JPH0441993B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Application Field) The present invention relates to bacteria of the genus Pseudomonas that produce lipase. (Prior Art) Lipase uses triglyceride as a substrate and hydrolyzes it into fatty acids and glycerin using oxygen. Also,
It is also known that esters are synthesized from fatty acids and alcohols by reducing the water content in the reaction water. At present, many attempts are being made to utilize lipases industrially, and in particular, the introduction of lipases into fatty acid production processes is expected. That is, the process of industrially decomposing fats and oils to produce fatty acids is currently carried out by a chemical hydrolysis method under high temperature and high pressure. but,
By introducing an oxygen reaction, the reaction can proceed at room temperature and pressure, so unsaturated fatty acids, which are often broken down by chemical hydrolysis methods, can be obtained without decomposition and in large quantities. energy consumption can be saved. In addition, it is also possible to utilize the substrate specificity of oxygen reactions and use inexpensive fats and oils as raw materials to improve them into high value-added fats and oils using transesterification. However, beef tallow and lard, which are the main raw materials for fatty acid production, are mainly composed of long-chain saturated fatty acids and are solid at room temperature. For this purpose, the oil or fat must be reacted at a temperature above its melting point, or it must be liquefied by adding an organic solvent and then reacted. Therefore, lipases used for fatty acid production are either thermostable and have optimal reactivity at high temperatures, or are resistant to organic solvents and cannot proceed in their presence. must not. Although the search for such a lipase has been actively conducted, an enzyme suitable for the demand at an industrial level has not been obtained to date. In terms of thermostable lipases, it has been reported that bacteria of the genus Pseudomonas can produce these lipases. That is, Pseudomonas KWI−56
(Pseudomonas sp KWI-56) is a lipase produced by Pseudomonas sp.
269985) Pseudomonas mephitica variutus lipolyteica
var.lipolytica), the optimum temperature for action is 70℃,
Lipase that does not deactivate even after heat treatment at 60℃ for 14 hours (Special Publication No. 50-25553), Pseudomonas
Produced by Pseudomonas fragi,
Lipase that retains 95% or more activity even after heat treatment at 75-80℃ for 20 minutes at 70℃ (Agric.Biol chem.1977, Vol. 41, 1353-1358), Pseudomonas, Fluorescens, Biotype I
(Pseudomonas fluorescens), the optimum temperature for action is 67℃, and even by heat treatment at 60℃ for 20 hours.
Lipase retaining 86.9% activity (Unexamined Japanese Patent Publication No. 1983-
58885) and other reports are known. In addition, regarding the optimum pH for action, many lipases have an optimum pH for action near neutrality, and Pseudomonas fragi has an optimum pH for action in the alkaline region.
Agric.Biol.Chem.1977, vol. 41, 1353-1358),
Achromobacter sp. (Japanese Unexamined Patent Publication No. 48-68792)
sp.: Japanese Patent Publication No. 52-21387), Humicola lanuginosa (Japanese Patent Publication No. 48-1989)
62990), Mucor Ribolytics (Mucor
lipolyticus: Agric.Biol.Chem.1973, 37 volumes,
2791-2796) are known. Some lipases produced by fungi are known to have an optimal pH for action in acidic regions, but none are known to have a pH below 5. (Problems to be solved by the invention) From the perspective of industrial use, the pH of reactions using lipases is not necessarily neutral, and there is a need for lipases that have an optimal pH for action in acidic or alkaline regions. . Furthermore, it would be more advantageous if the lipase also had heat resistance. (Means for solving the problem) As a result of a wide range of natural searches for microorganisms that produce heat-resistant lipase, the present inventors discovered the KWI-4 strain belonging to the genus Pseudomonas isolated from soil in Atsugi City, Kanagawa Prefecture. produced lipases with optimum operating pHs at PH4 and PH8, and that they were both thermostable lipases with optimum operating temperatures between 70 and 80°C. The mycological properties of the strain of the present invention are shown below. In order to study this mycological property, "classification and identification of microorganisms" is required.
(written by Takeharu Hasegawa, Gakkai Publishing Center), ``Guidelines for Medical Bacteria Identification'' (written by ST Cowan, translated by Toshikazu Sakazaki, published by Kindai Publishing), and ``Course on New Bacterial Culture Culture'' (written by Toshikazu Sakazaki, published by Kindai Publishing). A different method and medium composition were used. (a) Morphology Cell shape and size: Bacillus 2 microns long and 1 micron wide Cell dimorphism: Solitary or in short chains Motility: Yes, with one polar flagellum Spores: None Gram staining: Negative Anti-acidity: None (b) Growth condition Juicy agar plate culture: Round, round shape, smooth and glossy surface. Brown with a slight yellow tinge. Juice agar slant culture: filamentous, growth is normal, surface is smooth and shiny. No pigment formation. Brown with a slight yellow tinge. Broth liquid culture: Normal growth, turbidity, no pigment production. Meat juice gelatin puncture culture: gelatin is not liquefied. Litmus milk: No change. (c) Physiological properties Nitrate reduction: negative. Denitrification reaction: negative. MR test: negative. VP test: negative. Indole production: negative. Hydrogen sulfide formation: negative. Starch hydrolysis: negative. Utilization of citric acid: Coser's medium; negative. Christensen's medium; positive. Use of inorganic nitrogen sources; use sodium nitrate and ammonium sulfate. Production of pigment: Pseudomonas F agar, Pseudomonas P agar (manufactured by Difco), no production of pigment observed. Urease: Positive. Oxidase: positive. Tacalase: positive. Growth range PH: Grows at 4.5 to 8.5. Temperature: Grows below 37℃. No growth is observed at 38℃. Attitude towards oxygen: aerobic. O-F test: Acid production aerobically. Presence or absence of acid and gas production from sugars. Based on the Hugh-Leifson method. D-glucose, D-mannose, D-fructose, D-galactose, lactose, and glycerin do not generate gas but do generate acid. Neither gas nor sugar is produced from L-arabinose, D-xylose, maltose, sucrose, trehalose, D-sorbitol, D-mannite, inosites, and starch. Accumulation of poly-β-hydroxybutyric acid: negative. Decomposition of brotocatechinic acid: ortho form. Gluconic acid oxidation: positive. 21 Arginine dihydrase dest: negative. 22 Lysine decarboxylation: negative. 23 Lipase production: Positive. 24 Utilization of carbonate compounds: According to the method of Stanier et al. It grows on glucose, L-arginine, L-alanine, D-alanine, inulin, and DL-β-hydroxybutyric acid, but not on glycine, itaconic acid, arabinose metaconate, maltose, sorbitose, and L-threonine. Based on the above mycological properties, Virgie's manual
Bergeys'g Manual of Systematic Bacteriology
As a result of a search based on Pseudomonas putida Biovar B (Bacteriology), it almost matched Pseudomonas putida biovar B. However, while the conventional Pseudomonas putida biovar B can assimilate glycine and issurin DL-β-hydroxybutyrate, this strain cannot assimilate it. Furthermore, while the conventional Pseudomonas butida biovar B is positive for arginine dihydrase est, this strain is negative. Furthermore, the bacteria of the genus Pseudomonas that produce known thermostable lipases, namely the aforementioned Pseudomonas mehuiteica varietus lipolyteica
Pseudomonas frangii, Pseudomonas
Compared to Fluorescens biotype I,
Differences are observed at least in the following mycological properties.

【table】

[Table] Based on the above findings, this strain was determined to be a new strain even though it has a very close taxonomic relationship with Pseudomonas putida biovar B, and was named Pseudomonas KWI-4 strain. This bacterial strain was deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry on June 16, 1989. The microorganism accession number is FERM P-10784. (Action and Effect) Thermostable lipase can be produced using this strain. The culture conditions are as follows. First, regarding the medium composition, this strain inducibly produces lipase only when fats and oils such as olive oil are present in the medium. Therefore, as a carbon source, we use fats and oils such as olive oil, or substances that this strain can assimilate, such as glycerin and various sugars.
It is sufficient to use one with an appropriate amount of fat added. Nitrogen sources include ammonium sulfate, meat extract,
Polypeptone, soybean flour, etc. can be used. Furthermore, as inorganic salts, potassium, sodium, phosphoric acid,
It is necessary to add various salts such as magnesium and calcium. Adjust the pH to 7.0 with the medium composition described above and store at 30°C.
If culture is carried out aerobically, the amount of lipase produced in the medium reaches its maximum within 1 to 2 days after the start of culture. After removing the bacterial cells from the obtained culture solution by centrifugation, the supernatant can be used as an enzyme solution. Furthermore, the supernatant may be used after partial purification. That is, cooled acetone was added to the supernatant at a final concentration of 80% (V/
V) and collect the precipitate by centrifugation or the like. Furthermore, this precipitate may be dissolved in an appropriate buffer and used as an enzyme solution. Next, some properties of the thermostable lipase produced by the strain will be described below. The lipase activity was measured using the Yamada-Machida method (Journal of the Japanese Society of Agricultural Chemistry, 36, 860~
864, 1962). That is, using 2% olive oil/polyvinyl alcohol emulsion as a substrate, 1 unit (hereinafter referred to as U) was the amount of enzyme that liberated 1 micromole of fatty acid per minute at 37°C. Action Hydrolyzes triglyceride as a substrate into fatty acids and glycerin. Optimum temperature for action. As shown in Figure 1, the optimum temperature for action is 80°C. Heat resistance Figure 2 shows enzyme solutions heat-treated at various temperatures for 12 hours. Figure 3 shows the enzyme solution heat-treated at a temperature of 60°C for various times. The residual activity is more than 58% after heat treatment at 60°C for 12 hours, and 36% after heat treatment at 60°C for 24 hours. Optimum PH for action As shown in Figure 4, the optimum PH for action is 8, but a peak can also be seen at PH4. Next, the present invention will be explained in detail with reference to Examples. (Example) Meat extract 1% (W/W), polypeptone 1%
A liquid medium consisting of (W/W), 0.5% (W/W) sodium chloride, and 1% (W/W) olive oil was adjusted to pH 7.0 using an aqueous sodium hydroxide solution.
50 ml was added to a 500 ml Sakaguchi flask and sterilized under pressure using an autoclave. This medium was inoculated with Pseudomonas strain KWI-4, and shake culture was performed for 50 hours at 30° C. and 150 revolutions per minute using a rotary shaker. The lipase activity of the culture solution after completion of the culture was 18.4 U/ml. moreover,
Bacterial cells were removed from this culture solution by centrifugation to obtain a supernatant. The lipase activity of the supernatant is
It was 8.1U/ml.

[Brief explanation of drawings]

FIG. 1 is a diagram showing the optimum temperature for action of lipase produced by Pseudomonas strain KWI-4. Activity measurement reactions were carried out for 20 minutes under each temperature condition.
FIG. 2 is a diagram showing the heat resistance of the lipase. The enzyme solution was heat-treated at each temperature for 12 hours, and the residual activity was determined. FIG. 3 is also a diagram showing the heat resistance of the same lipase. The enzyme solution was heat-treated at a temperature of 60°C for various hours, and the residual activity was determined. Figure 4 shows the optimal action of the same lipase.
It is a figure showing PH.

Claims (1)

[Claims] 1 Belongs to the genus Pseudomonas,
Pseudomonas with lipase production ability
KWI-4 strain.