JPH0755147B2 - New lipase - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a novel lipase.

[Conventional technology]

Conventionally, hydrolysis of industrial fats and oils has been performed by a high temperature and high pressure hydrolysis method, but this method is a process under high temperature and high pressure conditions and requires high energy. On the other hand, the lipase hydrolysis method using an enzyme is an effective energy-saving process that can carry out the reaction under mild conditions at room temperature and atmospheric pressure. In order to industrially perform lipase fat and oil hydrolysis, a lipase having a high hydrolysis rate (90% or more) and heat resistance of fats and oils, especially high melting point fats and oils such as beef tallow and palm oil, is required.

Hitherto, a lipase produced by the yeast Candida rugosa has been known as a lipase for fat and oil hydrolysis (Meito industrial lipase OF). This lipase has the ability to decompose 90% or more of various oils and fats including olive oil at 30 ° C, but it is unstable and easily deactivated at 45 ° C and above, and is not suitable for hydrolysis of high melting point oils such as palm oil. Not suitable.

As a lipase for hydrolyzing fats and oils that has heat resistance, a lipase produced by C. monteus Mefitica (public announcement)
50-25553) and lipase produced by C. fluorescens (publication number 57-52835) are known, but the former requires calcium chloride to obtain a high hydrolysis rate (coconut oil 92%) ( Kosugi et al. J. Ferment. Techno
L.49,968 (1971)), the addition of calcium chloride has a drawback that metal soap is generated after decomposition and the recovery of fatty acids is deteriorated. In the latter case, beef tallow at 50 ℃
It decomposes 94.9% with 389 unit / g-oil lipase (public announcement)
57-39638), 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 120 hours to obtain a decomposition rate of 96.9%.
It takes a long time to hydrolyze beef tallow using this enzyme, which is not suitable for industrial use.

In addition, it is known that the lipase produced by C. sudomonas strain UKO-816 (publication number 58-146277) has thermostability, but this lipase is an ultra-high molecular weight polymer with a molecular weight of 20 million or more. However, it has a drawback that the specific activity (lipase activity per unit weight) is low.

[Problems to be solved by the invention]

Not only beef tallow but also palm oil having a higher melting point and a higher saturated fatty acid content tends to be used as a raw material for the hydrolysis of industrial fats and oils. Industrial lipases are required to perform high hydrolysis (90% or more) of palm oil and beef tallow in as short a time as possible and have high thermal stability.

[Means for solving problems]

Then, the inventors of the present invention have conducted extensive studies for the purpose of providing a lipase having a high hydrolysis rate and heat resistance suitable for the hydrolysis of industrial fats and oils, and as a result, the microorganisms isolated from the soil in Kai Town, Haga-gun, Tochigi Prefecture The present invention has been completed by finding that a lipase that fulfills the above purpose is present in the product.

That is, the present invention provides a novel lipase having the physicochemical properties described below.

In the present invention, the following method can be adopted for producing lipase. That is, it is a method of culturing a bacterium belonging to the genus Cichudomonas and having the above-mentioned lipase-producing ability, and collecting the above-mentioned lipase from the culture. The bacterium used in the present invention is a bacterium belonging to the genus Cydomonas capable of producing the lipase of the present invention, and any bacterium can be used. As a specific example of the bacterium, a new strain C. sudomonas KSM-16 strain isolated from the soil of Haga-gun, Tochigi prefecture by the present inventors can be mentioned.

The mycological properties of this strain are shown below.

(A) Morphology Cell shape and size Rod-shaped Width 0.75-1.0 μm Length 2.3-2.5 μm Motility There is one or more pen hairs.

No spores Gram stain Negative (B) Growth condition in each medium Meat juice agar plate culture Round, milky white colony formation, surface, smooth, glossy, full edge, bulge Meat agar slope culture Normal meat juice liquid culture at 30 ° C for 2 days Pellicle formation, uniform turbidity Juice gelatin stab culture No liquefaction, litmus milk coagulation and litmus reduction.

(C) Physiological properties Reduction of nitrate + denitrification - MR test - VP Test - generation of indole - generation of hydrogen sulfide - Hydrolysis of starch - utilizing NH ₄ + NO ₃ + dye use of citric acid Koser + Christensen + inorganic nitrogen sources Generation of KingA-KingB-urease-oxidase + catalase + Growth range Growth 42 ℃ +, 45 ℃-, 5 ℃-Growth temperature range 20-43 ℃, Growth optimum temperature range 30-37 ℃ Attitude toward oxygen Aerobic OF test 0 (Oxidative) Presence or absence of acid and gas production from sugars Acid L-arabinose-D-xylose + D-glucose + D-mannose + D-fructose + D-galactose + maltose + sucrose + lactose + trehalose + D-sorbit + D-mannitol + inosit + glycerin + starch-salicin-rafuinose-or less No gas was produced from the above sugars.

(D) Utilization of carbohydrate D-glucose + D-xylose + D-ribose + L-rhamnose-levulinic acid-D-tartaric acid-meso-tartaric acid + meso-erythritol-adnitol-mesaconic acid-citraconic acid-L-valine + m -Hydroxybenzoic acid-2,3-butanediol + betaine + arginine + Based on the above mycological properties, Verger's manual. of.
Datenative. Bacteriology 8th Edition (Berge
y ′s manual of determinative bacterio−logy 8th.e
As a result of searching by d.), Pseudomonas cepacia can be mentioned as a strain similar to this bacterium. So, there are Syudomonas sebacia (ATCC10
586) was cultivated under the same conditions as this bacterium and compared for each property, it was found that each property was different as shown below.

Based on these differences in various properties, this lipase-producing bacterium was judged to be a new strain that is different from any known bacterium, and was named C. sudomonas sp. KSM-16. Deposited. The microorganism deposit number is Microorganism Research Institute No. 8538.

In order to produce the lipase of the present invention using the above KSM-16 strain, the KSM-16 strain grows satisfactorily, and the carbon source, nitrogen source, inorganic salt and the like necessary for producing the lipase of the present invention smoothly are provided. It is carried out by culturing this in a medium containing it and collecting it from the culture solution.

The composition of the medium is not particularly limited as long as it is one that is usually used for culturing microorganisms belonging to the genus C. pseudomonas. For example, various inorganic nitrogens, various organic nitrogens, and combinations thereof can be used as the nitrogen source. In particular, peptone, meat extract, urea, etc. are effective as a single system. As the carbon source, sugars such as glucose, various oils and fats such as olive oil, fatty acids, fatty acid esters, and fatty acid derivatives can be used. Ammonium sulfate, phosphoric acid 1 as inorganic salts
It is effective to add potassium, dipotassium phosphate, sodium nitrate, magnesium sulfate and the like. In addition, if necessary, various organic and inorganic substances necessary for growth of bacteria and production of enzymes can be added.

Liquid culture is suitable for culture. The culturing temperature can be 20 to 42 ° C as mentioned above, and 30 to 37 ° C is particularly preferable. Also, by culturing aerobically, about 1-2
The production of lipase reaches the highest in a day. To obtain lipase from the thus obtained culture solution, cells are separated by centrifugation and filtration, and then the supernatant is precipitated with an organic solvent (acetone, ethanol, etc.), salting out with ammonium sulfate, or Ultrafiltration membrane (Amicon cut-off molecular weight 30000)
Thus, it can be concentrated and recovered.

To purify the recovered lipase of the present invention, the concentrated solution of the culture solution was ammonium sulfate precipitated, and the enzyme solution obtained by ethanol precipitation was treated with DE.
A generally known protein production method such as an ion exchanger such as AE-cellulose or a gel filtration method using Sephadex can be used. The enzyme solution having an increased specific activity obtained by these purification methods can be freeze-dried to obtain an enzyme powder.

The lipase of the present invention thus obtained has the following physicochemical properties.

1. Action Palm oil, olive oil, beef tallow, coconut oil, reaction temperature 60 ℃,
Decomposes by 90% or more in 24 hours. Especially palm oil decomposes by 92% or more.

2. Substrate specificity Decomposes various glycerides and esters.

Especially, it selectively decomposes saturated fats and oils.

Type of fats and oils Decomposition rate (50 ℃, 24 hours, 1000unit / g-oil) Palm oil 91.7% Olive oil 82.1% Palm oil 73.1% Beef tallow 89.1% 3. Stable pH range pH 3-12, (remaining after 30 ℃, 24hr) Activity, Fig. 1) Optimum pH range of action pH 6-7.5 (Fig. 2) 4. Optimum temperature range of action 65-80 ℃ (Fig. 3) Optimum temperature 70 ℃ 5. Molecular weight SDS-polyacrylamide gel electrophoresis 29, 000 according to gel filtration (Sephadex G-75) method.

6. Sugar content Color development by orcinol reaction + 7. Deactivation condition by pH, temperature etc. 30 ℃ and 50 ℃ Almost no deactivation after 3 hours heat treatment, but about 40% after 70 ℃ 2 hours heat treatment Deactivate (Fig. 4). 70 by adding 1 mM calcium chloride to the enzyme solution
The heat inactivation at 2 ° C for 2 hours is reduced to 30%, and the heat stability is increased. FIG. 5 shows the residual activity ratio after heat treatment at each temperature of 10-80 ° C. for 10 minutes.

8. Inhibition, activation and stabilization No influence of bile acid Influence of inorganic salts The enzyme reaction is performed at 50 ° C for 30 minutes under the condition that each metal salt coexists in the reaction system at a concentration of 1 mM. Each activity was expressed as 100.

No metal salt ………… 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 9. Titration method Dispense 9 ml of olive oil into a 150 ml cup. Next 0.1M
Take 14 ml of phosphate buffer (pH 7.0) and heat to 50 ° C. Stir the reaction mixture uniformly with a magnetic stirrer (800rp
The reaction is started by adding the enzyme solution while
(0 min) After reacting, add acetone and ethanol (1: 1) mixture
Add 20 ml to stop the reaction. Tween 80 (Twee
n80), 1 ml of 10% solution was added, and the fatty acid produced by the reaction was adjusted to pH.
Automatic neutralization titrator using electrodes (Mettler DL40RC)
Titrate with 0.5 N potassium hydroxide (alcoholic) solution. The enzyme titer was defined as 1 unit of the amount of enzyme that liberates 1 micromol of fatty acid per minute.

[Operation and effect of the invention]

INDUSTRIAL APPLICABILITY The lipase of the present invention has an excellent property that it shows extremely high hydrolyzing ability for industrial fats and oils in a short time and has high thermal stability.

Therefore, if the lipase of the present invention is used, industrial fats and oils can be hydrolyzed industrially under mild conditions in the normal temperature and normal pressure range.

〔Example〕

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

Example 1 Peptone (manufactured by Difco) 4%, glucose 0.5%, K ₂ HPO ₄ 1.
_{0%, MgSO 4 · 7H 2} O0.05%, after the liquid medium 100ml consisting of 0.5% oleic acid and 121 ° C. 15 minutes autoclaved put into 500ml Sakaguchi flask, shaking 30 ° C. 24 hours in advance at the same medium 1 ml of the preculture liquid of the cultivated C. sp. KSM-16 strain was inoculated and shake-cultured at 30 ° C for 48 hours. The activity of the supernatant, which was obtained by centrifuging the culture medium to remove bacterial cells, is 20 unit / ml.
It was.

Example 2 Urea 0.4%, 0.5% _{glucose, K 2 HPO 4 0.1%,} MgSO 4 · 7H
₂ 100% liquid medium consisting of 0.05%, yeast extract 0.1% and oleic acid 0.5% was put into a 500 ml Sakaguchi flask and sterilized under pressure steam at 121 ° C for 15 minutes, and then cultured in the same medium at 30 ° C for 24 hours with shaking. 1 ml of a pre-cultured solution of SPKSM-16 strain was inoculated and shake-cultured at 30 ° C. for 3 days. The lipase activity of the culture supernatant was 140 unit / ml.

Example 3 Peptone 4%, glucose 0.5%, KH ₂ PO ₄ 0.1%, MgSO ₄
・ 21 media consisting of 0.05% 7H ₂ O and 0.5% oleic acid
After sterilizing by autoclaving for 15 minutes at 121 ° C for 15 minutes in a jar jar fermenter, 100 ml of culture solution of C. pseudomonas sp. KSM-16 strain that had been shake-cultured in the same medium for 24 hours at 30 ° C was added at 30 ° C and 300 rpm. It was cultured for 48 hours. After culturing, the bacterial cells were removed by centrifugation, and the activity of the culture supernatant 1.5 was 30 unit / ml.
It was. The culture supernatant was concentrated to 300 ml by ultrafiltration (Diaflow membrane fractionation molecular weight of 10,000 manufactured by Amicon) and freeze-dried to obtain 10 g of crude enzyme powder. The enzyme powder had an activity of 3000 unit / g.

Example 4 Using the crude enzyme powder obtained in Example 3, palm oil, coconut oil,
Beef tallow and olive oil were decomposed. 1 g of oil was shaken in an Erlenmeyer flask with 1000 units of enzyme and oil-water (1: 5) to hydrolyze. The decomposition rate of each oil after 48 hours was 92% for palm oil.
%, Coconut oil 90.9%, beef tallow 90.1%, and olive oil 86.3%.

Example 5 Palm oil was hydrolyzed using the crude enzyme powder obtained in Example 3. In an Erlenmeyer flask, 160 units of enzyme for 1 g of oil,
The mixture was hydrolyzed by adding oil and water (1: 1) and shaking. After 24 hours, the decomposition rate of palm oil was 92%.

Reference example Obtaining microorganisms (1) Collect the soil around Kai Town, Haga-gun, Tochigi Prefecture, place 1 teaspoon of soil in a test tube containing 10 ml of sterilized physiological saline, and even suspend and disperse with a vortex mixer for 1 minute. It was
After leaving still for about 1 hour, 0.2 ml of the supernatant was sampled, uniformly applied on the surface of the separated agar medium prepared by the following method with a ladle stick, and then cultured at 30 ° C.

(Preparation of agar medium for separation) 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 Separately sterilize tributyrin and agar from the above composition,
After sterilization, these were combined aseptically, tributyrin was evenly dispersed with a shaker, and then a fixed amount was dispensed into a sterilized dish under heat retention to give an opaque plate agar medium.

(2) Next, among the colonies grown by the above culture, one platinum loop of a colony that decomposes tributyrin in the periphery to form a transparent ring is diluted 100 times with sterile physiological saline, and one platinum loop of this diluted solution is used as described above. The agar medium having the same composition as the agar medium for separation was lined on the surface and cultured at 30 ° C. for 3 days. It was confirmed by visual and microscopic observation that the resulting colonies did not differ from each other. Further, the above colonies were subjected to the same operation again to reconfirm the unity. The properties and physiological properties of the above strain on each medium are as described above.

(3) Next, 10% glycerol aqueous solution (2 ml) in which one platinum loop was sterilized from the strain on the above purely cultured slant medium
Suspend in a vial for freezing and store frozen at -80 ° C. Thus, after 3 months of cryopreservation, 1 platinum loop of the suspension obtained by rapid thawing was revived on ordinary agar medium, and the results of examining the properties and physiological properties on each medium under the same conditions as above, No change from before freezing was observed.

[Brief description of drawings]

Fig. 1 shows the lipase of the present invention at 30 ° C in the pH range of 3 to 13
It is a figure which shows the residual activity after a time process. FIG. 2 is a drawing showing the specific activity of the lipase of the present invention in the range of pH 3-8. FIG. 3 is a drawing showing the specific activity of the lipase of the present invention under each temperature condition. FIG. 4 is a drawing showing the relationship between the treatment time and the residual activity of the lipase of the present invention under various temperature conditions. FIG. 5 is a drawing showing the residual activity of the lipase of the present invention when treated under various temperature conditions for 10 minutes.

Claims (2)

[Claims] 1. A novel lipase having the following physicochemical properties. Action Palm oil, olive oil, beef tallow, coconut oil, reaction temperature 60 ℃,
Decomposes by 90% or more in 24 hours. Especially palm oil decomposes by 92% or more. Substrate specificity Decomposes various glycerides and esters. Especially, it selectively decomposes saturated fats and oils. Stable pH range pH3-12 Range of optimum pH of action pH6-7.5 Range of optimum temperature of action 65-80 ℃ Optimum temperature 70 ℃ Molecular weight SDS-Polyacrylamide gel electrophoresis 29000, gel filtration (Sephadex G-75) method According to 30,000. Sugar-containing color development due to orcinol reaction + 2. A Pseudomonas sp.
p.) The novel lipase according to claim 1, which is obtained by culturing KSM-16 (Microtechnology Research Institute No. 8538) and collecting from the culture.