Levodione reductase

Details Levodione reductase Levodione reductase

2000-01-31

2002-10-15

Marx, Irene (Department: 1651)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Oxidoreductase

C435S170000, C435S171000

Reexamination Certificate

active

06465228

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to a novel enzyme, in particular a levodione reductase (hereinafter referred to as LR), a process for producing the enzyme, and a process for producing (4R, 6R)-4-hydroxy-2,2,6-trimethylcyclohexanone (hereinafter referred to as actinol) from (6R)-2,2,6-trimethylcyclohexane-1,4-dione (hereinafter referred to as levodione) utilizing the enzyme.
BACKGROUND OF THE INVENTION
Actinol is an important intermediate for the production of zeaxanthin. European Patent Application Nos. 98115564.1 and 99115723.1 disclose processes for the manufacture of actinol. Such processes include contacting levodione with a microorganism selected from Cellulomonas, Corynebacterium, Planococcus and Arthrobacter which is capable of the selective asymmetric reduction of levodione to actinol. The resulting actinol from the reaction mixture is recovered therefrom.
Corynebacterium aquaticum
AKU611 (FERM BP-6448) was found to be one of the best microorganism strains for this purpose.
Corynebacterium aquaticum
AKU611 has the following taxonomical properties:
1)
Growable temperature
15-40° C.
2)
Optimum temperature for growth
  30° C.
3)
Obligatory aerobic and gram negative
microorganism
4)
Spore formation
None
5)
Polymorphism and traditional rod-cocus
cycles may be observed during cultivation
6)
Motility
None
Moreover, the strain
Corynebacterium aquaticum
AKU611 was identified as having these characteristics based on assimilation of various carbon sources by the Biolog System (Biolog Inc., Hayward, Calif., see also Nature Vol. 339, 157-158, May 11, 1989) as follows: 96-well microtiter-plates were inoculated with
Corynebacterium aquaticum
cells and incubated for 24 hours at 28° C. Each well contained one of the 96 types of carbon sources in BUGM+B medium (Biolog Universal Growth Media+Blood) (Biolog Inc.).
After incubation, the strain showed the following assimilation of carbon sources:
C Source
Assimilation
C Source
Assimilation
A1
−
A2
−
A3
−
A4
−
A5
−
A6
−
A7
−
A8
+
A9
+
A10
−
A11
−
A12
+
B1
−
B2
+
B3
−
B4
−
B5
+
B6
−
B7
+
B8
−
B9
+
B10
+
B11
+
B12
−
C1
−
C2
+
C3
−
C4
+
C5
+
C6
+
C7
−
C8
+
C9
−
C10
−
C11
−
C12
−
D1
−
D2
−
D3
+
D4
−
D5
+
D6
−
D7
−
D8
+
D9
−
D10
−
D11
+
D12
+
E1
−
E2
−
E3
+
E4
−
E5
−
E6
−
E7
−
E8
−
E9
−
E10
−
E11
−
E12
−
F1
−
F2
−
F3
−
F4
−
F5
−
F6
+
F7
−
F8
−
F9
−
F10
−
F11
−
F12
−
G1
−
G2
−
G3
−
G4
−
G5
−
G6
−
G7
−
G8
−
G9
−
G10
−
G11
−
G12
−
H1
−
H2
−
H3
−
H4
−
H5
−
H6
−
H7
−
H8
−
H9
−
H10
−
H11
−
H12
−
In the table, “+” indicates that the carbon source was assimilable and “−” indicates that it was not assimilable.
The alpha-numeric codes set forth above are defined in the table below:
A1
Water
A2
&agr;-cyclodextrin
A3
&bgr;-cyclodextrin
A4
Dextrin
A5
Glycogen
A6
Inulin
A7
Mannan
A8
TWEEN ® 40
A9
TWEEN ® 80
A10
N-acetyl-D-glucosamine
A11
N-acetyl-D-mannosamine
A12
Amygdalin
B1
L-arabinose
B2
D-arabitol
B3
Arbutin
B4
Cellobiose
B5
D-fructose
B6
L-fucose
B7
D-galactose
B8
D-galacturonic acid
B9
Gentiobiose
B10
D-gluconic acid
B11
&agr;-D-glucose
B12
m-inositol
C1
&agr;-D-lactose
C2
Lactulose
C3
Maltose
C4
Maltotriitrose
C5
D-mannitol
C6
D-mannose
C7
D-melezitose
C8
D-melibiose
C9
&agr;-methyl-D-galactoside
C10
&agr;-methyl-D-galactoside
C11
3-methyl-glucose
C12
&agr;-methyl-D-glucoside
D1
&bgr;-methyl-D-glucoside
D2
&agr;-methyl-D-mannoside
D3
Palatinose
D4
D-psicose
D5
D-raffinose
D6
L-rhamnose
D7
D-ribose
D8
Salicin
D9
Sedoheputulosan
D10
D-sorbitol
D11
Stachyose
D12
Sucrose
E1
D-tagatose
E2
D-trehalose
E3
Turanose
E4
Xylitol
E5
D-xylose
E6
acetic acid
E7
&agr;-hydroxybutyric acid
E8
&bgr;-hydroxybutyric acid
E9
&ggr;-hydroxybutyric acid
E10
p-hydroxy-phenylacetic acid
E11
&agr;-keto-glutaric acid
E12
&agr;-keto-valeric acid
F1
Lactamide
F2
D-lactic acid methyl ester
F3
L-lactic acid
F4
D-malic acid
F5
L-malic acid
F6
methyl pyruvate
F7
Monomethyl succinate
F8
propionic acid
F9
Pyruvic acid
F10
Succinamic acid
F11
Succinic acid
F12
N-acetyl-L-glutamic acid
G1
Alaninamide
G2
D-alanine
G3
L-alanine
G4
L-alanyl-glycine
G5
L-asparagine
G6
L-glutamic acid
G7
glycyl-L-glutamic acid
G8
L-pyloglutamic acid
G9
L-serine
G10
Putrscine
G11
2,3-butanediol
G12
Glycerol
H1
Adenosine
H2
2′-deoxy-adenosine
H3
Inosine
H4
Thymidine
H5
Uridine
H6
Adenosine-5′-monophos-
phate
H7
thymidine-5′-monophos-
H8
Uridine-5′-monophosphate
phate
H9
Fructose-6-phosphate
H10
Glucose-1-phosphate
H11
Glucose-6-phosphate
H12
DL-&agr;-glycerol phosphate
SUMMARY OF THE INVENTION
One embodiment of the present invention provides an isolated and purified enzyme having levodione reductase activity wherein the enzyme includes the following physico-chemical properties:
(a) a molecular weight of about 142,000 to about 155,000±10,000;
(b) a nicotinamide adenine dinucleotide (AND/NADH) co-factor;
(c) a substrate specificity for levodione;
(d) an optimum temperature of about 15° C. to about 20° C. at a pH of about 7.0;
(e) an optimum pH of about 7.5; and
(f) wherein the enzyme is activated by K
+
, Cs
+
, Rb
+
, Na
+
and NH
4
+
.
A process for producing an enzyme having levodione reductase activity is also provided wherein the enzyme has the following physico-chemical properties: a molecular weight of about 142,000 to about 155,000±10,000, a nicotinamide adenine dinucleotide (AND/NADH) co-factor, a substrate specificity for levodione, an optimum temperature of about 15° C. to about 20° C. at a pH of about 7.0, an optimum pH of about 7.5, and wherein the enzyme is activated by K
+
, Cs
+
, Rb
+
, Na
+
and NH
4
+
. This process includes cultivating cells of a Corynebacterium in an aqueous nutrient medium under aerobic conditions; and disrupting the cells to form a cell free extract containing the enzyme.
A process is also provided for producing actinol from levodione. This process includes forming a reaction mixture containing levodione and (i) a cell-free extract derived from Corynebacterium containing a levodione reductase or (ii) a levodione reductase having the following physico-chemical properties: a molecular weight of about 142,000 to about 155,000±10,000, a nicotinamide adenine dinucleotide (AND/NADH) co-factor, a substrate specificity for levodione, an optimum temperature of about 15° C. to about 20° C. at a pH of about 7.0, an optimum pH of about 7.5, and which enzyme is activated by K
+
, Cs
+
, Rb
+
, Na
+
and NH
4
+
; adding a reduced form of nicotinamide adenine dinucleotide to the reaction mixture; and isolating actinol from the reaction mixture.
An embodiment provides an isolated and purified levodione reductase derived from
Corynebacterium aquaticum
AKU611 (FERM BP-6448) cells having the following properties:
(a) a molecular weight of about 142,000 to about 155,000±10,000,
(b) a AND/NADH cofactor;
(c) a substrate specificity for levodione;
(d) an optimum temperature of about 15° C. to about 20° C. at a pH of about 7.0;
(e) a optimum pH of about 7.5; and
(f) the levodione reductase being activated by K
+
, Cs
+
, Rb
+
, Na
+
and NH
4
+
.
DETAILED DESCRIPTION OF THE INVENTION
A purified LR sample prepared according to the Examples presented below has the following physico-chemical properties:
1) Enzyme Activity
The novel LR of the present invention catalyzes the reduction of levodione to actinol in the presence of a co-factor according to the following formula:
Levodione+NADH⇄Actinol+AND
It has been determin

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