JPS62294082A - Enzyme and its production - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

Detailed Description of the Invention 3. Detailed Description of the Invention The present invention relates to MjiJ, L-sorbosone dehydrogenated N, and a method for producing the same.

The L-sorbosone dehydrogenase provided by the present invention is produced by L-sorbosone dehydrogenase in the presence of nicotinamide 7-denine dinucleotide (hereinafter referred to as NAD) or nicotinamide adenine dinucleotide phosphate (hereinafter referred to as NADP) as a coenzyme. Sorboson's 2-keto L-chloric acid (hereinafter referred to as 2
-KGA). 2-KGA is
It is an important precursor for the production of vitamin C.

The reaction that converts L-sorbosone to 2-KGA in microorganisms is known. The production of 2-KGA·＼ from noshin rubosone using cell-free extracts of microorganisms has been reported in various publications. U.S. Patent No. 3,907
．． Publication No. 639 describes Acetobacter (AceLob).
acLcr), Pseudomonas (P scudomo
nas)i, Escherichia (E 5cherichia)
) genus, Serratia genus, Bacillus (
Bacillus S), Staphyla coccus (S La
phylococcus) is Aerobacter (Ae
robacLer), Alcaligenes (A Ical)
iHenes) fi, Penicillium
It has been reported that all organisms belonging to the family Iuconobacter, the genus Candida, and the genus Gluconobacter undergo the above-mentioned transformation.

Kitamura et al.
onobacter melanogenes) I
L-Sorbosone oxidation 6F discovered from FO3293
reported that neither electron acceptors nor coenzymes are required for the expression of enzymatic activity (European Journal Op. , J.

Appl, Microbiol, 2.1.197
5. Furthermore, Makover et al.
reported the presence of L-sorbosone dehydrogenase in certain fractions of Gluco7pacta melanodenes IFO3293 and ATCC21812.
Page 1485, 1975: B 1otechnol
, Bioeng.

17.1485.1975). The authors also pointed out that nicotinamide 7-denine dinucleotide or nicotinamide adenine dinucleotide phosphate did not act as a coenzyme for the enzyme.

However, as mentioned above, NAD or NAD as a coenzyme
To date, there has been no disclosure of a vi-produced enzyme having the activity of oxidizing L-sorbosone to 2-KGA in the presence of P.

The present invention was completed based on the discovery that an enzyme isolated and purified from the cytoplasmic fraction of cells of a specific microorganism catalyzes the oxidation of L-sorbosone to 2-KGA.

The object of the present invention is to use NAD or N, %r as complement lJ elements.
l D /A left pull r 'r 7 1/ 11-71
7%/M9-V/'! A/.

An object of the present invention is to provide a novel L-sorbosone dehydrogenase that catalyzes the oxidation of L-sorbosone. Another purpose is to identify all organisms belonging to the genus Gluconobacter or their mutant strains that have the ability to produce L-sorbosone dehydrogenation in their cells. ! L
, the cells are disrupted, and a cell-free extract of the disrupted cells, preferably from the cytoplasm of the microorganism, is dehydrogenated.
An object of the present invention is to provide a novel method for producing a single-rubosone dehydrogenase, which is characterized by isolating and purifying G.

The physicochemical properties of the purified sample of the novel 1,-Sorbosone dehydrogenase obtained in the Examples described below are as follows.

(1) Enzyme activity The L-sorbosone dehydrogenase of the present invention has N as a coenzyme.
2-K L-sorbosone in the presence of AD or NADP
The reaction mode for catalyzing the reaction of converting into GA is as shown below.

■, - Sorboson + NDA or NAD I) -1
2-KGA 10 Reduced NAD or NADP - Measurement of Enzyme Activity Enzyme activity is determined by measuring the increase in absorbance of reduced NAD or NADP at 340 nm. The reaction solution contained 11 g of Sorboson, 0.2 mg of NAD in 50a + M potassium phosphate 41 equilibrium solution (ρ) 17.0') 0.41s + I, and the reaction was as follows:
Started by addition of enzyme. Reduced NAD or N
The production rate of ADP was measured at 30° C. using a spectrophotometer (Ebicon 810, manufactured by Kontron). The amount of enzyme required to produce 1 μM of reduced NAD or NADP at 30° C. for 1 minute was defined as 1 unit.

Requirements for the Issode Exploration - The requirements for the i-enzyme required for the expression of the present enzyme activity were investigated under the above-mentioned standard measurement conditions. As shown in table fjS1,
NAD could also serve as a punishment for NADP and others.

Milk 1 kg 4 No additives 0 NAD 100 NADP 35 7 bottles The activity against NAD is expressed as 100 ■) Substrate specificity The substrate specificity of this enzyme is determined by using the activity measurement method described in (1) for the substrate sorbosone. The reaction was carried out by replacing the various substances shown in Table 2. As shown in the results of 2nd R, L-sorbosone showed the highest activity against this enzyme. It showed oxidizing activity against glyoxal, glycolaldehyde, glutaraldehyde, pyropionaldehyde, menalglyoxal, acetaldehyde, and romannose.

No. 1 L-Sorboson 100 Glyoxal 33,3 Glycolaldehyde 23.3 Glutaraldehyde
16.7 Propion 7 Rudehyde
13.3 Methylglyoxal 10.0
77taldehyde 8. OD-mannose 4.9, benzaldehyde O glyoxytttO gelcolic acid OD-glucorone OD-fructose
OD-glucose
OL-Sorbose O-Hydroxyacetone O-Hydroxypyruvate Displayed as ■ Optimum pH Using potassium phosphate am solutions of various pH and ammonium buffer solution, purified enzyme L-sorbosone theory hydrogen #
The elementary activity and the effect of pH on the activity were investigated, and the results are shown in the tIS3 table.6LL 8.0 9>Fllh') 7 Mumu, which showed the highest activity at a pH of about 9.0, tNllrffiH3'
4.76.5 71.4
7.0 100,07・5
163.38.0
269.48, ONH401+
/NH4Cl, tli solution 93.99.0
326,510.0
, 1 bottle Displayed with activity at pH 7.0 as 100 (4) pH stability quality! J/j cables were added to pine kilvane buffer solutions (mixture of 0.1 M citric acid and 0.2 M disodium phosphate) at various pHs and left at 4° C. for 24 hours. The residual activity was measured under the nA measurement conditions shown in 0) above.

The measurement results are shown in Table 4. The purified enzyme has a pH of 6.0-
It was relatively stable at 8.0.

11 to 4 61.6 5 76.8 6 88.9 8 94.9 The activity at pH 7 is expressed as 100. After treatment for 10 minutes, the mixture was immediately cooled with ice water.

Residual activity was measured under the standard measurement conditions shown in (1) above. The measurement results are shown in Table 5.

The purified enzyme was stable up to 50°C and lost approximately 80% activity at 60°C.

11 Table 20℃ No. 10 100 30 II 96.440
7 92.9 5 Q it 82 , 160
II 21.4 bottles The activity at 20°C is expressed as 100 (6) The enzyme activity of L-Sorbosone dehydrogenase at the optimum temperature is shown in J. Yuki (1)
The measurement was carried out at a temperature of 25°C to 60°C under the standard measurement conditions shown in . The obtained results are shown in the MS6 table.

The activity of the enzyme of the invention increased with increasing temperature up to 60°C.

25 62.5 quintillion 30℃
The activity at is expressed as 100 σ) Molecular weight
, 0, and developed with the same buffer solution. 190°000±20.0001
．． : [Enzyme activity was found in the corresponding fraction.

(8) Measurement of Michaelis constant WL (K16) Under standard measurement conditions, various N
Measuring the oxidation rate versus AD or NADP concentration;
The Km constant was determined. As a result, the m constants for NAD and NADP are 4.55 x 10''SM, respectively.
and 1.60×10 −5 M.

(9) Effect of metal ions The effects of various metal ions on enzyme activity were investigated using the measurement method described above. The results obtained are shown in Table 7. Mg2+ and Fe2+ promote enzyme activity, but
Cu”+ inhibited.

Daidoshijin – Additive-free 100Mg”
10 113F'e"
1 116Mo”
10 96Cu”
0.1 0-* Activity without additive is expressed as 100 (10) Influence of inhibitors Using the measurement method described above, the influence of various inhibitors on # talkability was investigated. The results obtained are shown in Table 8.

N-ethylmaleimide strongly inhibited enzyme activity.

11 Flame N-ethylmaleimide 10 Ha-monoiodoacetic acid 5 57.7 Sodium anode 5 100 Dithiothreitol 5 119.2 No additives
-100 The activity is expressed as 100 when no additives are used. It can be purified by methods such as chromatography, del filtration, gel electrophoresis, salting out, dialysis, and combinations thereof.

The L-sorbosone dehydrogenase provided by the present invention is
It can be prepared by culturing a suitable microorganism, disrupting the cells, and separating and purifying a cell-free extract of the disrupted cells, preferably from the cytoplasm of the microorganism.

The microorganism used in the present invention is a microorganism belonging to the genus Gluconobacter or a mutant strain thereof.

For example, all strains belonging to the latest classification, Gluconobacter, belong to the species Gluconobacter oxydans. The shape of a strain belonging to Gluco/Bacter oxydans!
The biological and biological Vf signatures are described in the Pernoise Manual of Systematic Bacteriology (Bernois Manual of Systematic Bacteriology).
gey's Manual of SysLema
ticB ncterioloBy)Vol,
I% PIS pages 275-278, 19
1984 and F”, Gossele
), International Journal of Systematic Bacteriology (1nternaLion), etc.
al J, System+B acterio
Vol. 33, pp. 65-81, 1983
It was written in 2013.

The microorganisms belonging to the genus Gluconobacter used in the present invention can be isolated from nature or are available from depositories. Mutant strains derived therefrom can also be used in the present invention.

The mutant strains used in the present invention are generated by UV, X- or gamma-irradiation of wild plants, or by contacting them with nitrite or other suitable mutagens, or by natural mutation. It can be obtained by isolating a clone. Mutations of these wild flavors or mutant strains thereof can be effected for that purpose by any method familiar per se to the person skilled in the art. Many of these methods are described in various publications, such as "Chemical Mutagen," edited by Dejima, Yoshihichi, and Kata, published by Kodansha Jens, 1973.

The mutant strain of the present invention can also be obtained by cell fusion of a strain belonging to the species Gluconobacter oxydans in combination with mutant X4 induction and/or cell fusion.

The most preferred strains in the present invention are Gluconobacter oxydans UV-10, Gluco/Bacter oxydans E-1, and Gluconobacter oxydans H-
2, Gluconobacter oxidans l, -8, etc. These strains have been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology under the following accession numbers. The domestic deposit of the following microorganisms was transferred to the international deposit based on the Butabest Treaty on January 29, 1988, and each microorganism has the following accession number.

Gluconobacter oxydans us-io:r microtechnical laboratory PtrJ8422J (FERN-PNo. 84
22), [Electrical Engineering Research Article ttS1267 No. J (FERM BP
-1267) Gluconobacter oxidans E-1: FERM BP Gluconobacter Φ Oxyguns H-2: “Hikou Research Institute, 8th
No. 354 J (FERM-P No. 8354), “Kikoken Joyo No. 1266 J (FERM-BP Gluconobacter Oxidans L-8 “Kikou Kenjoyo No. 8355
No. J (FERM-P No. 8355), [FERMBP No. 1268 J (FERM-P No. 8355),
It can be cultured under aerobic conditions in a liquid medium supplemented with suitable nutrient sources. Cultivation can be carried out at pH 4.0 to about 8.0, preferably pH 4.5 to 6.5. Cultivation time varies depending on the microorganism and nutrient medium used, but is preferably about 10 to 6.5. It is 100 hours. The suitable temperature range for culturing is approximately 10°C to 40°C;
Preferably it is 25°C-35°C.

The medium usually contains an assimilable carbon source, e.g. glycerin,
D-mannitol, D-sorbitol, erythritol, ribitol, xylitol, arabitol, inositol, and lucitol, D-ribose, D-fructose, D-7cose, D-glucose, gluconate, L-
sorbose, maltose and sucrose, preferably L-sorbose, D-sorbitol or glycerin; extinguishable nitrogen sources such as peptone, yeast extract,
Organic substances such as soy flour and corn steep liquor; inorganic substances such as ammonium sulfate, ammonium chloride and potassium nitrate; vitamins and trace elements etc.
! It is necessary to include the source.

An embodiment for isolating and purifying L-sorbosone dehydrogenase from microorganisms after culturing will be briefly described below.

(1) Culture 1 by centrifugation! Harvest cells from the fluid.

(2) Wash the collected cells with distilled water, physiological saline or a mild solution with an appropriate pH.

(2) Suspend the washed cells in a buffer solution and disrupt them using a homogenizer, sonicator, lysozyme treatment, etc. to obtain a cell disruption solution.

(4) Isolate and purify L-sorbosone dehydrogenase from a cell-free extract of disrupted cells, preferably a cytosolic fraction of a microorganism.

L-Sorbosone dehydrogenase provided by the present invention is useful as a catalyst for producing 2-KGA from L-Sorbosone. One reaction is NAD* or NA
It must be carried out in the presence of DP in a solution such as potassium phosphate buffer, ammonium buffer, etc. at a p) (value of about 5° to about 10.0. The preferred temperature range for carrying out the reaction is , about 20°C to about 70°C.9
The reaction gives the most favorable results when H and temperature are set at about pH 8.0-9.0 and 60 °C, respectively; the concentration of L-sorbosone in solution can be varied depending on other reaction conditions. Generally speaking, about 10-100
g/jt is preferred, and about 30-40 g/jt is most preferred.

In the reaction, the enzyme can also be used in a state immobilized on a suitable carrier. Any commonly known means for fixing #wires can be used. For example, #element may be directly bonded to a resin film, fine particles, etc. having a functional group, or a difunctional group such as gel talquardehyde can be used. It may be bonded to fllffl via a bridging compound having the following.

The present invention will be explained below with reference to Examples.

Example I Preparation of L-sorbosone dehydrogenase (1) Gluconobacter-φoxyguns LJV-1
An agar slant culture of Glufubacter oxydans UV-10 (Hui Technological Research Institute No. 1267) was cultured at No.

The cells were inoculated into test tubes containing 3B medium 51 and cultured on a reciprocating shaker at 27°C for 3 days. The medium contained 7.0% L-sorbose, 0.05% glycerin, 1.5% yeast extract (manufactured by Orient), MgSO4.7H200,
Contains 25% Ca COy 1 and 0%. Culture 1 mock I was grown in the same medium 5001 as above containing 50j1.
Seed #C in an Erlenmeyer flask and cultured on a rotary infiltration chamber (180r, p, m,) at 30°C for 3 days.

The obtained culture 8001 was transferred to No. 3B medium 20! 6 fermenters inoculated into 301 fermenters containing
It was stirred at 0 r, p, + a, and aerated at 1 v, v, m (=volume of air/volume of medium 7 min). 40 o'clock party *
a. Cultures were collected to collect bacterial cells.

jH1 solution, 1,500 r, p, m, (365Xg
) for 10 minutes to remove calcium carbonate, and
The cells were centrifuged at 8°000 r, p, m (10,000×g) to obtain bacterial cells. The obtained bacterial mass was reduced to 0.85%.
Washed once with physiological saline. In this way, cultivation 1! 374 g of wet bacterial cells were obtained from the liquid 201.

(2) Production of Cytoplasmic Fraction (a) 94 g of the Gluconobacter oxydans face piece obtained in step (1) above was washed twice with physiological saline. The washed bacterial cells were added to 10+sM potassium phosphate buffer solution 470
1 and glass peas (0,1-φ) using a Guino Mill (Willi A, Patsuko 7 En Co., Ltd. > m-body crusher) at 2,000 r, pom.

for 4 minutes and crushed. 4.000r, p, listed (1
After centrifuging at 800 xg for 10 minutes to remove viable cells, the cell-free extract was centrifuged at 40.000 r, p, s+, (
The mixture was centrifuged at 80 eo o o xg for 1 hour. The resulting supernatant was collected as a cytoplasmic fraction (470ml).

(3) Diethylamide/ethylcellulose Cl-6B
Column chromatography All operations were performed at 4°C. The cytoplasmic fraction 4701 of Gluconobacter oxydans uv-io was
Dialysis was performed for 15 hours against M potassium phosphate buffer solution (pb 7°0). The dialyzed cytoplasmic fraction was equilibrated with quethylaminoethyl (hereinafter referred to as DEAE) in the same buffer solution as used during dialysis.
(referred to as ) onto a cellulose CL-6B column. Thereafter, the column was washed with the same buffer solution as above, and # element was eluted by a linear gradient elution method at a sodium chloride concentration of 0 to 0.2M in the buffer solution. Approximately 0. The enzyme activity was found in the solution eluted with iM sodium chloride. Next, the active fractions were pooled and dialyzed against 10+M potassium phosphate 41 buffer solution (pH 7.0) for 5 hours.

(4) DEAE Sephadex A-40 column chromatography The dialyzed solution was equilibrated with the same buffer W solution as above.
It was subjected to EAE Sephadex A-50 column chromatography (2,5φX13ea+).

The column was washed with a*Sa as described above, and the enzyme eluate was prepared by linear concentration gradient elution up to a sodium concentration of 0°2M in the tIL buffer solution. Collect the active fraction,
Dialysis was performed against 10+sM potassium phosphate buffer solution (pH 7.0).

6) Blue Sepharose Cl-6B column chromatography, then the dialyzed solution was purified using Blue Sepharose CL-6B column chromatography.
After washing the column (1.5 φ x 10 cm) to the baseline with the same buffer as above, the enzyme was eluted by a linear concentration gradient elution method with a sodium chloride concentration of up to 0.6M. The enzyme activity was eluted at 0.25M sodium chloride. The specific activity near the peak is summarized in Table 9.

1 Disaster Friends Cell TM6600,0 324.3 49,1
100DEAH-Sepharose CL-QB 33B, 3 239
．． 7 708,5 73.9 Blue Ceph (6) Purification of Purified Enzyme In order to check the purity of the purified enzyme, polyacrylamide del electrophoresis (Separated Gernia, 5% acryl 7mide, electrophoresis conditions: 20 mA, 4° C1arvf) was performed. The enzyme showed a single band and was prepared using a 0.05 M phosphate buffer solution 1 (pH 7,
It was confirmed that this protein had enzymatic activity by staining with 0)50 a+I for 30 minutes.

In order to check the purity and molecular weight of the enzyme, 5DS-polyacrylamide del electrophoresis (delta: 12° 5% acrylamide, electrophoresis conditions: 20 mA, room temperature, 3 hours)
I did it.

As a result, this enzyme has so,ooo±5,000 (text σ
As a standard sample of 0 molecular weight, 7 male 7 orylace B (MW, 92,500 ), good serum albumin (
MW, 6 (3,200), egg albumin (MW, 45.
000), carbonic anhydrase (MW, 31,
000), Soy Bean Trypton Inhibitor (MW1
21.500) and lysozyme (MW, 14,40
0) was used.

■ Confirmation of reaction product Contains 32# element 0,2 ml, 0,5 M potassium phosphate buffer solution a (pH 7,0) 0,1 ml, 0.5 M Sorboson 0°11.14 M NADo, 4 ml The reaction solution, made up to a total volume of 1-1 with distilled water, was heated at 30°C.
The reaction product reacted for 60 minutes was analyzed by thin layer chromatography and high performance liquid chromatography. As a result, the product has 2-KGA compared to the standard.
It was identified that

Example 2 In the same manner as described in Example 1, Gluconobacter oxydans E-1 (Kikoken Joyori No. 1265) and Gluconobacter oxydans H-2 (Kikoken Joyori No. 126) were prepared.
No. 6) and Gluconobacter oxidans-8 (
L-sorbosone dehydrogenase was isolated from Kikoken Joyori No. 1268) and its physicochemical properties were measured. As a result, the # cords obtained from these strains showed the same properties as the enzyme obtained from Gluconobacter oxydans UV-10 (Kikoken Joyori No. 1267).

Example 3 Gurhubacter oxydans UV-1 obtained by the method described in step (1) and step (2) of Example 1
100 ml of cell-free extract (total enzyme activity: 115 units) of 0 (Chokoken Joyori No. 1267), 50 ml of 0.5 M potassium phosphate t1gi solution [(pH 7, 0), 10%
L-Sorbosone solution 501m! and 300 liters of water with gentle stirring.
Cultured at ℃. As a result, 2-keto L-chloric acid
It was produced at a rate of '700 wag/hr.

Representative Patent Attorney Hiroyoshi Kodejima, °'1 Procedural Amendment (self-imposed September 1, 1985 Kunio Ogawa, Commissioner of the Patent Office 1, Indication of Case 1988 Patent Application No. 137818 2, Invention Name: Enzyme and its manufacturing method 3. Relationship with the case of the person making the amendment Patent applicant: 4. Agent: 107 5. Date of amendment order: None. "Detailed Description of the Invention" column (1) The statement of the scope of the claims (page 1, line 5 of the specification to page 3, line 5) is corrected as shown in the attached sheet.

(2) In the third line from the bottom of page 6 of the specification, rNDAJ is corrected to rNADJ.

(3) On page 7, line 8, the text "Ebicon 810," is corrected to r Ebicon 810.1.

(4) In the third line from the bottom of page 8, the text ``propionaldehyde, methylglyoxal'' is corrected to ``rpropionaldehyde, methylglyoxal''.

(5) On the second line from the bottom of page 8, [Roman North J is corrected to read rD-Mannose 1.

(6) On page 14, line 3, correct the text ``Measure the oxidation rate,'' to ``Measure the oxidation rate,'' to 1.

(7) On page 18, line 12, "Gluconobacter oxydans US-10J is corrected to r-Gluconobacter oxydans UV-10J."

(8) On the 6th line from the bottom of page 19, it says ``The culture can be carried out for as long as possible.'' It can be done twice. Correct the culture time to 1.

(9) On page 20, line 8 of the same document, the phrase "extinguishable" is corrected to read "1" which is extinguishable.

(10) On page 21, line 3 from the bottom, it says “about 30-40g/
” is corrected to r about 30-40g, #J.

(11) "Made by Orient" in the 4th line from the bottom of page 22
Correct the statement to read "r" made by Oriental Co., Ltd.

(12) ro from just below No. 24 to row 6, 2M still there.”
Correct the statement as ro, 1 up to 2M.

(13) On page 29, line 6 of the same page, the text “Cultivated.”
I shook it. ” he corrected.

Above (Attachment) [Claims] r(1) Catalyzes the oxidation of L-sorbomun to 2-keto L-chloric acid in the presence of nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate as a coenzyme. , and the following physicochemical properties a) Optimum pH: Approximately 9 b) Optimum temperature: Approximately 60°C C) Molecular weight: 190,000 ± 20.000 (50,0
00±5, consisting of four subunits with a molecular weight of OOO) d) A novel L-sorbosone dehydrogenase with inhibition HCu'' and N-ethylmaleimide.

(2) It has a novel ability to produce L-sorbosone dehydrogenase within the m cell, and Gluconobacter
r) Cultivating a microorganism belonging to the genus or a mutant strain thereof, disrupting the cells, and further isolating and contracting L-sorbosone dehydrogenase from a cell-free extract of the disrupted cells, preferably from the cytoplasm of the microorganism. A novel method for producing L-sorbosone dehydrogenase, which is characterized by the following characteristics:

(3) The microorganism is Gluconobacter oxidans (Gl
The novel L-microorganism according to claim 2, which is a microorganism belonging to the species
Method for producing Sorbosone dehydrogenase.

(4) The microorganism is Gluconobacter oxidans UV-
10 (Mechanical Engineering Research Institute No. 8422: Engineering Research Institute No. 1267
No.) Gluconobacter oxydans E-1 (Kikoken Bacteria No. 8353: Fiber Engineering Research Institute No. 1265), Gluconobacter oxydans H-2 (Kikoken Bacteria No. 8354)
No.: Microtechnical Research Institute No. 1266) and Gluconobacter
・The novel L-8 according to claim 3, which is a type of microorganism selected from the group consisting of Oxidans L-8 (Kikoken Bokuyori No. 8355: Kaikoken Joyori No. 1268). Method for producing Sorbosone dehydrogenase.

(5) Method G of oxidizing L-sorbosone to 2-KGA, in the presence of NAD or NADP as a coenzyme,
A method characterized in that the oxidation is catalyzed by L-sorbosone dehydrogenase according to claim 1, j

Claims (5)

[Claims] (1) Catalyzes the oxidation of L-sorboline to 2-keto-L-chloric acid in the presence of nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate as a coenzyme, and has the following physicochemical properties a) Optimum pH: Approximately 9 b) Optimum temperature: Approximately 60°C c) Molecular weight: 190,000±20,000 (50,0
d) Inhibition: A novel L-sorbosone dehydrogenase with inhibition by Cu^2^+ and N-ethylmaleimide. (2) It has a novel ability to produce L-sorbosone dehydrogenase in cells, and Gluconobacter (Gluconobacter)
r) Cultivating a microorganism belonging to the genus or its mutant strain, disrupting the cells, and further isolating and purifying L-sorbosone dehydrogenase from a cell-free extract of the disrupted cells, preferably from the cytoplasm of the microorganism. A novel method for producing L-sorbosone dehydrogenase, characterized by: (3) The microorganism is Gluconobacter oxydans (Gl
The novel L-microorganism according to claim 2, which is a microorganism belonging to the species
Method for producing Sorbosone dehydrogenase. (4) The microorganism is Gluconobacter oxydans UV-
10 (Feikoken Jyoyori No. 8422: Microtechnical Research Institute No. 1267
No.) Gluconobacter oxydans E-1 (Feikoken Bacteria No. 8353: Fiber Science and Technology Research Institute No. 1265), Gluconobacter oxydans H-2 (Fiber Science Laboratories No. 3354)
A type of microorganism selected from the group consisting of Gluconobacter oxydans L-8 (Feikoken Bacteria No. 8355: Fiber Science Institute No. 1268) A novel method for producing L-sorbosone dehydrogenase according to claim 3. (5) A method for oxidizing L-sorbosone to 2-KGA, characterized in that the oxidation is catalyzed by L-sorbosone dehydrogenase according to claim 1 in the presence of NAD or NADP as a coenzyme. How to do it.