthesized by ATG:biosynthetics GmbH (Merzhausen, (G)) and delivered in a Blue Screen pBSK-vector.
• the ⁇ -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.
• T4 ligase Fermentas, Burlington (CA)
• Electroporation was used to introduce the isolated plasmid DNA into the cell.
• 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.
• 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.
• electroporation the electrocompetent cells were thawed on ice and transferred to pre-cooled 2 mm electroporation cuvettes (PEQLAB Biotechnologie GmbH (Er Weg (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.
• thermoshaker Eppendorf, Hamburg (G)
• 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.
• GC gas chromatography
• HPCL High Performance Liquid Chromatography
• 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)
• Nitrogen was used as carrier gas at a flow rate of 1.5 mL/min. 1 of the sample volume was injected for analysis.
• 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.
• 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.
• 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..
• 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 ⁇ , 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)).
• 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).
• 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.
• 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
• E. coli strains transformed using plasmid pCOM 10_acido 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.
• 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.
• E. coli BL21 DE3
• paCYCDuet::TA, pCom10_acido E. coli BL21 (DE3) (paCYCDuet::TA, pCom10_acido) harboring the ⁇ -transaminase gene (PCT/EP2008/067447) and the ⁇ - laurolacatam hydrolase from Acidovorax sp. T31 was used.
• 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.
• 50 mM potassium phosphate buffer pH 7.4 solutions of 50 mM K 2 HP0 4 and 50 mM KH 2 P0 4 were 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.
• 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 NaHC0 3 and 100 mM NaHC0 3 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.
• ALSME applied concentration in assay: 1 ,0 mM
• 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.
• laurolactam the biotransformation samples were additionally analyzed by GC-MS which confirmed that laurolactam was the formed product.
• 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.

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