JPH11253157A - Microbe having ability for producing new lipase, lipase, its production and its use - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a lipase capable of catalyzing a strong random ester- interchange reaction usable for modification of an oil and fat. SOLUTION: This lipase has the following properties: (1) causing a strong randomization reaction in the ester-interchange reaction of an oil and fat; (2) having the optimum pH of 8.0-8.5; (3) having the optimum temperature of 60 deg.C; (4) having a molecular weight of 31,000±2,000 and (5) having the isoelectric point of 4.9±0.1. The production method comprises culturing microorganisms belonging to Burkholderia gladioli and having ability for producing the lipase to provide the lipase, and gathering the produced lipase. The randomly ester- interchanged oil and fat are obtained by allowing the lipase to act on the oil and fat.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel lipase, a bacterium producing the same, a method for producing the lipase, and use thereof. More particularly, it relates to a lipase obtained by culturing a bacterium belonging to Burkholderia gladioli.

[0002]

2. Description of the Related Art The transesterification of fats and oils is roughly classified into a chemical method and an enzymatic method. The random transesterification reaction is mainly carried out using a chemical catalyst. But,
This method requires washing with water to remove the catalyst after the completion of the reaction, and the operation is very troublesome. Besides,
At that time, a hydrolysis reaction occurred to generate a fatty acid, leading to a decrease in yield. Therefore, a lipase that catalyzes a random transesterification reaction under the condition that the hydrolysis of fats and oils does not occur (without adding water) has been demanded.

[0003] The lipases hitherto found require a very high unit of activity when randomization occurs in the transesterification of fats and oils. For example, "Basic and Application of Lipase" (Koshobo, July 1991, No. 295
P. 296) shows a random transesterification reaction using a lipase of the genus Candida. A method in which a large amount of enzyme of at least 10% is added to the fat or oil of the substrate and the reaction is carried out at 40 ° C for 66 hours. It was not a method that could be industrialized at all. In addition, Japanese Patent Application Laid-Open No. 8-214890 discloses a transesterification reaction of fats and oils using a lipase derived from the genus Alcaligenes, but the enzyme activity was not sufficient. For this reason, the random transesterification of fats and oils could not be performed. Under such circumstances, there has been a demand for a lipase which causes efficient transesterification of fats and oils which cannot be solved by existing lipases.

[0004]

DISCLOSURE OF THE INVENTION The present inventors have conducted a search for a novel lipase-producing bacterium exhibiting an efficient transesterification reaction of fats and oils and succeeded in finding a microorganism producing the desired lipase. Thus, the present invention has been completed. That is, an object of the present invention is to provide a novel lipase which exhibits an efficient random transesterification of fats and oils.

It is another object of the present invention to provide a bacterium belonging to the genus Burkholderia gladioli which has the ability to produce such a lipase. It is another object of the present invention to provide a method for producing lipase, which comprises culturing such a bacterium to produce lipase. Still another object of the present invention is to provide a method for producing a random transesterified fat by allowing the lipase to act on the fat.

[0006]

Means for Solving the Problems The present invention has the following properties: (1) causes a strong randomization reaction in transesterification of fats and oils, (2) optimum pH 8.0-8.5, (3) optimum Temperature 60
° C, (4) molecular weight 31,000 ± 2,000, and (5) isoelectric point
A lipase characterized by having 4.9 ± 0.1.

[0007] The present invention also relates to the above-mentioned method for producing a lipase, which comprises culturing a bacterium belonging to Burkholderia gladioli having the ability to produce the lipase, producing the lipase, and collecting the lipase. A method for producing a lipase is provided. According to an embodiment of the present invention, there is provided a method for producing a lipase, wherein the bacterium is Burkholderia gladioli BG-QLM-1 (see below). The invention further provides
Provided is a method for producing a random transesterified fat, which comprises causing the lipase to act on fats and oils to obtain a randomly transesterified fat and oil.

[0008] The present invention further provides a bacterium belonging to Burkholderia gladioli, which has the ability to produce the above lipase. The present invention also relates to a Burkholderia gladioli BG-QLM-1 capable of producing the lipase.
A strain or a variant thereof is provided. In the present specification, “random transesterification” means transesterification in which the fatty acid composition at each position is the same. Further, “randomization” means that the fatty acid composition at each position becomes the same.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION The lipase-producing bacterium of the present invention is newly discovered from soil and isolated by a single-cell separation method, and has the following bacteriological properties. a) Morphological properties: Cell shape and size: bacilli, 0.5 × 1-3 μm. 2. Cell polyplasia: rare, but rarely long. 3. Mobility: Yes. 4. Flagella formation: Extreme flagella. 5. Spores: do not form.

B) Growth state in each medium: Gravy agar plate: Translucent gray-yellow, slightly glossy round colonies are formed. The surface is smooth and the periphery is wavy. No diffusible dye is observed. 2. Gravy agar slant medium: It grows like a thread or a wart, and the growing part is yellow-white, and when exposed to light, it becomes translucent and has a gray resin-like color. 3. Broth liquid medium: Growth is good, but the medium is not very turbid and the upper layer remains almost transparent, and white cells precipitate in the lower layer. 4. Broth gelatin stab culture: Grows in thread form, liquefies first in the form of a crater, and later liquefies in layers. 5. Litmus milk: Produces alkali and gradually liquefies.

C) Physiological properties: Gram staining: negative. 2. Acid resistance: None. 3. Urease: negative. 4. Oxidase: positive. 5. Catalase: positive. 6. OF test: no oxidation, no gas generation. 7. Attitude to oxygen: aerobic. 8. Growth range: pH 5-9, temperature 15-37 ° C. 9. Indole production: negative. Ten. Methyl red test: negative.

11. VP test: negative. 12． Utilization of citric acid: positive. 13. Production of hydrogen sulfide: slightly positive. 14. Denitrification reaction: negative. 15． Nitrate reduction: negative. 16． Use of inorganic nitrogen sources: nitrate positive, ammonium salt positive. 17． Gelatin liquefaction: positive. 18． Starch hydrolysis: negative. 19. Hydrolysis of milk casein: positive. 20. GC content of intracellular DNA: 67-69%. twenty one. Quinone molecular species: Q8.

22. Alcaligenes latus IAM
12599, 10 against DNA of Pseudomonas stutzeri IFO3773
Shows no more than% homology. Burkholderia cepacia 1
It shows 40-45% homology to 0JCM5506 DNA. Two
3． Assimilation: positive: saccharose, maltose, lactose, trehalose, glucose, mannose, galactose, fructose, xylose, sorbitol, manitol, inositol, L-arabinose, glycerol; negative: starch, dextrin, raffinose.

On the basis of the above results, the Burges Manual of Deterministic Bacteriology (B
ergey's manual of determinative bacteriology ninth
edition), it turned out to belong to the genus Burkholderia. What is Burkholderia cepacia?
It was identified as a bacterium belonging to Burkuholderia gladioli based on the difference in DNA homology and the reducing property of nitrate, and the reducing and other physiological properties of nitrate. Burkholderia gladioli BG-
The strain was named QLM-1 strain. Such a strain was deposited with the Research Institute of Biotechnology, Industrial Technology Institute of the Ministry of International Trade and Industry on January 22, 1998, and given the accession number FERM P-16600.

The bacterium belonging to the Burkholderia gladioli of the present invention is not particularly limited, as long as it has the ability to produce a lipase having the above-defined properties. Among them, there are Burkholderia gladioli BG-QLM- Mutants derived from one strain are also included. The mutation includes a natural mutation or an artificial mutation caused by a chemical mutagen, ultraviolet light or the like.

To cultivate Burkholderia gladioli according to the present invention, a conventional nutrient medium can be used, provided that the medium contains a carbon source, a nitrogen source, inorganic salts and the like appropriately. Either can be used. As the carbon source, saccharose, maltose, lactose, trehalose, glucose, mannose, galactose, fructose, xylose, sorbitol, mannitol, inositol, L-arabinose, glycerol and other sugars, alcohols, organic acids, olive oil, soybean oil and other fats and oils , And combinations thereof. As the nitrogen source, organic nitrogen such as corn steep liquor, soy flour, peptone, meat extract, yeast extract, wheat gluten and the like, and inorganic nitrogen such as ammonium sulfate, ammonium nitrate and urea can be used. As the inorganic salts, salt, potassium chloride, magnesium sulfate, monopotassium phosphate, dipotassium phosphate, ferrous sulfate and the like can be used.
In order to further promote growth and enhance enzyme production, a surfactant such as EMULGEN, TRITON, SPAN, or TWEEN may be added.
The culture is carried out in an aerobic culture such as aeration and agitation in a liquid medium containing such components at 15 to 35 ° C. and a pH of 5 to 9 for 1 to 4 days.

In order to collect lipase from the culture solution, the culture solution is separated from the cells by filtration or centrifugation, and the liquid is subjected to ammonium sulfate salting out, solvent precipitation using alcohol, acetone, or the like, and separation and concentration using an ultrafiltration membrane. An enzyme can be obtained by a known method. In addition, to extract lipase accumulated in the cells, cell disruption, autolysis,
After a cell-free enzyme solution is obtained by a known method such as sonication, it can be purified in the same manner as the extracellular enzyme.
In order to obtain a highly purified enzyme preparation, conventional methods such as ultrafiltration concentration, acetone precipitation, ammonium sulfate precipitation, ion exchange chromatography, hydrophobic chromatography, adsorption chromatography, and gel filtration chromatography have been used. Purification may be performed by a conventional enzyme purification method.

The physicochemical properties of the enzyme of the present invention are described below. 1. Action: The enzyme of the present invention catalyzes strong randomization in transesterification of fats and oils. The randomization rate of the enzyme of the present invention is usually 70% or more, preferably 80% or more, and more preferably 90% or more. 2. Optimal action pH: Measured in the same manner as the activity measurement method described below in the range of pH 5 to 10 using olive oil as a substrate.
The relationship between the reaction pH and the relative activity was as shown in FIG. 1, and the optimum pH was 8 to 8.5. 3. Optimal temperature: 30 to 8 at pH 8.0 using olive oil as substrate
The activity was measured in the range of 0 ° C in the same manner as the activity measurement method described later.
The relationship between the reaction temperature and the relative activity was as shown in FIG. 2, and the optimum temperature was around 60 ° C.

4. Molecular weight: The molecular weight obtained by SDS-polyacrylamide gel electrophoresis is 31,000 ± 2,000
Met. 5. Isoelectric point: Isoelectric point measured by polyacrylamide gel electrophoresis was 4.9 ± 0.1. The transesterification reaction conditions using the lipase of the present invention can be appropriately selected. The method will be described below. As the raw material oil used in the method of the present invention, for example, soybean oil, rapeseed oil,
Vegetable fats such as rice oil, corn oil, cottonseed oil, palm oil, palm fractionated oil, palm kernel oil, coconut oil, cacao fat, monkey fat, etc., animal and vegetable fats such as fish oil, lard, tallow fat, milk fat, and fractionated fats thereof. Examples thereof include hardened oils, synthetic fats and oils such as trilaurin, triolein, tripalmitin, and tristearin, and fatty acids and fatty acid esters thereof. The transesterification of the present invention includes, for example, transesterification of fats and oils, fats and fatty acids, and fats and oils and fatty acid esters.

The lipase used in the present invention is not particularly limited in its purity and form, and may be any of a crude product, a partially purified product, and a purified product. For example, an enzyme precipitated with a solvent such as acetone or ethanol, diatomaceous earth, an ion exchanger, an enzyme immobilized on an adsorption resin, or the like can be used. The water content of these enzymes is desirably 10% or less. The reaction using the above lipase may be a batch system or a continuous system, but the production of a hydrolyzate is suppressed by reducing the substrate moisture to 1% or less. The amount of the enzyme used for the fat or oil may be 50 to 10,000 ppm based on the raw fat or oil. The reaction can be carried out without a solvent or in a non-polar solvent such as hexane, but is preferably carried out without solvent. Although the reaction temperature can be appropriately selected, the reaction is carried out at 30 to 100 ° C, preferably 35 to 70 ° C.

In fact, lipase of the present invention, lipase QL and lipase PL, which are lipases of the genus Alcaligenes, known as lipases having strong random transesterification ability, are mixed in an equal mixture of soybean oil and palm oil (both from Meito Sangyo Co., Ltd. )) Were added at equal weights and stirred at 60 ° C. for 24 hours to carry out a random transesterification reaction, analyze fats and oils, and measure the reaction rate and the randomization rate (two-position reaction rate). Table 1 shows the results. .

[0022]

[Table 1]

¹⁾ Reaction rate: (composition% of individual triglyceride of reaction oil-composition% of individual triglyceride of base oil)
(Composition% of individual triglycerides in chemically catalyzed reaction oil-Composition% of individual triglycerides in feedstock) x 100. ²⁾ Randomization rate (2-position reaction rate): (composition% of palmitic acid at 2-position of reaction oil-composition% of palmitic acid at 2-position of feedstock) ÷ (composition of palmitic acid at 2-position of chemical catalytic reaction oil) Composition%-Composition% of palmitic acid at 2 positions of feedstock) x 1
00.

From the results shown in Table 1, it can be seen that the lipase produced according to the present invention has several times stronger random transesterification activity than conventionally known random transesterification lipase. The present invention will be more specifically described by the following examples, which are merely illustrative and not intended to limit the present invention.

[0025]

EXAMPLES The activities of the hydrolysis activity and transesterification activity used in the examples were measured by the following methods. a) Hydrolytic activity: The method for measuring the activity of industrial lipase described in JIS K601-88, and the method for measuring by adding an emulsifier
Performed according to (Method B). Olive oil substrate (phosphate buffer,
After the enzyme was reacted at 37 ° C. for 10 minutes at pH 7.0), the amount of the enzyme that released 1 μmol of fatty acid per minute was defined as 1 unit.

B) Transesterification activity: An equal weight mixture of trilaurin and tricaprylin was precisely weighed into a vial, and
Melted at ° C. The reaction was started by adding 10 mg of lipase powder and
The mixture was stirred for 10 minutes, and 10 µl of the reaction solution was taken. 0.2 ml of a mixture of acetone-methanol-2N sulfuric acid (50:50:10) and hexane
The reaction was stopped by adding 0.2 ml. 1 μl of this hexane layer was analyzed by gas chromatography. Column is 3% Dexsil
300 on 100/200 supelcoport, 2.6 mm × 0.5 m (manufactured by Spelco), the temperature was raised from 280 ° C. to 320 ° C., and the carrier gas was helium at 50 ml / min. One unit is 1 μmol per minute
Of dicaproylmonolaurin was used.

Example 1 Lipase from Berk-Folderia gladioli BG-QLM-1 strain
100 ml of a liquid medium (pH 6.0) containing corn steep liquor 2%, polypeptone 0.1% and dipotassium phosphate 0.1% as a pre-culture medium was placed in a 500-ml Sakaguchi flask of a culture supernatant containing a protease activity . After steam sterilization, Bark Folderia Gladioli BG-QLM-1
One loopful of the strain (FERM P-16600) was inoculated. Shaking culture was performed at 25 ° C. for 24 hours to obtain a pre-culture solution. In a 2 liter (1 liter) jar, 4.0% Kinako, 1% corn steep liquor, 0.2% dipotassium phosphate, 0.4% soybean oil, Emulgen 320P (Kao
A liquid medium consisting of 0.3% was added, and after sterilizing with steam, the above pre-cultured liquid was inoculated, and stirred at 30 ° C., 600 rpm with aeration at 600 rpm.
Aeration culture was performed at 0.5 vvm for 48 hours. After the cultivation, the culture was centrifuged to remove the solids containing the bacterial cells, thereby obtaining a culture supernatant. The hydrolysis activity of the resulting culture supernatant was 3,000 units (U) / ml.

Example 2 Purification of Lipase Ammonium sulfate was added to 1 L of the culture supernatant obtained in Example 1 so as to be 35% saturated, dissolved, and left at 5 ° C. for 6 hours. The resulting precipitate was collected by centrifugation and dissolved in buffer. DEAE-Toyopearl equilibrated solution obtained
(Manufactured by Tosoh Corporation) for adsorption. The active fraction eluted by the salt concentration gradient was octyl sepharose CL-4B.
(Pharmacia) The column was adsorbed on a column, eluted with a concentration gradient of ammonium sulfate, and further subjected to gel filtration using Sephadex G100 (Pharmacia) to obtain a purified enzyme having a purity of 95% or more. The purification magnification was 50 times, the recovery rate of the purified enzyme from the culture supernatant was 20%, and the specific activity was 500 U / mg.

Example 3 Purification of lipase To 1 liter of the culture supernatant obtained in Example 1, 4 liters of acetone cooled to -20 ° C. were added, and the formed precipitate was collected. The lipase was further purified by drying in a dryer for 24 hours. The hydrolysis activity of this acetone powder was 200,000 U / g, and the transesterification activity was 5,000 U / g. On the other hand, lipase QL (derived from the genus Alcaligenes) has a hydrolysis activity of 30,000 U / g and a transesterification activity of 1,000 U / g.
The lipase PL (derived from the genus Alcaligenes) has a hydrolysis activity of 100,000 U / g and a transesterification activity of 1,000 u / g.
Therefore, it was revealed that the lipase of the present invention has a transesterification activity several times higher than that of lipases QL and PL, which are conventionally considered to have very high transesterification activity.

Example 4 Random transesterification of various fats and oils 100 g of various fats and oils was added to 50 mg of Burkholderia gladioli lipase obtained in Example 3, and the mixture was stirred at 60 ° C. for 24 hours. After the reaction, measure the reaction rate and randomization rate,
The results are shown in Table 2.

[0031]

[Table 2]

The results in Table 2 show that a random transesterification reaction occurs for various fats and oils.

Example 5 Random transesterification reaction using immobilized lipase One liter of the culture supernatant obtained in Example 1 was concentrated 10 times with an ultrafiltration membrane. 100 g of Radiolite # 3000 (manufactured by Showa Chemical Industry Co., Ltd.) was added to the mixture, mixed and dried under vacuum. The transesterification activity of this immobilized enzyme was 500 U / g. The enzyme was packed in a column and the fats and oils used in Example 4 were heated at 60 ° C.
The solution was passed through the column with SV3. When the solution was passed through SV3, the reaction rate was 100% and the randomization rate was 90% or more.

[0034]

Industrial Applicability The lipase of the present invention catalyzes a strong random transesterification reaction and can be used for reforming fats and oils. Since these transesterified oils do not use a chemical catalyst, they are useful as safe transesterified fats.

[Brief description of the drawings]

FIG. 1 is a view showing the optimum pH of the lipase of the present invention.

FIG. 2 is a view showing an optimum temperature of the lipase of the present invention.

────────────────────────────────────────────────── ─── Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI // (C12N 9/20 C12R 1:01) (C12N 1/20 C12R 1:01) (72) Inventor Takashi Yamashita Hachioji, Tokyo 1159-1-507, Uchikoshi-cho, City (72) Inventor Haruo Machida 2-24-4 Asahigaoka, Hino-shi, Tokyo

Claims (6)

[Claims] 1. The following properties: (1) a strong randomization reaction occurs in transesterification of fats and oils, (2) optimum pH 8.0-8.5, (3) optimum temperature 60
° C, (4) molecular weight 31,000 ± 2,000, and (5) isoelectric point
A lipase having 4.9 ± 0.1. 2. The method for producing a lipase according to claim 1, wherein the lipase is Burkholderia gradioli.
A method for producing a lipase, comprising culturing a bacterium belonging to L. ladioli ) capable of producing the lipase, producing the lipase, and collecting the lipase. 3. The bacterium is Burkholderia gladioli BG.
The method according to claim 2, wherein the strain is -QLM-1 strain. 4. A method for producing a random transesterified fat, which comprises causing the lipase according to claim 1 to act on a fat or oil to obtain a randomly transesterified fat or oil. 5. A bacterium belonging to Burkholderia gladioli capable of producing the lipase according to claim 1. 6. Burkholderia gladioli BG-QLM-1
The bacterium according to claim 5, which is a strain or a mutant thereof.