JPS6317432B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel D-sorbitol dehydrogenase and a method for producing the same. Conventionally, as an enzyme having a dehydrogenating effect on D-sorbitol, for example, L-iditol dehydrogenase (system name: L-
Iditol: NAD oxidoreductase, EC, 1, 1,
1, 14), aldose reductase (aldose reductase)
reductase, strain name Polyol: NADP
oxidoreductase, EC, 1, 1, 1, 21), mannitol dehydrogenase, strain name D-mannitol: cytochrome
oxidoreductase, EC, 1, 1, 2, 2), etc. are well known, and their reaction progress is
NAD (nicotinamide adenine dinucleotide),
It is essential that NADP (nicotinamide adenine dinucleotide phosphate) or cytochrome be present as an electron acceptor. The present inventors have continued research on the aerobic metabolism of microorganisms for many years, and as a result, we have found that it has extremely high specificity for sorbitol, and that NAD or NADP
It was discovered that microorganisms produce a new D-sorbitol dehydrogenase that cannot use D-sorbitol as an electron acceptor, and based on this, further research led to the completion of the present invention. The present invention provides (1) D-sorbitol dehydrogenase and (2) Gluconobacter, Acetobacter, Serratia, Proteus, Bacillus, Staphylococcus, Brevibacterium, Pseudomonas or Klebsiella D-sorbitol dehydrogenase, which is characterized by culturing D-sorbitol dehydrogenase-producing bacteria belonging to the genus in a medium, producing and accumulating D-sorbitol dehydrogenase in the culture, and collecting it from the culture. This is a method for producing enzymes. The microorganisms used in producing the enzyme of the present invention include Gluconobacter genus, Acetobacter genus, Serratia genus,
Genus Proteus, Bacillus
Genus, Staphilococcus,
Any microorganism belonging to the genus Brevibacterium, Pseudomonas, or Klebsiella that dehydrogenates D-sorbitol can be used. Specific examples of the above microorganisms include G.suboxydans var.α IFO3254, G.albidus IFO3250,
Gluconobacter capsiulatus (G.
cupsulatus) IFO3462, G.oxydans IFO3189, G.industrius
IFO3260, Gluconobacter gluconicus (G.
gluconics) IFO3171, G.cerinus (G.cerinus) IFO3265, Gluconobacter dioxyacetonix (G.
dioxyacetonucus) IFO3271, G.melanogenus (G.melanogenus) IFO3292,
Gluconobacter liquefaciens (G.
liquefaciens) IFO12388, Acetobacter aurantius (A.aurantius) IFO3245, Acetobacter acetigenes (A.acetigenes)
IFO3277, Acetobacter lancens (A.
rancens) IFO3298, A.aceti IFO3281, A.ascendens IFO3188, S.marcescens IFO3052, P.vulgaris IFO3167, Bacillus subtilis (B. subtilis) IFO3013, Staphylococcus aureus (S. aureus)
IFO3060, Brevibacterium ammoniagenes (B.ammoniagenes) IFO12072, P.aeruginosa (P.aeruginosa) IFO3445, Klebsiella pneumoniae (K.pneumoniae)
Examples include IFO3317. The microorganisms with the above IFO numbers are listed on the List of Cultures published by the Fermentation Research Institute.
1978 6th edition (Institute for Fermentation)
Osaka List of Cultures 1978 sixth edition)
It can be obtained from the Fermentation Research Institute. In general, Gluconobacter, Acetobacter, Serratia, Proteus, Bacillus, Staphylococcus, Brevibacterium, Pseudomonas, or Klebsiella are characterized by Easily changed, such as X-ray irradiation, UV irradiation, radiation irradiation, artificial mutagens (e.g. nitrosoguanidine, ethyleneimine, etc.)
It can be easily mutated by artificial mutation means using .
Even among such mutant bacteria, any strain capable of producing D-sorbitol dehydrogenase can be used in the method of the present invention. Any carbon source that can be assimilated by these microorganisms can be used as a carbon source for culturing these microorganisms, but usually starches, sugars such as hexoses, pentoses, etc. Alcohols such as glycerol, sorbitol, and ethanol can also be used. Further, as nitrogen sources, in addition to various organic compounds such as peptone, yeast extract, cottonseed flour, soybean meal, and corn steep liquor, inorganic compounds such as nitrates and ammonium salts, urea, and aqueous ammonia can also be used. The medium contains carbon sources, nitrogen sources, various metal salts necessary for growth, such as potassium, sodium, magnesium, manganese, iron, copper, zinc, cobalt, etc., and various vitamins necessary for growth, such as pantothenic acid. B vitamins, amino acids, nucleobases, etc. can also be added. In order to produce this enzyme more efficiently, it is desirable to add substrates such as sorbitol and mannitol to the medium at some stage of culture as enzyme production promoting substances. Also, the pH of the medium
It is desirable to use an acid or alkali to adjust the pH to around 5 to 7. In order to culture this enzyme-producing microorganism in a medium, either solid culture or liquid culture can be adopted, but it is preferable to perform aeration-stirring culture. The culturing time may be set to the time at which the production of the enzyme is maximized, but usually about 10 to 100 hours is sufficient. The culture temperature is preferably about 25 to 35°C. After culturing, the microbial cells are filtered, centrifuged,
Collect bacteria by means such as coagulation and sedimentation, or use commonly used bacterial cell destruction methods such as French press, ultrasonication, trituration, and freeze-thaw methods, and if necessary, use SH-protection such as mercaptoethanol. After disrupting the bacterial cells in the presence of the agent, centrifugation is performed to collect the disrupted bacterial cell fraction. Since the enzyme activity is contained in this fraction, it can be suspended as is and used as a crude enzyme, but for further purification it is necessary to use a surfactant such as Triton X-100 (manufactured by Wako Pure Chemical Industries, Ltd.). ) and stir to solubilize the enzyme, and then the enzyme protein can be purified and collected using a combination of conventional enzyme purification methods such as cellulose column chromatography, gel filtration, salting out, dialysis, and fractional precipitation. can. Furthermore, the enzyme activity is suitably measured as follows. Namely, 0.1 ml of enzyme solution, 0.1 ml of 1 molar sorbitol, 0.1 ml of 0.1 mol potassium erythyanide, and 0.5 pine kilvaine buffer (PH4.5).
ml and distilled water 0.2 ml (however, as a control, use the same amount of distilled water instead of sorbitol). 5g of ferric iron, 3g of sodium lauryl sulfate, 95ml of 85% phosphoric acid and 0.5ml of distilled water (1), 3.5ml of distilled water
Stop the reaction by adding 100% of the reaction mixture, and after allowing it to stand for 10 minutes, measure the absorbance of the reaction solution at 660 nm. Enzyme activity is expressed as 1 unit, which is the amount of enzyme required to reduce 2 micromoles of ferricyanide per minute of the above reaction. The physicochemical properties of D-sorbitol dehydrogenase obtained by the methods of Examples 1 to 3 are shown below. (a) Action: Dehydrogenates D-sorbitol and converts it to L-sorbose. (b) Substrate specificity: Mainly dehydrogenates D-sorbitol. Very little dehydrogenation of D-mannitol. (c) Optimum PH range: PH4.5±0.5 (d) Optimal temperature range: 25℃±5℃ (e) Stability: PH5.0, 5℃, does not lose activity even if left for 24 hours. (f) Inactivation conditions: Inactivation occurs when heated to 45°C or higher for 10 minutes in acetate buffer (PH5.0). (g) Inhibition: The activity is inhibited by p-chloromeric yuribenzoate. (h) Stabilization: Stabilized by the presence of D-sorbitol. (i) Molecular structure: molecular weights of approximately 56,000 units, approximately 43,000 units, and approximately 16,000 units (respectively SDS
-based on gel electrophoresis). It has a flavin molecule and a cytochrome molecule as prosthetic molecules in its molecule. (j) Visible absorption spectrum: λ _nax : 417 nm, 523 nm, 552 nm. _λnio : 481nm, 540nm. Shoulder: approx. 530nm. (k) Electron acceptor: ferricyanide, 2,6-dichlorophenol indophenol or phenazine methosulfate. Since this D-sorbitol dehydrogenase has extremely high substrate specificity, it is extremely useful as an enzyme for quantifying D-sorbitol in industries such as the production of D-sorbitol from glucose or the production of L-sorbose from D-sorbitol. Not only that, if this enzyme is immobilized on a suitable carrier, D
- Continuous production of L-sorbose from sorbitol is also possible, and it can be said that it is a useful enzyme from an industrial standpoint. Next, the present invention will be explained in more detail with reference to Examples and Experimental Examples. Example 1 (Production of D-sorbitol dehydrogenase) Sodium gluconate 1%, D-sorbitol 1%, yeast extract 0.2%, polypeptone 0.3% (PH
After dispensing 100 ml of the medium with the composition (weight/volume) consisting of
A loopful of a slant culture of IFO3254 (suboxydans var. α) is inoculated and cultured for 40 hours at 30°C on a rotary shaker at 200 rpm. During cultivation, in addition to measuring enzyme activity using the method described above, growth rate was determined by measuring the turbidity of the culture medium using a Klett-Summerson photometer, and protein content was measured using the Lowry method (Journal). of Biological Chemistry, Volume 193, Page 265,
1951, Journal of Biological Chemistry). The culture results are shown in Table 1.

[Table] Example 2 (Promotion of enzyme production) Medium containing 1% glycerol and 0.5% yeast extract
(A), a medium containing 1% D-sorbitol and 0.5% yeast extract (B), and a medium containing 0.7% D-sorbitol, 0.7% glycerol, and 0.3% yeast extract (C) in the same manner as in Example 1. After culturing for 24 hours, the activity of the grown cells is measured, and when the activity in medium (A) is 100, an activity of 250 is obtained in medium (B) and 167 in medium (C). Example 3 (Collection of novel sorbitol dehydrogenase) (1) First step: Cells collected from the culture solution (6000 ml) cultured for 25 hours in Example 1 were added to 0.01 M acetate buffer containing 2 mM mercaptoethanol. (PH
5.0) (200ml), then crush the bacterial cells with a French press, and from the crushed liquid,
Obtain cell-free extract by centrifugation at 7000 rpm for 10 minutes. This extract is further subjected to ultracentrifugation at 68,000 xg for 90 minutes, and a membrane fraction is collected as a precipitate. (2) Second step: After suspending the membrane fraction in the above buffer solution to a protein content of 10 to 30 mg/ml,
0.1MD-sorbitol, 0.1M potassium chloride,
Add 1% Triton X-100 and stir for 2 hours.
Thereafter, the supernatant fraction obtained by performing ultracentrifugation in the same manner as above is used as the solubilized enzyme fraction. (3) Third step: Add polyethylene glycol 6000 to this solubilized enzyme fraction to a concentration of 20%.
After standing overnight, the precipitate was collected by centrifugation at 10,000 rpm for 10 minutes, and the precipitate was suspended in the above buffer containing 0.1% Triton X-100 and 0.05 MD-sorbitol. (4) Fourth step: After charging this suspension to a DEAE-cellulose column (3 cmφ x 30 cm),
If you wash the column with the above buffer containing 0.1% Triton . Meanwhile 28~
Fraction 32 is the most active fraction. (5) Fifth step: Collect the active fraction from the cellulose column and add polyethylene glycol 6000 to
Add to a concentration of 10%, leave overnight, centrifuge to obtain a supernatant, and add ethylene glycol 6000 to a concentration of 20%. After standing again overnight, centrifugation is performed at 10,000 rpm for 10 minutes, and the resulting precipitate is collected as enzyme protein. The yield and yield of activity during this period are shown in Table 2. In other words, from the membrane fraction in the first step, the yield
At 32%, it is possible to obtain an enzyme preparation that is 132 times more purified.

[Table] Second process

Solubilized enzyme fraction 666
4660 7 113

[Table] Experimental Example 1 (Substrate specificity) Among the activity measurement methods described in the text using the purified enzyme preparation obtained in Example 3, the reaction was performed by replacing the substrate with various substances shown in Table 3. As a result of examining the substrate specificity, as shown in the results in Table 3, D
- Shows extremely high specificity for sorbitol.

[Table] Experimental example 2 (enzyme stability) Using the purified enzyme obtained in Example 3,
Temperature stability was determined by measuring the residual activity after treatment at a temperature of 10°C for 10 minutes, and PH stability was determined by measuring the residual activity after being left at 5°C overnight in a pine kil vane buffer solution with a pH of 3 to 7.5. The results shown in Tables 4 and 5, respectively, are obtained.

【table】

[Table] Experimental Example 3 (Inhibitor) Various compounds shown in Table 6 were added to the enzyme solution at a concentration of 1mM, and after being left at 25℃ for 10 minutes,
When the residual activity was measured by the above activity measuring method, the results shown in Table 6 were obtained.

[Table] Experimental example 4 (Influence of coexisting substances) In the above activity measurement method, the final concentration in the reaction solution
Add the substances listed in Table 7 to a concentration of 100 mM, conduct the reaction, and examine the effect on the reaction to obtain the results shown in Table 7.

[Table] Display experiment example 5 (electron acceptor) When potassium ferricyanide is used as an electron acceptor, the activity measurement method described above is used, and when 2,6-dichlorophenol indophenol is used,
The reaction composition was 1.0 ml of 0.1 M phosphate buffer (PH6), 0.1 ml of 10 mM dichlorophenol indophenol, 0.1 ml of 1 M sorbitol, 0.1 ml of 3 mM phenazine metasulfate, 0.1 ml of enzyme solution, and 1.6 ml of distilled water.
The amount of enzyme required to reduce 1 micromole of dichlorophenol indophenol per minute is one unit.
5) 1.0ml, 30mM phenazine metasulfate 0.1ml,
1MD - Sorbitol 0.1ml, enzyme solution 0.1ml, distilled water
The reaction was carried out at 25℃ for 5 minutes with 1.7ml of reaction composition.
The amount of oxygen required during that time was measured using an oxygen electrode, and the amount of enzyme required to consume 0.5 micromol of oxygen per minute was measured as one unit, and the maximum reaction rate (V _nax ), substrate and electron acceptor Find the Michaelis constant (Km) for , and obtain the results in Table 8.

[Table] Experimental Example 6 (Physical and chemical properties) When the purified enzyme obtained in Example 3 was treated with the commonly performed SDS-polyacrylamide gel electrophoresis method, the molecular weights were 56,000, 43,000, and 16,000.
It is divided into three subunits. In addition, this enzyme preparation was diluted with pine kilvane buffer to give a protein concentration of 0.38 mg/ml, and some D-
When sorbitol and ubiquinone are added and the absorption spectrum in the visible region is measured, the spectrum shown in FIG. 1 is obtained. Experimental Example 7 (Optimal pH and temperature for reaction) In the activity measurement method described above, the pH of the buffer solution was set at 3 to
When reacting at 7.5, the optimum pH is 4.5.
Similarly, when the buffer pH was set to 4.5 and the reaction temperature was set to 20 to 50°C, the optimum temperature was 25°C.
It is ℃. Experimental Example 8 (Ultracentrifugation) The final sample obtained in Example 2 was used as the protein amount.
As a result of ultracentrifugation analysis at a concentration of 6 mg/ml, as shown in the reference image (ultracentrifugation - sedimentation image, image after 60000 rpm, 100 minutes), the sample used was a single protein component. You can see that it's getting better. (In the reference figure, indicates the red band of cytochrome,
represents the peak of D-sorbitol dehydrogenase, and represents the peak of coexisting Triton X-100, respectively. ) Example 4 (Production of enzymes by various microorganisms) Various microorganisms are cultured under the culture conditions used in Example 1, and specific D-sorbitol dehydrogenase activity is measured. That is, 1 ml of each enzyme-containing solution produced by various microorganisms, 1 molar concentration of sorbitol
After reacting at 25°C for 5 minutes with a composition of 0.1 ml, 0.1 morph potassium erythyanide, 0.5 ml pine kilvaine buffer (PH4.5), and 0.2 ml distilled water, immediately add 0.5 ml ferric sulfate-dupanol reagent. , add 3.5 ml of distilled water to stop the reaction, leave it for 10 minutes, and then drain the reaction solution.
Measure the absorbance at 660nm. As a control, the corresponding identical enzyme-containing solution 0.1
ml was subjected to reaction, post-treatment, and absorbance measurement in the same manner as above. However, in the reaction, the same amount of distilled water is used instead of sorbitol. Enzyme activity (reaction rate) is shown in Table 9 as the specific activity by subtracting the control value from the enzyme activity value, where 1 unit is the amount of enzyme required to reduce 2 micromoles of ferricyanide per minute of the above reaction. .

[Table] Thorium

【table】 [Brief explanation of the drawing]

FIG. 1 shows the visible region absorption spectrum of D-sorbitol dehydrogenase of the present invention.

Claims (1)

[Claims] 1. D-sorbitol dehydrogenase having the following properties: (a) Action: Dehydrogenates D-sorbitol and converts it to L-sorbose. (b) Substrate specificity: Mainly dehydrogenates D-sorbitol. Very little dehydrogenation of D-mannitol. (c) Optimum PH range: PH4.5±0.5 (d) Optimal temperature range: 25℃±5℃ (e) Stability: PH5.0, 5℃, does not lose activity even if left for 24 hours. (f) Inactivation conditions: Inactivation occurs when heated to 45°C or higher for 10 minutes in acetate buffer (PH5.0). (g) Inhibition: The activity is inhibited by p-chloromeric yuribenzoate. (h) Stabilization: Stabilized by the presence of D-sorbitol. (i) Molecular structure: molecular weights of approximately 56,000 units, approximately 43,000 units, and approximately 16,000 units (respectively SDS
-based on gel electrophoresis). It has a flavin molecule and a cytochrome molecule as prosthetic molecules in its molecule. (j) Visible absorption spectrum: λ _nax : 417 nm, 523 nm, 552 nm. _λnax : 481nm, 540nm. Shoulder: approx. 530nm. (k) Electron acceptors: ferricyanide, 2,6-dichlorophenol, indophenol or phenazine methosulfate. 2. Culture D-sorbitol dehydrogenase-producing bacteria belonging to the genus Gluconobacter, Acetobacter, Serratia, Proteus, Bacillus, Staphylococcus, Brevibacterium, Pseudomonas or Klebsiella in a medium. A method for producing D-sorbitol dehydrogenase having the following properties, which comprises producing and accumulating D-sorbitol dehydrogenase in a culture and collecting it from the culture. (a) Action: Dehydrogenates D-sorbitol and converts it to L-sorbose. (b) Substrate specificity: Mainly dehydrogenates D-sorbitol. Very little dehydrogenation of D-mannitol. (c) Optimum PH range: PH4.5±0.5 (d) Optimal temperature range: 25℃±5℃ (e) Stability: PH5.0, 5℃, does not lose activity even if left for 24 hours. (f) Inactivation conditions: Inactivation occurs when heated to 45°C or higher for 10 minutes in acetate buffer (PH5.0). (g) Inhibition: The activity is inhibited by p-chloromeric yuribenzoate. (h) Stabilization: Stabilized by the presence of D-sorbitol. (i) Molecular structure: molecular weights of approximately 56,000 units, approximately 43,000 units, and approximately 16,000 units (respectively SDS
-based on gel electrophoresis). It has a flavin molecule and a cytochrome molecule as prosthetic molecules in its molecule. (j) Visible absorption spectrum: λ _nax : 417 nm, 523 nm, 552 nm. _λnax : 481nm, 540nm. Shoulder: approx. 530nm. (k) Electron acceptors: ferricyanide, 2,6-dichlorophenol, indophenol or phenazine methosulfate.