CA2127544A1 - The enzymatic oxidation of (d)-2-hydroxy carboxylic acids to 2-keto carboxylic acids - Google Patents
The enzymatic oxidation of (d)-2-hydroxy carboxylic acids to 2-keto carboxylic acidsInfo
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- CA2127544A1 CA2127544A1 CA002127544A CA2127544A CA2127544A1 CA 2127544 A1 CA2127544 A1 CA 2127544A1 CA 002127544 A CA002127544 A CA 002127544A CA 2127544 A CA2127544 A CA 2127544A CA 2127544 A1 CA2127544 A1 CA 2127544A1
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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
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
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- 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
- C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
- C12P41/002—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by oxidation/reduction reactions
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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
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
- C12P7/26—Ketones
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Abstract
A process for the production of 2-ketonic carboxylic acids from (D)-2-hydroxycarboxylic acids using a biocatalyst, the electrons produced during the oxidation of (D)-2-hydroxycarboxylic acids being transferred to a quinone derivative.
Description
~ 12 7 ~ ~ ~1 o. z . OOS0/43032 The enzymatic oxidation of_~D~-2-h~droxy carboxylic acid~ to 2-keto carboxvlic acids The in~ention relatos to an Lmproved proce~3 for preparing 2-~eto carboxylic acids from (D)-2-hydroxy carboxylic acid~ using a biocatalyst and to the prepara-tion of mixtures of ~L)-2-hydroxy carboxylic acid~ and 2-~eto carboxylic acids from (D,L)-2-hydroxy carboxylic acids.
Reductions carried out by microorganism~ which are provided with the reducing equivalent~ via an exter-nal electron carrier which i9 regenerated electrochemi-cally ar~ known. It i8 po~sible to employ for thi~ a large number of microorgani~m~ and, a~ electron carrier~, a number of dye~ and other ~ubst~nce~ (DE-A 32 26 888 and DE-A 33 32 562).
EP-A 260 611 di~clo~es the oxidation of (D)-2-hydroxy carboxylic acid~ to 2-keto carboxylic acid~ by redox enzyme~ which tran~fer the reducing equivalents u~der anaerobic condition~ to a Yiologen aR external redox mediator. Thiq patent application mention~ the two ~iologen~CAV(1,1'-dicarboxamidomethyl-4,4'-dipyridinium dication) and CYY (1,1'-d~cy~nomethyl-4,4~-dipyridinium dication) as redox mediators which can be regenerated electrochemically at an anode, with atmo pheri~ oxygen, with iron(III) compound~ or with hydrogen peroxide.
However, on reaction of (D,~)-lactic acid or (D,L)-2- -hydroxyglutar~te it i~ pos~ibl~ to i~ola~ only the (L) for~s which do not react. Pyr~vic acid and 2-ketoglutar-a~e are metabolized in the proce~s and cannot be i~olated tSkopa~ et al., Ang~w. 99, (19873 139-141 In add~tion, the viologens CAV and CYV have~
con~iderable toxicity; furthermore, they are not suffic-iently stable in the pH range used for the reaction.
~ an ob~ect of the prQsent invention to provide a proces for prep~ring 2-keto carboxylic acids - which doe~ not have the abovementioned di~advantage~.
We have found that this ob~ect i9 achieved by a 21 ~75~14
Reductions carried out by microorganism~ which are provided with the reducing equivalent~ via an exter-nal electron carrier which i9 regenerated electrochemi-cally ar~ known. It i8 po~sible to employ for thi~ a large number of microorgani~m~ and, a~ electron carrier~, a number of dye~ and other ~ubst~nce~ (DE-A 32 26 888 and DE-A 33 32 562).
EP-A 260 611 di~clo~es the oxidation of (D)-2-hydroxy carboxylic acid~ to 2-keto carboxylic acid~ by redox enzyme~ which tran~fer the reducing equivalents u~der anaerobic condition~ to a Yiologen aR external redox mediator. Thiq patent application mention~ the two ~iologen~CAV(1,1'-dicarboxamidomethyl-4,4'-dipyridinium dication) and CYY (1,1'-d~cy~nomethyl-4,4~-dipyridinium dication) as redox mediators which can be regenerated electrochemically at an anode, with atmo pheri~ oxygen, with iron(III) compound~ or with hydrogen peroxide.
However, on reaction of (D,~)-lactic acid or (D,L)-2- -hydroxyglutar~te it i~ pos~ibl~ to i~ola~ only the (L) for~s which do not react. Pyr~vic acid and 2-ketoglutar-a~e are metabolized in the proce~s and cannot be i~olated tSkopa~ et al., Ang~w. 99, (19873 139-141 In add~tion, the viologens CAV and CYV have~
con~iderable toxicity; furthermore, they are not suffic-iently stable in the pH range used for the reaction.
~ an ob~ect of the prQsent invention to provide a proces for prep~ring 2-keto carboxylic acids - which doe~ not have the abovementioned di~advantage~.
We have found that this ob~ect i9 achieved by a 21 ~75~14
- 2 - o.Z. 0050/43032 proce~s for prepar~ng 2-keto carboxylic acid~ from (D) -2-hydroxy carboxylic acid~ using a biocataly~t, in which the electrons produced in the oxidation of (D)-2-hydroxy carboxylic acid~ are tran~ferred to a quinone derivati~e.
We have al~o found that mixtures of (L)-2-hydroxy carboxylic acid~ and 2-keto carboxylic acid~ can be prepared from (D,$)-2-hydroxy carboxylic acids by thi~
process .
It i~ po~ible by the proce~ according to the invention very generally to oxidize (D)-2-hydroxy carboxylic acid~ to the corresponding 2-keto carboxylic acid~. Thus, for example, the 2-hydroxy carboxylic acids described by Schummer et al. (Tetrahedron 47 (1991) 9019-9034) can be u~ed as ~ub~trate. Particularly ~uitable (D)-2-hydroxy carboxyl~c acid~ are (D)-glycerate, (D)-gluconate, (D)-galactonate, (D)-gulonate, (D)~ribonate, (D)-xylonato and 'L)-mannonate, (L3-arabinonate and lactobionic acid (4-p-D-galactosyl-D-gluconate). There i5 no con~er~ion of, for example, (L)-gluconate because C-2 ha~ the (L) configuration. The procesQ is very particu-larly ~uitable for preparing pyruvic acid or the salt~
thereof (pyruvate~) from (D)-lactic acid or the ~alt~
thereof. Application of the proce~ to (D,L)-lactic acid results in a mixture of pyruvate and (L)-lactic acid, which can easily b~ ~epara~ed chemically.
The biocat~ly~ts employed in the proce~s can be micro~rgani~m~ or enzy~e preparation~ prepared therefrom.
Mlcroorganism~ are prefer~bly used a~ biocataly~t~, and tho~e of th~ genus Proteu~, for example Proteus vulgari~
DSM 30118 or Prot~u~ mirabilis DSN 30115, of the genu~
Propionibaeterium, for example Propionibacterium acidi-propioniei DSM 20272, of the genu~ Clo~tridium, for example Clostridium homopropionicum sp. nov. DSM 5847, or of the genu~ Paraeoeeu~, for example Paracoecu~ denitri-fican~ DSM 413, are p~rtieularly preferred.
Suitable mieroorgani~m~ can ea~ily be id~ntified by, for ex~mple, te~ting their ability to oxidize ~ t .. ".,.,. ".7",".. ~ ,"~; ~ "" -~,,,~ ,";~;~ "~- ",, `
2 ~ ~ g ~
_ 3 _ O.Z. 0050/43032 (D)-lactate to pyruvate.
The ~nzyme preferably used is the 2-hydroxy-carboxylate-viologen oxidoreducta~e from Proteus vulgari~
or Proteu~ m~rab~
The microorganiqms can be cultured separately and, where appropriate after storage, added as cell suspension or as enzyme preparation anaerobically to the reaction ~olution. It ~ also po~ible to add the sub-~trate direct to the medium at the ~tart or fini~h of the cultivation of the microorgani~m, ~o that it i8 converted after completion of the growth phase. Thi~ procedure i~
particulsrly ~uitable when a chemical electron acceptor ~uch as dimethyl sulfoxide i~ u~ed.
The 2-keto carboxylic acid prepared by the process according to the invention may, for example in the ca~e of pyruvate, be further metabolized by the microorganism~ used. It is therefore expedient to inhibit the catabolism of the pyruvate or of onic acids ~uch as (D)-gluconate in the microorganism~. This can be effec-ted, for example, chemically by metal-complexing sub~tan-ce~ ~uch as ethylenediaminetetraacetic acid (EDTA) or antibiotics ~uch a~ tetracycline. EDTA i~ preferably used, the concentration~ being, as a rule, from 0.01 to 10 m~
The electron~ produced in the oxidstion of the (D)-2-hydroxy carboxylic acid~ are transferred to a mediator.
Suitable mediators ar~ the following group~ of substancess 1. Quinone dyes such as anthraquinones or naphtho-quinones, eg. anthraquinone~ulfonic acid~, hydroxy-naphthoquinone~, 2. Viologen dye~, eg. benzylviologen, CAV,
We have al~o found that mixtures of (L)-2-hydroxy carboxylic acid~ and 2-keto carboxylic acid~ can be prepared from (D,$)-2-hydroxy carboxylic acids by thi~
process .
It i~ po~ible by the proce~ according to the invention very generally to oxidize (D)-2-hydroxy carboxylic acid~ to the corresponding 2-keto carboxylic acid~. Thus, for example, the 2-hydroxy carboxylic acids described by Schummer et al. (Tetrahedron 47 (1991) 9019-9034) can be u~ed as ~ub~trate. Particularly ~uitable (D)-2-hydroxy carboxyl~c acid~ are (D)-glycerate, (D)-gluconate, (D)-galactonate, (D)-gulonate, (D)~ribonate, (D)-xylonato and 'L)-mannonate, (L3-arabinonate and lactobionic acid (4-p-D-galactosyl-D-gluconate). There i5 no con~er~ion of, for example, (L)-gluconate because C-2 ha~ the (L) configuration. The procesQ is very particu-larly ~uitable for preparing pyruvic acid or the salt~
thereof (pyruvate~) from (D)-lactic acid or the ~alt~
thereof. Application of the proce~ to (D,L)-lactic acid results in a mixture of pyruvate and (L)-lactic acid, which can easily b~ ~epara~ed chemically.
The biocat~ly~ts employed in the proce~s can be micro~rgani~m~ or enzy~e preparation~ prepared therefrom.
Mlcroorganism~ are prefer~bly used a~ biocataly~t~, and tho~e of th~ genus Proteu~, for example Proteus vulgari~
DSM 30118 or Prot~u~ mirabilis DSN 30115, of the genu~
Propionibaeterium, for example Propionibacterium acidi-propioniei DSM 20272, of the genu~ Clo~tridium, for example Clostridium homopropionicum sp. nov. DSM 5847, or of the genu~ Paraeoeeu~, for example Paracoecu~ denitri-fican~ DSM 413, are p~rtieularly preferred.
Suitable mieroorgani~m~ can ea~ily be id~ntified by, for ex~mple, te~ting their ability to oxidize ~ t .. ".,.,. ".7",".. ~ ,"~; ~ "" -~,,,~ ,";~;~ "~- ",, `
2 ~ ~ g ~
_ 3 _ O.Z. 0050/43032 (D)-lactate to pyruvate.
The ~nzyme preferably used is the 2-hydroxy-carboxylate-viologen oxidoreducta~e from Proteus vulgari~
or Proteu~ m~rab~
The microorganiqms can be cultured separately and, where appropriate after storage, added as cell suspension or as enzyme preparation anaerobically to the reaction ~olution. It ~ also po~ible to add the sub-~trate direct to the medium at the ~tart or fini~h of the cultivation of the microorgani~m, ~o that it i8 converted after completion of the growth phase. Thi~ procedure i~
particulsrly ~uitable when a chemical electron acceptor ~uch as dimethyl sulfoxide i~ u~ed.
The 2-keto carboxylic acid prepared by the process according to the invention may, for example in the ca~e of pyruvate, be further metabolized by the microorganism~ used. It is therefore expedient to inhibit the catabolism of the pyruvate or of onic acids ~uch as (D)-gluconate in the microorganism~. This can be effec-ted, for example, chemically by metal-complexing sub~tan-ce~ ~uch as ethylenediaminetetraacetic acid (EDTA) or antibiotics ~uch a~ tetracycline. EDTA i~ preferably used, the concentration~ being, as a rule, from 0.01 to 10 m~
The electron~ produced in the oxidstion of the (D)-2-hydroxy carboxylic acid~ are transferred to a mediator.
Suitable mediators ar~ the following group~ of substancess 1. Quinone dyes such as anthraquinones or naphtho-quinones, eg. anthraquinone~ulfonic acid~, hydroxy-naphthoquinone~, 2. Viologen dye~, eg. benzylviologen, CAV,
3. Triphenylmethane dye , eg. benzaurin, aurin, 2~ 7~ 14 _ 4 - O.Z. 0050/43032
4. Phthalocyanine~, eg. Fe, Cu or Co phthalocyanine~,
5. Methine dye~, eg. a~traphloxine,
6. Pyrrole dye~ or porphyrin derivative~, eg. metal chel~te complexe~ of the~e compounds, S 7. Pteridines or pteridones, 8. Flavin~, eg. scriflavin, lumifla~in, 9. Imidazole derivatives, eg. metronidazole, 10. Complexes of m~tal~ o~ group VIB, VIIB and VIII, eg.
ferrocenemonocarboxylic acid~, 11. Thiolate~ with metal8 of group VIB, VIIB and VIII, 12. Thiol~, eg. dihydrolipoic acid, dithiothrei~ol, glutathione, 13. Indophenols, eg. 2,6-dichlorophenolindophenol, 14. Indigo dy~, eg. indigotetrasulfonate, 15. Ncphthi~zine~, eg. ro indulin 2G, 16. Phena~ine~, eg. phena2~ne atho ulfate, pyocyanine, 17. Phe~o~hiazine8, eg. ~hionlna, teluidine blue O, 18. Phenoxazine~, 9g . re~orufin, 19. Chelate compl~xe8 of me~al~ of group VIII, eg.
ethylenediamine~etraacetic acid (EDTA) with iron (II/III~, ~ 2 .~ ~ ~4 _ 5 _ o.Z. 0050/43032 20. Mediator sy~tema compo~ed of combinations of groups 1 to 19, eg. thionine and iron(II/III) EDTA.
The mediators from group 1 ar~ preferably used, and the anthraquinone~ulfonic acids, eg. anthraquinon~-2-~ulfonic acid or anthraquinone-2,6-di~ulfonic acid, are particularly ~uitable.
The mediators are u~ually employed in catalytic amounts when the electron~ are transferred from the mediator to an electron acceptor.
~owever, if the mediator~ are al~o u~ed a~
electron acceptor, they are expediently employed in equimolar amounts to the substrate to be oxidized. ~' The mediator which ha~ been reduced in the proce~s can be reoxidized and thu~ employed anew in the proce~
The reaction can be repre~ented a~ follows~
P. vulgari~
R-CHOH-cOOH + Med(Os~ , > R-CO-COO~ + Med(r,d~ ~, + 2 H~ (Eq- 1) ~' Med~r~ + E~o~) > Med~Os~ + EA~.
(Eq. 2) R-CHOH-COO~ + EA(o~) - > ~-CO-CO~ + EP~.d~
+ 2 ~+ (Eq. 3)' The,reoxidation of the mediator (Med) or electron accRptor (EA) (Eq. 2) can take plac~ in a vari~ty of ways. The mod~ of reoxidation cho~n in each ca~e dep~nds on which mediator i~ u~ed. Th~ mediator can b~ regenera-ted continuously during the reaction. Another option i~
to oxidize the m~diator or electron acceptor, which has been employed~in equL~olar amount~, after completion of the reaction~ This i~ particularly advantag~ou~ when the reduced electron acceptor i~ volatile during the rQaction or can be removed from the reaction mixture by di~tilla-tion.
The following processe~'are suitable for re-generating the mediators: -`- - 6 - O.Z. 0050/43032 1. The mediator i~ pumped through an electrochemical cell and reoxidized at the anode. The microorganisms can either be al~o circulated through the electro-chemical cell or, more advantageously, be retained by a membrane in the form of a hollow fiber bundle or aq immobilizate on sintered glass, in which case the half-life of the enzyme activity in the micro-organism~ is considerably increased.
2. The mediator i~ regenerated by blowing oxygen or air into the reaction ~olution. This can be done by pumping the reaction solution, with or without microorgani~m~, through a hollow fiber bundle which is exposed to oxygen from the outside. However, in thi~ case it is advanta~eous to retain the micro-organisms a~ in proce~s 1. It is important in this met;lod that the amount of oxygen-blown in i~ only ~ust sufficient to regenerate ~he amount of reduced mediator. A con~tant small concentration of reduced mediator i~ advantageous in thi~ ca~e (anaerobic condition~) bec~use the enzyme is then not damaged by oxygen.
3. The mediator i~ regenerated by a second enzyme in Proteu~ vulgari~ or Proteu~ mirabilis and another electron acceptor.
~xample~ of suit~ble elGctron acceptors are the following group~:
1. Sulfoxides, eg. dime~hyl ~ulfoxide (DNSO), diethyl sulfoxida, tetr~methylene sulfoxide, DL-methionine ~ulfoxid~, 2. N-oxide_, eg. ~-me~hylmorpholins N-oxide, trim~thylamine N-oxide, pyridine N-oxide, 3. Fumarate, f~l ~ 7 ~i~ 4 _ 7 _ o.z. 0050/43032 4. Nitrate, 5. Chlorate, 6. Thio~ulfate, tetrathiosulfate.
The N-oxide~, especially N-methylmorpholine N-oxide, have prov~n to be particularly suitable electron acceptors. Th~ N-methylmorpholin3 produced in ths re~ction c~n be oxidized b~ck to N-methylmorpholine N-oxide in aqueou~ solution by reacting it, for exampl~, with peroxide~ ~uch a~ hydrogen peroxicl~ or percarboxylic acid~ or with oxyg~n (Houben-Weyl, ~ethoden der organi chen Chemie, ~ol. 16a I (1990) ~04-~20).
Tho el~ctron accaptor can b~ added either in equi-molar amount~ or in catalytic amount~ to the ~ub-3trate and then regenerat~d outside the r~action mix~ure for example dim~thyl ~ulfide (from dimethyl ~ulfoxide) reAdily escap~s from the ~queou~ reaction qolution and can be conveEted back by catalytic ox~dation with atmo~ph~ric oxygen in~o the oxide (DMSO) which can be returned ~o th~ r~actlon 501u~
tion.
4. -ThQ medintor i5 reg~neratsd by a second miero-organi~, e~. Paracoccu~ deni~rificans ~5M 413.
Suit~bl~ electron acceptors in thi~ ~a3e are nitrat~, nitrite and nitrou~ oxide. The product of reduction in thi~ case i~ molecular nitrogen which Qscape~ a~ ga~ from the reaction ~olu~ion.
Reoxidation proce~ses 1 to 4 are ~itable for batch, ~emibatch and continuous opexation.
Temper~tures generally suitable for ~he indivi-dual processe~ under anaerobic condition~ are from 10 to ~12 7 ~) 4 ~
- 8 - O.Z. OOS0/43032 50, preferably 25 to 40C. The pH i~ expediently mea~ured during the reaction and kept con~tant in the range f rom 8.5 to 9.5 by addition of sodium hydroxide solut~on or hydroc~lor~c acid where appropriate. The D-~actic acid or S D-lactate (pH 8-9) concentration i~ usually from 0.01 to O.7 M (1-63 gtl). The required amount of D-lactic acid can be added all at once at the outset or in several portions during the reaction. ~he pre~ence of L-lactic acid doe~ not interfere; L-lactic acid i~ not converted into pyruvic acid and therefore remains unchanged.
D-lactic acid c~n al~o be introduced into the reaction ~olution as ~lt. It i~ po~ible to u~e, inter alia, the lithium, ~odium, pota~ium and ammonium ~alt~.
Th~ resulting pyruvic acid or pyruvate c~n be qu~ntified by conventional methods (enzymatic detection with L-LDH/NADH) tR. Czok, W. Lamprecht in H.U. 8ergmeyers Methoden der enzymatiw hen Analy~e. 3rd edltion, Vol. I/II, 1407-1411, Verlag Chemie, Weinheim (1974)]. For thi~, the ~ample~ are mixed 1:1 with per-chloric acid (7~), centrifu~ed, heated at 95-C for 1 h and, after cooling, appropriately diluted and analyzed.
It i~ advi~able to ~top the react~on as soon a~
the pyruvic acid concentration ha~ re~ched it~ maximum.
The re~ulting pyruvic acid can then be removed and purified by con~entional proce~e~ (~ee DE-A 37 33 157)~.
The proce~s according to ~he in~ention makas it po~ble to prep~re 2-keto car~oxylic acid~ from (D)-2-hydroxy c~r~oxylic acid~ in high yield without the final product being metabolized, which frequently interfere~ in other biocatalytic proce~es.
The invention i~ illustrated by the following Example~:
EXA~PLE 1 Cultivation of Protous vulgari~ with relatively high wet weight of bacteria per liter of medium without los~ of enzyme activity.
Gluco~e medium (medium A):
212 ;7~4~
_ g _ O.Z. 0050/43032 Glucose 10.0 g/l Dipotas~ium hydrogen pho~phate 5.1 g/l Yea~t 0.5-2.5 g/l Tryptone 5.0 g/l S Sodium formate 1.0 g/l*
Ammonium chloride 0.17 g/l Magnesium sulfate (x 7 H20) 0.05 g/l Mhngane~e ~ulfate ~x H20) 0.4 mg/l~ ;
Iron sulfate (x 7 H2O) 0.4 mg/l*
Disodium molybdate 13.7 mg/l Disodium ~elen~te 0.263 mg/l~ ;
Calc$um chloride (x 2 H2O) 40.0 mg/l*
p-Aminobenzoic acid 0.4 mg/l~
Biotin 20.0 vg/l~
Water ad 1 1 The glucose and the dipota~sium hydrogen pho -phate were sterilized (20 min at ~21-C~ then flu~hed with -~
nitrogen) separate from the rema~nder of the medium and added after cooling. The pH of the medium was 7.5-8Ø
If the microorgsnism~ are used only for the oxidation it is possible to omit the constituent~ identi-f~ed by an asterisk. This wa~ a~sociated with no los~ of enzyme acti~ity (2-hydroxy-carboxylate-viologen oxido-reductase) or a lower yield of cells per liter. The medium was inoculated with 0.5-1.0 percent of a Proteu'~
vulgaris preculture. Cultivation was carried out at 37-C, controlling the pH nt 7.2 with 2-6 N ~odium hydroxide solution (about 6 g of ~od~um hydroxide per 1 of medium o were requ~red) unt11, after lS-18 h, th~ ~tations~J
growth phase ~tartsd. The wet weight of bacteria wa~ 6-
ferrocenemonocarboxylic acid~, 11. Thiolate~ with metal8 of group VIB, VIIB and VIII, 12. Thiol~, eg. dihydrolipoic acid, dithiothrei~ol, glutathione, 13. Indophenols, eg. 2,6-dichlorophenolindophenol, 14. Indigo dy~, eg. indigotetrasulfonate, 15. Ncphthi~zine~, eg. ro indulin 2G, 16. Phena~ine~, eg. phena2~ne atho ulfate, pyocyanine, 17. Phe~o~hiazine8, eg. ~hionlna, teluidine blue O, 18. Phenoxazine~, 9g . re~orufin, 19. Chelate compl~xe8 of me~al~ of group VIII, eg.
ethylenediamine~etraacetic acid (EDTA) with iron (II/III~, ~ 2 .~ ~ ~4 _ 5 _ o.Z. 0050/43032 20. Mediator sy~tema compo~ed of combinations of groups 1 to 19, eg. thionine and iron(II/III) EDTA.
The mediators from group 1 ar~ preferably used, and the anthraquinone~ulfonic acids, eg. anthraquinon~-2-~ulfonic acid or anthraquinone-2,6-di~ulfonic acid, are particularly ~uitable.
The mediators are u~ually employed in catalytic amounts when the electron~ are transferred from the mediator to an electron acceptor.
~owever, if the mediator~ are al~o u~ed a~
electron acceptor, they are expediently employed in equimolar amounts to the substrate to be oxidized. ~' The mediator which ha~ been reduced in the proce~s can be reoxidized and thu~ employed anew in the proce~
The reaction can be repre~ented a~ follows~
P. vulgari~
R-CHOH-cOOH + Med(Os~ , > R-CO-COO~ + Med(r,d~ ~, + 2 H~ (Eq- 1) ~' Med~r~ + E~o~) > Med~Os~ + EA~.
(Eq. 2) R-CHOH-COO~ + EA(o~) - > ~-CO-CO~ + EP~.d~
+ 2 ~+ (Eq. 3)' The,reoxidation of the mediator (Med) or electron accRptor (EA) (Eq. 2) can take plac~ in a vari~ty of ways. The mod~ of reoxidation cho~n in each ca~e dep~nds on which mediator i~ u~ed. Th~ mediator can b~ regenera-ted continuously during the reaction. Another option i~
to oxidize the m~diator or electron acceptor, which has been employed~in equL~olar amount~, after completion of the reaction~ This i~ particularly advantag~ou~ when the reduced electron acceptor i~ volatile during the rQaction or can be removed from the reaction mixture by di~tilla-tion.
The following processe~'are suitable for re-generating the mediators: -`- - 6 - O.Z. 0050/43032 1. The mediator i~ pumped through an electrochemical cell and reoxidized at the anode. The microorganisms can either be al~o circulated through the electro-chemical cell or, more advantageously, be retained by a membrane in the form of a hollow fiber bundle or aq immobilizate on sintered glass, in which case the half-life of the enzyme activity in the micro-organism~ is considerably increased.
2. The mediator i~ regenerated by blowing oxygen or air into the reaction ~olution. This can be done by pumping the reaction solution, with or without microorgani~m~, through a hollow fiber bundle which is exposed to oxygen from the outside. However, in thi~ case it is advanta~eous to retain the micro-organisms a~ in proce~s 1. It is important in this met;lod that the amount of oxygen-blown in i~ only ~ust sufficient to regenerate ~he amount of reduced mediator. A con~tant small concentration of reduced mediator i~ advantageous in thi~ ca~e (anaerobic condition~) bec~use the enzyme is then not damaged by oxygen.
3. The mediator i~ regenerated by a second enzyme in Proteu~ vulgari~ or Proteu~ mirabilis and another electron acceptor.
~xample~ of suit~ble elGctron acceptors are the following group~:
1. Sulfoxides, eg. dime~hyl ~ulfoxide (DNSO), diethyl sulfoxida, tetr~methylene sulfoxide, DL-methionine ~ulfoxid~, 2. N-oxide_, eg. ~-me~hylmorpholins N-oxide, trim~thylamine N-oxide, pyridine N-oxide, 3. Fumarate, f~l ~ 7 ~i~ 4 _ 7 _ o.z. 0050/43032 4. Nitrate, 5. Chlorate, 6. Thio~ulfate, tetrathiosulfate.
The N-oxide~, especially N-methylmorpholine N-oxide, have prov~n to be particularly suitable electron acceptors. Th~ N-methylmorpholin3 produced in ths re~ction c~n be oxidized b~ck to N-methylmorpholine N-oxide in aqueou~ solution by reacting it, for exampl~, with peroxide~ ~uch a~ hydrogen peroxicl~ or percarboxylic acid~ or with oxyg~n (Houben-Weyl, ~ethoden der organi chen Chemie, ~ol. 16a I (1990) ~04-~20).
Tho el~ctron accaptor can b~ added either in equi-molar amount~ or in catalytic amount~ to the ~ub-3trate and then regenerat~d outside the r~action mix~ure for example dim~thyl ~ulfide (from dimethyl ~ulfoxide) reAdily escap~s from the ~queou~ reaction qolution and can be conveEted back by catalytic ox~dation with atmo~ph~ric oxygen in~o the oxide (DMSO) which can be returned ~o th~ r~actlon 501u~
tion.
4. -ThQ medintor i5 reg~neratsd by a second miero-organi~, e~. Paracoccu~ deni~rificans ~5M 413.
Suit~bl~ electron acceptors in thi~ ~a3e are nitrat~, nitrite and nitrou~ oxide. The product of reduction in thi~ case i~ molecular nitrogen which Qscape~ a~ ga~ from the reaction ~olu~ion.
Reoxidation proce~ses 1 to 4 are ~itable for batch, ~emibatch and continuous opexation.
Temper~tures generally suitable for ~he indivi-dual processe~ under anaerobic condition~ are from 10 to ~12 7 ~) 4 ~
- 8 - O.Z. OOS0/43032 50, preferably 25 to 40C. The pH i~ expediently mea~ured during the reaction and kept con~tant in the range f rom 8.5 to 9.5 by addition of sodium hydroxide solut~on or hydroc~lor~c acid where appropriate. The D-~actic acid or S D-lactate (pH 8-9) concentration i~ usually from 0.01 to O.7 M (1-63 gtl). The required amount of D-lactic acid can be added all at once at the outset or in several portions during the reaction. ~he pre~ence of L-lactic acid doe~ not interfere; L-lactic acid i~ not converted into pyruvic acid and therefore remains unchanged.
D-lactic acid c~n al~o be introduced into the reaction ~olution as ~lt. It i~ po~ible to u~e, inter alia, the lithium, ~odium, pota~ium and ammonium ~alt~.
Th~ resulting pyruvic acid or pyruvate c~n be qu~ntified by conventional methods (enzymatic detection with L-LDH/NADH) tR. Czok, W. Lamprecht in H.U. 8ergmeyers Methoden der enzymatiw hen Analy~e. 3rd edltion, Vol. I/II, 1407-1411, Verlag Chemie, Weinheim (1974)]. For thi~, the ~ample~ are mixed 1:1 with per-chloric acid (7~), centrifu~ed, heated at 95-C for 1 h and, after cooling, appropriately diluted and analyzed.
It i~ advi~able to ~top the react~on as soon a~
the pyruvic acid concentration ha~ re~ched it~ maximum.
The re~ulting pyruvic acid can then be removed and purified by con~entional proce~e~ (~ee DE-A 37 33 157)~.
The proce~s according to ~he in~ention makas it po~ble to prep~re 2-keto car~oxylic acid~ from (D)-2-hydroxy c~r~oxylic acid~ in high yield without the final product being metabolized, which frequently interfere~ in other biocatalytic proce~es.
The invention i~ illustrated by the following Example~:
EXA~PLE 1 Cultivation of Protous vulgari~ with relatively high wet weight of bacteria per liter of medium without los~ of enzyme activity.
Gluco~e medium (medium A):
212 ;7~4~
_ g _ O.Z. 0050/43032 Glucose 10.0 g/l Dipotas~ium hydrogen pho~phate 5.1 g/l Yea~t 0.5-2.5 g/l Tryptone 5.0 g/l S Sodium formate 1.0 g/l*
Ammonium chloride 0.17 g/l Magnesium sulfate (x 7 H20) 0.05 g/l Mhngane~e ~ulfate ~x H20) 0.4 mg/l~ ;
Iron sulfate (x 7 H2O) 0.4 mg/l*
Disodium molybdate 13.7 mg/l Disodium ~elen~te 0.263 mg/l~ ;
Calc$um chloride (x 2 H2O) 40.0 mg/l*
p-Aminobenzoic acid 0.4 mg/l~
Biotin 20.0 vg/l~
Water ad 1 1 The glucose and the dipota~sium hydrogen pho -phate were sterilized (20 min at ~21-C~ then flu~hed with -~
nitrogen) separate from the rema~nder of the medium and added after cooling. The pH of the medium was 7.5-8Ø
If the microorgsnism~ are used only for the oxidation it is possible to omit the constituent~ identi-f~ed by an asterisk. This wa~ a~sociated with no los~ of enzyme acti~ity (2-hydroxy-carboxylate-viologen oxido-reductase) or a lower yield of cells per liter. The medium was inoculated with 0.5-1.0 percent of a Proteu'~
vulgaris preculture. Cultivation was carried out at 37-C, controlling the pH nt 7.2 with 2-6 N ~odium hydroxide solution (about 6 g of ~od~um hydroxide per 1 of medium o were requ~red) unt11, after lS-18 h, th~ ~tations~J
growth phase ~tartsd. The wet weight of bacteria wa~ 6-
7 g~l (dry weight 1.2-1.4 g/l).
The acti~ity of the 2-hydroxy-carboxylate-~iolo-gen oxidoreductase was about 2.0 unit~ of lactate dehydrogena~e/mg of protein (about 1,OOQ unit~/g dry weight of bacteria)~ and that of dimethyl ~ulfoxide reductase wa~ ~bout 0.1 units of DMSO reducta~e/mg of protein (about 50 unit~/g dry weight of bacteria).
- .~
212 7~ ~4 - 10 - O.Z. 0050/43032 Cultivation of Proteus vulgari~ and Proteus mir~bilis with ralatively high enzym6 activitie~
(D,L)-Lactate medium (medium B):
As m~dium A but without gluco~e and with 7.0 g/ 1 ~odium (D,L~-lactat~ or 3.5 g/l ~odium L-lactate (D-lac~ate i9 not metabolized) and 0.05-0.1 M (3.9-7.8 g/l) dimethyl ~ulfoxide (which wa~ added after the medium had cooled). Th~ pH of the medium wa~ 7.5-8.5. The medium was inoculated with 0.5-l.0 percent of a Proteus vulgari~ preculture. The culture reached the 3tationary growth pha~e after 18-20 h at 37-C. The wet weight of bacteria wa~ about 2.5 g/l, and th~ dry w~ight was O.S g/l. The corre~ponding figure~ for Proteu~ mirabilis were twice the~e. Similar activitie~ are obtained with 50 mM pyruvate or fum~rate a~ electron acceptors.
The activity of the 2-hydroxy-carboxylate-violo-gen oxidoreductase wa~ about 4.0 -unit~ of lactate dehydrogena~e/mg of protein (about 2,000 units/g dry weight of bacteria), and th~t of dim~thyl sulfoxide reductase wa~ about 0.4 unit~ of DMSO reducta~etmg of protein (about 200 unit~/g dry weight of bacteria). The corre~ponding figure~ for Proteu~ mir~bili~ were likewi~e 4.0 unit~ of lactate dehydroganase/mg of protein. The figura for d~methyl ~ulfoxid~ r~ducta~e reached 1.9 U/mg5 of protein tabout 950 unit~/g dry weight of bacteri~).
Cultlv~tion of Paracoccu~ d~nitrific~ns Gluco~e m~dium (med~um C):
Gluco~ 9.0 g/l Pota~sium n~trate 10.0 g/l Disodium hydrogen pho~phate 2.7 g/l Pota~sium dihydrogen pho~phate 4.1 g/l Yea~t 0.1 ~ g/l Ammonium chloride 1.6 g/l Nagnesium ~ulfate (x 7 H20) 0.25 g/l Di~odium molybdate (x 2H2O) 0.15 g/l Manganese ~ulfate (x H2O) 0.1 mg/l 212 7~
~ O.Z. 0050/43032 ~
~.' C~lcium chloride (x 2 H2O) 20.0 mg/l Iron ammonium citrate 20.0 ~M
water ad 1 1 Medium C wa~ sterilized like medium A. The iron S ammonium citrate was added after cooling. The pH of the medium wa~ about 7.3. Th~ medium wa~ inoculated with 1.0 percent of a Paracoccus denitrifican~ preculture. The culture reached the start of the stationary growth phase after 20-24 h at 35-C without pH control. The wet w~ight of bacteria wa~ ~bout 8 g/l, and the dry weight was 1.6 g/l.
EXAMPL~ 3 ~
Production of pyruvate in an electrochemical cell with -Proteu~ vulgariJ with and without EDTA
35.5 mmol of D-lactate, 0.21 mmol of anthra-quinone-2,6-di~ulfonic acid, 0.35 mmol of EDTA and 362 mg of Proteu~ vulgar~s cell~ (dry weight) were added to 70 ml of anolyte (deionized w~ter) in an electrochemical flow-through cell with a graphite felt anode (and cathode). The reaction solution wa~ circulated through the slectrochemical cell at a flow rate of 18 l/h by a peri~taltic pu~p. A 90 ml stirrQd ves~l wa~ u~ed for volume ad~u~tment. The potential in the electrochemical cell wa~ kept con~tant at -300 mV vs. SCE, and a maximum 2S current of 0.5 A wa~ reached. The temperature wa~
35~0.S-C. The p~ W~8 kept con3tant at 8.S wi~h 4 ~ æodium hydroxid~ ~olution.
After 21.5 h, 94.6% (33.6 mmol) of the D-lactate had b~en oxidiz~d to pyruvate. 5.0~ (1.8 mmol) of the D-lactate w~ still present. The con~er~ion calculat~d from the con~umption of ~odiu~ hydroxide ~olution w~s ~ 100%
and from the current con~ump~ion wa~ 94.9~.
The analogou~ experiment without addition of EDTA
~how~ that 75.2% (26.7 mmol) of the D-lactate were oxidized to pyruv~te after 21.5 h. 13.0% (4.6 mmol) of the D-lactate were ~till pre~ent. The co~ver~ion c~lcu-lated from the con~umption of sodium hydroxide ~olution . ~12f34/i - 12 - O.Z. 0050/43032 w~ 108%, and that from the current con~umption wa~
2 100%.
Production of pyruv~te in an electrochemical cell with S Proteus vulg~ri~ in a hollow fiber reactor 129 mmol of D-lactate, 2.65 mmol of anthraquin-one-2,6-di~ulfonate and 0.27 mmol of EDTA were dissolved in 265 ml of deionized water in a stirred vessel, and 165 mg of Proteus vulgaris cells (dry weight) were added.
Part of the reaction volume was continuously removed through a membrane in the foDm of a hollow fiber bundle and waD pumped through an electrochemical cell to reoxid-ize the anthraquinone-2,6-disulfonic acid and returned to the reaction chamber. The pH wa~ kept con~tant with sodium hydroxide solution. The temperature was 3510.5-C.
The potential in the electrochemical cell was -300 mV v~.
SCE. 95~ of the D-lactat~ had been oxidized to pyruvate after 143 h. 1.7 mmol (1.3~) of the D-lactate were ~till present.
EXAMPLE S
Production of pyruvate with Proteu~ vulgaris and regeneration of the mediator with oxygen 50.4 mmol of D-lactate, 1.0 mmol of anthraquin-one-2,6-disulfonate and 0.32 mmol of EDTA were di~olved in 100 ml of deionized water in a ~tirred ve3sel, a~d 362 mg of Proteus vulgari~ cell8 (dry weight) w~re added.
Part-of ~he react~on volume wa~ continuously circulated at a flow rate of 15 l/h through a hollow fiber bundle wh1ch was exposed externally to a gaqe pres~ure of 0.5-0.8 bar of oxygen. The temperature was 35~0.5C. The pH
w~ kept ccnstant with 4 N ~odium hydroxide solution. ~5%
(22.7 mmol) of the D-lactate had been oxidizad to pyruv-ate after 29 h. Be~ide~ 33~ (16.4 mmol) of D-lactate it was also pos~ible to detect S.9 mmol (11.7~) o acetate.
Production of pyruvate with Proteus vulgaris and Para-coccus denitrificans and nitrate as electron acceptor 212'7~
- 13 - O.Z. 0050/43032 12.5 mmol of D-lactate, 0.05 mmol of CAV, 0.5 "ol of EDTA and 12.5 mmol of nitrste were dissolved in 50 ml of 0.3 M tri~/HCl buffer pH 8.5 in a ~tirred vessel, and gO mg of Proteu~ vulgari~ cellq (dry weight) and 300 mg of Paracoccus denitrificans cell~ (dry weight) were added. Th~ temperature wa~ 35+0.5-C. The pH was kept constant at 8.5 with 1 N hydrochloric acid. The oxidation of D-lactate to pyruvate wa~ 83~ (10.4 mmol) after 8.7 h and 100% (12.5 mmol) after 20.7 h.
Pyruvate production from D-lactate with N-methyl-morpholine N-oxid~ a~ electron acceptor and Prot~u~
vulg~ri~
Proteu~ vulgaris was cultivatad in medium B as de~cribed in Example 1.
36.4 mmol of D-lactate, 0.35 mmol of anthraquinone-2,6-di~ulfonate, 0.70 mmol of EDTA and 37.0 mmol of N-m~thylmorpholin~ N-oxide were dissolved ~n 70 ml of deionized water in a stirred vesQel, and 181 mg of Proteus vulgaris cells (dry weight~ were added. The temperature wa~ 40~0.5-C. The pH wa~ kept constant at
The acti~ity of the 2-hydroxy-carboxylate-~iolo-gen oxidoreductase was about 2.0 unit~ of lactate dehydrogena~e/mg of protein (about 1,OOQ unit~/g dry weight of bacteria)~ and that of dimethyl ~ulfoxide reductase wa~ ~bout 0.1 units of DMSO reducta~e/mg of protein (about 50 unit~/g dry weight of bacteria).
- .~
212 7~ ~4 - 10 - O.Z. 0050/43032 Cultivation of Proteus vulgari~ and Proteus mir~bilis with ralatively high enzym6 activitie~
(D,L)-Lactate medium (medium B):
As m~dium A but without gluco~e and with 7.0 g/ 1 ~odium (D,L~-lactat~ or 3.5 g/l ~odium L-lactate (D-lac~ate i9 not metabolized) and 0.05-0.1 M (3.9-7.8 g/l) dimethyl ~ulfoxide (which wa~ added after the medium had cooled). Th~ pH of the medium wa~ 7.5-8.5. The medium was inoculated with 0.5-l.0 percent of a Proteus vulgari~ preculture. The culture reached the 3tationary growth pha~e after 18-20 h at 37-C. The wet weight of bacteria wa~ about 2.5 g/l, and th~ dry w~ight was O.S g/l. The corre~ponding figure~ for Proteu~ mirabilis were twice the~e. Similar activitie~ are obtained with 50 mM pyruvate or fum~rate a~ electron acceptors.
The activity of the 2-hydroxy-carboxylate-violo-gen oxidoreductase wa~ about 4.0 -unit~ of lactate dehydrogena~e/mg of protein (about 2,000 units/g dry weight of bacteria), and th~t of dim~thyl sulfoxide reductase wa~ about 0.4 unit~ of DMSO reducta~etmg of protein (about 200 unit~/g dry weight of bacteria). The corre~ponding figure~ for Proteu~ mir~bili~ were likewi~e 4.0 unit~ of lactate dehydroganase/mg of protein. The figura for d~methyl ~ulfoxid~ r~ducta~e reached 1.9 U/mg5 of protein tabout 950 unit~/g dry weight of bacteri~).
Cultlv~tion of Paracoccu~ d~nitrific~ns Gluco~e m~dium (med~um C):
Gluco~ 9.0 g/l Pota~sium n~trate 10.0 g/l Disodium hydrogen pho~phate 2.7 g/l Pota~sium dihydrogen pho~phate 4.1 g/l Yea~t 0.1 ~ g/l Ammonium chloride 1.6 g/l Nagnesium ~ulfate (x 7 H20) 0.25 g/l Di~odium molybdate (x 2H2O) 0.15 g/l Manganese ~ulfate (x H2O) 0.1 mg/l 212 7~
~ O.Z. 0050/43032 ~
~.' C~lcium chloride (x 2 H2O) 20.0 mg/l Iron ammonium citrate 20.0 ~M
water ad 1 1 Medium C wa~ sterilized like medium A. The iron S ammonium citrate was added after cooling. The pH of the medium wa~ about 7.3. Th~ medium wa~ inoculated with 1.0 percent of a Paracoccus denitrifican~ preculture. The culture reached the start of the stationary growth phase after 20-24 h at 35-C without pH control. The wet w~ight of bacteria wa~ ~bout 8 g/l, and the dry weight was 1.6 g/l.
EXAMPL~ 3 ~
Production of pyruvate in an electrochemical cell with -Proteu~ vulgariJ with and without EDTA
35.5 mmol of D-lactate, 0.21 mmol of anthra-quinone-2,6-di~ulfonic acid, 0.35 mmol of EDTA and 362 mg of Proteu~ vulgar~s cell~ (dry weight) were added to 70 ml of anolyte (deionized w~ter) in an electrochemical flow-through cell with a graphite felt anode (and cathode). The reaction solution wa~ circulated through the slectrochemical cell at a flow rate of 18 l/h by a peri~taltic pu~p. A 90 ml stirrQd ves~l wa~ u~ed for volume ad~u~tment. The potential in the electrochemical cell wa~ kept con~tant at -300 mV vs. SCE, and a maximum 2S current of 0.5 A wa~ reached. The temperature wa~
35~0.S-C. The p~ W~8 kept con3tant at 8.S wi~h 4 ~ æodium hydroxid~ ~olution.
After 21.5 h, 94.6% (33.6 mmol) of the D-lactate had b~en oxidiz~d to pyruvate. 5.0~ (1.8 mmol) of the D-lactate w~ still present. The con~er~ion calculat~d from the con~umption of ~odiu~ hydroxide ~olution w~s ~ 100%
and from the current con~ump~ion wa~ 94.9~.
The analogou~ experiment without addition of EDTA
~how~ that 75.2% (26.7 mmol) of the D-lactate were oxidized to pyruv~te after 21.5 h. 13.0% (4.6 mmol) of the D-lactate were ~till pre~ent. The co~ver~ion c~lcu-lated from the con~umption of sodium hydroxide ~olution . ~12f34/i - 12 - O.Z. 0050/43032 w~ 108%, and that from the current con~umption wa~
2 100%.
Production of pyruv~te in an electrochemical cell with S Proteus vulg~ri~ in a hollow fiber reactor 129 mmol of D-lactate, 2.65 mmol of anthraquin-one-2,6-di~ulfonate and 0.27 mmol of EDTA were dissolved in 265 ml of deionized water in a stirred vessel, and 165 mg of Proteus vulgaris cells (dry weight) were added.
Part of the reaction volume was continuously removed through a membrane in the foDm of a hollow fiber bundle and waD pumped through an electrochemical cell to reoxid-ize the anthraquinone-2,6-disulfonic acid and returned to the reaction chamber. The pH wa~ kept con~tant with sodium hydroxide solution. The temperature was 3510.5-C.
The potential in the electrochemical cell was -300 mV v~.
SCE. 95~ of the D-lactat~ had been oxidized to pyruvate after 143 h. 1.7 mmol (1.3~) of the D-lactate were ~till present.
EXAMPLE S
Production of pyruvate with Proteu~ vulgaris and regeneration of the mediator with oxygen 50.4 mmol of D-lactate, 1.0 mmol of anthraquin-one-2,6-disulfonate and 0.32 mmol of EDTA were di~olved in 100 ml of deionized water in a ~tirred ve3sel, a~d 362 mg of Proteus vulgari~ cell8 (dry weight) w~re added.
Part-of ~he react~on volume wa~ continuously circulated at a flow rate of 15 l/h through a hollow fiber bundle wh1ch was exposed externally to a gaqe pres~ure of 0.5-0.8 bar of oxygen. The temperature was 35~0.5C. The pH
w~ kept ccnstant with 4 N ~odium hydroxide solution. ~5%
(22.7 mmol) of the D-lactate had been oxidizad to pyruv-ate after 29 h. Be~ide~ 33~ (16.4 mmol) of D-lactate it was also pos~ible to detect S.9 mmol (11.7~) o acetate.
Production of pyruvate with Proteus vulgaris and Para-coccus denitrificans and nitrate as electron acceptor 212'7~
- 13 - O.Z. 0050/43032 12.5 mmol of D-lactate, 0.05 mmol of CAV, 0.5 "ol of EDTA and 12.5 mmol of nitrste were dissolved in 50 ml of 0.3 M tri~/HCl buffer pH 8.5 in a ~tirred vessel, and gO mg of Proteu~ vulgari~ cellq (dry weight) and 300 mg of Paracoccus denitrificans cell~ (dry weight) were added. Th~ temperature wa~ 35+0.5-C. The pH was kept constant at 8.5 with 1 N hydrochloric acid. The oxidation of D-lactate to pyruvate wa~ 83~ (10.4 mmol) after 8.7 h and 100% (12.5 mmol) after 20.7 h.
Pyruvate production from D-lactate with N-methyl-morpholine N-oxid~ a~ electron acceptor and Prot~u~
vulg~ri~
Proteu~ vulgaris was cultivatad in medium B as de~cribed in Example 1.
36.4 mmol of D-lactate, 0.35 mmol of anthraquinone-2,6-di~ulfonate, 0.70 mmol of EDTA and 37.0 mmol of N-m~thylmorpholin~ N-oxide were dissolved ~n 70 ml of deionized water in a stirred vesQel, and 181 mg of Proteus vulgaris cells (dry weight~ were added. The temperature wa~ 40~0.5-C. The pH wa~ kept constant at
8.5~0.1 with 2 N hydrochloric acid. After 4.0 h, more than g9.5~ (36.3 mmol) of the D-lactate had been oxidized to pyruvate.
Production of pyruvate with Proteus vulgari~ and dimethyl sulfo~ide a~ electron acceptor 35.5 mmol of D-lactate, 0.21 mmol of anthra-quinone-2,6-di~ulfonic acid and 0.21 mmol of EDTA were di~olvod in 70 ml of deionized water in a stirred ve~sel, and 362 mg of Proteu~ vulgari~ cells (dry weight) were added. 36.0 mmol of dimethyl ~ulfoxide were added in ~everal portion~ during the reaction. The ~mperatura wa~
38+0.5C. The pH was kept con~tant at 8.5 with 2 N ~odium hydroxide solution. After 8.5 h, 95.5~ (33.9 mmol) of the D-lactat~ had been oxidized to pyruvate. The conver~ion~
with Proteu~ mirabili~ were about 1.5 t~me~ fa~ter.
~1~ ia~
- 14 - O.Z. 0050/43032 After addition of a further 35.5 mmol (~ 71.0 mmol) of D-lactate and 36.0 mmol of dLmethyl sulfoxide it was po~sible to detect 50.9 mmol (71.7%, O.64 ~) of pyruvatQ and S mmol (7.0%) of D-lactate after 29 h. It is assumed that about 20~ of the pyruvate had polymerized tCopper et al., Chem. Rev. 83 (1983) 321-358~.
EXAMPL~ 9 Production of pyruvate with Proteus vulgaris from D-l~ct~te in the presence of L-lactate and dimethyl ~ulf-oxide as electron acceptor 35.S mmol of D-lactate, 35.5 mmol of L-lactate, 0.21 mmol of anthr~quinone-2,6-di~ulfonic acid and O.21 mmol of EDTA were dis~olved in 70 ml of deionized water in a stirred ve~sel, and 550 mg of Proteus vulgari~
cells (dry weight) were add~d. 36.0 mmol of dimethyl ~ulfoxide were added in several portion~ during the reaction. The temperature wa~ 38~0.5-C. The pH was kept con~tant at 8.5 with 2 N ~odium hydroxide solution. After 3.6 h, 100% (35.5 mmol) of the D-lactat~ had been oxid-ized to pyruvate. By contrast, all the L-lactate was ~till pre~ent.
Production of pyruv~te with biocatalyst (Proteus vulgari~) employed sevor~l tim~s and dimethyl ~ulfoxide a~ electron acceptor :
- 50 ml of a reaction ~olution which conta~ned 26.0 mmol of D-lactate, 0.15 mmol of anthraguinone-2,6-di~ulfonic acid and 0.25 mmol of EDTA w~re added to 720 mg of Proteu~ vulgsri~ cell~ (dry weight) in a stirrQd ~e8%~l . 26.0 m~ol of dim~thyl ~ulfoxide were added in several portion~ during the reaction. The t~mperature w~ 38~0.5C~ The pH wa~ kept ~onstant at 8.5 with 2 N qodium hydroxide ~olu~ion. The reaction wa~
stopped at a D-lactat~ concentration of s 2.0%. The Proteu~ vulqari~ cell~ were removed from the reaction ~olution by centrifugation, and a further 50 ml of 212'75~
- 15 - O.Z~ OOS0/43032 ~eaction solution wQre added. The biocatalyst was em-ployed 6 times in ~ucce~ion. The result~ are shown in the following Table:
Use I Dry weight Time Pyruvate D-lactate tmgi th' tmmol] ~mmol]
1st 1 720 1.9 25.8 0.20 2nd j 670 4.0 j26.3 10.06 3rd 600 6.3 25~9 0.34 ...
4th S40 8.5 27.0 0.33 5th 1 500 11.3 25.5 0.43 6th ¦ 480 21.5 25.3 0.40 The ~tated amount~ of pyru~ate and D-lactate relate to the reaction solution after centxifugation.
Part of the Proteu~ vulgari~ cell~ w~ lost from each ~atch owing to ~ampling and through ~eparation from the reaction ~olution. Part of the reaction ~olution remain~
in tho bacterial biomass in each batch, which explain-~
why the amount of pyru~ate i~ ~o~ewhat larger in some batches.
Production of pyruvats with Rroteu~ vulgaris in th~
culture medium 2 1 of medium A without glucose, 100 mmol of D-lact~te (no L-lactate) and 200 mmol of dim~thyl sul-foxide wera inoculat~d with 1.0 percent of a Proteus vulgar~ pr~culture. ~he growth and reaction temperature wa~ 37~1~C; the pH wa~ 7.3-7.8. Aftar 21.5 h, 60.6 mmol (60.6%) of pyruvate, 24.8 mmol (24.8~) of D-lactate and 16.8 mmol (16.8~) of acetate wer~ det~cted. Dimethyl ~ulfoxide wa~ no long~r present.
Production of pyrUVatQ with Proteus vulgaris and stoi-chiometric mediator a~ electron acceptor 17.0 mmol of D-lactate, 17.0 mmol of 21~. s ~4~
- - 16 - O.z. 0050/43032 anthrawequinone-2,6-disulfonic acid and 0.35 mmol of EDTA
were dis~olved in 70 ml of deionized water in a stirred ve~sel, and 360 mg of Proteu~ vulgaris cell~ (dry weight) were added. The temperature wa~ 40~0.5-C. The pH was kept S con~tant at 8.5 with 4 N ~odium hydroxide solution. After 2.0 h, 99.0% (16.8 mmol) of the D-lactate had been oxidized to pyruvate. 70% (12 mmol) of the anthraquinone-2,6-di~ulfonie aeid were reeovered by centrifuging the reaetion solution at s 4-C and were re-u~ed. ~he remai-ning 30% (5 mmol) remained di~solved in the reaetion~olution.
Produetion of pyruvate with Proteu~ vulgari~ cell~
immobilized on ~intered gla~ Rasehig ring~
98.9 mmol of D-laetate, 1.25 mmol of anthra-quinone-2,6-di~ulfonate and 0.25 mmol of EDTA were di~olved in 250 ml of deionized w~ter in a fixed bed eireul~tion reaetor, and 28.2 mg of Proteus vulgari~
eell~ (dry weight, immobilized on ~intered gla~s Ra~ehi~
ring~) were employed. The reaetion volume wa~ eireulated at a flow r~te of 20 l/h through an eleetroehemieal eell.
The pH was kept eon~tant with 2 N ~odium hydroxide solution. The temperature wa~ 35~0.5-C. The potential of the eleetroehemieal eell wa~ -300 mV v~. SCE. After 116.5 h, 64.4~ (63.7 m~ol) of the D-laetate had been oxidized to pyruvate.
- EXANPL~ 14 Con~er~ion of a rae~mie 2-hydroxy earboxylie acid with Proteu~ vulgari~
40 ml of anolyte eontain~d 5 mg of EDTA (to prevent deearboxylation of the resulting 2-oxo-4-phenyl-- 3-butenoie a~id by divalen~ metal ions), 17.7 mmol of D,L-2-hydroxy-4-phenyl-3E-butenoate and `O.64 mmol of anthraquinone-2,6-diRulfonic acid. The temperature wa~
37~0.5-C, and the rate of pumping through the electro-chemical cell wa~ ad~usted to 18 l/h. At a potential of -527 mV v~. SCE in the electrochemical cell, part of the ~i7~4 - - 17 - O.Z. 0050/43032 oxidized anthraquinone-2,6-di~uifonic acid wa~ reduced (the polarity of the cathode and anode wa~ rever~ed, which removed the di~olved oxygen in the anolyte and thu~ produced an anaerobic reaction solution). A poten-tial of -327 m~ v~. SCE wa~ then ~et up, and 450 mg of Proteu~ vulgaris cell~ (dry weight) were added in the form of a ~uspen~ion, and a maximum current of 0.1 A wa~
reached. After 56 h, the current wa~ zero becAu~e all the ~ubstrate had reacted. 8.5 ml of 2 N ~odium hydroxide ~olution were needed for titration of the anolyte (pH
8.5) up to the end of the reaction.
Conver~ions from HP~Cs 8.2 mmol (92.6%) of 2 oxo-4-phenyl-3-butenoic acid 7.7 mmol (87.0%) of L-2-hydroxy-~-phenyl-3E-butenoate Calculated from current con~umptions 8.5 mmol (96.2%) Calculated from NaOH consumptions 8.5 mmol (96.2%) The 2-keto carboxylic acid wa~ ~eparated from the L-2-hydroxy carboxylic acid by crystallization in diethyl ether and chloroform and by MICC (multi layer coil chromatography).
EXAMPL~ 15 Production of pyruvate from D-la~tate with Propioni-bacterium acidi-propionici in an electrochemical ceIl (example of a microor~anism ot~r than Proteus w lg~ri~) - 17.5 mmol of D-lactate, O.21 mmol of anthra-quinone-2,6-di~ulfonic ~cid, 0.35 m~ol of EDTA and 1 g of Proplonibacterium acidi-propionic~ cell~ (dry weight) were added to 70 ml of anolyte (de~onized w~ter) in an electrochemical flow-through c~ll with a graphite felt anode (and cathode). The reaction solution w~ circulated through the electrochemical cell at a flo~ rate of 18 l/h by a peristaltic pump. A 90 ml s~irred ve~el wa~ used for volum~ ad~ustment. The potential in the electrochemi-cal cell w~ kept con~tsnt at -300 mV v~. SCE, and a maximum current of 37 mA WaQ reached. The temperature wa~
2 1 2~
- 18 - O.Z. OOS0/43032 37+0.5C. The pH was kept con~tant at 8.5 with 4 N ~odium hydrox~de solution.
After 70 h, 96.6~ (16.9 mmol) of the ~-lactate had been oxidi2ed to pyruvate. D-~actate wa~ no longer detectable ( 5 0 . O1 mmol). No propionic acid had been produced.
Isolation of the pyruvate from the reaction ~olution~
The reaction ~olution~ were acidified to pH s 2.0 with perchloric acid and centrifuged to remove protein.
The ~upernatant wa~ then boiled for 1 h, cooled, sodium chloride was addod (~alting out) and the mixture was extracted with ether ~n a perforator for 6-8 h. The extract wa~ concentrated undor reduced pre~ure and diYsolved in about 100 ml of cold water (s 4-C) (for about 0.15 mol of pyruvic acid). Th~ solution was ad~u~ted to pH 6.0 w~th sodium hydroxide ~olution, and ethanol wa~ ~lowly added. Th~ precipitated sodium pyruv-ate wa~ then filtered off and freeze-dried for 24 h. The yield i about 95-97~ of ~odium pyruvate ~Price and Levintow, Biochem. Prepar. 2 (19S2) 22].
EXAMPL~ 17 Reaction of a racemic 2-hydroxy carboxylic acid with Proteu~ mirabilis and dimet ffl l sulfoxide a~ electron acceptor 12.5 mmol of (D,L)-2-hydroxy-4-phenyl-3E-bu~eno-at~,-0.5 m~ol of anthraquinone-2,6-d~ulfonic acid and 6.5 mmol of dime~hyl sulfoxide wera di~solved in 50 ml of deionized watsr in a 3tirred ve~sel, and 170 mg of Proteu~ mirabili~ cell~ (dry weight) w~re added. The temperature wa~ 40~0.5C. Th~ pH was k~pt con~tant at 8.5. The oxidation of (D)-2-hydroxy-4-phe~yl-3E-butenoate to 2-oxo-4-phenyl-3E-butenoate was 81% (5.1 mmol~ after 6 h and > 99~ (6.2 mmol) after 23.S h. B~ contra~, all the (L)-2-hydroxy-4-phenyl-3E-butenoate wa~ still pre~ent.
2 1 2 '~
- 19 - O.Z. OOS0/43032 Production of 2-oxo-(D)-gluconste from (D)-gluconate with Proteu~ mirabilis and dimethyl sulfoxide a~ electron acceptor 10.0 mmol of (D~-gluconate, 0.05 mmol of anthra-quinone-2,6-disulfonic acid, 0.25 mmol of EDTA and lS.0 mmol of dimethyl sulfoxide were di~olved in 50 ml of deionized water in a ~tirred ve~el, and 800 mg of Proteu~ mirabili~ cell~ (dry weight) were added. The temperaturQ was 40~0.5-C. ~he pH was kept constant at
Production of pyruvate with Proteus vulgari~ and dimethyl sulfo~ide a~ electron acceptor 35.5 mmol of D-lactate, 0.21 mmol of anthra-quinone-2,6-di~ulfonic acid and 0.21 mmol of EDTA were di~olvod in 70 ml of deionized water in a stirred ve~sel, and 362 mg of Proteu~ vulgari~ cells (dry weight) were added. 36.0 mmol of dimethyl ~ulfoxide were added in ~everal portion~ during the reaction. The ~mperatura wa~
38+0.5C. The pH was kept con~tant at 8.5 with 2 N ~odium hydroxide solution. After 8.5 h, 95.5~ (33.9 mmol) of the D-lactat~ had been oxidized to pyruvate. The conver~ion~
with Proteu~ mirabili~ were about 1.5 t~me~ fa~ter.
~1~ ia~
- 14 - O.Z. 0050/43032 After addition of a further 35.5 mmol (~ 71.0 mmol) of D-lactate and 36.0 mmol of dLmethyl sulfoxide it was po~sible to detect 50.9 mmol (71.7%, O.64 ~) of pyruvatQ and S mmol (7.0%) of D-lactate after 29 h. It is assumed that about 20~ of the pyruvate had polymerized tCopper et al., Chem. Rev. 83 (1983) 321-358~.
EXAMPL~ 9 Production of pyruvate with Proteus vulgaris from D-l~ct~te in the presence of L-lactate and dimethyl ~ulf-oxide as electron acceptor 35.S mmol of D-lactate, 35.5 mmol of L-lactate, 0.21 mmol of anthr~quinone-2,6-di~ulfonic acid and O.21 mmol of EDTA were dis~olved in 70 ml of deionized water in a stirred ve~sel, and 550 mg of Proteus vulgari~
cells (dry weight) were add~d. 36.0 mmol of dimethyl ~ulfoxide were added in several portion~ during the reaction. The temperature wa~ 38~0.5-C. The pH was kept con~tant at 8.5 with 2 N ~odium hydroxide solution. After 3.6 h, 100% (35.5 mmol) of the D-lactat~ had been oxid-ized to pyruvate. By contrast, all the L-lactate was ~till pre~ent.
Production of pyruv~te with biocatalyst (Proteus vulgari~) employed sevor~l tim~s and dimethyl ~ulfoxide a~ electron acceptor :
- 50 ml of a reaction ~olution which conta~ned 26.0 mmol of D-lactate, 0.15 mmol of anthraguinone-2,6-di~ulfonic acid and 0.25 mmol of EDTA w~re added to 720 mg of Proteu~ vulgsri~ cell~ (dry weight) in a stirrQd ~e8%~l . 26.0 m~ol of dim~thyl ~ulfoxide were added in several portion~ during the reaction. The t~mperature w~ 38~0.5C~ The pH wa~ kept ~onstant at 8.5 with 2 N qodium hydroxide ~olu~ion. The reaction wa~
stopped at a D-lactat~ concentration of s 2.0%. The Proteu~ vulqari~ cell~ were removed from the reaction ~olution by centrifugation, and a further 50 ml of 212'75~
- 15 - O.Z~ OOS0/43032 ~eaction solution wQre added. The biocatalyst was em-ployed 6 times in ~ucce~ion. The result~ are shown in the following Table:
Use I Dry weight Time Pyruvate D-lactate tmgi th' tmmol] ~mmol]
1st 1 720 1.9 25.8 0.20 2nd j 670 4.0 j26.3 10.06 3rd 600 6.3 25~9 0.34 ...
4th S40 8.5 27.0 0.33 5th 1 500 11.3 25.5 0.43 6th ¦ 480 21.5 25.3 0.40 The ~tated amount~ of pyru~ate and D-lactate relate to the reaction solution after centxifugation.
Part of the Proteu~ vulgari~ cell~ w~ lost from each ~atch owing to ~ampling and through ~eparation from the reaction ~olution. Part of the reaction ~olution remain~
in tho bacterial biomass in each batch, which explain-~
why the amount of pyru~ate i~ ~o~ewhat larger in some batches.
Production of pyruvats with Rroteu~ vulgaris in th~
culture medium 2 1 of medium A without glucose, 100 mmol of D-lact~te (no L-lactate) and 200 mmol of dim~thyl sul-foxide wera inoculat~d with 1.0 percent of a Proteus vulgar~ pr~culture. ~he growth and reaction temperature wa~ 37~1~C; the pH wa~ 7.3-7.8. Aftar 21.5 h, 60.6 mmol (60.6%) of pyruvate, 24.8 mmol (24.8~) of D-lactate and 16.8 mmol (16.8~) of acetate wer~ det~cted. Dimethyl ~ulfoxide wa~ no long~r present.
Production of pyrUVatQ with Proteus vulgaris and stoi-chiometric mediator a~ electron acceptor 17.0 mmol of D-lactate, 17.0 mmol of 21~. s ~4~
- - 16 - O.z. 0050/43032 anthrawequinone-2,6-disulfonic acid and 0.35 mmol of EDTA
were dis~olved in 70 ml of deionized water in a stirred ve~sel, and 360 mg of Proteu~ vulgaris cell~ (dry weight) were added. The temperature wa~ 40~0.5-C. The pH was kept S con~tant at 8.5 with 4 N ~odium hydroxide solution. After 2.0 h, 99.0% (16.8 mmol) of the D-lactate had been oxidized to pyruvate. 70% (12 mmol) of the anthraquinone-2,6-di~ulfonie aeid were reeovered by centrifuging the reaetion solution at s 4-C and were re-u~ed. ~he remai-ning 30% (5 mmol) remained di~solved in the reaetion~olution.
Produetion of pyruvate with Proteu~ vulgari~ cell~
immobilized on ~intered gla~ Rasehig ring~
98.9 mmol of D-laetate, 1.25 mmol of anthra-quinone-2,6-di~ulfonate and 0.25 mmol of EDTA were di~olved in 250 ml of deionized w~ter in a fixed bed eireul~tion reaetor, and 28.2 mg of Proteus vulgari~
eell~ (dry weight, immobilized on ~intered gla~s Ra~ehi~
ring~) were employed. The reaetion volume wa~ eireulated at a flow r~te of 20 l/h through an eleetroehemieal eell.
The pH was kept eon~tant with 2 N ~odium hydroxide solution. The temperature wa~ 35~0.5-C. The potential of the eleetroehemieal eell wa~ -300 mV v~. SCE. After 116.5 h, 64.4~ (63.7 m~ol) of the D-laetate had been oxidized to pyruvate.
- EXANPL~ 14 Con~er~ion of a rae~mie 2-hydroxy earboxylie acid with Proteu~ vulgari~
40 ml of anolyte eontain~d 5 mg of EDTA (to prevent deearboxylation of the resulting 2-oxo-4-phenyl-- 3-butenoie a~id by divalen~ metal ions), 17.7 mmol of D,L-2-hydroxy-4-phenyl-3E-butenoate and `O.64 mmol of anthraquinone-2,6-diRulfonic acid. The temperature wa~
37~0.5-C, and the rate of pumping through the electro-chemical cell wa~ ad~usted to 18 l/h. At a potential of -527 mV v~. SCE in the electrochemical cell, part of the ~i7~4 - - 17 - O.Z. 0050/43032 oxidized anthraquinone-2,6-di~uifonic acid wa~ reduced (the polarity of the cathode and anode wa~ rever~ed, which removed the di~olved oxygen in the anolyte and thu~ produced an anaerobic reaction solution). A poten-tial of -327 m~ v~. SCE wa~ then ~et up, and 450 mg of Proteu~ vulgaris cell~ (dry weight) were added in the form of a ~uspen~ion, and a maximum current of 0.1 A wa~
reached. After 56 h, the current wa~ zero becAu~e all the ~ubstrate had reacted. 8.5 ml of 2 N ~odium hydroxide ~olution were needed for titration of the anolyte (pH
8.5) up to the end of the reaction.
Conver~ions from HP~Cs 8.2 mmol (92.6%) of 2 oxo-4-phenyl-3-butenoic acid 7.7 mmol (87.0%) of L-2-hydroxy-~-phenyl-3E-butenoate Calculated from current con~umptions 8.5 mmol (96.2%) Calculated from NaOH consumptions 8.5 mmol (96.2%) The 2-keto carboxylic acid wa~ ~eparated from the L-2-hydroxy carboxylic acid by crystallization in diethyl ether and chloroform and by MICC (multi layer coil chromatography).
EXAMPL~ 15 Production of pyruvate from D-la~tate with Propioni-bacterium acidi-propionici in an electrochemical ceIl (example of a microor~anism ot~r than Proteus w lg~ri~) - 17.5 mmol of D-lactate, O.21 mmol of anthra-quinone-2,6-di~ulfonic ~cid, 0.35 m~ol of EDTA and 1 g of Proplonibacterium acidi-propionic~ cell~ (dry weight) were added to 70 ml of anolyte (de~onized w~ter) in an electrochemical flow-through c~ll with a graphite felt anode (and cathode). The reaction solution w~ circulated through the electrochemical cell at a flo~ rate of 18 l/h by a peristaltic pump. A 90 ml s~irred ve~el wa~ used for volum~ ad~ustment. The potential in the electrochemi-cal cell w~ kept con~tsnt at -300 mV v~. SCE, and a maximum current of 37 mA WaQ reached. The temperature wa~
2 1 2~
- 18 - O.Z. OOS0/43032 37+0.5C. The pH was kept con~tant at 8.5 with 4 N ~odium hydrox~de solution.
After 70 h, 96.6~ (16.9 mmol) of the ~-lactate had been oxidi2ed to pyruvate. D-~actate wa~ no longer detectable ( 5 0 . O1 mmol). No propionic acid had been produced.
Isolation of the pyruvate from the reaction ~olution~
The reaction ~olution~ were acidified to pH s 2.0 with perchloric acid and centrifuged to remove protein.
The ~upernatant wa~ then boiled for 1 h, cooled, sodium chloride was addod (~alting out) and the mixture was extracted with ether ~n a perforator for 6-8 h. The extract wa~ concentrated undor reduced pre~ure and diYsolved in about 100 ml of cold water (s 4-C) (for about 0.15 mol of pyruvic acid). Th~ solution was ad~u~ted to pH 6.0 w~th sodium hydroxide ~olution, and ethanol wa~ ~lowly added. Th~ precipitated sodium pyruv-ate wa~ then filtered off and freeze-dried for 24 h. The yield i about 95-97~ of ~odium pyruvate ~Price and Levintow, Biochem. Prepar. 2 (19S2) 22].
EXAMPL~ 17 Reaction of a racemic 2-hydroxy carboxylic acid with Proteu~ mirabilis and dimet ffl l sulfoxide a~ electron acceptor 12.5 mmol of (D,L)-2-hydroxy-4-phenyl-3E-bu~eno-at~,-0.5 m~ol of anthraquinone-2,6-d~ulfonic acid and 6.5 mmol of dime~hyl sulfoxide wera di~solved in 50 ml of deionized watsr in a 3tirred ve~sel, and 170 mg of Proteu~ mirabili~ cell~ (dry weight) w~re added. The temperature wa~ 40~0.5C. Th~ pH was k~pt con~tant at 8.5. The oxidation of (D)-2-hydroxy-4-phe~yl-3E-butenoate to 2-oxo-4-phenyl-3E-butenoate was 81% (5.1 mmol~ after 6 h and > 99~ (6.2 mmol) after 23.S h. B~ contra~, all the (L)-2-hydroxy-4-phenyl-3E-butenoate wa~ still pre~ent.
2 1 2 '~
- 19 - O.Z. OOS0/43032 Production of 2-oxo-(D)-gluconste from (D)-gluconate with Proteu~ mirabilis and dimethyl sulfoxide a~ electron acceptor 10.0 mmol of (D~-gluconate, 0.05 mmol of anthra-quinone-2,6-disulfonic acid, 0.25 mmol of EDTA and lS.0 mmol of dimethyl sulfoxide were di~olved in 50 ml of deionized water in a ~tirred ve~el, and 800 mg of Proteu~ mirabili~ cell~ (dry weight) were added. The temperaturQ was 40~0.5-C. ~he pH was kept constant at
9.3~0.1. After ~ 20 h, 99~ (9.9 mmol) of the ~D)-glucon-ate had been oxidized to 2-oxo-(D)-gluconate.
Claims (2)
1. A process for preparing 2-keto carboxylic acids from (D)-2-hydroxy carboxylic acids using a biocatalyst, which comprises transferring the electrons which are produced in the oxidation of (D)-2-hydroxy carboxylic acids to a quinone derivative.
2. A process for preparing mixtures of 2-keto carboxylic acids and (L)-2-hydroxy carboxylic acids from (D,L)-2-hydroxy carboxylic acids using a biocatalyst, which comprises transferring the electrons which are produced in the oxidation of (D)-2-hydroxy carboxylic acids to a quinone derivative.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DEP4205391.9 | 1992-02-21 | ||
| DE4205391A DE4205391A1 (en) | 1992-02-21 | 1992-02-21 | PROCESS FOR THE ENZYMATIC OXIDATION OF (D) -2-HYDROXYCARBONE ACID TO 2-CETOCARBONE ACIDS |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2127544A1 true CA2127544A1 (en) | 1993-09-02 |
Family
ID=6452303
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002127544A Abandoned CA2127544A1 (en) | 1992-02-21 | 1993-02-09 | The enzymatic oxidation of (d)-2-hydroxy carboxylic acids to 2-keto carboxylic acids |
Country Status (10)
| Country | Link |
|---|---|
| EP (1) | EP0627007B1 (en) |
| JP (1) | JPH07503852A (en) |
| KR (1) | KR950700422A (en) |
| AT (1) | ATE180017T1 (en) |
| CA (1) | CA2127544A1 (en) |
| DE (2) | DE4205391A1 (en) |
| DK (1) | DK0627007T3 (en) |
| ES (1) | ES2133384T3 (en) |
| TW (1) | TW369563B (en) |
| WO (1) | WO1993017120A1 (en) |
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|---|---|---|---|---|
| EP0978567B1 (en) * | 1997-12-29 | 2005-03-09 | Sanyo Shokuhin Co., Ltd. | Process for producing optically active alcohols |
| TWI275645B (en) * | 2000-02-16 | 2007-03-11 | Daicel Chemical Industries Ltd. | (R)-2-octanol dehydrogenases, methods for producing the enzymes, DNA encoding the enzymes, and methods for producing alcohols using the enzymes |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| DE3226888A1 (en) * | 1982-07-17 | 1984-01-19 | Basf Ag, 6700 Ludwigshafen | METHOD FOR CARRYING OUT ELECTROMICROBIAL REDUCTIONS |
| DE3631228A1 (en) * | 1986-09-13 | 1988-03-24 | Basf Ag | METHOD FOR CARRYING OUT ENZYMATIC OXIDATIONS |
| FR2614038B1 (en) * | 1987-04-17 | 1989-08-04 | Centre Nat Rech Scient | ELECTROENZYMATIC PROCESS FOR THE PRODUCTION OF ENANTIOMERIC CONTROLLED PURITY COMPOUNDS |
-
1992
- 1992-02-21 DE DE4205391A patent/DE4205391A1/en not_active Withdrawn
-
1993
- 1993-02-09 KR KR1019940702904A patent/KR950700422A/en not_active Application Discontinuation
- 1993-02-09 CA CA002127544A patent/CA2127544A1/en not_active Abandoned
- 1993-02-09 AT AT93903936T patent/ATE180017T1/en not_active IP Right Cessation
- 1993-02-09 EP EP93903936A patent/EP0627007B1/en not_active Expired - Lifetime
- 1993-02-09 ES ES93903936T patent/ES2133384T3/en not_active Expired - Lifetime
- 1993-02-09 DE DE59309577T patent/DE59309577D1/en not_active Expired - Lifetime
- 1993-02-09 JP JP5514490A patent/JPH07503852A/en active Pending
- 1993-02-09 WO PCT/EP1993/000310 patent/WO1993017120A1/en active IP Right Grant
- 1993-02-09 DK DK93903936T patent/DK0627007T3/en active
- 1993-02-11 TW TW082100958A patent/TW369563B/en active
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| WO1993017120A1 (en) | 1993-09-02 |
| EP0627007B1 (en) | 1999-05-12 |
| DK0627007T3 (en) | 1999-11-01 |
| EP0627007A1 (en) | 1994-12-07 |
| KR950700422A (en) | 1995-01-16 |
| ATE180017T1 (en) | 1999-05-15 |
| DE4205391A1 (en) | 1993-08-26 |
| DE59309577D1 (en) | 1999-06-17 |
| ES2133384T3 (en) | 1999-09-16 |
| TW369563B (en) | 1999-09-11 |
| JPH07503852A (en) | 1995-04-27 |
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