JPS6057830B2 - Method for producing glycerol dehydrogenase - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for efficiently producing glycerol dehydrogenase using a bacterial strain capable of producing glycerol dehydrogenase. Filamentous fungi belonging to the genus Staphylococcus, Micrococcus,
The present invention relates to a method for producing glycerol dehydrogenase produced by bacteria belonging to the genus Aeromonas and Corynebacterium.

Glycerol dehydrogenase [1.1.1.6] (hereinafter abbreviated as GDH) has been known for a long time to be present in bacterial cells such as Aerobacter aerogenes, Escherichia coli, and Bacillus subtilis. There is.

In addition, other microorganisms belonging to the genus Proteus, Erwinia, and Serratia (Special Public Interest
0-21553), or caused by microorganisms belonging to the genus Achromobacter, Arthrobacter, Previbacterium, and Pseudomonas (Japanese Patent Application Laid-Open No. 12788-1988)
It has been known. In recent years, the properties of enzymes, particularly their high specificity, have attracted attention, and in the field of clinical testing, test reagents that utilize enzyme methods are attracting a great deal of interest due to their simplicity and simplicity. G.D.
It is known that H can also be used for quantifying triglycerides in body fluids by conjugating with lipoprotein lipase, or for measuring the activity of phosphatase that uses glycerophosphate as a substrate. It is strongly desired to provide a method to do so. In order to respond to such prospects, the present inventor conducted a wide search for the enzyme activity in the microbial world, and found that filamentous fungi belonging to the genus Aspergillus, Penicillium, Neurospora, and Fusarium, or the genus Staphylococcus, Micrococcus, and Aeromonas , marked GDH in bacteria belonging to the genus Corynebacterium
It was discovered that production capacity exists, and the present invention was completed.

Bacterial strains that can be used in the present invention include, for example, Aspergillus Oryzae I
FO4l77, Aspergillus Sawyer (AspsOj
ae) IF'04386, Penicillium Notatum (
PenniclllumrlOtatum)IF'0
4640, Penicillium expansum (Pen.e
xpanslOn) IF'06096, Neurospora crossa (NeLlr″0sp0racrassa)
IFO6O68, Fusarium Crumorum (Fusar
iunlculmOrum) IFO59O2 and other filamentous fungi such as Staphylococcus aureus
IFO3O36, Micrococcus Variance (MicrOcOccusvariance)
ans) IFO3765, Aeromonas hydrophila, (AerOmOnashydOrOphlla) 1
F01297 & Micrococcus luteus (MicrO
cOccusluteus) FOl27O8, Corynebacterium
In addition to these bacteria, filamentous fungi such as Aspergillus, Penicillium, Neurospora, and Fusarium, and Staphylococcus, Micrococcus, Aeromonas, and Corynebacterium, etc. belongs to bacteria,
Any bacteria that produces GDH can be used in the method of the present invention.

According to the present invention, filamentous fungi belonging to the genera Aspergillus, Pencilium, Neurospora, and Fusarium and bacteria belonging to the genera Staphylococcus, Micrococcus, Aeromonas, and Corynebacterium are cultured in a nutrient medium. As a result, GDH is produced and accumulated in the bacterial cells, and enzyme powder can be obtained by extraction, purification, and drying using known methods and improved methods that will be developed in the future. More specifically, the GDH-producing bacteria of each of the above genera are grown in an appropriate nutrient medium, such as a medium containing appropriate carbohydrates, nitrogen sources, and inorganic salts, or in the case where GDH is an inducible enzyme, in order to increase the enzyme production ability. The cells are cultured in a medium containing glycerol and organic promoters to accumulate GDH in the cells, and the carbohydrates include glucose, galactose, mannose, fructose, sucrose,
Sugars such as lactose, maltose, waste molasses, and starch hydrolysates, and sugar alcohols such as glycerol, sorbitol, and mannitol can be used. As the nitrogen source, yeast-processed scratches, peptone, meat-processed scratches, corn steep liquor, chilitin hydrolyzate, defatted soybeans, ammonium salts, and the like are used. Inorganic salts include metal salts such as sodium, manganese, magnesium, calcium, cobalt, nickel, and zinc, as well as sulfuric acid, phosphoric acid,
Salts such as hydrochloric acid and nitric acid can be used. Good organic accelerators include yeast, peptone, meat, and corn steep liquor. The bacterial cells containing GDH thus obtained are separated by filtration or centrifugation, suspended in an appropriate buffer, crushed by known methods such as grinding, sonication, mechanical compression, or autolysis. Extract the enzyme.

After separating insoluble matter from the extract by filtration or centrifugation, enzyme products are prepared from the resulting solution or supernatant by salting out with ammonium sulfate, Glauber's salt, etc., or solvent precipitation using acetone, alcohol, etc. get. In order to obtain a more highly purified enzyme preparation, an adsorption/elution method using ion exchange, a gel filtration method, or the like may be used. The activity of GDH produced by bacteria is dependent on glycerol and N. When reacting with AD as a substrate, the generated N,
ADHf) Calculated by measuring the increase in adsorption degree at 340r1m with a spectrophotometer.

That is, 800 μmol of glycerol, N, ADO. 5
Reaction mixture containing 30 μmol of carbonate buffer (PH9.O)2. Mix 0.1ml of enzyme solution with buL and heat at 25°C.
The increase in ultraviolet absorption at a wavelength of 340 rpm was measured for 4 minutes from the start of the reaction, and the increase in absorbance per minute was calculated from the linear part.

That is, as a control, the same operation was performed with the above composition using water instead of glycerol, and 340r1m of the test solution was used.
Subtract that of the control from the increase in absorbance of . As the molecular extinction coefficient of NADH at 340r1m (ε=6.2
2 x 103), the amount of N and ADH produced is determined from the subtracted absorbance value, and based on this, the enzyme titer in the sample is calculated. However, in the case of GDH produced by filamentous fungi,
Since NADP is used as a coenzyme, GDH of filamentous fungi is measured in exactly the same manner as the above-mentioned measurement method except that NADP is used instead of NAD. The enzyme titer is expressed as 1 μmol NADH and NADPH per minute under the above conditions.
The amount of enzyme that produces 1 unit is used.

Table 8 shows the culture titer and specific activity of GDH produced by filamentous fungi and bacteria for preliminary testing of the present invention.

The fungal culture composition for this preliminary test is shown in Table 1, and the titer assay method is shown in Table 2. The physicochemical properties of GDH obtained by the present invention are shown using Staphylococcus aureus IF'0306 source (purified enzyme with a specific activity of 100 units/M9 obtained in Example 1 described later) as a representative example. show. (1) Action This enzyme dehydrogenates glycerol in the presence of NAD and catalyzes the reaction that produces dihydroxyacetone and NADH. (2) Substrate specificity In addition to glycerol, this enzyme dehydrogenates glycerol and catalyzes the reaction to produce dihydroxyacetone and NADH. It can also act on 2,3-butanediol, glycero-d-monochlorohydrin, etc., but alcohols such as methanol, ethanol, propanol, etc. cannot be dehydrogenated (see Table 4).

Furthermore, when N,ADP was used as a coenzyme, there was no activity at all compared to N,AD.

Further, the KTrl values for glycerol and NAD were 1.09 x 10-2M and 8.93 x 10-5M, respectively. (3) Optimal PH and stable PH range The optimal PH of this enzyme is 9. (The optimum pH for the reverse reaction is around 6.).

The stable pH range of this enzyme is 6. O
~10.0. Furthermore, the most stable pH is 7.0
ft, 100% at 55℃ and 1 dust treatment at the same pH.
activity remains. (4) Range of action process The optimum temperature for action of this enzyme is around 45 to 50°C under the activity measurement conditions described above.

(5) Disqualification conditions due to PHL temperature, etc. This enzyme has a pH of 7. In the case of a powder treatment, it is stable up to 60°C and is completely inactivated at 75°C.

(6) Effects of various drugs This enzyme activity is affected by Cu2+ ion, Zn2+ ion, and Zn2+ as shown in Table 5.
ion, Cd2+ ion.

On the other hand, as shown in Table 6, significant activation was observed by K+, Rb+, and NH4+. No inhibition was observed with metal chelating agents such as EDTA, but significant inhibition was observed with metal chelating agents such as O-phenanthroline. Also P
Since the activity is markedly inhibited by SH inhibitors such as CMB, it is inferred that the SH group is involved in the expression of the activity of this enzyme.

(7) Molecular weight The molecular weight of this enzyme was approximately 3
It is calculated to be 90,000, and by electrophoresis using SDS, it is calculated to be about 42,000.

From these, it is inferred that this enzyme consists of w subunits. Next, the present invention will be explained by examples.

Example 1 Glycerol 1.0% (wt%, same below), polypeptone 1.5%, dipotassium phosphate 0.3%, sodium chloride 0.2%, magnesium sulfate (7H20) 0.02
%, yeast extract 0.1% (PH7.O~7.2
) mixture 20e was placed in a 30e capacity jar fermenter and sterilized with steam at 120'C for three times to obtain a culture medium.

On the other hand, Staphylococcus aureus IFO3O was cultured with shaking at 28°C for 4 hours using the same composition medium.
Aseptically inoculating 250 nL of a 6O culture into the medium,
Culture with aeration at 28°C for 2 incubations.

The culture solution 40e is treated with a continuous centrifuge, the bacterial cells are collected, and the bacterial cells are dissolved in 0.1M phosphate buffer (PH7.O) 5e.
Suspend in This suspension is subjected to an ultrasonic crusher to crush the bacterial cells. After crushing, impurities are removed by centrifugation, streptomycin sulfate is added to the resulting supernatant at a concentration of 1%, and the resulting insoluble substances are removed by centrifugation. First, ammonium sulfate is added to the resulting supernatant to achieve a 40% saturation, and insoluble matter is removed as a precipitate by centrifugation. Ammonium sulfate is further added to the supernatant to achieve a final saturation of 60%, and GDH is recovered as a precipitate. The activity recovery rate as a precipitate was 90%, and the specific activity had increased by 7 times. The obtained precipitate was added to 600ml of 0.1M phosphate buffer (
PH7. Dissolve in O), put in a cellophane tube,
．． Dialyze for 1 hour against 01M potassium phosphate buffer. The obtained desalted enzyme solution was preliminarily diluted with 0.01M phosphate buffer (PH”7).
．． 0) to adsorb GDH. Furthermore, after washing away impurities with the buffer solution used for equilibration of the resin, 0.25r! The same buffer solution containing salt in No. 4 and 0
．． A concentration gradient was created with the same buffer in which 4M salt was dissolved, and GDH was eluted while gradually increasing the salt concentration. The eluted GDH active fraction was collected and treated with ammonium sulfate.
Salting out fractionation is carried out as above at 5-56% saturation. 55
GDH precipitated with saturated ammonium sulfate was collected by centrifugation and added to a small amount of 0.1M phosphate buffer (PH7.O).
Sephadex G-2 buffered with the same buffer after dissolving in
Molecular sieve desalting is performed in Step 5, and the resulting desalted solution is freeze-dried.

Thus about 1.44y of GDH with specific activity 2 & 7 units/M9
is obtained. The activity recovery rate from the sterile extract was 59%, and the increase in specific activity reached 18%. Example 2 Micrococcus and Variance IF'C3765 were used as the bacterial strain, and glucose 1% was used as the medium.
Polypeptone 1.5%, dipotassium phosphate 0.3%, potassium sodium chloride 0.2%, magnesium sulfate (7H20
) 0.02%, yeast scratches 0.1% (PH7.O~7.
A solution 20e consisting of 2) was placed in a 30e capacity jar fermenter, sterilized under the same conditions as in Example 1, and cultured with shaking for 2 generations in a medium of the same composition in advance for 4 m. Culture of Stococcus palliances IFO3765 25
0wLι was inoculated aseptically and cultured with aeration for 2 hours for 24 days.

The obtained culture solution 401 is collected and suspended in the same manner as in Example 1, and then the bacterial cells are disrupted using a DYNO-MILL (manufactured by Wiley.K.Butskofen, Switzerland).

After grinding, insoluble matter is removed by centrifugation, streptomycin sulfate is added to the resulting supernatant to a concentration of 1%, impurities are removed as a precipitate by centrifugation, and the supernatant is Ammonium sulfate is added to give a final saturation of 35%, impurities are removed as a precipitate by centrifugation, and ammonium sulfate is added to the supernatant to give a final saturation of 65%, and GDH is recovered as a precipitate. The activity recovery rate of this precipitate was 85%, and the specific activity increased 7 times. The obtained precipitate was dialyzed and treated with an ion exchanger in the same manner as in Example 1, and freeze-dried GDH with a specific activity of about 25.0 $/WIg was about 1.
Got 2y. Example 3 Pheromonas hydrophila IFOl as the strain used
2978, Micrococcus luteus IFOl27O8
, Corynebacterium. Faciance IFOl2O77
Culture and purification were carried out in the same manner as in Example 1 to obtain lyophilized GDH with the following specific activity.

Example 4 The bacterial strain used was Aspergillus. Orysee IFO4l77
, Aspergillus sojae IFO488 Penicillium. Notatum IFO464O) Neurospora cratusa IFO6OB) Fusarium culmorum IFO59O
2 was cultured and purified in the same manner as in Example 1 to obtain lyophilized GDH with the following specific activity.

Claims (1)

[Claims] 1 Glycerol dehydrogenase-producing bacteria belonging to the genus Aspergillus, Penicillium, Neurospora, Fusarium, Staphylococcus, Micrococcus, Aeromonas, and Corynebacterium are cultured in a nutrient medium, and from the culture A method for producing glycerol dehydrogenase, which comprises collecting glycerol dehydrogenase.