JPH088859B2 - Shade Monas KWI-56 strain - Google Patents

Description

TECHNICAL FIELD The present invention relates to a bacterium of the genus Pseudomonas that produces thermostable lipase.

(Prior Art) Lipase is an enzyme that hydrolyzes fatty acid and glycerin using triglyceride as a substrate. Further, a reaction of synthesizing an ester from a fatty acid and an alcohol when the water content in the reaction water is reduced is also known.

At present, industrial use of lipase has been actively tried, and introduction of lipase into fatty acid production processes is particularly 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. However, by introducing an enzymatic reaction, the reaction can proceed at room temperature and atmospheric pressure, so unsaturated fatty acids that were often decomposed by the chemical hydrolysis method can be obtained without decomposition. It can save a lot of energy consumption. In addition, by utilizing the substrate specificity of the enzymatic reaction and using the transesterification reaction,
It is also possible to improve rare oils and fats by using inexpensive oils and fats as raw materials.

However, beef tallow and lard, which are the main raw materials for fatty acid production,
It is mainly composed of long-chain saturated fatty acids and is solid at room temperature. For this purpose, the fats and oils must be reacted at a temperature above the melting point or liquefied by adding an organic solvent and then reacted. Therefore, the lipase used for fatty acid production must have heat resistance and have optimal reactivity at high temperature, or must have resistance to organic solvents and be able to proceed the reaction in the presence thereof. I won't. Although the search for such lipase has been actively conducted, no enzyme suitable for the demand on an industrial level has been obtained until now.

In terms of thermostable lipase, it has been reported that Pseudomonas bacteria can produce these lipases.

That is, Pseudomonas mephitica Var.lipoly
tica) produced a lipase that does not inactivate even when heat-treated at optimal temperatures of 70 ℃ and 60 ℃ for 14 hours (Japanese Patent Publication No.
25553), Pseudomonas fra
gi) produces lipase (Agric.Biol.Chem. 41: 1353-1358, 1977) that retains 95% or more of activity even after heat treatment at optimum temperature of 75-80 ° C and 70 ° C for 20 minutes.
Year), Pseudomonas. Report on lipase produced by Fluorescens biotype I (Pseudomonas fluorescens) that retains 86.9% activity even after heat treatment at optimal temperature of 67 ° C and 60 ° C for 20 hours (JP-A-57-58885) It has been known.

(Problems to be Solved by the Invention) However, since lipases produced by the above-mentioned bacteria are not sufficient as the number of types of lipases, many other bacteria producing various thermostable lipases are required.

(Means for Solving Problems) As a result of broadly searching for a microorganism that produces a thermostable lipase from the natural world, the present inventors have found that KWI-56 strain belonging to the genus Pseudomonas isolated from the soil of Atsugi City, Kanagawa Prefecture. However, they have found that it produces a lipase that has extremely high thermostability and an optimum reaction temperature of 70-80 ℃.

The mycological properties of the strain of the present invention are shown below. For the examination of this mycological property, "Classification and Identification of Microorganisms" (Takeharu Hasegawa, Academic Publishing Center), "Medical Bacteria Identification Handbook"
(ST Cowan, translated by Riichi Sakazaki, Modern Publishing), the method described in "New Bacterial Medium Science Course" (Riichi Sakazaki Modern Publishing),
The medium composition was used.

a) Morphology Cell shape and size: length 2.2-3.0 microns, width 0.5
~ 0.7 micron bacillus cell dimorphism: single or short chain motility: yes, with one polar flagella spore: none gram stain: negative anti-acidic: none b) growth condition broth agar plate culture: round, and Circular, smooth and glossy surface. White with a slight yellow tinge.

Broth agar slant culture: filamentous, normal growth, smooth and shiny surface. No pigment formation. White with a slight yellow tinge.

Broth liquid culture: normal growth, turbidity, no pigment formation.

Meat broth gelatin stab culture: normal growth, liquefaction.

Litmus milk: slightly alkaline, liquefied.

c) Physiological properties Nitrate reduction: positive.

Denitrification reaction: Negative.

MR test: negative.

VP test: negative.

Indole formation: negative.

Hydrogen sulfide formation: Slightly positive.

Starch hydrolysis: negative.

Utilization of citric acid: Coser's medium; positive.

Christensen's medium; positive.

Use of inorganic nitrogen source: Do not use sodium nitrate but use ammonium sulfate.

Pigment formation: No pigment formation was observed in Pseudomonas F agar, Pseudomonas P agar (manufactured by Difco), Crigler's medium (without indicator), and TSI agar medium (without indicator).

Urease: Positive.

Oxidase: positive.

Catalase: positive.

Growth range Grows at pH: 4.5-8.5. Optimal for 5.5 to 7.0.

Temperature: Grows at 15-37 ℃. No growth is observed at 10 ° C and 40 ° C. Optimally around 33 ° C.

Attitude toward oxygen: Aerobic OF test: Acid is aerobically produced.

Presence or absence of acid and gas generation from sugars By Hugh-Leifson method.

No gas is generated from L-arabinose, D-xylose, D-glycose, D-mannose, D-fructose, D-galactose, maltose sucrose, lactose, trehalose, D-sorbit, D-mannitol, inosit and glycerin. It produces an acid. Neither gas nor sugar is produced from starch.

Accumulation of poly-β-hydroxybutyric acid: positive.

Degradation of protocatechuic acid: ortho type.

Glycolic acid oxidation: positive.

Arginine decarboxylation: negative.

Lysine decarboxylation: Positive.

Lipase production: positive.

Utilization of carbon compounds: By the method of Stanier et al.

Glycose, galactose, lactose, arabinose, maltose, sorbitol L-sulonine, L-arginine, L-alanine, D-alanine, acetamide, DL-
It grows on β-hydroxybutyric acid, but not on glycine, inulin, itaconic acid, and metaconic acid.

Based on the above-mentioned mycological properties, Burj's manual lobe.
Definite. Bacteriology 8th Edition (Berge
y's Manual of Determinative Bacteriolgy 8ed) and Bargey's manual. of. Systematic.
Bacteriology (Bergey's Manual of Systematic Ba
cteriology), Pseudomonas.
It almost coincided with Sepasia. However, while conventional Pseudomonas cepacia can grow at 40 ° C, this strain can grow only at a temperature of 37 ° C or lower. Also,
Conventional Pseudomonas. Although cepacia produces a non-fluorescent pigment, no pigment production can be seen in this strain.

Further, known Pseudomonas bacteria producing thermostable lipase, that is, Pseudomonas. Mephitica. Varietas. Lipolitica, Pseudomonas. Frazzy, Pseudomonas fluorescens. Even when compared with biotype I, there are at least the following differences in mycological properties.

From the above findings, this strain is Pseudomonas. Although it was judged to be a new strain despite having a taxonomical relationship very close to that of Sepacia, it was named Pseudomonas strain KWI-56. This strain was deposited on October 15, 1987 at the Institute for Microbial Engineering, Ministry of International Trade and Industry, Institute of Industrial Technology. The microorganism accession number is Mikori Kenjoyori No. 3178 (FERM BP-3178).

(Action and effect) This strain can be used to produce a thermostable lipase. The culture conditions are as follows.

First, regarding the medium composition, this strain inducibly produces lipase only when oils and fats such as olive oil are present in the medium. Therefore, fats and oils such as olive oil may be used as the carbon source, or substances such as glycerin and various sugars that can be assimilated by the strain of the present invention may be used with an appropriate amount of fats and oils added. Ammonium sulfate, meat extract, polypeptone, soybean flour, etc. can be used as the nitrogen source. Further, it is necessary to add each salt such as potassium, sodium, phosphoric acid, magnesium and calcium as an inorganic salt.

If the pH is adjusted to 7.0 with the medium composition described above and the culture is performed aerobically at 30 ° C., the lipase production amount in the medium becomes maximum within 1 to 2 days after the start of the culture. After the cells are removed from the resulting culture solution by centrifugation, the supernatant can be used as an enzyme solution. Also, the supernatant may be used after partial purification. That is, the final concentration of cooled acetone was 80% (V /
V) and collect the precipitate by centrifugation. Further, 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. In addition, the measurement of the lipase activity was carried out by Yamada Kazu Machida method (Journal of the Japanese Society of Agricultural Chemistry, Vol. 36, pages 860-864, 1962). That is, using 2% olive oil and polyvinyl alcohol emulsion as a substrate, the amount of enzyme that liberates 1 micromol of fatty acid in 1 minute at 37 ° C. was 1 unit (hereinafter referred to as U).

Action Triglycerides are used as substrates to hydrolyze fatty acids and glycerin.

Optimum temperature of action As shown in FIG. 1, the optimum temperature of action is 70 ° C to 80 ° C.

Heat resistance Figure 2 shows the enzyme solution heat-treated for 12 hours at each temperature. Figure 3 shows the enzyme solution heat-treated at a temperature of 60 ° C for each hour. The residual activity is 85% or more after heat treatment at 60 ° C for 24 hours, and 28% after heat treatment at 74 ° C for 12 hours.

 Next, the present invention will be described in detail with reference to examples.

(Example) A liquid medium consisting of 1% meat extract (W / W), 1% polypeptone (W / W), 0.5% sodium chloride (W / W) and 1% olive oil (W / W) was added to an aqueous sodium hydroxide solution. Using pH 7.
Adjust to 0, add 50 ml to a 500 ml Sakaguchi flask,
It was autoclaved and autoclaved. The Pseudomonas KWI-56 strain was inoculated into this medium, and shake culture was carried out for 50 hours under the conditions of 30 ° C. and 150 revolutions per minute using a rotary shoe. The lipase activity of the culture solution after the culture is
It was 34.8 U / ml. Further, cells were removed from this culture solution by centrifugation to obtain a supernatant. The supernatant had a lipase activity of 30.8 U / ml.

[Brief description of drawings]

FIG. 1 is a graph showing the optimal action excess of lipase produced by Pseudomonas KWI-56 strain. The activity measurement reaction was performed for 20 minutes at each temperature condition. FIG. 2 is a diagram showing heat resistance of the lipase. Heat the enzyme solution at each temperature for 12 hours,
The residual activity was determined. FIG. 3 also shows the heat resistance of the lipase. The enzyme solution was heat-treated at a temperature of 60 ° C. for each hour to determine the residual activity.

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Claims (1)

[Claims] 1. Pseudomonas KWI-5 belonging to the genus Pseudomonas and having the following mycological properties.
6 strains. a) Morphology Cell shape and size: length 2.2-3.0 microns, width 0.
5 to 0.7 micron bacillus Cell dimorphism: Single or short chain Motility: Yes, with one polar flagellum Spore: None Gram stain: Negative anti-acidity: None b) Growth condition Meat agar plate culture: circular, Totsu-circular, smooth and glossy surface. White with a slight yellow tinge. Broth agar slant culture: filamentous, normal growth, smooth and shiny surface. No pigment formation. White with a slight yellow tinge. Broth liquid culture: normal growth. Turbidity, no pigment formation. Meat broth gelatin stab culture: normal growth, liquefaction. Litmus milk: slightly alkaline, liquefied. c) Physiological properties Nitrate reduction: positive. Denitrification reaction: Negative. MR test: negative. VP test: negative. Indole formation: negative. Hydrogen sulfide formation: Slightly positive. Starch hydrolysis: negative. Utilization of citric acid: Coser's medium; positive. Christensen's medium; positive. Use of inorganic nitrogen source: Do not use sodium nitrate but use ammonium sulfate. Pigment formation: No pigment formation was observed in Pseudomonas F agar, Pseudomonas P agar (manufactured by Difco), Crigler's medium (without indicator), and TSI agar medium (without indicator). Urease: Positive. Oxidase: positive. Catalase: positive. Growth range Grows at pH: 4.5-8.5. Optimal for 5.5 to 7.0. Temperature: Grows at 15-37 ℃. No growth is observed at 10 ° C and 40 ° C. Optimally around 33 ° C. Attitude toward oxygen: aerobic. OF test: Acid is generated aerobically. Presence or absence of acid and gas generation from sugars By Hugh-Leifson method. L-arabinose, D-xylose, D-glycose,
D-mannose, D-fructose, D-galactose, maltose, sucrose, lactose, trehalose, D-sorbit, D-mannitol, inosit. No gas is generated from glycerin, but acid is generated. Neither gas nor sugar is produced from starch. Accumulation of poly-β-hydroxybutyric acid: positive. Degradation of protocatechuic acid: ortho type. Glycolic acid oxidation: positive. Arginine decarboxylation: negative. Lysine decarboxylation: Positive. Lipase production: positive. Utilization of carbon compounds: By the method of Stanier et al. Glycose, galactose, lactose, arabinose, maltose, sorbitol, L-threonine, L-
It grows on arginine, L-alanine, D-alanine, acetamide, DL-β-hydroxybutyric acid, but glycine,
Does not grow on inulin, itaconic acid, or metaconic acid.