HK1005888B

HK1005888B - Biocatalytic process for the production of l-(-)-carnitine from crotonobetaine and strains of proteeae for use in said process

Info

Publication number: HK1005888B
Application number: HK98104940.6A
Authority: HK
Inventors: Shapiro Stuart; Bernardini Manrico; J. Sih Charles
Original assignee: Sigma-Tau Industrie Farmaceutiche Riunite S.P.A.
Priority date: 1990-05-14
Filing date: 1998-06-05
Publication date: 1999-01-29

Description

The present invention relates to a process for manufacturing L-(-)-carnitine (2) from crotonobetaine (1) via the action of an enzyme produced by a microorganism, this enzyme catalyzing the stereospecific hydration of crotonobetaine to L-(-)-carnitine.

The present invention also relates to novel Proteeae strains suitable for use in this process.

As known, carnitine contains a single centre of asymmetry and therefore exists as two enantiomorphs, designated D-(+)-carnitine and L-(-)-carnitine. Of these, only L-(-)-carnitine is found in living organisms, where it functions as a vehicle for transporting fatty acids across mitochondrial membranes. Whilst L-(-)-carnitine is the physiologically-active enantiomer, for some years racemic DL-carnitine had been used as a therapeutic agent. It is now recognised, however, that D-(+)-carnitine is a competitive inhibitor of carnitine acyltransferases, and that it can diminish the level of L-(-)-carnitine in myocardium and skeletal muscle.

It is therefore essential that only L-(-)-carnitine be administered to patients undergoing haemodialysis treatment or treatment for cardiac or lipid metabolism disorders.

Various chemical procedures have been proposed for the industrial-scale production of carnitine. These procedures are not stereospecific, leading to racemic mixtures of D-(+)- and L-(-)-isomers. It thus becomes necessary to apply methods of resolution in order to separate the enantiomeric constituents of the racemate. The aforementioned synthetic chemical procedures are complex and costly, and in all cases result in the production of equimolar quantities of D-(+)-carnitine and L-(-)-carnitine.

Several microbiological methods have recently been proposed for producing L-(-)-carnitine, either de novo from common fermentation ingredients or via a stereospecific transformation of achiral betaine derivatives. Regarding the former methods, Japanese patent 71567/1984 (TAKEDA) describes the elaboration of L-(-)-carnitine by the mould Emericella quadrilineata when the organism is cultivated in a complex medium. Most of the latter procedures are predicated upon the stereospecific hydration of crotonobetaine to L-(-)-carnitine, and differ principally by virtue of the particular microorganism employed to accomplish the biotransformation of interest.

Most of the organisms described in the relevant patent literature belong to the family Enterobacteriaceae [e.g., EP 121,444 (HAMARI), EP 122,794 (AJINOMOTO), EP 148 132 (SIGMA-TAU), DDRP 221,905 (KARL-MARX-UNIVERSITÄT), JP Application 61/234788 (SEITETSU)] including Proteus mirabilis, although non-enterobacteria have also been employed [e.g., EP 158,194 and EP 195,944 (LONZA) which utilise strains of Achromobacter xylosoxydans, and JP Application 60/275181 (Biol K.K. & CHO KASEHIN K. K.) for which optimal results were reported using the mould Penicillium citrinum].

Substances other than crotonobetaine that have been tried as precursors for the microbial production of L-(-)-carnitine include 3-deydrocarnitine [e.g., JP 272086/1987 (AJINOMOTO)] and carnitine nitrile [EP 319,344 (KYOWA HAKKO KOGYO)].

However, the microbiological procedures proposed to date have not proven practicable for manufacturing L-(-)-carnitine on an industrial scale for one or more of the following reasons:

(i) the yield of L-(-)-carnitine is extremely low;
(ii) the precursor substrate is extremely unstable;
(iii) the microorganisms must be cultivated in a costly nutritive medium;
(iv) the micoorganisms can only tolerate low concentrations [up to 2-3% (w/v)] of crotonobetaine;
(v) side reactions, such as the reduction of crotonobetaine to gamma-butyrobetaine or the oxidation of L-(-)-carnitine to 3-dehydrocarnitine, reduce the final yield of L-(-)-carnitine.

The crotonobetaine used in the procedures described below consisted of ca. 89% (^w/_w) crotonobetaine inner salt (desalinated crotonobetaine) + ca. 11% (^w/_w) water (determined using Karl Fischer reagent), corresponding to approximately one molecule of water per molecule of crotonobetaine. Consequently, this substrate shall henceforth be referred to as "crotonobetaine monohydrate", although "monohydrate" is not intended to imply a definitive structural relationship between crotonobetaine and water.

Elsewhere, when the term "crotonobetaine inner salt" is used, it refers to 100% pure crotonobetaine inner salt (anhydrous desalinated crotonobetaine).

Four new strains of Proteus mirabilis, capable of hydrating crotonobetaine to L-(-)-carnitine on biotransformation media wherein the concentration of crotonobetaine inner salt is about 10-12% (^w/_v), were isolated and biotyped (see below). These new strains do not require expensive nutrients or additives for either growth or biotransformation; rather, both cell growth and biotransformation can be achieved using simple, inexpensive media. Within the limits of detectability of the analytical methods employed the new strains of P. mirabilis of the present invention do not catalyse side reactions, such as those indicated in the aforecited point (v), that reduce the final output of L-(-)-carnitine.

Four P. mirabilis isolates were found capable of converting 12% (^w/_v) crotonobetaine monohydrate (744.4 mM) to L-(-)-carnitine in molar yields exceeding 40%.

The four P. mirabilis isolates (designated in-house as Sigma-Tau SP237 subclone NACit I1, Sigma-Tau BT 1IV, Sigma-Tau GP 1AVI, and Sigma-Tau R 2AIX) were deposited on 21 April 1989 with the Agricultural Research Service Culture Collection, Northern Regional Research Centre, United States Department of Agriculture, 1815 North University Street, Peoria, Illinois 61604 U.S.A., and were assigned the codes NRRL B-18480, NRRL B-18481, NRRL B-18482, and NRRL B-18483, respectively, by the Agricultural Research Service. The isolation method and morphological, and physiological/biochemical properties of the four strains are described below.

( i ) Proteus mirabilis NRRL B-18480 (Sigma-Tau SP237 subclone NACit I1).

The organism was originally recognised morphologically as a contaminant on a plate of Acinetobacter lwoffii. The organism was subsequently purified using a medium (henceforth referred to as NACit + cb) consisting of nutrient broth + 0.8% (^w/_v) sodium chloride + 0.5% (^w/_v) citric acid monohydrate + 1.5% (^w/_v) crotonobetaine monohydrate + 2% agar, pH adjusted to 6.8 with potassium hydroxide.

The organism was maintained by daily transfer (37°C) on NACit + cb; it was typed by the American Type Culture Collection (ATCC) (Rockville, Maryland, U.S.A.), and by the Industrial Microbiology Laboratories at Sigma-Tau Industrie Farmaceutiche Riunite S.p.A. (Pomezia, Italy) using morphological examination in conjunction with API 20 E (version C), API ZYM (version B), and API 50 CHE (version D) strips (API System S. A., Montalieu-Vercieu, France), and other biochemical/physiological tests as deemed necessary.

Morphological Properties

Gram stain: negative Shape: bacillus Dimensions: 0.9 x 4 »m Flagella: peritrichous Motility: present, with swarming Chromogenesis: negative Sporulation: negative

Physiological/Biochemical Properties

Requirement for >CO₂: negative Requirement for ≧0.5% NaCl: negative Voges-Proskauer (room temperature): weak Voges-Proskauer (37°C): doubtful Methyl red: positive Production of H₂S (triple sugar iron agar): negative Growth in Simmons citrate medium: negative Utilisation of citrate: negative Utilisation of L-tartrate: negative Utilisation of mucate: negative Growth on acetate as sole carbon source: negative Growth on malonate as sole carbon source: negative Growth on MacConkey agar: positive Liquefaction of gelatine: negative Amino acid auxotrophy: L-histidine Pectin hydrolysis: negative Acid phosphatase (naphthol AS-BI phosphate, pH 5.4): negative Acid phosphatase (β-naphthyl phosphate, pH 5.4): positive Alkaline phosphatase (β-naphthyl phosphate, pH 8.5): positive Esterase (β-naphthyl butyrate): negative Esterase (β-naphthyl caprylate): negative Lipase (β-naphthyl myristate): negative Hydrolysis of Tween® 20: positive Hydrolysis of Tween® 80: negative Arginine dihydrolase: negative Arginine utilisation (Møller's medium): negative Ornithine decarboxylase: negative Lysine decarboxylase: negative Production of indole: negative Tryptophane deaminase: positive (weak) Phenylalanine deaminase: positive Urease: positive Trypsin: negative Chymotrypsin: negative Cystine arylamidase: negative Leucine arylamidase: positive Valine arylamidase: negative Catalase: positive Oxidase: negative Reduction of nitrate to nitrite: positive Growth in the presence of KCN: positive O-F Glucose, oxidative: positive O-F Glucose, fermentative: positive α-Galactosidase: negative β-Galactosidase: negative β-Glucuronidase: negative α-Glucosidase: negative β-Glucosidase: negative N-Acetyl-β-glucosaminidase: negative α-Fucosidase: negative α-Mannosidase: negative

Antibiotic Sensitivities

Antibiogrammes were obtained on Mueller-Hinton agar [Mueller, J. H., and Hinton. J., "A protein-free medium for primary isolation of the gonococcus and meningococcus", Proc. Soc. Exp. Biol. Med., 48: 330(1941)] supplemented with 0.15% starch [Olsen, M. A., and Scott, W. J., "Influence of starch in media used for the detection of heated bacterial spores", Nature, 157: 337(1946)] (14 h, 37°C), using Oxoid antibiotic susceptibility discs in accordance with the Kirby-Bauer procedure [Bauer, A. W., Kirby, W. M. M., Sherris, K. C., and Turck, M., "Antibiotic susceptibility testing by a standardized single disk method", Amer. J. Clin. Pathol., 45: 493(1966)].

Antibiotic (amount)	Diameter (mm) of inhibition zone	Interpretation
amikacin (30 »g)	17	susceptible
ampicillin (10 »g)	23	susceptible
benzylpenicillin (10 IU)	22	susceptible
cefuroxime (30 »g)	28	susceptible
cephalothin (30 »g)	18	susceptible
chloramphenicol (30 »g)	20	susceptible
colistin (10 »g)	11	susceptible
gentamicin (10 »g)	17	susceptible

One the basis of these results, the isolate corresponds to a strain of Proteus mirabilis according to the classification scheme of the ATCC; moreover, according to the ATCC, Proteus mirabilis NRRL B-18480 (Sigma-Tau SP237 subclone NACit I1) is unique and does not correspond to any other organism in their culture collection.

It has been observed that extensively-purified single clones of P. mirabilis NRRL B-18480 that are urease-positive (ure⁺) can give rise to urease-negative (ure⁻) clones at a low frequency; the morphological-physiological/biochemical profile and the antibiogramme of these ure⁻ variants are otherwise identical to those of their ure⁺ progenitors [cf. Farmer, J. J., III, Hickman, F. W., Brenner, D. J., Schreiber, M., and Rickenbach, D. G., "Unusual Enterobacteriaceae: 'Proteus rettgeri' that 'change' into Providencia stuartii", J. Clin. Microbiol., 6: 373 (1977); Collins, C. M., and Falkow, S., "Genetic analysis of an Escherichia coli urease locus: evidence of DNA rearrangement", J. Bacteriol., 170: 1041(1988)]. Moreover, the capacity of the ure⁻ variants to convert crotonobetaine to L-(-)-carnitine is unchanged. The reverse phenotypic transformation, i.e., the spontaneous appearance of a ure⁺ strain of P. mirabilis NRRL B-18480 from a ure⁻ antecedent, has not been observed by us (cf. Lewis, A. D., and Rosen. I. G., "Characterization of a Proteus rettgeri that transfers genes for urease production and lactose fermentation", American Society for Microbiology Annual Meeting, 1973, Abstract G 218).

( ii ) Proteus mirabilis NRRL B-18482 (Sigma-Tau GP 1AVI).

The organism was isolated from faeces obtained from a guinea-pig in the animal collection of Sigma-Tau Industrie Farmaceutiche Riunite S.p.A.

Fresh faecal matter (0.5-1.0 g) was incubated for 24 h without agitation in 4 mL of sterile brain-heart infusion (BHI) (Difco) + 1.5% (^w/_v) crotonobetaine monohydrate (pH 6.8, 37°C). Loopfuls of broth were taken from just below the meniscus and streaked onto plates of the medium described by Xilinas, M. E., Papavissiliou, J. T., and Legakis, N.J. ["Selective medium for growth of Proteus, J. Clin. Microbiol., 2: 459 (1975)], hereafter referred to as "Greek agar". After 48 h at 37°C, single colonies were picked and purified by repeated restreaking of single clones on plates of Greek agar + 1.5% (^w/_v) crotonobetaine monohydrate (pH 6.8, 37°C, 48 h) and MacConkey agar CS (Difco) + 1.5% (^w/_v) crotonobetaine monohydrate (pH 6.8, 37°C, 24 h).

The organism was maintained by daily transfer (37°C) on Greek agar + 1.5% (^w/_v) crotonobetaine monohydrate. It was typed in the Industrial Microbiology Laboratories at Sigma-Tau Industrie Farmaceutiche Riunite S.p.A. using morphological examination in conjunction with the API 20 E, API ZYM, and API 50 CHE strips, and other biochemical/physiological tests as deemed necessary.

Morphological Properties

Gram stain: negative Shape: bacillus Dimensions: 0.8 x 2»m Motility (NACit + cb; BHI + 1.5% (^w/_v) crotonobetaine monohydrate + 2% agar, pH 6.8): present, with swarming Chromogenesis: negative Sporulation: negative

Physiological/Biochemical Properties (37°C)

Requirement for >CO₂: negative Voges-Proskauer: weak Production of H₂S (triple sugar iron agar): positive Growth in Simmons citrate medium: negative Growth on MacConkey agar: positive Liquefaction of gelatine: negative Amino acid auxotrophy: none Acid phosphatase (naphthol AS-BI phosphate, pH 5.4): positive (weak) Acid phosphatase (β-naphthyl phosphate, pH 5.4): positive Alkaline phosphatase (β-naphthyl phosphate, pH 8.5): positive Esterase (β-naphthyl butyrate): positive (weak) Esterase (β-naphthyl caprylate): negative Lipase (β-naphthyl myristate): negative Arginine dihydrolase: negative Ornithine decarboxylase: positive Lysine decarboxylase: negative Production of indole: negative Tryptophane deaminase: positive Phenylalanine deaminase: positive Urease: positive Trypsin: negative Chymotrypsin: negative Cystine arylamidase: negative Leucine arylamidase: positive Valine arylamidase: negative Oxidase: negative O-F glucose, oxidative: positive O-F glucose, fermentative: positive α-Galactosidase: negative β-Galactosidase: negative β-Glucuronidase: negative α-Glucosidase: negative β-Glucosidase: negative N-Acetyl-β-glucosaminidase: negative α-Fucosidase: negative α-Mannosidase: negative

Antibiotic Sensitivities

Antibiogrammes were obtained exactly as described for P. mirabilis NRRL B-18480 (Sigma-Tau SP237 subclone NACit I1).

Antibiotic (amount)	Diameter (mm) of inhibition zone	Interpretation
amikacin (30 »g)	17	susceptible
ampicillin (10 »g)	22	susceptible
benzylpenicillin (10 IU)	18	intermediate
cefuroxime (30 »g)	22	susceptible
cephalothin (30 »g)	21	susceptible
chloramphenicol (30 »g)	20	susceptible
colistin (10 »g)	/	resistant
gentamicin (10 »g)	17	susceptible

On the basis of these results, the isolated organism corresponds to a strain of Proteus mirabilis according to the classification scheme of the API system.

( iii ) Proteus mirabilis NRRL B-18481(Sigma-Tau BT 1IV).

The organism was isolated from faeces obtained from a rabbit in the animal collection of Sigma-Tau Industrie Farmaceutiche Riunite S.p.A. exactly as described for Proteus mirabilis NRRL B-18482(Sigma-Tau GP 1AVI). The organism was maintained by daily transfer (37°C) on Greek agar + 1.5% (^w/_v) crotonobetaine monohydrate. It was typed in the Industrial Microbiology Laboratories of Sigma-Tau Industrie Farmaceutiche Riunite S.p.A. using morphological examination in conjunction with the API 20 E, API ZYM, and API 50 CHE strips, and other biochemical/physiological tests as deemed necessary.

Morphological Properties

Gram stain: negative Shape: bacillus Dimensions: 0.8 x 2 »m Motility (NACit + cb; BHI + 1.5% (^w/_v) crotonobetaine monohydrate + 2% agar, pH 6.8): present, with swarming Chromogenesis: negative Sporulation: negative

Physiological/Biochemical Properties (37°C)

Requirement for >CO₂: negative Voges-Proskauer: negative Production of H₂S (triple sugar iron agar): positive Growth in Simmons citrate medium: negative Growth on MacConkey agar: positive Liquefaction of gelatine: positive Amino acid auxotrophy: none Acid phosphatase (naphthol AS-BI phosphate, pH 5.4): positive (weak) Acid phosphatase (β-naphthyl phosphate, pH 5.4): positive Alkaline phosphatase (β-naphthyl phosphate, pH 8.5): positive Esterase (β-naphthyl butyrate): positive (weak) Esterase (β-naphthyl caprylate): negative Lipase (β-naphthyl myristate): negative Arginine dihydrolase: negative Ornithine decarboxylase: positive Lysine decarboxylase: negative Production of indole: negative Tryptophane deaminase: positive Phenylalanine deaminase: positive Urease: positive Trypsin: positive Chymotrypsin: negative Cystine arylamidase: negative Leucine arylamidase: positive Valine arylamidase: negative Oxidase: negative O-F Glucose, oxidative: positive O-F Glucose, fermentative: positive α-Galactosidase: negative β-Galactosidase: negative β-Glucuronidase: negative α-Glucosidase: negative β-Glucosidase: negative N-Acetyl-β-glucosaminidase: negative α-Fucosidase: negative α-Mannosidase: negative

Antibiotic Sensitivities

Antibiotic (amount)	Diameter (mm) of inhibition zone	Interpretation
amikacin (30 »g)	17	susceptible
ampicillin (10 »g)	20	susceptible
benzylpenicillin (10 IU)	16	intermediate
cefuroxime (30 »g)	20	susceptible
cephalothin (30 »g)	21	susceptible
chloramphenicol (30 »g)	18	susceptible
colistin (10 »g)	/	resistant
gentamicin (10 »g)	17	susceptible

( iv ) Proteus mirabilis B-18483 (Sigma-Tau R 2AIX).

The organism was isolated from faeces obtained from a rat in the animal collection of Sigma-Tau Industrie Farmaceutiche Riunite S. p. A. exactly as described for Proteus mirabilis NRRL B-18482(Sigma-Tau GP 1AVI). The organism was maintained by daily transfer (37°C) on Greek agar + 1.5% (^w/_v) crotonobetaine monohydrate. It was typed in the Industrial Microbiology Laboratories of Sigma-Tau Industrie Farmaceutiche Riunite S. p. A. using morphological examination in conjunction with the API 20 E, API ZYM, and API 50 CHE strips, and other biochemical/physiological tests as deemed necessary.

Morphological Properties

Gram stain: negative Shape: bacillus Dimensions: 0.8 x 3 » Motility (NACit + cb; BHI + 1.5% (^w/_v) crotonobetaine monohydrate + 2% agar, pH 6.8): present, with swarming Chromogenesis: negative Sporulation: negative

Physiological/Biochemical Properties (37°C)

Requirement for >CO₂: negative Voges-Proskauer: negative Production of H₂S (triple sugar iron agar): positive Growth in Simmons citrate medium: negative Growth on MacConkey agar: positive Liquefaction of gelatine: positive Amino acid auxotrophy: none Acid phosphatase (naphthol AS-BI phosphate, pH 5.4): negative Acid phosphatase (β-naphthyl phosphate, pH 5.4): positive Alkaline phosphatase (β-naphthyl phosphate, pH 8.5): positive Esterase (β-naphthyl butyrate): positive (weak) Esterase (β-naphthyl caprylate): negative Lipase (β-naphthyl myristate): negative Arginine dihydrolase: negative Ornithine decarboxylase: positive Lysine decarboxylase: negative Production of indole: negative Tryptophane deaminase: positive Phenylalanine deaminase: positive Urease: positive Trypsin: negative Chymotrypsin: negative Cystine arylamidase: negative Leucine arylamidase: positive Valine arylamidase: negative Oxidase: negative O-F Glucose, oxidative: positive O-F Glucose, fermentative: positive α-Galactosidase: negative β-Galactosidase: negative β-Glucuronidase: negative α-Glucosidase: negative β-Glucosidase: negative N-Acetyl-β-glucosaminidase: negative α-Fucosidase: negative α-Mannosidase: negative

Antibiotic Sensitivities

Antibiotic (amount)	Diameter (mm) of inhibition zone	Interpretation
amikacin (30 »g)	> 30	susceptible
ampicillin (10 »g)	> 30	susceptible
benzylpenicillin (10 IU)	25	susceptible
cefuroxime (30 »g)	> 30	susceptible
cephalothin (30 »g)	> 30	susceptible
chloramphenicol (30 »g)	/	resistant
colistin (10»g)	/	resistant
gentamicin (10 »g)	> 30	susceptible

COMPARATIVE BIOTRANSFORMATION DATA FOR THE SYNTHESIS OF L-(-)-CARNITINE FROM CROTONOBETAINE BY Proteus mirabilis ISOLATES IN SHAKEN FLASKS

Proteus mirabilis NRRL B-18480 (Sigma-Tau SP237 subclone NACit I1) was maintained by dally transfer on plates of NACit + cb; incubation temperature, 37°C. Proteus mirabilis NRRL B-18481 (Sigma-Tau BT 1IV), P. mirabilis NRRL B-18482 (Sigma-Tau GP 1AVI), and P. mirabilis NRRL B-18483 (Sigma-Tau R 2AIX) were maintained by daily transfer on plates of Greek agar + 1.5% (^w/_v) crotonobetaine monohydrate, pH 6.8; incubation temperature, 37°C.

Loopfuls of the foregoing strains were inoculated into 250-mL Erlenmeyer flasks, each containing 50 mL of brain-heart infusion + 1.5% (^w/_v) crotonobetaine monohydrate, pH 6.8. The flasks were shaken (200 rpm, 1-inch orbital eccentricity, 37°C) for 17 h, then the cells were collected by centrifugation (10 min, 12,000 xg, 4°C), and washed once with cold physiological saline (50 mL). Washed cell pellets were resuspended in non-sterile biotransformation medium (50 mL) consisting of 25 mM potassium phosphate + 1% (^w/_v) glycerol + 12% (^w/_v) crotonobetaine monohydrate, pH 6.0. Biotransformation reactions were shaken as above at 33°C for 96 h; during this period aliquots (100 »L) were withdrawn and assayed enzymically for L-(-)-carnitine by the method of Pearson, D. J., Chase, J. F. A., and Tubbs, P. K. ["The assay of (-)-carnitine and its O-acyl derivatives", Meth. Enzymol., 9: 612 (1966)].

The following results were obtained (corrected for evaporation over the period 0-96 h):

mM L-(-)-CARNITINE ACCUMULATED
h
5	104	80
24	260	184
48	285	257
72	299	286
96	340	315

h
5	84	102
24	229	258
48	254	295
72	307	292
96	311	296

% TURNOVER OF EXOGENOUS CROTONOBETAINE TO L-(-)-CARNITINE*
h
5	14	11
24	35	25
48	38	34
72	40	39
96	46	42

h
5	11	14
24	31	35
48	35	40
72	41	39
96	42	40

ALTERNATIVE COMPLEX MEDIA SUPPORTING HIGH "CROTONOBETAINE HYDRATASE" ACTIVITY IN Proteus mirabilis NRRL B-18480 (Sigma-Tau SP237 subclone NACit I1)

Proteus mirabilis NRRL B-18480 (Sigma-Tau SP237 subclone NACit I1) was maintained by daily transfer on plates of NACit + cb; incubation temperature, 37°C. Loopfuls of this strain were inoculated into a 250-mL Erlenmeyer flask containing 50 mL of diverse media:

(a) Brain-heart infusion (Difco) + 1.5% (w/v) crotonobetaine monohydrate, pH 6.8.
(b) 0.9% Nutrient broth (Difco) + 0.9% yeast exact (Difco) + 0.9% Soytone (Difco) + 0.2% glucose + 0.5% sodium chloride + 0.25% dibasic sodium phosphate (anhydrous) + 1.5% (w/v) crotonobetaine monohydrate, pH 6.8.
(c) 1.35% Nutrient broth + 1.35% Soytone + 0.2% glucose + 0.5% sodium chloride + 0.25% dibasic sodium phosphate (anhydrous) + 1.5% (w/v) crotonobetaine monohydrate, pH 6.8.
(d) 1.35% Soytone + 1.35% yeast extract + 0.2% glucose + 0.5% sodium chloride + 0.25% dibasic sodium phosphate (anhydrous) + 1.5% (w/v) crotonobetaine monohydrate, pH 6.8.
(e) 1.35% Nutrient broth + 1.35% yeast extract + 0.2% glucose + 0.25% sodium chloride + 0.25% dibasic sodium phosphate (anhydrous) + 1.5% (w/v) crotonobetaine monohydrate, pH 6.8.
(f) 0.9% Nutrient broth + 0.9% Soytone + 0.9% yeast extract + 0.5% sodium chloride + 0.25% dibasic sodium phosphate (anhydrous) + 1.5% (w/v) crotonobetaine monohydrate, pH 6.8.
(g) 0.9% Nutrient broth + 0.9% Soytone + 0.9% yeast extract + 0.2% glucose + 0.25% dibasic sodium phosphate (anhydrous) + 1.5% (w/v) crotonobetaine monohydrate, pH 6.8.
(h) 0.9% Nutrient broth + 0.9% Soytone + 0.9% yeast extract + 0.2% glucose + 0.5% sodium chloride + 1.5% (w/v) crotonobetaine monohydrate, pH 6.8.
(i) Brain-heart infusion, pH 6.8.

The flasks were shaken (200 rpm, 1-inch orbital eccentricity, (37°C) for 17 h, then the cells from each flask were harvested separately by centrifugation (10 min, 12.000 xg, 4°C), and washed once with cold physiological saline (50 mL). Washed cell pellets were resuspended in non-sterile biotransformation medium (50 mL) consisting of 25 mM potassium phosphate + 1% (^w/_v) glycerol + 12% (^w/_v) crotonobetaine monohydrate, pH 6.0. Biotransformation reaction mixtures were shaken as above at 33°C for 96 h; during this period aliquots (100 »L) were withdrawn and assayed enzymically for L-(-)-carnitine as previously described.

mM L-(-)-CARNITINE ACCUMULATED
h
5	85	130	80	140	164	91	130	104	18
24	232	244	173	256	256	232	217	244	71
48	278	285	243	279	266	272	222	271	113
72	334	323	255	299	299	317	252	283	125
96	338	347	268	315	323	323	259	297	129

% TURNOVER OF EXOGENOUS CROTONOBETAINE TO L-(-)-CARNITINE*
h
5	11	17	11	19	22	12	17	14	2
24	31	33	23	34	34	31	29	33	10
48	37	38	33	37	36	37	30	36	15
72	45	43	34	40	40	43	34	38	17
96	45	47	36	42	43	43	35	40	17

BIOTRANSFORMATION OF CROTONOBETAINE TO L-(-)-CARNITINE BY Proteus mirabilis NRRL B-18480 IN A BENCHTOP FERMENTOR

The biotransformation of crotonobetaine inner salt to L-(-)-carnitine was accomplished using cells of P. mirabilis NRRL B-18480 in a 2-L benchtop fermentor, as follows:

The bacterium was maintained by daily transfer on slants of brain-heart infusion agar (Difco) + 1.5% (^w/_v) crotonobetaine inner salt; incubation temperature, 37°C.

The contents of a 24-h slant were inoculated into a baffled 1000-mL Erlenmeyer flask containing 250 mL of brain-heart infusion (Difco) + 1.5% (^w/_v) crotonobetaine inner salt. The flask was continuously shaken (170 rpm, 2-inch orbital eccentricity, 37°C) for 6 h, after which time the broth was used to inoculate six second-stage cultures, each containing 250 mL of brain-heart infusion + 1.5% (^w/_v) crotonobetaine inner salt, in baffled 1000-mL Erlenmeyer flasks. These flasks were shaken as described, above for ca. 15 h; the cells were then recovered by centrifugation (20 min. 8.500 xg, 4°C) and resuspended in a biotransformation medium (1 L) consisting of 25 mM potassium phosphate + 0.5% (^w/_v) glycerol + 10% (^w/_v) crotonobetaine inner salt. A 2-L Biolafitte fermentor was charged with the cell-containing biotransformation medium and operated for 24 h under the following conditions: agitation rate: 150 rpm air flow rate: 1v/v/m initial pH: ∼6.8 working volume: 1 L temperature: 35°C.

A 10% (^w/_v) glycerol solution was continuously pumped into the fermentor at a rate of 5 mL/h.

Unreacted crotonobetaine and L-(-)-carnitine concentrations were monitored by high-pressure liquid chromatography (HPLC): column: 10-» Partisil SCX, 4 mm x 250 mm (PoliConsult Scientifica s.r.l., Rome, Italy) column temperature: 35°C injection volume: 10 »L mobile phase: 75 mM KH₂PO₄-CH₃CN (4:6), pH unadjusted detector: refractive index retention times (min): L-(-)-carnitine, 8.52 crotonobetaine, 9.81 gamma-butyrobetaine, 12.08.

At 22 h, the biotransformation medium was collected and filtered.

The filtrate, analyzed enzymically for L-(-)-carnitine as previously described, was found to contain 48.0 mg/mL L-(-)-carnitine and 57.3 mg/mL unreacted crotonobetaine.

Molar turnover of crotonobetaine to L-(-)-carnitine: 43%. Yield of L-(-)-carnitine: 100%.

ISOLATION OF L-(-)-CARNITINE FROM THE BIOTRANSFORMATION FLUID.

The filtered biotransformation fluid was successively passed through columns of Amberlite^R ion-exchange resins, viz. IRA-402 basic resin (OH-form) and IRC-50 acid resin (H⁺ form), to remove inorganic cations and anions. The eluate was concentrated in vacuo (50°C), and the concentrate treated with iso butanol three times to azeotropically lower the water content to 5-10% (^w/_w).

The resulting mixture was crystallised using 3 parts (^w/_v) of iso butanol, to yield a precipitate containing 4 parts of L-(-)-carnitine and 1 part of crotonobetaine (^w/_w) [HPLC]. After three successive crystallisations, a solid consisting of L-(-)-carnitine with <0.5% (^w/_w) of crotonobetaine (HPLC) was obtained.

By this procedure, ca. 50% (^w/_w) of the L-(-)-carnitine present in the biotransformation medium was recovered [ ]²⁵ = -31.5°. The mother liquors were collected and recycled in succeeding biotransformation.

Claims

Proteus mirabilis NRRL B-18480 and its descendants, mutants, and derivatives that retain the progenitor's capacity to transform crotonobetaine to L-(-)-carnitine.
Proteus mirabilis NRRL B-18481 and its descendants, mutants, and derivatives that retain the progenitor's capacity to transform crotonobetaine to L-(-)-carnitine.
Proteus mirabilis NRRL B-18482 and its descendants, mutants, and derivatives that retain the progenitor's capacity to transform crotonobetaine to L-(-)-carnitine.
Proteus mirabilis NRRL B-18483 and its descendants, mutants, and derivatives that retain the progenitor's capacity to transform crotonobetaine to L-(-)-carnitine.
Descendants, mutants, and derivatives of the Proteus mirabilis strains of claims 1, 2, 3 and 4 that retain their capacity to transform crotonobetaine to L-(-)-carnitine either as free cells or as immobilized cells.
A microbiological process for producing L-(-)-carnitine that comprises:
(a) preparing a surface culture on a solid medium of a microorganism capable of stereoselectively hydrating crotonobetaine to L-(-)-carnitine;

(b) preparing a catalytically-active biomass by inoculating a liquid medium containing crotonobetaine with the culture obtained in step (a);

(c) separating the biomass from the liquid medium;

(d) suspending the separated biomass in a biotransformation medium containing al least 10% (^w/_v) of crotonobetaine inner salt;

(e) recovering L-(-)-carnitine from the biotransformation fluid; wherein said microorganism is selected from the group consisting of Proteus mirabilis NRRL B-18480, Proteus mirabilis NRRL B-18481, Proteus mirabilis NRRL B-18482, Proteus mirabilis NRRL B-18483.
The process of claim 6, wherein the biotransformation medium contains 10-12% (^w/_v) crotonobetaine inner salt.
The process of claim 6, wherein the solid medium of step (a) contains crotonobetaine.
The process of claim 6, wherein the separation step (c) is accomplished by centrifugation.
The process of claim 6, wherein the separation step (c) is accomplished by filtration.