AU625525B2 - A method of discontinuous production of l-carnitine by a microbiological process - Google Patents
A method of discontinuous production of l-carnitine by a microbiological process Download PDFInfo
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- AU625525B2 AU625525B2 AU59837/90A AU5983790A AU625525B2 AU 625525 B2 AU625525 B2 AU 625525B2 AU 59837/90 A AU59837/90 A AU 59837/90A AU 5983790 A AU5983790 A AU 5983790A AU 625525 B2 AU625525 B2 AU 625525B2
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- carnitine
- butyrobetaine
- betaine
- crotonobetaine
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- 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
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/007—Carnitine; Butyrobetaine; Crotonobetaine
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- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
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Abstract
The process starts from gamma -butyrobetaine and/or crotonobetaine.
Description
Ir~ To: The Commissioner of Patents "iiiiiii Li i T e s o e1 it t L- t 1 1 COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952-62 COMPLETE SPECIFICATION
(ORIGINAL)
Form r? 1 FOR OFFICE USE: Application Number: Lodged: Class Int. Class Complete Specification Lodged: Accepted: Published: Priority: Related Art: TO BE COMPLETED BY APPLICANT Name of Applicant: Address of Applicant: Actual Inventor: Address for Service: TO BE COMPLETED BY APPLICANT LONZA LTD Gampel/Valais, Switzerland Frans HOEKS care of R.K. MADDERN ASSOCIATES, 345 King William Street, Adelaide, South Australia, 5000 Complete Specification for the invention entitled: "A METHOD OF DISCONTINUOUS PRODUCTION OF L-CARNITINE BY A MICROBIOLOGICAL PROCESS" The following statement is a full description of this invention, including the best method of performing it known to mn. us.
Signatur). of declarant) Unterschrifen)de Hans Peter irter Beat Rauber Dkaranten. t o (Vice-Directo) (Confidential other sinature is e l er p 3emerkung. h erlatubign g dr Unterschrift Ist nic benoetigt.
I.I
la- A method of discontinuous production of L-carnitine by a microbiological process Description The invention relates to a novel method of producing Lcarnitine by a microbiological process.
L-carnitine is an essential substance for human metabolism, e.g. for breaking down fatty acids. Artifically produced Lcarnitine is therefore used in pharmaceutical preparations as an active principle against corresponding deficiency o diseases.
B t 0 It is known to produce L-carnitine from y-butyrobetaine.
a 0 S The y -butyrobetaine, in the presence of sodium-2- 0'015 oxoglutarate, a reducing agent, a source of iron ions and atmospheric oxygen as a hydroxyl-group donor, is brought into contact with a hydroxylase enzyme, released from spores of Neurospora crassa (US-PS 4 371 618). This process has the disadvantage of requiring a number of co-factors, which have to be externally supplied. In the reaction, for example, stoichiometric quantities of 2-oxoglutarate are oxidatively decarboxylated to succinate. Fe 2 is required as an 02 activator, ascorbate is needed to keep the iron ion in the reduced form, and catalase is needed to destroy the traces of harmful H202 which are produced.
Lindstedt et al, IBiochemistry 6, 1262-1270 (1967) "The Formation and Degradation of Carnitin in Pseudomonas"] isolated a microorganism of the genus Pseudomonas, which is grown by using y-butyrobetaine as a source of C and N. The first reaction in the breakdown process was the hydroxylation of y-butyrobetaine to L-carnitine, which was now 2 produced as an intermediate and then completely catabolised to C0 2
H
2 0 and NH 3 The hydroxylase involved, obtained from the bacteria, also has the aforementioned disadvantageous need for co-factors when used for production of L-carnitine [Lindstedt et al, Biochemistry 16, 2181-2188 (1977) "Purification and Properties of y -Butyrobetaine Hydroxylase from Pseudomonas sp. AK It is also known from US-PS 4 708 936 to produce L-carnitine by a continuous microbiological process. Disadvantages have been found, however, in that in this continuous process the stability of the strain has to be very high (more than 1000 h), and relatively low limits are set to the concentration of o 15 product in the medium. The product/educt ratio is also relatively low.
0o 00 An object of the invention is to avoid the disadvantages of known methods and provide a method of enantioselective microbiological production of high yields of L-carnitine from crotonobetaine and/or y-butyrobetaine.
According to the present invention, there is provided a method of discontinuous production of L-carnitine by microbiological 25 processing of crotonobetaine and/or y -butyrobetaine, wherein the Y -butyrobetaine and/or crotonobetaine, together with betaine 4, as a source 6f C and N and an additional carbon source,are added to a culture medium containing a microorganism of the.
genus Pseudomonas, Rhizobium, Agrobacterium, E.coli or a yeast of the genus Saccharomyces, and L-carnitine is separated after the maximum L-carnitine concentration has been obtained.
Advantageously, use is made of microorganisms of the genus Rhizobium, preferably the strain HK 1331b deposited on 8.2.1985 at the Deutsche Sammlung von Mikroorganismen (DSM, 3 Gesellschaft fOr Biotechnologische Forschung mbH, r Griesebachstr. 8, D-3400 G6ttingen, No. DSM 3225, and the strain HK 13 deposited on 23.1.1984 at the same place, no.
DSM 2903.
Strains of microorganisms altered by genetic engineering are also suitable for the process.
In contrast to the systems known in the prior art, the microorganisms according to the invention use H20 instead of 02 as a hydroxyl group donor, as we have found from our own investigations using H2 1 8 0 and 1802.
0 o The selection and characterisation of these preferred microorganisms are described in EP-A 0 158 194, the contents of which are incorporated herein by reference.
The method according to the invention of producing Lcarnitine is preferably carried out as follows. Productive biomass is produced in a first or "batch" phase. To this end, one of the aforementioned strains is cultivated in the manner set out in EP-A 0 158 194, in a I lr sterilized mineral medium, preferably containing vitamins [Kulla et al, Arch. Microbiol. 135, 1 (1983)] at 20 to 400C, preferably at 30 0 C, advantageously at a pH between 6 to 8, preferably 7, and for 5 to 80 hours, preferably 15 to hours. The medium advantageously contains 0.01 to 10 wt.%, preferably 0.01 to 5 wt.% of choline, glutamate, acetate, I dimethylglycine or betaine as a growth substrate. It is particularly preferable to use betaine together with glutamate or another carbon source in proportions of 0.02 to 5 wt.% each.
The starting compounds to be converted, i.e. Tbutyrobetaine, crotonobetaine or mixtures thereof, are also
H
4 supplied in the batch phase in proportions of 0.01 to preferably 0.1 to 5 of the reaction medium.
The y-buty.robetaine or crotonobetaine can be present partly in the form of the hydrochloride salt or as a free internal salt.
The biomass cultivated in the batch phase can be used to inoculate other cultures. The other cultures advantageously have the same composition as the pre-cultures.
in the method according to the invention the biomass *t cultivated in the batch phase is the starting point for Lcarnitine production in the "fed-batch" phase.
The culture medium in the "fed-batch" phase is substantially the same as in the batch phase.
The "fed-batch" phase is characterised in that the educt crotonobetaine and/or y -butyrobetaine, betaine and an 20 additional carbon source are added in measured quantities to the culture solution.
The carbon sources can be compounds conventionally used by the experts and known in the literature e.g. for Rhizobium strains [The prokaryotes, Chapter 67, The genus Rhizobium, Springer Verlag 1981, page 825].
The carbon sources are advantageously sugars such as glucose or fructose, sugar alcohols such as glycerol, or organic acids such as acetic acid.
Preferably glucose or glycerol is used.
1 4 L1 L--iiii ii. i- 5 The molar ratio of carbon to nitrogen (C to N) is advantageously chosen between more than 5 mol C to 1 mol N and 10000 mol C to 1 mol N, preferably between 10 mol C to 1 mol N and 100 mol C to 1 mol N.
At very high carbon-nitrogen ratios, it may be necessary to add a nitrogen source in addition to betaine. The additional nitrogen source can be a known substance conventionally used by the experts, e.g. ammonium.
Additional nutrients such as a sulphur source, a phosphorus source, trace elements, vitamins or complex nutrients such as meat extracts, yeast extracts or swollen maize water, can a.o be added in measured amounts.
4 1 The educts Y-butyrobetaine or crotonobetaine are 15 advantageously added in amounts such that their concentration a 4 in the culture medium is advantageously between 0.005 and S preferably between 0.02 and 2%.
The carbon/nitrogen source is added at a rate depending on the amount of biomass in the bioreactor. One known method of determining the concentration of biomass is to measure the dry weight of the culture solution. Depending on the dry weight and the desired carbon/nitrogen ratio, the carbon Ssource and betaine are added to the fermenter at a rate of advantageously 0.01 to 100, preferably 0.1 to 10 mol carbon per kg dry weight in the fermenter per hour.
It is advantageous to operate at a temperature range of to 400C and a pH between 6 and 8.
After cultivation in the "fed-batch" phase, usually for to 250 hours, a concentation of L-carnitine of more than 6% can be obtained in the culture.
m 6 In an alternative modification of the process, part of the culture solution can be drawn off after fermentation and additional "fed-batch" cultivation can be started with the remaining part, once additional culture medium has been added.
This "repeated fed-batch" process can increase the volumetric productivity of fermentation.
The culture can be lightened by temporarily demineralizing and purifying the y-butyrobetaine and/or crotonobetaine, using ion exchangers or electrodialysis.
Production of L-carnitine from a culture solution is S known e.g. from EP-A 195 944 and can be carried out by freeing the solution from charged particles (cations and anions) after separating the biomass e.g. by centrifuging, 6 ultrafiltration or microfiltration in a laboratory electrodialysis plant. The end point of demineralization can be determined by measurements of conductivity. In the process the salts migrate into the flow of concentrate, whereas the L-carnitine remains as an internal salt ("betaine") in the flow of diluate. The resulting yields of L-carnitine in the diluate after, demineralization can be more than As an alternative to electrodialysis, L-carnitine can be demineralized by using a strongly acid cation exchanger in the H form [compare J. P. Vandecasteele, Appl. Environ. Microbiol.
39, 327 (1980)]. The solution is made to flow over an ion exchange column until the ion exchanger is exhausted and Lcarnitine breaks through. The anions go as free acids into the flow, whereas the cations remain on the ion exchanger.
After the ion exchanger has been washed to neutrality with water, the L-carnitine can be eluted with aqueous ammonia L -i .L 7 solution. The resulting yields of L-carnitine in the ammoniacal eluate can be more than The dilute L-carnitine solutions obtained by electrodialysis or by ion exchange can be concentrated by evaporation or reverse osmosis and then dewatered azeotropically.
The L-carnitine thus obtained can be converted into pure white L-carnitine by subsequent recrystallization advantageously from isobutanol/acetone, methanol, ethanol or n-butanol; or from a combination of solvents in which Lcarnitine is only slightly soluble, e.g. acetone, ethyl acetate, -y-butyl acetate, isobutyl methyl ketone or a acetonitrile, preferably isobutanol, and additional treatment with active carbon. This method can be used to I obtain L-carnitine with specific rotations of [*]25D -30.5 to -31.0, c 1 in HzO [the value in the literature is Strack et al, Hoppe-Seyler's Z.f. physiolog.Chem., 318 (1960), 129], the content being more than 99% (HPLC).
Example 1 i A 0.3 1 preculture of the strain HK 1331b was cultivated in the following nutrient medium at 30*C, pH 7.0 for 24 hours: Composition of nutrient medium L-glutamate 2 g Betaine 2 g Y-butyrobetaine 2 g Buffer solution 100 ml Mg-Ca-Fe solution 25 ml Solution of trace elements 1 ml 4 -8- Vitamin solution Iml with water to 1 Buffer solution Na2 S04 ig Na2 HP04 .2H2 0 25.08 9 KH2 P04 10 9 NaCl 30 g with water to 1 1 Mg-Ca-Fe solution MgCl2 .6H20 16 g CaC12.2H20 0.58 g FeCl3 .6H20 0.032 g with water to 1 1 Solution of trace elements ZnSO4.7H20 100 mg MnC12.4H20 30 mg H3 B03 300 mg 1 ltCOC12 .6H2 0 200 mg CUC12.2H20 10 mg NiCl2 .6H20 22 mg Na2 M004 .2H2 0 30 mg with wa'ter to 1 1 Vitamin solution Pyridoxal.HC1 10 mg Riboflavin 5 mg Nicotinamide 5 mg Thiamin.HCI 5 mg Biotin 2 mg Sodium pantothenate 5 mg 9 p-aminobenzoic acid 5 mg Folic acid 2 mg Vitamin B 12 5 mg with water to 1 1 Using the preculture, 5 litres of nutrient medium having the same composition were inoculated in the fermenter and cultivated at 30 0 C and pH 7 for 24 hours. The pH was kept constant at 7.0 by adding 8% phosphoric acid.
a) "Fed-batch" operation was chen started. Two solutions having the following composition were continuously added in 0 0 9 measured amounts.
0 0 0 Composition of carbon-nitrogen feed 4 5 Betaine 100 g a 0 4 SGlucose 135 g with water to 1 1 Carbon/nitrogen ratio 10.3 1 0Ba"'0 Composition of the -butyrobetaine feed -butyrobetaine 300 g 4, with water to 1 1 The solution containing the carbon and nitrogen source was added at rate of 4.5 ml/h, corresponding to a specific feed rate of 4 mol C/kg dry weight/h at the beginning of the "fed-batch" phase. The dry weight was determined in the usual manner Appl. Microbiol. Biotechnol 28 [1988] 109f.). The concentration of y-butyrobetaine and Lcarnitine was determined by HPLC. The y-butyrobetaine solution was added at a rate such that the concentration of y-butyrobetaine in the fermenter was between 0.05 and 150 hours after inoculating the fermenter, the L- 10 carnitine concentration was 6.4% and the concentration of unreacted X-butyrobetaine was 0.29%. This corresponded to conversion of y-butyrobetaine.
b) "Fed-batch" operation was carried out as per a) using the same composition of the carbon/nitrogen feed and the fbutyrobetaine feed.
The solution containing the carbon and nitrogen source was added at a rate of 4.5 ml/h, corresponding to a specific feed rate of 4 mol C/kg dry weight/h at the beginning of the "fed-batch" phase. The dry weight was determined in conventional manner Appl. Microbiol. Biotechnol 28 [1988] 109f.). The concentration of T-butyrobetaine and Lcarnitine was determined by HPLC. The y-butyrobetaine solution was added in a proportion such that the concentration of '-butyrobetaine in the fermenter was between 0.05 and 0.15 155 hours after inoculating the fermenter, the concentration of L-carnitine was 6.3% and the concentration of unreacted y-butyrobetaine was 0.13%.
This corresponded to 98% conversion of y-butyrobetaine.
Isolation of L-carnitine Pure L-carnitine could be isolated from the solution, which contained 64 g/l L-carnitine, 2.9 g/1 butyrobetaine and inorganic salts, by a method as set out in EP-A 195 944.
The product after the purification stage by recrystallization was 56.3 g of white L-carnitine, HPLC 99%, specific rotation 2 oD (c 1, i ii 11 Example 2 300 ml of the nutrient medium described in Example 1 but containing 2 g/1 crotonobetaine instead of T-butyrobetaine, was inoculated with the strain HK 1331b and cultivated at pH 7 for 24 hours.
Using the preculture, 5 litres of nutrient medium were inoculated as in Example 1 and cultivated at 30°C and pH for 24 hours. The pH was kept constant at 7.0 by adding 8% phosphoric acid. "Fed-batch" operation was then started.
As described in Example 1, the carbon and nitrogen source and a solution having the following composition were Scontinuously added.
Composition of crotonobetaine feed Crotonobetaine 300 g with water to 1 litre.
o *IA.0 The solution containing the carbon and nitrogen source was added at a rate of 4.5 ml/h, corresponding to a specific feed rate of about 4 mol C/kg dry weight/h at the beginning of the "fed-batch" phase. The sample was treated and Sanalysed in the same manner as described in Example 1. The crotonobetaine was added at a rate such that the concentration of crotonobetaine in the fermenter was between 6 i t0 0.05 and 0.5 150 hours after inoculation of the S" fermenter, the concentration of L-carnitine was 6.1% and the concentration of unreacted crotonobetaine was 0.17%. This corresponded to 95% conversion of crotonobetaine 12 Isolation of L-carnit 4 ne Pure L-carnitine could be prepared from the solution, which contained 61 g/l L-carnitine, 1.7 g/l crotonobetaine and inorganic salts, by a method as set out in EP-A 195 944.
The product after recrystallization was 52 g of white L-carnitine having the same specifications as in Example 1.
I
i 4
II
4 4 4 (4 4 4 I
Claims (7)
1. A method of discontinuous production of L-carnitine by microbiological processing of crotono- betaine and/or Y -butyrobetaine, wherein Y -butyro- betaine and/or crotonobetaine, together with betaine as a source of carbon and nitrogen and an additional carbon source, are added to a culture medium containing a microorganism of the genus Pseudomonas, Rhizobium, Agrobacterium, or E.coli or a yeast of the genus Saccharomyces, and L-carnitine is separated after a 1-10 maximum L-carnitine concentration has been obtained.
2. A method according to claim 1, wherein the crotonobetaine and/or Y -butyrobetaine are added in proportions such that their concentration in the l culture medium is between 0.005 and
3. A method according to claim 1 or 2, wherein the betaine and the additional carbon source are added in proportions such that the molar ratio of carbon to nitrogen is from more than 5 to 1 to 10000 to 1. o 1 i 1 ,yur uAy laon ot y-butyrobetaine to L-carnitine, which was 14
4. A method according to claim 1, wherein a sugar, sugar alcohol or an organic acid is used as the additional carbon source.
A method according to claim 4, wherein glucose or glycerol is used as the additional carbon source.
6. A method according to claim 1, wherein betaine and the carbon source are added at a rate of between 0.01 and 100 mol of carbon per kg dry weight in the a o C 0 fermenter per hour. 0 0 0
7. A method of discontinuous production of o L-carnitine by microbiological processing of crotonobetaine and/or y -butyrobetaine, substantially as hereinbefore described in exampleL 1 or 2. oo Dated this 26th day of July, 1990. a LONZA LTD S, By their Patent Attorneys R K MADDERN ASSOCIATES a a U 0. i k Si ASA ABSTRACT i I e00 o 000 £a a o O 0 0 0 00 o0 0 0 00 0 0 0 0 00 00 0 0 0 0 A method of discontinuous production of L-carnitine by microbiological processing, comprising adding y -butyrobetaine and/or crotonobetaine, together with betaine as a source of carbon and nitrogen and an additional carbon source, to a culture medium containing a microorganism of the genus Pseudomonas, Rhizobium, Agrobacterium, or E.coli or a yeast of the genus Saccharomyces, and separating L-carnitine from h'b medium after a maximum L-carnitine concentration has been obtained. 0404 0 4 44 47
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CH281389 | 1989-07-28 | ||
| CH2813/89 | 1989-07-28 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| AU5983790A AU5983790A (en) | 1991-01-31 |
| AU625525B2 true AU625525B2 (en) | 1992-07-16 |
Family
ID=4242178
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| AU59837/90A Expired AU625525B2 (en) | 1989-07-28 | 1990-07-26 | A method of discontinuous production of l-carnitine by a microbiological process |
Country Status (15)
| Country | Link |
|---|---|
| EP (1) | EP0410430B1 (en) |
| JP (1) | JPH0751071B2 (en) |
| AT (1) | ATE132535T1 (en) |
| AU (1) | AU625525B2 (en) |
| BR (1) | BR9003672A (en) |
| CA (1) | CA2021869C (en) |
| DD (1) | DD296702A5 (en) |
| DE (1) | DE59010027D1 (en) |
| DK (1) | DK0410430T3 (en) |
| ES (1) | ES2081878T3 (en) |
| FI (1) | FI102083B1 (en) |
| IE (1) | IE902689A1 (en) |
| IL (1) | IL95196A (en) |
| MX (1) | MX170707B (en) |
| NO (1) | NO178235C (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| IT1261230B (en) * | 1993-04-08 | 1996-05-09 | Sigma Tau Ind Farmaceuti | IMPROVED PROCEDURE FOR THE PREPARATION OF L - (-) - CARNITINA STARTING FROM ITS PRECURSORS WITH OPPOSED CONFIGURATION. |
| AU7854694A (en) * | 1993-10-08 | 1995-05-04 | Lonza A.G. | Genes for butyrobetaine/crotonobetaine-l-carnitine metabolism and their use for the microbiological production of l-carine |
| WO2006051387A1 (en) * | 2004-11-09 | 2006-05-18 | University Of Stellenbosch | Method of producing a carnitine-synthesising micro-organism |
| KR100713103B1 (en) * | 2005-07-07 | 2007-05-02 | 씨제이 주식회사 | - - A microorganism of Enterobacteriacae genus haboring genes associated with L-carintine biosynthesis and method of producing L-carnitine using the microorganism |
| EP2325164A1 (en) | 2009-11-18 | 2011-05-25 | Lonza Ltd. | Methods for the production of l-carnitine |
| US8604237B2 (en) | 2009-11-18 | 2013-12-10 | Lonza Ltd | Methods for the production of L-carnitine |
| KR102329115B1 (en) | 2014-05-30 | 2021-11-19 | 엘지전자 주식회사 | Laundry Treating Apparatus |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS59192095A (en) * | 1983-04-13 | 1984-10-31 | Ajinomoto Co Inc | Preparation of l-carnitine |
| FI86889C (en) * | 1984-03-29 | 1992-10-26 | Lonza Ag | Process for the preparation of L-carnitine in a microbiological manner |
| CH664374A5 (en) * | 1985-02-27 | 1988-02-29 | Lonza Ag | METHOD FOR PRODUCING L-CARNITIN BY A MICROBIOLOGICAL WAY. |
-
1990
- 1990-07-24 MX MX021712A patent/MX170707B/en unknown
- 1990-07-24 IE IE268990A patent/IE902689A1/en not_active IP Right Cessation
- 1990-07-24 CA CA002021869A patent/CA2021869C/en not_active Expired - Lifetime
- 1990-07-24 JP JP2196017A patent/JPH0751071B2/en not_active Expired - Fee Related
- 1990-07-25 DK DK90114287.7T patent/DK0410430T3/en active
- 1990-07-25 EP EP90114287A patent/EP0410430B1/en not_active Expired - Lifetime
- 1990-07-25 AT AT90114287T patent/ATE132535T1/en not_active IP Right Cessation
- 1990-07-25 DE DE59010027T patent/DE59010027D1/en not_active Expired - Lifetime
- 1990-07-25 ES ES90114287T patent/ES2081878T3/en not_active Expired - Lifetime
- 1990-07-26 IL IL9519690A patent/IL95196A/en active IP Right Grant
- 1990-07-26 AU AU59837/90A patent/AU625525B2/en not_active Expired
- 1990-07-26 FI FI903742A patent/FI102083B1/en active IP Right Grant
- 1990-07-27 BR BR909003672A patent/BR9003672A/en unknown
- 1990-07-27 NO NO903338A patent/NO178235C/en not_active IP Right Cessation
- 1990-07-27 DD DD90343118A patent/DD296702A5/en not_active IP Right Cessation
Also Published As
| Publication number | Publication date |
|---|---|
| FI903742A0 (en) | 1990-07-26 |
| DK0410430T3 (en) | 1996-01-29 |
| NO178235C (en) | 1996-02-14 |
| BR9003672A (en) | 1991-08-27 |
| ATE132535T1 (en) | 1996-01-15 |
| NO178235B (en) | 1995-11-06 |
| AU5983790A (en) | 1991-01-31 |
| DE59010027D1 (en) | 1996-02-15 |
| NO903338D0 (en) | 1990-07-27 |
| IL95196A0 (en) | 1991-06-10 |
| FI102083B (en) | 1998-10-15 |
| IE902689A1 (en) | 1991-02-27 |
| FI102083B1 (en) | 1998-10-15 |
| JPH0751071B2 (en) | 1995-06-05 |
| DD296702A5 (en) | 1991-12-12 |
| NO903338L (en) | 1991-01-29 |
| EP0410430A2 (en) | 1991-01-30 |
| CA2021869A1 (en) | 1991-01-29 |
| ES2081878T3 (en) | 1996-03-16 |
| JPH0376591A (en) | 1991-04-02 |
| CA2021869C (en) | 2000-09-05 |
| MX170707B (en) | 1993-09-08 |
| IL95196A (en) | 1995-05-26 |
| EP0410430A3 (en) | 1991-08-21 |
| EP0410430B1 (en) | 1996-01-03 |
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