JPS6012027B2 - Novel glycerol dehydrogenase and its production method - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel glycerol dehydrogenase and a method for producing the same. More specifically, the present invention relates to a novel glycerol dehydrogenase having the following properties, and also to a method for efficiently producing the enzyme from a new strain belonging to the genus Bacillus that has the ability to produce the enzyme. Conventionally, glycerol dehydrogenase [ECI. 1.1.6] (hereinafter abbreviated as GDH) has been known for a long time to be present in microbial cells such as Aerobacter aerogenes, Escherichia coli, and Bacillus subtilis. In addition, microorganisms belonging to the genus Proteus, Erwinia, and Serratia (Special public interest
No. 0-21553) or caused by microorganisms belonging to the genus Achromobacterium, Arthrobacterium, Brevibacterium, and Pseudomonas (Tokusei Sho 53-12788
No.) is known. In recent years, the properties of enzymes, particularly their high substrate specificity, have attracted attention, and in the field of clinical testing, enzyme-based test reagents have attracted a great deal of attention due to their simplicity. GDH is also known to be conjugated with lipoprotein lipase and can be used to quantify triglycerides in body fluids or to measure the activity of phosphatase that uses glyceric acid as a substrate. It is strongly desired to provide a method. In order to meet this demand, the present inventors conducted a wide search in the natural world for bacterial strains that have the enzyme activity, and as a result, they found that G-1, which belongs to the genus Bacillus and was isolated from the soil under Akashi City, has a particularly remarkable ability to produce GDH. I found it. In addition, as a result of intensive research on the GDH produced by this strain,
The molecular weight of the enzyme was measured using Cephadex G-200 and was 85,000±5,000, which is smaller than the molecular weight of all conventionally known GDHs, which are more than 100,000, and is extremely characteristic in terms of molecular size. The present invention was completed based on the discovery that the present invention has the following properties. That is, the present invention is a novel glycerol dehydrogenase having at least the following properties 11 to 1. m Molecular weight: 85,000 soil 5,000 (cephadex G
-200 gel filtration method)'2} Optimum pH for action: 9
．． 5 to 10.0 (2500 reactions in 0.1M glycine-Kq-KOH buffer) Stable pH range: pH 5.0 to
9.0 (500, 15 hours processing)

[4) Optimum temperature for action:
Around 45q0 (in 0.1M glycine-KCI-KOH buffer, pH 9.5) {5} Stable temperature range: 50oo or less (in 0.09M potassium monophosphate buffer, pH 7.5
15 minutes treatment) {Km value for 6'glycerol: 1
．． Value of K for 33×10-2M and NAD: 1.2
8 x 10-4M Furthermore, the present invention involves culturing in a medium a bacterium that produces a novel glycerol dehydrogenase belonging to the genus Bacillus and having the properties {1} to '6}, and from the culture, the above properties can be obtained. This is a method for producing a novel glycerol dehydrogenase comprising [1} to {6}. The novel GDH-producing bacterial strain G-1 according to the present invention has the following cocoon-like properties. A form (Grows on 1' broth agar medium, the form of the fungus is catalpa-like, 1.2-1.5 x 3-5 Buddhas in size, usually 5-6 lotuses, In the late stage of culture, it shows a single morphological change. There is no cell pleomorphism. ■ No motility '3' Spore formation (0.5 x 0.8 oval, present in the center) ) {41 Gram stainability is enteric B growth stage {1} Broth agar plate culture Translucent yellow-white, glossy, circular, and) raised colonies with round periphery, diffusive, pigmented does not produce. '21 Growth on broth agar slant is good, with yellowish-white color. Forms an expanded shape. {31 Culture growth in broth liquid medium is good, no film is formed on the surface, and the medium becomes cloudy. {Grows on top of 4' broth agar puncture culture and in the puncture hole. [51 Meat juice gelatin puncture culture grows on the surface and liquefies gelatin. C Physiological properties ○' Reduction of nitrate: Negative ■ M Old test: Negative {3' VP test: Negative {41 Indole formation: Negative ■ Hydrogen sulfide formation: Negative Starch hydrolysis: Positive/Positive {7 ) Utilization of citric acid: Positive shelf Utilization of inorganic nitrogen source: Positive leg Pigment production: No pigment was produced in King A and B media, and no water-soluble pigment was produced in broth agar, broth gelatin, and potato dextrose media. unacceptable. Skin Urease: Negative 00 Oxidase: Negative 02 Catalase: Positive/Sexy 03 Attitude towards oxygen: Aerobic and facultative anaerobic chest
Growth pH: 5-9, especially grows well at 6.5-7.5. 03 Growth temperature: 15-40qo, especially 25-30qo
It grows well in 08 0-F Test: Positive/Positive 07) Sugar Utilization: Sugar utilization was investigated using Hugh Lifeson Tower. Acid production L-arabinose + D-xylose D-glucose +
D-mannose - D-fructose + D-lactose - maltose + low sugar + milk sugar - trehalose - D-sorbitol - D-mannitol - no production of gas ■ In the presence of 0.001% lysozyme Growth in: Does not grow. ■ Egg-York reaction (lecithinase activity): Negative
version) [(Mr. r bag y' s man female lofDeterm
inative mother cteriolo plow) (8th edition, 1
974)], the strain was found to be an amphoteric strain with positive Gram staining, form endospores, be catalase positive, and absorb carbohydrates by Atsushi Rakunagi. It is determined that it belongs to the genus Bacillus based on its properties such as its ability to decompose and produce acid. Furthermore, the acetylmethyl carbinol reaction (VP reaction) is positive, and the presence or absence of assimilation of various sugars is similar to Bacillus and Megaterium. However, it was identified as a new strain belonging to the genus Bacillus because it clearly differs from Bacillus megaterium in its characteristics, such as not producing 2S, showing a positive M old reaction, and growing under facultative anaerobic conditions. be done. This strain has been submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology with Microbial Accession No. 5059 (hereinafter abbreviated as Chokoken Bacteria No. 505).
It has been deposited as. The novel GDH according to the present invention is obtained by using the novel GDH-producing strain belonging to the genus Bacillus in a nutrient medium.
Preferably, by culturing in a medium supplemented with glycerol to increase enzyme production ability, the enzyme is produced and accumulated in the bacterial cells, and enzyme powder can be obtained by extraction, purification, and drying using known methods. . To explain more specifically, G-
One strain is cultured in a suitable nutrient medium, such as a medium containing suitable rice bran, Murohata source, inorganic salts, and glycerol and organic promoters to increase enzyme production ability, and new GDH is accumulated in the bacterial body. Here, as the lees, sugars such as glucose, galactose, fructose, sucrose, lactose, blackstrap molasses, and starch hydrolysates, and sugar alcohols such as glycerol, sorbitol, and mannitol can be used. As a nitrogen source, yeast extract,
Bebutone, meat extract, cornstarch liquor, casein hydrolyzate, defatted soybeans, ammonium salts, etc. are used. Examples of inorganic salts that can be used include metal salts such as manganese, sodium, potassium, magnesium, calcium, iron, cobalt nickel, and zinc, as well as salts such as sulfuric acid, phosphoric acid, hydrochloric acid, and nitric acid. Manganese in particular is a metal salt essential for growth. If it is lacking, growth will not occur.Suitable growth-promoting substances include yeast extract, malt extract, meat extract, veptone, cornstarch liquor, etc.In the present invention, glycerol and glycerol are particularly used as enzyme inducers. A large amount of new GDH can be generated by adding the contained substances and other derivatives to the culture medium.These additives may be added from the beginning of the culture or during the culture.The amount added is usually Good results can be obtained by adding glycerol at a ratio of about 2% to the medium.The medium should be kept near neutral, and aerobic culture such as aerobic culture should be carried out at around 30°C for 1 to 2 hours. The thus obtained novel GDH-containing bacterial cells are separated by sieving or centrifugation, suspended in an appropriate buffer, and then subjected to methods such as grinding, sonication, mechanical compression, or autolysis. The enzyme is extracted by crushing it by a known method. Unportable substances are separated from the extract by sieving or centrifugation to obtain a cell-free enzyme solution, and then salting out with ammonium sulfate, main sulfur, etc., or acetone, alcohol, etc. Enzyme preparations were obtained using known methods such as solvent precipitation using ion exchange.To obtain more highly purified enzyme preparations, adsorption lacquering methods and gel filtration methods using ion exchange may be used. .
The activity of the novel GDH is calculated by measuring with a spectrophotometer the increase in absorbance of the generated NADH in 34 dishes when glycerol and NAD are reacted as substrates. That is, glycerol 3. 1 h mol, NAD 8.0 mol, glycine-KOH buffer (pH 9.3) 0.3 h
Mix 2.9 molar of the reaction mixture with 0.1 molar of the enzyme solution, react at 25°C, and measure the increase in ultraviolet absorption at a wavelength of 34 m for 4 minutes from the start of the reaction. The increase in absorbance per minute is calculated from That is, as a control, the same operation is performed with the above composition but using water instead of glycerol, and the increase in absorbance at 340 nm of the test solution is subtracted from that of the control. Using (6.22 x 1 p) as the molecular extinction coefficient of NADH at 34 degrees, calculate the amount of NADH produced from the subtracted absorbance value, and calculate the enzyme titer in the sample based on this. do.
The enzyme titer is expressed as 1 mole of N per minute under the above conditions.
The amount of enzyme that produces ADH is taken as one unit. The physicochemical properties of the novel GDH obtained by the present invention are shown for the enzyme produced by strain G-1. m 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 can act on 1,2-propanediol, 2,3-butanediol, glycerol-Q-monochlorohydrin, etc., but it also acts on methyl alcohol, ethyl alcohol, propyl alcohol, etc. Alcohols such as alcohols do not dehydrogenate. The value of K for glycerol is 1.33×10-2M,
The value of K for MAD is 1.28x10-4M. The relative activity for each substrate is shown in Table 1. Table 1 '31 Optimum carp and stable pH range The optimal pH of this enzyme was 9.9% under the above activity measurement conditions.
5 to 10.0 (see Figure 1). The stable matrix of this enzyme is pH 5.0 ~ after treatment at 5℃ for 1 hour.
9.0 (see Figure 2). Furthermore, the most stable fence is around 7.5 (0.09M potassium-phosphate buffer),
At this pH, almost 100% even after treatment at 50oo for 15 minutes.
activity remains. '41 Range of optimal temperature for action The optimal temperature for action of this enzyme is around 45 qo under the following activity measurement conditions (see Figure 3). '5} Inactivation conditions due to pH, temperature, etc.
7.5 (0.08 M potassium-phosphate buffer), it is stable up to 50% when treated powder by powder, and is completely inactivated at 70°C (see Figure 4). '6} Effects of various drugs This enzyme activity is affected by Cu + ions, ZnH ions, CdH ions,
ions, PbH ions, Ag+ ions, and Hg++ ions. On the other hand, Ca++ ions, FeH ions, FeH ions,
Activation is observed by Mn++ ions and Co++ ions, and significant inhibition is observed by metal chelating agents such as EDTA. Furthermore, since the activity is markedly inhibited by SH inhibitors such as PCMB, it is inferred that the SH group is involved in the expression of the activity of this enzyme. Next, Table 2 shows the effects of each drug. Table 2 Husband p-Ch. Molecular Weight of RomerCuribenZOate The molecular weight of this enzyme is calculated to be 85,000 and 5,000 by the Sephadex G-200 gel transfer method (see Figure 5). In the figure, A is Chymotrypsis/-gen A (derived from bovine liver) (molecular weight 25,000), B is ovalbumin (molecular weight 4,500
0), C is bovine serum albumin (molecular weight 67,000), D
represents GDH of the present invention, E represents globulin (human origin) (molecular weight 16,000), and F represents secondary GDH (derived from Bacillus megaterium). Next, the novel method for producing GDH according to the present invention will be explained using an example using the G-1 strain. Example 1 Glycerol 2%, Polybeptone (Daigo Nutrition) 2%,
KH2P040.4%, K2HP042.5%, yeast extract (manufactured by Oriental Yeast) 0.1%, MnC12.4
Ratio 0 0.005%, M is 04.7 days 20 0.005
%, FeC12・4 days 200.005%, CaC】2・
2mo0 0.005%, antifoaming agent Adekanol LG-12
6 (manufactured by Asahi Denka) 0.04% (pH 7.5) culture medium consisting of 50 skins was placed in a 500-year-old Sakaguchi flask, 12
After autoclaving at 0oo for 10 minutes, one platinum loop was aseptically inoculated with strain G-1 (Choken Bacteria No. 505y), and 2 at 30oo.
The cells were cultured under shaking for 4 hours (14 $. Pm. 7c in shaking). The new GDH was produced and accumulated within the bacterial cells, and its productivity was 4.2 units per Z of culture solution. In addition, when the bran is used as glucose (other compositions are the same as above), 0.
It was 3 units. Example 2 A 130-liter medium was prepared with the medium composition shown in Example 1 (however, the glycerol concentration was 4%) and charged into a 200-liter fermenter. After steam sterilization at 120 oo for 10 minutes, add 1.3 〆 of the culture solution of strain G-1 (Choken Bacteria Accession No. 505), which had been previously cultured in a medium with the same composition for 30 oo for 2 hours. 1%)
Aseptically shield and aerate at 30oo for 2 hours (IV.
．． m) Brood frames (18 p.m.) were cultured. 122 hours of the culture solution was treated with a continuous centrifuge to collect bacterial cells, and the bacterial cells were suspended in 0.09 M phosphate buffer (pH 7.5) for 20 minutes. This suspension was applied to a grinder to grind the bacterial cells. After grinding, the residue was separated using diatoms as a transit aid, and ammonium sulfate was added to the resulting solution to a saturation of 65% to recover new GDH as a precipitate. The yield of the new GDH activity in this precipitate was 90%, and the specific activity was increased 7 times. The obtained precipitate was dissolved in 0.09M phosphate buffer (pH 7.5), and Sephadex G-25 (buffered with 0.02M phosphate buffer (pH 7.5)) was added.
Column (manufactured by Pharmacia, Sweden) packed with
The active fraction was collected by desalting. The obtained desalted enzyme solution was then equilibrated in advance with 0.09M phosphate buffer (pH 7.5) and applied to a DEAE-cellulose (Brown, USA) column (2-volume column).
After adsorbing the new GDH through water and washing with the same buffer, a concentration gradient was created with the same buffer containing 0.2M salt and the same buffer containing 0.8M salt, and the salt was gradually absorbed. New GDH was eluted with increasing concentration. The eluted new GDH active fraction was collected and ammonium sulfate was added to it to 67% saturation to precipitate the new GDH. The precipitate was collected by centrifugation (10,000 x 20 minutes) and added to 800' of 0.09M phosphate buffer (pH 7.5).
) and then adjust the pH to 6.0 with 20% (V/V) phosphoric acid.
In order to remove impurity proteins, the powder was left to stand at 370 for 4 minutes, and the resulting unlacquered matter was removed by centrifugation (10,000 x evening, 20 minutes) to obtain a supernatant liquid. The thus obtained clouding enzyme solution was then diluted with 0.01M phosphate buffer (pH 7.5).
Desalting was carried out using a Sephadex G-25 column (6 volume column) buffered with ) and the active fraction was collected. The obtained desalted enzyme solution was freeze-dried to obtain a new GDH specimen. The specific activity of this product is 15.0 units/double 9,
The yield from the culture solution was 28.9%, and the specific activity was increased 60 times.

[Brief explanation of the drawing]

FIG. 1 shows the pH activity curve of the enzyme of the present invention. FIG. 2 shows the pH stability curve of the enzyme of the present invention. FIG. 3 shows the temperature activity curve of the enzyme of the present invention. FIG. 4 shows the temperature stability curve of the enzyme of the present invention. FIG. 5 shows the results of molecular weight determination of the enzyme of the present invention by Sephadex G-200 gel filtration method. *1 Kaede *2 Diagram 3■ Tomomi diagram *6 Crab

Claims (1)

[Scope of Claims] 1. A novel glycerol dehydrogenase having at least the following properties (1) to (6). (1) Molecular weight: 85,000±5,000 (gel filtration method using Cephadex G-200) (2) Optimum pH for action
:9.5-10.0 (0.1M glycine-KCl-KO
(25°C reaction in H buffer) (3) Stable pH range: 5.
0 to 9.0 (5°C, 15 hours treatment) (4) Optimum temperature for action: around 45°C (in 0.1M glycine-KCl-KOH buffer, pH 9.5) (5) Stable temperature range: 50°C Below (in 0.05M potassium-fuel acid buffer, pH 7.5,
15 minutes treatment) (6) Km value for glycerol:
Km value for 1.33×10^-^2M and NAD:
1.28 x 10^-^4M2 A bacterium belonging to the genus Bacillus that produces a novel glycerol dehydrogenase having the following properties (1) to (6) was cultured in a medium, and the following properties ( 1) A method for producing glycerol dehydrogenase, which comprises collecting a novel glycerol dehydrogenase having the following. (1) Molecular weight: 85,000±5,000 (gel filtration method using Cephadex G-200) (2) Optimum pH for action
:9.5-10.0 (0.1M glycine KCl-KO
(25°C reaction in H buffer) (3) Stable pH range: 5.
0 to 9.0 (5°C, 15 hours treatment) (4) Optimum temperature for action: around 45°C (in 0.1M glycine-KCl-KOH buffer, pH 9.5) (5) Stable temperature range: 50°C (in 0.05M potassium-phosphate buffer, pH 7.5,
15 minutes treatment) (6) Km value for glycerol:
Km value 1 for 1.33×10^-^2M and NAD
．． 28×10^-^4M