DE4402127A1 - New carnitine racemase from E.coli and related plasmids - Google Patents

Abstract

Carnitine racemase (CR) encoded by the 891 nucleotide sequence given in the specification is new. The 297 amino acid sequence of CR is also given in the specification. Also new are the plasmids pT7 -5KE32, -5KE37 and -5KE36.

Description

The present invention relates to a new, not yet in the enzyme enzyme containing the nomenclature, the carnitine racemase, which caiD ko is dated, and its overexpression. The carnitine racemase is in the Able to convert D (+) - carnitine to the physiologically active L (-) - carnitine deln.

Characteristic of known technical solutions

L (-) - carnitine (3-hydroxy-4-trimethylamine butyrate) is the only one physiologically active form of carnitine. It is ubiquitous in the animal kingdom widespread and also occurs in plants. D-carnitine has so far been used never detected in living things. L-carnitine is long chain for transport activated fatty acids through the inner mitochondrial membrane into the mitochondrial matrix, where the β-oxidation of the fatty acids takes place, essential. With a lack of L (-) - carnitine due to disturbances in the Biosynthesis or in the resorption or microbial population of the The mitochondrial β-oxidation of fatty acids is inhibited and Carnitine deficiency syndromes in the form of muscular dystrophies, lipid stores myopathies etc. arise (overview: Pathology, 1985, Vol. 17, 116-169). Affected patients are treated with L (-) - carnitine. This also applies to patients with chronic renal failure. Because in the Past the necessary amounts of L (-) - carnitine are not always available , the recombinant D, L-carnitine was often used. D (+) - carnitine, the nonphysiological component of the racemate is not just in terms of ineffective on stimulating β-oxidation, but is also considered a competitive inhibitor for the various L-carnitine-specific Transport systems and enzymes considered (Life Science, 1981, Vol. 28, 2931-2938). D (+) - Carnitine is responsible for the occurrence of myasthenia grave-like symptoms responsible for the application of D, L-carnitine made (Lancet, 1979, Vol. I, 1041-1042). The use of the racemate is from this medical point of view no longer justifiable.

In the field of biotechnology, L (-) - carnitine has a growing one Meaning. A stimulating influence on the growth of various yeasts (JP-PS 73128.724) and various bacterial species (Acinetobacter, Pseudomonas, Enterobacteriaceae) (DE-PE 2 04 105), as well as for methylotro phe bacteria (DD-PS 2 11 039) was detected. A use for stimu  The production of monoclonal antibodies has recently been proposed struck (J. Biotech., 1991, Vol. 18, 173-176).

The discovery and overproduction of carnitine racemase represents one essential contribution to the improvement of the process (DD-PS 3 00 181) Conversion of D-carnitine into L-carnitine. It is important above all the possibility of this after chemical synthesis of D, L-carnitine and at D-carnitine resulting from racemate resolution is economically advantageous harder to use.

The microbiological conversion of D-carnitine was achieved for the first time Jung and Kleber (DD-PS 3 00 181). L-Carnitine is made from D-Carni tin with the help of whole cells or cell-free extracts of Escherichia coli 044 K74 won.

Since the discovery of L-carnitine in muscle, this sub has been won punching in complex isolation and cleaning processes from animal Material. In the 1950s, multi-step chemical synthesis was carried out works, but all lead to D, L-carnitine. To isolate the L-Iso To date, methods have been used which are based on the resolution of racemates fractional crystallization using optically active acids based (e.g. DD-PS 23217; DD-PS 93347; DE-OS 29 27 672; Biotechnol. Bioeng., 1984, vol. 26, 911-915; FEBS Lett., 1975, Vol. 54, 21-25; J. org. Chem., 1986, vol. 51, 2842-2844). All chemical syntheses with subsequent Racemate cleavage has the disadvantage that of the synthesized Substance itself can only be isolated up to a maximum of 50% as L (+) - carnitine. In order to overcome this disadvantage, stereospe have been used in recent years Specific syntheses from achiral precursors (e.g. Tetrahedron, 1992, Vol. 48, 319-324), in particular using stereospecific enzymatic Syntheses developed: For example, the reverse reaction of the L (-) - Carnitine dehydrogenase (EC 1.1.1.108) used to make 3-dehydro (oxo) carnitine Hydrogenate L-carnitine (US Pat. No. 4,221,869). L-carnitine can also be made 4-Butyrobetaine using 4-butyrobetaine hydroxylase (EC 1.14.11.1) won NEN (DE-OS 31 23 975). A microorganism or its produ graced L-carnitine amidase (EC 3.5.1.73) can be used to be selective L (-) - carnitinamide and / or the L portion of D, L-carnitinamide to L (-) - Car to hydrolyze nitin (EP 0501294 A1). Other procedures are based on the Use of crotonobetaine and 4-butyrobetaine as achiral starting ver binding (EP 0158194, EP 0195944). Especially different strains of Entero bacteriaceae are capable of producing L-carnitine under anaerobic conditions  Synthesize crotonobetaine or 4-butyrobetaine (DD-PS 2 21 905; JP-PS 6167,499; JP-PS 61,234,794; JP-PS 61,234,788). For industrial applications Most processes have disadvantages because either the initial measure materials are expensive and / or unstable, the enzymes used have inadequate activity or stability or use expensive coenzymes and unwanted by-products be formed. A detailed overview of the synthesis of L (-) - Car nitin from microorganisms and isolated enzymes can be found in Adv. Bio chem. Engine. Biotechnol., 1993, Vol. 50, 21-44.

Aim of the invention

The aim of the invention is a method that is economically advantageous Way the production of L-carnitine on microbiological or enzymati allowed ways from the waste product D (+) - carnitine.

State the nature of the invention

The invention has the task of converting D (+) - carnitine, which after Racemate resolution of chemically synthesized D, L-carnitine as waste pro product occurs in L (-) - Cernitin technologically simple and efficient shape. The protein used for this biotransformation, Carni tinracemase, it is still one in the enzyme gymnastics enzyme not listed, the first time in Escherichia coli 044 K74 postu was gated.

The object is achieved in that a strain is set under the cultivation conditions described below gene is able to overexpress carnitine racemase and D (+) - carnitine to convert to L (-) - carnitine.

The carnitine racemase, hereinafter referred to as CaiD and by the Plasmid pT7-5KE32 and pT7-5KE37 is encoded by the following featured:

• a) Reactivity:
  it racemizes D-carnitine to L-carnitine and vice versa,
• b) Inductors:
  it is an inducible enzyme in the wild strain Escherichia coli 044 K74 and is induced under anaerobic conditions by L-carnitine and crotonobetaine (but not by D-car nitin),
• c) Molecular weight:
  the molecular weight is about 32,000,
• d) Factor requirement:
  a low-molecular factor is essential for the activity of carnitine racemase, which is provided by the protein CaiE, which is encoded on the plasmids pT7-5KE32 and pT7-5KE36,
• e) Genetic organization:
  CaiD and CaiE are encoded together with the proteins CaiT, CaiA, CaiB and CaiC in the carnitine (cai) operon, which is involved in the carnitine metabolism in Escherichia coli 044 K74,
• f) Localization on the chromosome:
  the cai operon was located in the first minute of the Eschericia coli chromo soms,
• g) Nucleotide sequence and amino acid sequence:
  the nucleotide sequence and the amino acid sequence derived from it are available under the accession number X73904 ECCAI in the EMBL.

Any peptide and nucleotide sequence that has more than 70% homology with CaiD has a carnitine racemase activity.

The strain suitable for carrying out the invention is Escherichia coli K38, which carries the plasmid pGP1-2 and the plasmid pT7-5KE32. The Plasmid pGP1-2 codes for the RNA polymerase of phage T7, which is un control of the thermosensitive repressor of the phage lambda - through the same plasmid encodes - located and confers kanamycin resistance. The Plasmid pT7-5KE32 carries the Ge under the control of the T7 polymerase promoter ne caiD and caiE, those for the carnitine racemase CaiD and factor-forming Encode protein CaiE and confers ampicillin resistance.

The bacteria are cultivated submerged in complex or mini Painting medium with the addition of D, L-carnitine, L (-) - carnitine or crotonobetaine (1-50 mmol / l) (preferably 20 mmol / l) and kanamycin (preferably 20 µg / ml) and ampicillin (preferably 50 µ / ml). After an incubation time from 4-16 h (preferably 8 h) under anaerobic conditions at a Temperature of 20-34 ° C (preferably 30 ° C), the cells for un dangerous a temperature shock of 37-45 ° C (preferably 42 ° C) for 25 min exposed and for another 1-4 h (preferably 2 h) at 30-45 ° C (preferred 37 ° C) cultivated. The cells are then centrifuged  separation separated and in phosphate buffer pH 6-9 (preferably 7.5) recorded and disrupted using known methods (ultrasound, French Press, Alcoa).

Both the cell suspension and the crude protein extract are treated with D- Carnitine solutions contacted and activity determination of the car incubated nitin racemase at 37 ° C for 3-5 min. The cells are separated by Ab centrifuge or the protein by acid or heat precipitation or ultra centrifugation removed.

The advantages of the overexpression strain described are

• 1. Increased carnitine rcmease activity, which is a more effective turnover of Allow D (+) - carnitine to L (-) - carnitine.
• 2. About 10-15 percent of the cell protein is present as carnitine racemase, which facilitates purification using known techniques.

The overexpression strain will be explained using a few examples.

example 1

Escherichia coli K38 pT7-5KE32 is cultivated submerged in a sealed 250 ml jar filled to the brim with LB medium and which contains 20 mM D, L-carnitine. The inoculation takes place with 5 ml an aerobic preculture (DD₆₀₀ = 1.5) using the said culture mediums without added carnitine. After 8 h incubation at 30 ° C the culture Exposed to thermal shock at 42 ° C for 25 min. It becomes 2 h at 37 ° C incubated, then the bacteria for 15 min at 6000 × g and 4 ° C ge harvested and washed twice with phosphate buffer (67 mM, pH 7.5). The cell len are taken up in 10 ml of phosphate buffer (50 mM, pH 7.5) and by means of French press open minded. Undigested cells and cell debris are separated by centrifugation for 20 min at 25000 × g.

The cell-free extract obtained in this way becomes D (+) - carnitine up to one Concentration of 20 mmol / l added and the reaction mixture for 3 min Incubated at 37 ° C. The protein is precipitated with trichloroacetic acid and in The L (-) - carnitine formed survived enzymatically by means of the carnitine  acetyltransferase determined. The specific activity of carnitine racemase is 310 mU mg protein.

Example 2

The procedure is as set out in Example 1. Instead of the ash tree richia coli K38 pT7-5KE32 become the strains Escherichia coli K38 pT7-5KE36 and Escherichia coliK38 pT7-5KE37 cultured independently. The Cell-free extracts obtained are isolated for carnitine racemase activity tested, with no significant increase in this activity being measured. After mixing the two extracts in a ratio of 1: 1, a specific cal activity of carnitine racemase of 260 mU per mg protein determined.

Example 3

The nucleotide sequence of the carnitine racemase CaiD and the factor-forming pro Teins CaiE after sequencing the Carnitinoperon from Escherichia coli 044 K74 determined using the Sanger method and are in the EMBL available under accession number X73904 ECCAI. The nucleotide sequence becomes the amino acid sequence derived by computer analysis and homology used in protein sequence databases. CaiD shows homologies with enoyl-CoA 0hydratases / isomerases and CaiE with acetyltransferases.

Example 4

The position of the caiD and caiE genes is on the Escherichia coli chromosome localized in the first minute using the MapSearch program. This The result is confirmed by the Southern hybridization of the Plasmids pLC4-46 from the systematic gene bank by Clarke and Carbon, the carries the first minute of the Escherichia coli chromosome.  

Example 5

The relative molar mass of carnitine racemase is derived from the amino acid sequence calculated by CaiD and in gel electrophoresis in the presence of sodium do decyl sulfate (SDS) confirmed. It is 32,000.

Example 6

Escherichia coli K38 pT7-5KE32 is cultivated submerged in a sealed 250 ml jar filled to the brim with LB medium and which was 20 mM D, L-carnitine, 20 µg / ml kanamycin and 50 µg / ml Am pillicin contained. The inoculation was carried out with 5 ml of an aerobic preculture (DD₆₀₀ = 1.5) using the culture medium mentioned without carnitine sentence. After 8 h of incubation at 30 ° C, the culture is at 42 ° C for 25 min subjected to a thermal shock. Then 400 µg / ml rifampicin added and the microorganisms cultured at 37 ° C for 2 hours. The bacteria are harvested for 15 min at 6000 × g and 4 ° C and twice washed with phosphate buffer (67 mM, pH 7.5).

The cell suspension obtained in this way becomes D-carnitine up to a concentration of 20 mmol / l was added and the reaction mixture was incubated at 37 ° C. for 5 min beer. The cells are inactivated with trichloroacetic acid Supernatant formed carnitine determined enzymatically. The specific asset The carnitine racemase was 2.69 U per mg dry cell mass.

The strain E. coli 044 K74 used to carry out the method is stored in the DSM under number 8828.

Claims (9)

1. Carnitine racemase, characterized by the nucleotide sequence according to Appendix 1 and the amino acid sequence according to Appendix 2. 2. Carnitine racemase according to claim 1, which is genetically organized in such a way that CaiD and CaiE are coded together with the proteins CaiT, CaiA, CaiB and CaiC in the carnitine (cai) operon,
the cai operon is located on the E. coli chromosome,
whose nucleotide sequence has at least 70% homology with the nucleotide sequence SEQ ID NO1 according to Appendix 1, it is an inducible enzyme in the wild strain Escherichia coli 044 K74 and - induced by L-carnitine and crotonobetaine, but not by D-carnitine, under anaerobic conditions is decorated
it racemizes D-carnitine to L-carnitine and vice versa, for its reactivity a low-molecular factor is indispensable, which is provided by the protein CaiE, which is encoded on the plasmids pT7-5KE32 and pT7-5KE36 and its nucleotide sequence in Appendix 3 , its amino acid sequence is shown in Appendix 4,
their molecular weight is about 32,000,
it includes an amino acid sequence which has at least 70% homology with the amino acid sequence SEQ ID NO2. 3. Process for the preparation of carnitine racemase, characterized in that the strain Escherichia coli K 38, which the plasmid pGP1-2 as well as carrying the plasmid pT7-5KE32, submerged in a complex or minimal medium, the DL Carnitine, L-Carnitine or Crotonobetaine as well as Kanamycin and Ampicillin were added, is cultivated, by taking after an incubation period of 4 to 16 hours anaerobic conditions at a temperature of 20 to 34 Grade C gives the cells a temperature shock of 37 to 45 degrees C be exposed for a period of about 25 minutes and then continue at 30 to 45 degrees for 1 to 4 hours be cultivated, the cells are separated by centrifugation, are to be taken up in phosphate buffer and at a pH of 6 to 9 and be unlocked in a manner known per se from the cell suspension or the cell-free crude extract, the each contain the carnitine racemase, the latter isolated and cleaned in a manner known per se becomes. 4. Plasmid pT7-5KE32. 5. Plasmid pT7-5KE37. 6. Plasmid pT7-5KE36.   7. Use of the microorganisms K38 pT7-5KE32 and K38 pT7- 5KE37 as carnitine racemase producers. 8. Use of Carnitine Racemase to Convert D- to L- Carnitine. 9. Process for the preparation of L-carnitine from D-carnitine, characterized in that Carnitine racemase according to claim 1 together with that of CaiE produced cofactor in a reaction batch from 67 mmol / l phosphate buffer according to Sörensen, pH = 7.5, to which D-carnitine was added up to one Concentration of 20 mmol / l, incubated for 3 min at 37 degrees C, then centrifuged, the protein or the intact cells removed and the L (-) - carnitine formed from the reaction medium in itself is separated in a known manner.