JPS6322185A - Emzyme and its production - Google Patents

Abstract

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

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel coenzyme-independent L-sorbose dehydrogenase and a method for producing the same.

The L-sorbose dehydrogenase provided by the present invention catalyzes the oxidation of sorbose to L-sorbosone. L-Sorbosone is a precursor of 2-keto L-gulonic acid, an important intermediate in the production of vitamin C.

The reaction that converts L-sorbose to L-sorbosone in microorganisms is known. The production of L-sorbosone from L-sorbose using cell-free extracts of microorganisms has been reported in various publications. U.S. Patent No. 3.
912,592, Gluconobacter (G Iu
Conobacter) genus, Pseudomonas (Ps
eudomonas) genus, Acinetobacter (Ac1
netobacter genus, Bacillus
s) genus, S arcina! , S preptomyces , Serratia , Aerobacter (A
erobaeLor) genus, Mycobacterium (My
It has been reported that microorganisms belonging to the genus P. cobacterium and the genus P. cobacterium carry out the above-mentioned transformation. Makover (Makover etc.) is Pseudomonas putid (Pseudomonas putid)
Describe the basic properties of the enzyme of AT CC21812 (
Biotechnology and Bioengineering 17, pp. 349-359, 1975: B 1o
techno1. Bioeng, 17.148
5-1515.1975), but did not isolate or purify the enzyme; Kitamura et al.
Gluconobacter mel
Biotechnology and Bioengineering would like to report that the activity of L-sorbose dehydrogenase discovered from IF03293 (A.
17, pp. 349-359), but the enzyme has not been isolated.

As mentioned above, there has been no disclosure of a purified enzyme having activity for oxidizing L-sorbose to L-sorbosone.

The present invention was completed with the discovery that an enzyme isolated and purified from a cell-free extract of cells of a specific microorganism, preferably a membrane fraction, specifically catalyzes the oxidation of L-sorbose to L-sorbosone. It is something that

The purpose of the present invention is to produce L-sorbosone by acting on L-sorbose, and to develop a novel L-sorbosone with higher substrate specificity.
Its purpose is to provide sorbose dehydrogenase. Another purpose is to culture microorganisms belonging to the genus Gluconobacter or their mutant strains that have a new ability to produce sorbose dehydrogenase in cells, destroy the cells, and then perform cell-free extraction of the destroyed cells. An object of the present invention is to provide a novel method for producing L-sorbose dehydrogenase, which is characterized by isolating and purifying L-sorbose dehydrogenase from a cell membrane of a cell, preferably a microorganism.

The physicochemical properties of the purified sample of novel L-sorbose dehydrogenase obtained in the Examples described below are as follows.

(1) Enzyme activity measurement method The L-sorbose dehydrogenase of the present invention catalyzes the reaction of dehydrogenating L-sorbose to convert it to L-sorbosone in the presence of an electron acceptor. Shi-sorbose + electron acceptor → L-sorbosone deca-reduced electron acceptor This enzyme contains 2,6-cyclophenol indophenol (hereinafter abbreviated as DCIP), phenazine melisafate, and Wursters as electron acceptors. Blue, potassium ferricyanide, coenzyme Q or cytochrome C can be used, but oxygen cannot be used directly.

The enzyme activity was determined by measuring the decrease in the absorbance of DCIP at 600 nm at 25°C, and the amount of enzyme required to reduce 1 μM DCIP was reduced to 1 μM per minute.
Display as unit. The extinction coefficient of DCIP is 14.5mM-1 at pH 7.0. The basic composition of the reaction solution is shown below, and is prepared in advance before measuring enzyme activity.

Basic reaction solution 2 0.3% Triton X1003x (1 2,5mM DCI P 0.45ml distilled water
4.95 shoulders! A potassium phosphate buffer solution containing these at pH 7.0 is used as the basic reaction solution.

0.4 ml of this basic reaction solution, 1 M sorbose 0,1
Add xl, enzyme solution and distilled water to make a total volume of 0.5111. As a control, the same amount of distilled water was used instead of sorbose, and the 6 reactions measured in a quartz cell with a light length of 1 c1 were initiated by the addition of sorbose and its activity was
It is determined from the initial reduction rate of IP.

(2) Substrate specificity The substrate specificity of the purified enzyme was determined by using the activity measurement method described in (1), replacing the substrate sorbose with various substances shown in Table 1.Table 1 As shown in the results, it showed extremely high specificity for L-sorbose.

Table 1 Substrate Relative activity (%) 1L-Sorbose 100D-Glucose
OD-Mannitol OD
- Sorbitol OD - Fructose 0 Glycerol
O Adonitol O Meso-Erythritol O Na-Gluconic Acid
0Ca-idonic acid
0 Mannose 0 Xylose 0 Ethanol
0 lactose
0 seuucrose 0 galactose 0 cellobiose
O-inosylol
0Arabinose 05L-Activity towards sorbose is expressed as 100.

(3) Optimal pH The L-sorbose dehydrogenase activity of the purified enzyme and the effect of p on that activity were determined using pine kilvane buffers with various pH values.
The influence of H was investigated and shown in Table 2.

Table 2 pH relative activity (% Bi4.0 0 4.5 0 5.1 9.4 5.6 35.3 6.1 68.8 6.6 90.0 7.2 100 7.7 95. 0 8.1 85.3 The activity at pH 7.2 is expressed as 100. As shown in Table 2, high activity was shown between pH 6.5 and pH 8.0.

(4) Using a pH-stable purified enzyme, leave it at 4°C for 121 hours in a pine kilvane buffer solution with a pH of 4 to 8.0, and then measure its residual activity using the method shown in (1). , the results shown in Table 3 were obtained.

1-1-Minichi 4.0 0 4.5 0 5.0 0 5.5 0 6.0 23.8 6.5 60.0 7.0 100 7.5 75.9 ”Activity at pH 7.0 The purified enzyme, expressed as 100, was relatively stable at pH 6.5-8.0.

(5) Using a thermostable purified enzyme, treat it in 10 m M potassium phosphate M, 1 solution (pH 7,0) at a temperature of 0 to 65 °C for 5 minutes, and then immediately remove the remaining activity in ice water. It was measured by the method shown in (1). The results are shown in Table 4.

Table 4 30 97.9 36 86.6 41 69.2 46 47.5 55 14.7 Activity at 10°C is expressed as 100 As shown in Table 4, it is stable up to 30°C and about 50% at 46°C. %, and about 90% of the activity was lost at 65°C.

(6) L by the method shown in (1) at an optimum temperature of 20°C to 58°C.
-Measure the sorbose dehydrogenase activity and share the results with the fifth
Shown in the table.

1-Table 20 28.2 25 26.7 29 35.9 36 50.8 40 70.7 45 93.8 55 91.8 58 77.0 The activity of the enzyme of the present invention is expressed with the activity at 1148°C as 100. , increases as the temperature increases,
It reached its maximum at 48°C.

(7) Molecular weight This enzyme solution was mixed with 0.3% Triton X-100 and 0.2M
- After charging a Sephadex G-200 column (1-0 It was developed in

The molecular weight of this enzyme was calculated to be 210.000±20.000 by gel filtration. However, this enzyme is a membrane-bound protein that is solubilized from the cell membrane fraction by a surfactant, and the molecular weight determined above cannot be called the true molecular weight. The purified enzyme showed a single band, and its molecular weight was 58,000±5, OOO
and consists of the same subunits.

(8) Measurement of Michaeli constant (Km) According to the method shown in (2) above, 0.5mM-
The initial rate of the oxidation reaction was measured using L-sorbose at a concentration of 320 mM. L-sorbose concentration is approximately 200m
When M, the maximum reaction rate is shown, and its Michaelis constant (K
m value) was 102±10 mM in the presence of DCIP.

(9) Influence of metal ions The influence of various metal ions on the activity of this enzyme was investigated by the method shown in (1) above, and the measurement results are shown in Table 6. Co” and Fe” promote this enzyme activity, but Cu”
was inhibited.

Table 6 (mM) CaCl 2 0.4 100 0.8 100 CoCl・6H200,4109,2 0,8118,4 CaSO30,80 CuSO,・5H,OO,0830,9CuC12・2
H,OO,40 Cu(N○z)z ・H2O0,32,27F e(S
O4)z ・XH2O0,4121,30,9106,
4 Mg5Ox-7H,00,4100 0,8100 MgClx-68200,883,3 MnC12・4H200,4100 0,998,3 Mn SO4・HzO0,410O N a 2 M o O4・2 H200, 386-1
1,0669,6 TiCI 0.4
1000.8 100 1.8 100 ZnSo< ・7H200,4100 0,9100 NiSO,・6H200,4100 0,9100 No additive 100(1
0) Influence of inhibitors The influence of various inhibitors on the activity of this enzyme was investigated using the measurement method shown in (1) above, and the results are shown in Table 7. Quinine, monoiodoacetic acid, sodium fluoroacetic acid, and sodium fluoride inhibited this enzyme activity.

Table 7 EDTA O,9405,2
13,3 Quinine 1.85 04
．． 24 30.5 N-ethylmaleimide 1.85 0
4.24 0 Sodium Azide 1.85 0
4.24 0 Monoiodoacetic acid 1.85 0
4.24 34.6 9.37 68.5 PCMB (p-chloromer 0.02
0 Kyuribenzoate) 0.04 0
0.17 0 Na2HAs ・04 ・7HzO1,8504,24
0 Sodium fluoroacetic acid 1.85 0
4.24 8.6 9.37 26.5 Sodium fluoride 1.85 10
．． 34.24 10.3 9.37 22.1 Potassium cyanide 0.88
0(12) Purification method - Sorbose dehydrogenase can be purified by methods commonly used for enzyme purification or a combination thereof, such as ion exchange chromatography, liquid chromatography, etc. Examples include methods such as gel filtration, gel electrophoresis, salting out, and dialysis.

The L-sorbose dehydrogenase provided by the method of the present invention is prepared by culturing a suitable microorganism, disrupting the cells, and separating and purifying a cell-free extract of the disrupted cells, preferably from a membrane fraction of the microorganism. can do.

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

According to the latest classification, all strains belonging to Gluconobacter belong to the species Gluconobacter oxidans.

The morphological and biological characteristics of strains belonging to the species Gluconobacter oxydans were
of Systematic Bacteriology (Berg.
ey's M annual of Systema
tic Bacteriology) Vol.
I.

pp. 275-288, 1984 and F. Gossele et al., International Journal of Systematic Bacteriology (International J, Sy.
stem.

Bacteriol) Vol, 33, page 65-
81, 1983.

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

The mutant strain used in the present invention is obtained by irradiating the wild strain with ultraviolet rays and applying X! ! It can be obtained by irradiation or gamma irradiation, or by contacting with nitrite or other suitable mutagens, or by isolating clones resulting from natural mutation. Mutations of these wild strains or their mutants 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 "1 Chemical Mutagen", edited by Dejima, Yoshida, and Kata, published by Kodansha Science Publishing, 1973.

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

The most preferred strains in the present invention are Gluconobacter oxydans UV-10, Gluconobacter oxydans E-1, and Gluconobacter oxydans.
H-2, Gluconobacter oxydans L-8, etc. These strains have been deposited at the Institute of Microbial Technology, Agency of Industrial Science and Technology under the following accession numbers.

The domestic deposits of the following microorganisms were transferred to the International Deposit under the Budapest Treaty on January 29, 1985, and are given the following accession numbers.

Gluconobacter oxydans UV-10: "FERM-P No. 8422" (FERM-P No. 8433), [FERM-P No. 1267] (FERM BP-1267) Gluconobacter oxydans Dance E-1 = "FERM-P No. 8353", "FERM-P No. 1265" rFERM BP-1265> Gluconobacter oxydans H-2 = " Gluconobacter oxydans Shi-8 = “FERM-P No. 8354” (FERM-P No. 8354), “FeRM-P No. 1266” (FERM BP-1266) No. 8355 (FERM-P No. 8355), FERM-P No. 1268 (FERM BP-1268) Microorganisms are cultured under aerobic conditions in liquid medium supplemented with appropriate nutrients. The culture can be carried out at pH 4.0 to about 8.
．． 0, preferably pH 4,5-pH 6,5. Cultivation time varies depending on the microorganism and nutrient medium used, but is preferably about 10-100 hours. The suitable temperature range for culturing is approximately 10°C.
-40°C, preferably 25°C-35°C.

The medium contains assimilable carbon sources such as glycerin, D-
Mannitol, D-sorbitol, erythritol, ribitol, xylitol, arabitol, inositol, dolcitol, D-ribose, D-fructose, D
- fucose, D-glucose, gluconate, L-sorbose, maltose and sucrose, preferably L
- sorbose, D-sorbitol or glycerin; digestible nitrogen sources, organic substances such as peptone, yeast extract, soybean flour and corn steep liquor; inorganic substances such as ammonium sulfate, ammonium chloride and potassium nitrate; vitamins and It is necessary to include nutritional sources such as trace elements.

One embodiment for isolating and purifying L-sorbose dehydrogenase from microorganisms after culture is briefly described below.

(1> Collect cells from the culture solution by centrifugation.

(2) Wash the collected cells with distilled water, physiological saline or M@ warm solution with appropriate pH.

(3) The washed cells are suspended in a buffer solution and disrupted using a homogenizer, ultrasonicator, lysozyme treatment, etc. to obtain a cell disruption solution.

(4) L-sorbose dehydrogenase is obtained from a cell-free extract of disrupted cells, preferably a cell membrane fraction of a microorganism.

The L-sorbose dehydrogenase provided by the present invention is useful as a catalyst for producing L-sorbosone from L-sorbose. The reaction is carried out in the presence of an electron acceptor such as DCIP, phenazine mesosulfate, Wurster's Blue, potassium ferricyanide, Coni〉'zyme Q, or cytochrome C, potassium phosphate yI solution, Tris-
HCl [in a solution such as a buffer solution, a p of about 5.0 to about 10.0]
It must be done to H value. The preferred temperature range for conducting the reaction is about 20°C to about 60°C.

p) (and the temperature is set at approximately pH 6.5-8.0 and 50 °C, respectively, the reaction gives the most favorable results; the concentration of L-sorbose in the solution varies depending on the other reaction conditions. However, in general, it is about 110-1 (
)O/j! is preferred, and about 30-40y/1 is most preferred.

In the reaction, the enzyme can also be used in a state immobilized on a suitable carrier. - Any best known means of enzyme immobilization can be used. For example, the enzyme may be directly bonded to a resin membrane, fine particles, etc. having a functional group, or may be bonded to the resin via a bridging compound having a difunctional group such as glutardialdehyde.

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

Example I Preparation of sorbose dehydrogenase 1) Gluconobacter oxidans UV-10 (
Culture of Glucobacter oxydans UV-10 (Feikoken Joyori No. 1267) was inoculated into a test tube containing 5 ml of No. 3B medium. , and cultured on a reciprocating shaker for 3 days at 27°C. The medium contains 7.0% L-sorbose and 0.05% glycerin.
, yeast extract (manufactured by Orient) 1.5%, Mg5Q,
-71-(200,25% and CaCOs 1
.

Contains 0%. Add 1xl of culture to 50ml of the same medium as above.
Inoculate a 500 Erlenmeyer flask containing No. 11, and at 30°C.
The cells were cultured on a rotary shaker (180r, p, m,) for 3 days. 800xl of the resulting culture was added to No. 3B medium 2.
301 fermenters including 01 were inoculated. 30 fermenters
Stir at ℃, 400r, p, m, +lv, v, m, (=
Aeration was performed (volume of air/volume of medium 7 min). After culturing for 40 hours, the culture was harvested to collect the bacterial cells.

The culture solution was heated at 1,500r, p, m, (365xy).
Centrifuge for 0 minutes to remove calcium carbonate, and then centrifuge for 8,0 minutes.
Centrifugation was performed at OOr, p, m, (10,000xg) to obtain bacterial cells. The obtained bacterial mass was washed once with 0.85% physiological saline. In this way, wet bacterial cells 3742 were obtained from the culture solution 201.

(2) Preparation of membrane fraction Gluconobacter oxydans cells obtained in the first step (1) 77.51? was washed twice with physiological saline. The washed bacterial cells were suspended in 10mM potassium phosphate solution 230w1, and washed at 2.000 r, p using a Dynomill (Wheely A, manufactured by Barakotsuten Co., Ltd.) bacterial cell crusher containing glass beads (0, 1xiφ). , m, for 4 minutes and crushed. 4.0OOr, p, m, (1,8
After centrifuging for 10 minutes at
Centrifugation was performed for 1 hour at (Oxg). The resulting precipitate was collected as a membrane fraction (42y).

The membrane fraction was treated with 0.5% Toquene 80 and 0.2 ML-
It was suspended in buffer solution 60011 containing sorbose. After stirring the suspension for 3 hours, 40, OOOr, p, m,
The mixture was centrifuged at (80,00xg) for 1 hour. Subsequently,
The remaining membrane was treated with 0.3% Triton X-100 and 0.2
It was suspended in NIgI solution containing M L-sorbose. After stirring the suspension for 3 hours, 40,0OOr, p, m,
Centrifugation was performed for 1 hour at (80.0 OOx). The supernatant thus obtained contained L-sorbose dehydrogenase. The supernatant was treated with 0.3% Triton x-io.

and buffer solution 21 containing 0.2M L-sorbose
I underwent dialysis twice for one night.

(3) Diethylaminoethyl (hereinafter referred to as DEAE) cellulose column chromatography The dialysis solution (570 mffi) obtained in the above step was diluted to 0.
DEAE cellulose equilibrated with a buffer containing 3% Triton X-100 and 0.2M L-sorbose
A column (2.5φ x 5Qcx) was applied, the column was washed with the same buffer solution as above, and the enzyme was eluted with a linear concentration gradient of sodium chloride up to 0.5M.

(4) DEAE Sepharose column chromatography Enzyme fraction 100i1 obtained in the above step, 0.3%)
! J) 1 containing N-X-100 and 0.2M L-sorbose! A DEAE Sepharose column (1,5φ
x 30 cl). After washing the column with the same buffer solution, linear concentration gradient elution was performed with the sodium chloride concentration in the MWi solution up to 0.2M as shown in Figure 1.
L-sorbose dehydrogenase was obtained. 87th fraction - 89th fraction
Both fractions (15i+1) were pooled and used in the next purification step.

(5) Sephadex G-200 column chromatography A part of the enzyme fraction (20z1) was equilibrated with the same buffer solution as above using a Sephadex G-200 column (1,
It was applied to Oφx100CII and developed. The active fraction
Stored at -80°C. An outline of the enzyme purification process is shown in Table 8.

(6) Purity of the isolated enzyme In order to examine the purity of the purified enzyme, polyacrylamide gel electrophoresis (separation gel: 15% acrylamide, electrophoresis conditions: 20 mA, 4°C, 8 hours) was performed.Enzyme teeth,
By further staining for 30 minutes with 0.05M phosphate buffer solution (pH 7,0) containing 50mM L-sorbose, nitro blue tetrazolium 40xg/xi and phenazine mesosulphate 140xg/xi It was confirmed that this protein has enzymatic activity.

In order to investigate the molecular weight and molecular structure of this enzyme, SDS-polyacrylamide gel electrophoresis (separation gel running: 15% acrylamide, electrophoresis conditions: 20 mA, room temperature, 3 hours) was performed. As a result, this enzyme was 58,000±5,0
Phosphorylace B (MW, 92,500
>, calf serum albumin'y (MW, 66.200),
Ovalbumin (MW, 45,000), carbonic anhydrase (MW, 31,000), soy bean trypsin inhibitor (MW, 21.5000>) and lysozyme (MW, 14.400') were used.

(7) Confirmation of reaction products Nm Enzyme 0, 2 xl, 0.5 M IJ 7M force!
J'ym Wt solution (pH 7,0>0.05xl, L
A reaction solution containing 0.05 zl of M L-sorbose and 0.02 zf of 0.2 M phenazine mesosulfate and made up to 0.5xl with distilled water was reacted at 45° C. for 30 minutes.

The reaction products were analyzed by thin layer chromatography and high performance liquid chromatography. As a result, the product was identified as L-sorbosone by comparison with the standard.

Example 2 Gluconobacter
Oxidans E-1 (Feikoken Jyoki No. 1265), Gluconobacter oxydans H-2 (Feikoken Jyoyori No. 1)
No. 266) and Gluconobacter oxydans
8 (Feikoken Jokyo No. 1268) mustard-sorbose dehydrogenase was isolated and its physical properties were measured. the result,
The enzymes obtained from these strains showed the same properties as the enzyme obtained from Gluconobacter oxidans UV-10 (Feikoken Joyori No. 1267).

Example 3 Gluconobacter oxydans UV-1 obtained by the method described in steps (1) to (2) of Example 1
0 ('f&Koken Joyori No. 1267) cell-free extract (total enzyme activity: 123 units) 100z1.0.5M potassium phosphate buffer solution (pH 6,0) 50zi', 0.2
M phenazine meso sulfate 10R1 and distilled water 2
The reaction mixture solution containing 90z1 was shaken at 30° C. with gentle stirring. As a result, L-sorbosone was produced at a rate of 142 g/hr.

Example 4 Purified membrane-bound sorbose dehydrogenase (total enzyme activity: 120 units) obtained by the method described in Steps (1) to (5) of Example 1, 400zf, 0.5M potassium phosphate buffer solution ( pH6,0) 5011, LM L-
Sorbose solution 50! ! The reaction mixture solution containing 0.2M phenazine mesosulfate 10R1 was shaken at 30° C. with gentle stirring. As a result, L
- Sorbosone was produced at a rate of 244 wg/hr.

[Brief explanation of the drawing]

FIG. 1 is a chromatography of membrane-bound L-sorbose dehydrogenase on a DEAE Sepharose (Pharmacia Fine Chemicals CL-6B) column. Procedural proceedings official document (autograph) August 6, 1986 Kunio Ogawa, Director General of the Patent Office 1, Indication of the case 1988 Patent Application No. 137817 2, Name of the invention Enzymes and their manufacturing process 3, Amendments Name of patent applicant F. Hoffmann U. Roche Land Kompany Akchengesellschaft 4, Agent 〒107 6, Subject of amendment (attachment) [Claims Cor. (1) L -Acts on sorbose to produce L-sorbosone, has high substrate specificity, and has the following physicochemical properties a) Optimum pH: approximately pH 6,5-8, 0b) Optimum temperature: 45-55 C) Molecular structure: A novel coenzyme-independent L-sorbose dehydrogenation consisting of one type of subunit with a molecular cap of approximately 58,000±5,000 as measured by SDS-polyacrylamide gel electrophoresis. enzyme. (2) A microorganism belonging to the genus Gluconobacter or its mutant strain that has a novel ability to produce L-sorbose dehydrogenase in cells is cultured, the cells are destroyed, and the destroyed cells are further destroyed. A novel method for producing coenzyme-independent L-sorbose dehydrogenase, which comprises isolating and purifying L-sorbose dehydrogenase from a cell-free extract, preferably from a membrane fraction of a microorganism. (3) The microorganism is Gluconobacter oxydans (G
2. A method for producing a novel coenzyme-independent L-sorbose dehydrogenase according to claim 2, which is a microorganism belonging to the species 1uconobacter oxydans. (4) The microorganism is Gluconobacter oxydans UV
-10 (Microtechnical Research Institute No. 8422; Microtechnology Research Institute No. 126
No. 7), Gluconobacter oxydans E-1 (February 2011 No. 8353), Gluconobacter oxydans H-2 (February 8354) Gluconobacter oxydans L-8 (@Koken Bacteria No. 8355)
A method for producing a novel coenzyme-independent L-sorbose dehydrogenase according to claim 3, which is a type of microorganism selected from the group consisting of: (5) A method for oxidizing L-sorbose to L-sorbosone, characterized in that the oxidation is catalyzed by the L-sorbose dehydrogenase set forth in claim 1.'' ) Kunio Ogawa, Commissioner of the Patent Office, September 1, 1988 1. 8 indications 1988 Patent Application No. 137817 2 Title of the invention Enzymes and their manufacturing process 3 Relationship with the amended person case Patent Applicant 4, Agent 107 5, Date of amendment order None 6, Subject of amendment (1) ``In the 6th line of page 4 of the specification, it says ``r (Makover et al.''). ”. (2) On page 6, line 13 of the same page, ``phenazine melisafate'' is corrected to ``r-phenazine meso sulfate.'' (4) On page 14, line 9 of the same page, correct the phrase "sephadex without equilibration" to read "sephadex 1 with r-equilibration." (5) ) In the 3rd line from the bottom of page 17, the text "quinine," is corrected to r-quinine, 1. (6) In the 8th line of page 24, the text "after culture solution" is corrected to r after culture. (7) In the 5th line from the bottom of page 26, correct the phrase ``Made by Orient Co., Ltd.'' to r. Correct the phrase ``as a result,'' to ``as a result,''.

Claims (5)

[Claims] (1) Produces L-sorbosone by acting on L-sorbose, has high substrate specificity, and has the following physicochemical properties a) Optimal pH: approximately pH 6.5-8.0 b) Optimal Temperature: 45-55°C c) Molecular structure: A novel coenzyme-independent L consisting of one type of subunit with a molecular weight of approximately 58,000±5,000 as measured by SDS-polyacrylamide gel electrophoresis. - Sorbose dehydrogenase. (2) It has a novel ability to produce L-sorbose dehydrogenase in cells, and Gluconobacter (Gluconobacter)
r) Cultivating a microorganism belonging to the genus or a mutant strain thereof, disrupting the cells, and further isolating and purifying L-sorbose dehydrogenase from a cell-free extract of the disrupted cells, preferably a membrane fraction of the microorganism. A method for producing a novel coenzyme-independent L-sorbose dehydrogenase, characterized by: (3) The microorganism is Gluconobacter oxydans (Gl
A method for producing the novel coenzyme-independent L-sorbose dehydrogenase according to claim 2, which is a microorganism belonging to the species Uconobacteroxydans. (4) The microorganism is Gluconobacter oxydans UV-
10 (Feikoken Jyoyori No. 8422: Microtechnical Research Institute No. 1267
), Gluconobacter oxydans E-1 (FEI Bacteria No. 8353), Gluconobacter oxydans H-2 (FEI Bacteria No. 835)
Gluconobacter oxydans L-8 (No. 8355, No. 1268) A method for producing the novel coenzyme-independent L-sorbose dehydrogenase according to claim 3, which is a microorganism. (5) A method for oxidizing L-sorbose to L-sorbosone, characterized in that the oxidation is catalyzed by the L-sorbose dehydrogenase according to claim 1.