JPH0550268B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a novel microorganism belonging to the genus Pseudomonas.

[Conventional technology]

Industrial hydrolysis of fats and oils has conventionally been carried out by high-temperature, high-pressure hydrolysis, but this method is a process under high-temperature, high-pressure conditions and requires high energy. On the other hand, the lipase hydrolysis method that uses enzymes is an effective energy-saving process that can carry out the reaction under mild conditions in the normal temperature and pressure range. In order to carry out industrial lipase hydrolysis of fats and oils, a lipase that has a high hydrolysis rate (90% or more) of oils and fats, particularly target high-melting point oils such as beef tallow and palm oil, and is heat resistant is required.

Until now, lipase produced by the yeast Candida rugosa has been known as a lipase for hydrolyzing fats and oils (Lipase OF manufactured by Meito Sangyo). This lipase has the ability to decompose over 90% of various oils and fats including olive oil at 30℃, but it is unstable and easily deactivated at temperatures above 45℃.
It is not suitable for hydrolyzing high melting point oils such as palm oil.

As heat-resistant lipases for hydrolyzing fats and oils, the lipase produced by Pseudomonas mephiteica (public notice 1982-25553) and the lipase produced by Pseudomonas fluorescens (public notice 57-52835) are known. Calcium chloride is necessary to obtain a high hydrolysis rate (coconut oil 92%) (Kosugi et al. J. Ferment. Technol. 49,
968 (1971)), the addition of calcium chloride has the disadvantage that metal soap is produced after decomposition, impairing the recovery of fatty acids. The latter has a decomposition rate of 94.9% when using lipase at 389 units/g-oil of beef tallow at 50℃.
It decomposes (public notice 1983-39638), but it takes 70 hours to reach this decomposition rate. Furthermore, when beef tallow is hydrolyzed with 140 units of lipase per gram, the decomposition rate reaches 62% after 24 hours, and it takes a long time of 120 hours to achieve a decomposition rate of 96.9%. It takes a long time to hydrolyze beef tallow using this enzyme, so it is not suitable for industrial use.

In addition, it is known that the lipase produced by Pseudomonas UKO-816 strain (published in 146277, 1982) has heat resistance, but this lipase is an ultra-high molecular weight with a molecular weight of over 20 million. However, it has the disadvantage that specific activity (lipase activity per unit weight) is low.

[Problems to be Solved by the Invention] In addition to beef tallow, palm oil, which has a higher melting point and a higher content of saturated fatty acids, is often used as a raw material for hydrolyzing industrial fats and oils. Industrial lipases are required to hydrolyze palm oil and beef tallow to a high degree (90% or more) in as short a time as possible, and to have high thermal stability.

[Means for solving problems]

Therefore, the present inventors conducted a wide search for microorganisms with lipase-producing ability that exhibit high hydrolysis rate and heat resistance suitable for hydrolyzing industrial oils and fats, and as a result, Pseudomonas was isolated from soil in Ichigai-cho, Haga-gun, Tochigi. The inventors discovered that some microorganisms belonging to the genus have such abilities, and completed the present invention.

That is, the present invention belongs to the genus Pseudomonas,
A novel Pseudomonas sp. KSM-16 (FER) with lipase production ability suitable for hydrolyzing industrial oils and fats.
8538).

Pseudomonas sp. KSM-16 has the following mycological properties.

(A) Form Cell shape and size Can-shaped Width 0.75-1.0μm Length 2.3-2.5μm Motile There is more than one hair.

Spores None Gram staining negative (B) Growth status in each culture medium Juicy agar plate culture Forms round, milky white colonies, surface Smooth, glossy, all edges, bulges Juicy agar slant culture Ordinary broth liquid culture 30℃, bacteria grow in 2 days Membrane formation, uniformly cloudy Meat juice gelatin puncture culture Not liquefied Litmus milk Coagulated and reduced litmus.

(C) Physiological properties Reduction of nitrate + denitrification reaction − MR test − VP test − Formation of indole − Formation of hydrogen sulfide − Hydrolysis of starch − Utilization of citric acid Koser + Christensen+ Utilization of inorganic nitrogen sources NH ₄ + NO ₃ + Pigments Production of KingA−KingB− Urease − Oxidase + Catalase + Growth range Growth 42℃+, 45℃−, 5℃− Growth temperature range 20−43℃, Optimum growth temperature range 30−37℃ Attitude toward oxygen Aerobic OF test O (oxidative) Presence of acid and gas generation from sugars Acid L-arabinose - D-xylose + D-glucose + D-mannose + D-fructose + D-galactose + Maltose + Sucrose + Lactose + Trehalose + D-Sorbit + D-Mannite + Inosyte + Glycerin + Starch - Salicin - Raffinose - No gas was generated from the above sugars.

(D) Carbohydrate utilization D-glucose + D-xylose + D-ribose + L-rhamnose - levulinic acid - D-tartaric acid - meso-tartaric acid + meso-erythritol - adonitol - mesaconic acid - citraconic acid - L-valine + m -Hydroxybenzoic acid -2,3-butanediol + betaine + arginine + Based on the above mycological properties, Bergey's manual of determinative bacteriology 8th edition (Bergey's manual of determinative
bacteriology 8th.ed.), Pseudomonas cepacia was listed as a strain similar to this bacterium. Therefore, we cultured Pseudomonas cepacia (ATCC10586) under the same conditions as this bacterium and compared its properties.
It was found that they differ in each property as shown below.

Test items Pseudomonas sp. cepacia KSM-16 ATCC10586 Litmus milk Coagulation Liquefaction Carbohydrate assimilation Adonitol − + m-hydroxybenzoic acid − +
Pigment production - + (yellow brown) Based on the differences in these properties, this lipase-producing bacterium was judged to be a new strain different from any known bacteria, and was named Pseudomonas sp. KSM-16.
On December 2, 1960, it was deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry. Its microorganism accession number is 8538.

To produce lipase using the KSM-16 strain of the present invention, the KSM-16 strain must be grown in a medium containing the carbon source, nitrogen source, inorganic salts, etc. necessary for the successful production of lipase. This is carried out by culturing it and collecting it from the culture solution.

The composition of the medium is not particularly limited as long as it is normally used for culturing microorganisms belonging to the genus Pseudomonas. For example, various types of inorganic nitrogen, various types of organic nitrogen, and combinations thereof can be used as the nitrogen source. In particular, peptone, meat extract, urea, etc. are effective as single systems. Carbon sources include sugars such as glucose, various fats and oils such as olive oil,
Fatty acids, fatty acid esters, fatty acid derivatives, etc. can be used. As inorganic salts, it is effective to add ammonium sulfate, monopotassium phosphate, dipotassium phosphate, sodium nitrate, magnesium sulfate, and the like. In addition, various organic substances and inorganic substances necessary for growth of bacteria and production of enzymes can be added as necessary.

Liquid culture is suitable for culturing. As for the culture temperature, as mentioned above, it can be cultured at 20-42°C, preferably 30-37°C. Furthermore, by culturing aerobically, lipase productivity reaches its maximum in about 1 to 2 days. To obtain lipase from the culture solution obtained in this way, the bacterial cells are separated by centrifugation and filtration, and then the supernatant liquid is treated with an organic solvent (acetone,
precipitation with ethanol, etc.), salting out method with ammonium sulfate,
Or ultrafiltration membrane (manufactured by Amicon, molecular weight cutoff)
30,000) can be concentrated and recovered.

To purify the obtained lipase, the concentrated culture solution is subjected to ammonium sulfate precipitation, and the enzyme solution obtained by ethanol precipitation is subjected to commonly known protein purification methods such as gel filtration using an ion exchanger such as DEAE-cellulose or Cephadex. law is available. Enzyme solutions with increased specific activity by these purification methods can be freeze-dried to obtain enzyme powder.

The physicochemical properties of the lipase obtained using the KSM-16 strain of the present invention are as follows.

1 Action: Decomposes over 90% of palm oil, olive oil, beef tallow, and coconut oil in 24 hours at a reaction temperature of 60°C. In particular, palm oil is decomposed by more than 92%.

2 Substrate specificity Decomposes various glycerides, esters, etc.

In particular, it selectively decomposes saturated fats and oils.

Type of fat and oil Degradation rate (50℃, 24 hours, 1000unit/g-oil) Palm oil 91.7% Olive oil 82.1% Coconut oil 73.1% Beef tallow 89.1% 3 Stable PH range PH3-12, (30℃, after 24 hours) Residual activity, Figure 1) Optimal pH range for action: PH6-7.5 (Figure 2) 4. Range of optimal temperature for action: 65-80℃ (Figure 3) Optimal temperature: 70℃ 5. Potency measurement method: Divide 9ml of olive oil into 150ml cups. Note.
Next, take 14ml of 0.1M phosphate buffer (PH7.0) and
Warm to ℃. While stirring the reaction solution uniformly with a magnetic stirrer (800 rpm), add the enzyme solution to start the reaction, and after reacting for a certain period of time (30 minutes), add 20 ml of acetone and ethanol (1:1) mixture. stop the reaction. Tween 80 in this liquid
Add 1 ml of 10% (Tween 80) solution, and titrate the fatty acid produced by the reaction with a 0.5N potassium hydroxide (alcoholic) solution using an automatic neutralization titration device (Mettler DL40RC) using a PH electrode. For the enzyme titer, one unit was defined as the amount of enzyme that liberated 1 micromole of fatty acid per minute.

6 Conditions for deactivation due to PH, temperature, etc. Heat treatment at 30℃ and 50℃ for 3 hours causes almost no inactivation, but heat treatment at 70℃ for 2 hours causes about 40% deactivation.
It becomes inactive (Figure 4). 1mM calcium chloride in enzyme solution
By adding it, heat inactivation at 70°C for 2 hours is reduced to 30%, increasing thermal stability. Figure 5 shows the residual activity rate after heat treatment for 10 minutes at each temperature of 10-80°C.

7 Inhibition, activation and stabilization No effect of bile acids Effect of inorganic salts Under conditions where each metal salt coexists at a concentration of 1mM in the reaction system
Fermentation reaction was carried out at 50°C for 30 minutes, and each activity was expressed with the activity when no salt was added as 100.

No metal salts ……100 CuSO ₄ …… 32 MgSO ₄ …… 88 MgCl ₂ …… 83 KCl …… 96 NaCl …… 95 CaCl ₂ ……100 CoCl _{2 …… 71 HgCl 2 ……} 75 ZnCl ₂ …… 10 FeCl ₃ ...10 8 Molecular weight In SDS-polyacrylamide gel electrophoresis
29,000 According to the gel filtration (Sephadex G-75) method, it is 30,000.

9 Sugar content Sugar color development by orcinol reaction + [Action and effects of the invention] Pseudomonas sp. KSM-16 of the present invention
produces a lipase with a high hydrolysis rate and heat resistance suitable for hydrolyzing industrial oils and fats.

〔Example〕

Next, the present invention will be explained by giving reference examples and reference examples.

Example 1 (1) Soil around Ichikai-cho, Haga-gun, Tochigi was sampled, one teaspoon of the soil was placed in a test tube containing 10 ml of sterile physiological saline, and suspended and dispersed using a vortex mixer for 1 minute. After standing still for about 1 hour, the supernatant liquid was
0.2 ml was collected and applied uniformly on the surface of a separation agar medium prepared by the method described below using a Conrad stick, and then cultured at 30°C.

(Preparation of agar medium for isolation) Medium composition Tributyrin...0.1% (NH ₄ ) ₂ SO ₄ ...0.5% Soyton...0.5% Yeast extract...0.1% KH ₂ PO ₄ ...0.5% MgSO ₄・7H ₂ O...0.1% Agar...2.0% PH4.5 (prepared with HCl) Preparation method Among the above compositions, tributyrin and agar are sterilized separately, and after sterilization, they are combined aseptically and tributyrin is dispersed in a shearer. After this, a certain amount of the mixture was dispensed into a sterilized petri dish while being kept warm to form an opaque flat plate agar medium.

(2) Next, among the colonies grown by the above culture, one platinum loop of a colony that decomposes tributyrin and forms a transparent membrane around it is placed in sterile physiological saline.
Diluted 100 times, streaked 1 platinum loop of this diluted solution on an agar medium with the same composition as the above-mentioned separation agar medium, and
The cells were cultured at ℃ for 3 days. It was confirmed by macroscopic and microscopic observation that the multiple colonies produced were not different from each other. Furthermore, the above colony was subjected to the same operation again to reconfirm its unity. The properties and physiological properties of the above-mentioned strains on each medium are as described above.

(3) Next, one platinum loopful of the pure cultured bacterial strain on the slant medium was suspended in a freezing vial containing a sterilized 10% glycerin aqueous solution (2 ml) and stored frozen at -80°C. After 3 months of frozen storage, one platinum loop of the suspension obtained by rapid thawing was resuscitated on an ordinary agar medium, and the properties and physiological qualities on each medium were examined under the same conditions as above. No change was observed from before freezing.

Reference example 1 Peptone (manufactured by Difco) 4%, glucose 0.5%,
100 ml of a liquid medium consisting of 0.1% H ₂ HPO ₄ , 0.05% MgSO ₄ 7H ₂ O, and 0.5% oleic acid was placed in a 500 ml Sakaguchi flask and sterilized by autoclaving at 121°C for 15 minutes.
1 ml of a preculture of Pseudomonas sp. KSM-16, which had been previously cultured with shaking at 30°C for 24 hours in the same medium, was inoculated and cultured with shaking at 30°C for 48 hours. The activity of the supernatant obtained by centrifuging the culture solution and removing the bacterial cells is
It was 20 units/ml.

Reference example 2 Urea 0.4%, glucose 0.5%, K ₂ HPO ₄ 0.1%,
100 ml of a liquid medium consisting of 0.05% MgSO ₄ 7H ₂ O, 0.1% yeast extract, and 0.5% oleic acid was placed in a 500 ml Sakaguchi flask and sterilized by autoclaving at 121°C for 15 minutes.
1 ml of a preculture of Pseudomonas sp. KSM-16, which had been previously cultured in the same medium at 30°C for 24 hours with shaking, was inoculated and cultured with shaking at 30°C for 3 days. The lipase activity of the culture supernatant was 140 units/ml.

Reference example 3 Peptone 4%, glucose 0.5%, KH ₂ PO ₄ 0.1
%, MgSO ₄ 7H ₂ O 0.05%, and oleic acid 0.5% were placed in a 5-volume jar fermentor and sterilized by autoclaving at 121°C for 15 minutes. 100 ml of culture solution was added to the cultured Pseudomonas sp. KSM-16 strain and cultured at 30° C. and 300 rpm for 48 hours. After culturing,
The activity of culture supernatant 1.5, in which the bacterial cells were removed by centrifugation, was 30 units/ml. The culture supernatant was concentrated to 300 ml by ultrafiltration (Amicon Diaflow membrane, molecular weight cutoff 10,000), and then freeze-dried to obtain 10 g of crude enzyme powder. 3000 units/enzyme powder
The activity of g was observed.

Reference Example 4 Using the crude enzyme powder obtained in Reference Example 3, palm oil,
He decomposed coconut oil, beef tallow, and olive oil.
Hydrolysis was carried out by shaking 1 g of oil in an Erlenmeyer flask with 1000 units of enzyme and oil to water (1:5). The decomposition rate of each oil after 48 hours was 92% for palm oil and 92% for coconut oil.
90.9%, beef tallow 90.1%, and olive oil 86.3%.

Reference Example 5 Palm oil was hydrolyzed using the crude enzyme powder obtained in Reference Example 3. 160 units of enzyme per 1 g of oil was added to an Erlenmeyer flask at a ratio of oil to water (1:1) for hydrolysis by shaking. The decomposition rate of palm oil after 24 hours is
It was 92%.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the residual activity of lipase obtained using the KSM-16 strain of the present invention after treatment at 30°C for 24 hours in the pH range of 3 to 13. Figure 2 shows the pH of lipase obtained using the KSM-16 strain of the present invention.
It is a figure which shows the specific activity in the range of 3-8.
FIG. 3 is a drawing showing the specific activity of lipase obtained using the KSM-16 strain of the present invention under various temperature conditions. FIG. 4 is a drawing showing the relationship between treatment time and residual activity of lipase obtained using the KSM-16 strain of the present invention under various temperature conditions. Figure 5 shows
FIG. 2 is a diagram showing the residual activity of lipase obtained using the KSM-16 strain of the present invention when treated for 10 minutes under various temperature conditions.

Claims (1)

[Claims] 1. Pseudomonas having the following mycological properties:
Pseudomonas sp. KSM-16 (Microtechnical Research Institute No. 8538). (A) Morphology Cell shape and size: Canal width: 0.75-1.0 μm Length: 2.3-2.5 μm Motile: one or more hairs. Spores None Gram staining negative (B) Growth status in each medium Broth agar plate culture Forms round, milky-white colonies, surface Smooth, glossy, all edges, ridges Broth agar slant culture Normal Broth culture in broth liquid culture 30℃, 2 days Membrane formation, uniformly cloudy Meat juice gelatin puncture culture Not liquefied Litmus milk Coagulated and reduced litmus. (C) Physiological properties Reduction of nitrate + denitrification reaction − MR test − VP test − Formation of indole − Formation of hydrogen sulfide − Hydrolysis of starch − Utilization of citric acid Koser + Christensen+ Utilization of inorganic nitrogen sources NH ₄ + NO ₃ + Pigments Production of KingA−KingB− Urease − Oxidase + Catalase + Growth range Growth 42℃+, 45℃−, 5℃− Growth temperature range 20−43℃, Optimum growth temperature range 30−37℃ Attitude toward oxygen Aerobic OF test O (oxidative) Presence of acid and gas generation from sugars Acid L-arabinose - D-xylose + D-glucose + D-mannose + D-fructose + D-galactose + Maltose + Sucrose + Lactose + Trehalose + D-Sorbit + D-Mannite + Inosyte + Glycerin + Starch - Salicin - Raffinose - No gas was generated from the above sugars. (D) Carbohydrate utilization D-glucose + D-xylose + D-ribose + L-rhamnose - levulinic acid - D-tartaric acid - meso-tartaric acid + meso-erythritol - adonitol - mesaconic acid - citraconic acid - L-valine + m -Hydroxybenzoic acid - 2,3-butanediol + betaine + arginine +