Note: Descriptions are shown in the official language in which they were submitted.
~ 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
- 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,
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-
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~).
EXANPLE 2
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%.
EXAMPLE 4
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.
3S EXAMPLE 6
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.
EXAMPLE 7
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.
EXAMPLE 8
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.
EXAMPLE 10
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.
EXAMPLE 11
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.
EXAMPLE 12
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.
EXAMPLE 13
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.
EXAMPLE 16
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
EXAMPLE 18
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.
