JPS6012028B2 - Method for producing glycerol dehydrogenase - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention uses a bacterial strain capable of producing glycerol.
The present invention relates to a method for efficiently producing glycerol dehydrogenase, and more specifically to a method for producing glycerol dehydrogenase produced by bacteria belonging to the genus Cellulomonas.

Glycerol dehydrogenase [1.1.1.6] (hereinafter abbreviated as GDH) has long been known to be present in the bacterial bodies of Aerobacter aerogenes, Escherichia coli, Bacillus subtilis, etc. There is.

In addition, microorganisms belonging to the genus Proteus, Erpinia, and Serratia (Special public interest
0-121553), or microorganisms belonging to the genus Achromobacter, Arthrobacter, Brevibacterium, and Pseudomonas (JP-A-53-127). In recent years, the properties of enzymes, especially their high specificity, have attracted attention, and in the field of clinical testing, test drugs that use enzyme methods are becoming more and more popular.
It has attracted a lot of attention because of its simplicity. GDH
It is known that by conjugation with lipoprotein lipase, it can be used for quantifying triglycerides in body fluids or for measuring the activity of phosphatase that uses glycerophosphate as a substrate. It is strongly desired to provide the following. In order to meet this demand, the present inventors conducted a wide search for the enzyme activity in the microbial world.
It was discovered that DH production ability exists, and the present invention was completed.

Bacterial strains that can be used in the present invention include, for example, Cellulomonas
Flavigena (CCI1ulomonasf1avi)
a) All GH-producing bacteria that belong to the genus Cellulomonas can be used in the method of the present invention. According to the present invention, GDH is produced and accumulated in the bacterial cells by culturing bacteria belonging to the genus Cellulomonas in a nutrient medium, so that GDH can be extracted by known methods and improved methods that will be developed in the future. Enzyme powder can be obtained by purification and drying. To be more specific,
The Cellulomonas flavigena IF03748 was cultured in a suitable nutrient medium, such as a medium containing suitable sugars, chloride sources, inorganic salts, or when GDH is a inducing enzyme, glycerol and organic promoters to enhance enzyme production. GDH is accumulated in the bacterial cells by culturing in a medium containing glucose, galactose, mannose,
Bases such as fructose, sucrose, lactose, maltose, blackstrap molasses, and starch hydrolysates, as well as bran alcohols such as glycerol, sorbitol, and mannitol, can be used. As the room millet source, yeast extract, veptone, meat extract, corn steep liquor, casein hydrolyzate, defatted soybean, ammonium salt, etc. are used. As the inorganic salts, metal salts such as sodium, potassium, manganese, magnesium, calcium, cobalt, nickel, and zinc, and salts such as sulfuric acid, phosphoric acid, hydrochloric acid, and nitric acid can be used. Good organic promoters include yeast extract, veptone, meat extract, and corn steep liquor. The GDH-containing cocoons thus obtained are separated by filtration or centrifugation, suspended in an appropriate buffer, and then ground,
The enzymes are extracted by disruption by known methods such as sonication, mechanical compression or autolysis.

After separating insoluble matter from the extract by furnace filtration or centrifugation, enzyme labeling is performed from the resulting furnace liquid or supernatant by known methods such as salting out with ammonium sulfate, bitter salt, etc., or solvent precipitation using acetone, alcohol, etc. get goods. In order to obtain a more highly purified enzyme preparation, an adsorption/elution method using ion exchange, a gel filtration method, etc. may be used. The activity of GDH is calculated by measuring the increase in absorbance of the generated NADH at 34 m using a spectrophotometer when glycerol is reacted with NAD as a substrate.

That is, 300 fmol of glycerol, NADO. 5 molar ammonium buffer (PH9.0) 2.9 molar reaction mixture containing 3 molar of enzyme solution was mixed with 0.1 molar of the enzyme solution,
The reaction was carried out at 25°C, and the increase in ultraviolet absorption at a wavelength of 34 m 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 carried out with the above composition using water instead of glycerol, and the increase in absorbance at 34 m of the test solution was subtracted from that of the control. Using (6.2 x 1 pi) as the molecular extinction coefficient of NADH at 34 m, the amount of NADH produced is determined from the subtracted absorbance value, and based on this, the enzyme titer in the sample is calculated. The enzyme titer is expressed as one unit, which is the amount of enzyme that produces one French mole of NADH per minute under the above conditions.

The bacterial culture medium composition for this preliminary test is shown in Table 1.
The assay method for titer is shown in Table 2. Table 1 Media composition for preliminary test Table 2 Potency assay method for preliminary test Total reaction volume 3.0 Autumn Reaction temperature 25°C Potency display method △Abs/min (340 nm) Physical and chemical properties of GDH obtained by the present invention Characteristics of Cellulomonas
Flavigena IF03748 origin (Example 1 below)
The specific activity (13 mountain units/secret purified enzyme) obtained in the following is shown as a representative example.

'1' action This enzyme dehydrogenates glycerol in the presence of NAD and catalyzes the reaction that produces dihydroxyacetone and NADH.

[21 Substrate specificity This enzyme can act on 1,2-propanediol, ethylene glycol, meso-erythritol, etc. in addition to glycerol, but it cannot dehydrogenate alcohols such as methanol, ethanol, and propanol ( (See Table 3)
.

Furthermore, the K values for glycerol and NAD are each 1.1.
×10-2M and 9.0×10-5M.

Table 3 GDH '31 Normal pH and stable pH range The optimum seedling for this enzyme is 9. (optimum pH for reverse reaction)
is around 6.0).

The stable PH level of this enzyme is 5.5~ after being treated at 30℃ for 1 hour.
It is in the range of 10.0. Furthermore, the most stable pH is around 7.0, and 100% activity remains even after treatment for 5, 0, and 15 minutes. (2) Range of optimal temperature for action The optimal temperature for action of this enzyme is around 50 pm under the activity measurement conditions described above.

'51 Inactivation conditions due to pH, temperature, etc. When treated at pH 7.0 for 15 minutes, this enzyme has 60q0
It is stable until 70oo, and 77% is inactivated at 70oo.

'61 Influence of various drugs This enzyme activity is affected by Cu20 ions, Zn20 ions, Cd2
It is significantly inhibited by 10 ions, Co20 ions, and Ni20 ions.

On the other hand, significant activation was observed by K, Rd+, and NH4. Although no inhibition was observed with metal chelating agents such as EDTA, significant inhibition was observed with metal chelating agents such as O-phenanthroline.

The activity is also significantly inhibited by PCMB, boric acid, and monoodoacetic acid. '71 Molecular Weight The molecular weight of the present enzyme is calculated to be about 336,000±12,000, and is estimated to consist of about 42,000 to 43,000 sapunit 1.

Next, the present invention will be explained by examples.

Example 1 Glycerol 1.0% (weight% and below are the same), polybeptone 1.5%, dipotassium phosphate 0.3%, sodium chloride 0.2%, magnesium sulfate (740) 0.02%,
Add a mixture of 0.1% yeast extract (7.0 to 7.2) to 30 jars.
A medium was obtained by steam sterilization at 20°C for 30 minutes.

On the other hand, a culture of Cellulomonas flavigena IF03748, which had been cultured with shaking for 28 hours using the same composition column, was aseptically shielded on the column and incubated at 28°C.
Culture with aeration for 24 hours. Forty minutes of the culture solution is processed in a continuous centrifuge, the bacterial cells are collected, and the bacterial cells are suspended in a 0.1 M phosphate buffer (pH 7.0). This suspension is applied to a dyno mill to crush the bacterial cells. After crushing, insoluble matter is removed by centrifugation, and the resulting supernatant is placed in a cellophane tube and dialyzed for 1 hour with 0.01M potassium phosphate buffer. The obtained desalted enzyme solution was preliminarily adjusted to 0.0
D equilibrated with 1M phosphate buffer (pH 7.0)
It is passed through a column packed with EAB-cellulose (manufactured by Braun) to adsorb GDH. Furthermore, after washing away impurities with the buffer solution used to equilibrate the resin, a concentration gradient was created with the same buffer solution containing 0.29M sodium chloride and the same buffer solution containing 0.4M sodium chloride, and the sodium chloride was gradually added to the buffer solution. G while increasing the concentration
DH was eluted. Collect the eluted GDH active fraction,
A salting out fractionation is carried out at 35-50% saturation of ammonium sulfate. GDH precipitated with 50% saturated ammonium sulfate was collected by centrifugation and added to a small amount of 0.1M phosphate buffer (pH 7).
．． 0), dialyzed in the same manner as above, and the resulting desalted solution was mixed with hydroxyapatite (Hydrox apatite) equilibrated with 0.01M potassium phosphate buffer (Book 7.0).
GDH is adsorbed through a column packed with phosphate buffer (Book 7.0) 0.01-0.08M.
Collect the GDH activity fraction of the gradient.

Claims (1)

[Claims] 1. A method for producing glycerol dehydrogenase, which comprises culturing glycerol dehydrogenase-producing bacteria belonging to the genus Cellulomonas in a nutrient medium, and collecting glycerol dehydrogenase from the culture.