JPH06209772A - Production of lipase and lipase producing bacteria used therefor - Google Patents

Abstract

PURPOSE:To produce lipase by cultivating lipase producing bacteria having antibiotics resistance and collecting lipase from the cultured medium and to obtain the lipase producing bacteria used therefor. CONSTITUTION:Lipase production by cultivating lipase producing bacteria obtained by mutation of Pseudomonas bacteria as a mother stock and having antibiotics resistance, and collecting lipase from the cultured medium, and lipase producing bacteria used therefor are required in this patent. High production of lipase is attained by this lipase producing method and the lipase producing bacteria used therefor.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for efficiently producing a lipase useful as an industrial enzyme. More specifically, it relates to a method for efficiently producing lipase using a lipase-producing strain having antibiotic resistance. It also relates to antibiotic-resistant bacteria useful for the production of enzymes.

[0002]

BACKGROUND OF THE INVENTION Lipase is widely used as an enzyme for food processing, a medical enzyme as a digestive agent, a reagent enzyme for measuring blood lipids, and an industrial enzyme for hydrolysis and modification of fats and oils. Is used for. In recent years, lipase has been attracting attention as an enzyme for detergents because it is said to be effective in improving detergency.

Many strains of industrial lipase have been reported, and some of them have been used for industrial production. For example, those strains are Pseudomonas (Pseudomonas) genus Alcaligenes (Alcaligenes)
Genus, Achromobacter genus, Mucor ( M
ucor genus, Candida genus, Humicola ( Hum
It is known to be derived from the genus icola ). In addition, a strain of Pseudomonas to produce lipase Pseudomonas fluorescens (Ps. Fluorescens), Pseudomonas cepacia (Ps. Cepacia), Pseudomonas fragi (Ps. Fragi), Pseudomonas aeruginosa (Ps. Ae
ruginosa ), Pseudomonas alcaligenes ( Ps. alca
ligenes ).

[0004]

However, the productivity of lipase by these strains is generally extremely low, and it is not always satisfactory for industrial use. Therefore, in order to solve this problem, improvement of productivity by mutation is usually tried. A conventional method for improving productivity is a method of performing mutation treatment to select a highly productive strain from all surviving bacteria, but this method requires enormous time and labor. Therefore, establishment of a method for producing these lipases more efficiently is strongly demanded. As for other enzymes, a method for efficiently producing cellulase, protease or amylase using a strain resistant to vancomycin or ristocetin (Japanese Patent Laid-Open No. 1-222771), α-using a strain resistant to tunicamycin Method for producing amylase (T. Sasaki et. Al. , BIOCHEMICAL AND
BIOPHYSICAL RESEACH COMMUNICATIONS, 70 , 125-131, 1
976), but no such report has been made for lipase.

[0005]

[Means for Solving the Problems] As a result of intensive studies on the improvement of lipase productivity of lipase-producing bacteria, the present inventors found that if lipase-producing bacteria are conferred with antibiotic resistance,
The inventors have found that the lipase productivity is greatly improved and completed the present invention. For example, it was found that an artificial or natural mutant strain of the genus Pseudomonas, which has resistance to streptomycin, which is one of the antibiotics, produces a large amount of lipase in the medium. Incidentally, streptomycin resistance in the present invention, in the presence of streptomycin at a concentration that originally cannot grow in ordinary bacteria, for example, depending on the type of strain, generally tens of pp
It has the property of being able to grow even in the presence of about m of streptomycin.

Further, it was found that, for example, if the lipase-producing bacterium is given resistance to a cell wall synthesis inhibitor which is a kind of antibiotic, the lipase productivity is greatly improved. In addition, the cell wall synthesis inhibitor resistance in the present invention,
It has the property of being able to grow even in the presence of a cell wall synthesis inhibitor at a concentration at which an ordinary strain cannot grow normally, for example, in the presence of a cell wall synthesis inhibitor of about several tens ppm, although it varies depending on the type of strain.

That is, the present invention provides a method for producing lipase using a lipase-producing bacterium having antibiotic resistance. It also provides an antibiotic-resistant bacterium that is useful for the production of enzymes such as lipase.
In the present invention, bacteria having antibiotic resistance are, for example,
It is obtained as follows. That is, a lipase-producing bacterium (parent strain) to which resistance is to be imparted is grown in an appropriate medium, and the bacterium is irradiated with ultraviolet rays, X-rays or the like, or ethyl methane sulfonate (EMS), N-methyl-N′-nitro. -N-nitrosoguanidine (NTG), p-dimethylaminobenzenediazosulfonic acid, or other known mutagen, and appropriately washed and diluted to obtain a non-viable concentration (minimum inhibitory concentration (MIC)) of the parent strain. Antibiotics containing antibiotics, for example, streptomycin resistance and cell wall synthesis inhibitor resistance, which are grown by spreading on an agar medium containing several tens of ppm in the case of streptomycin and about 100 ppm in the case of cell wall synthesis inhibitor. Obtain colonies of substance resistant strains. Since many of the resistant strains thus obtained surprisingly produce a large amount of lipase as compared with the parent strain, a high lipase-producing bacterium can be obtained from these resistant strains.

[0008] Further, using the obtained resistant strain as a parent strain, a strain having a higher lipase productivity can be obtained by artificial or natural mutation. The mutant strains and the lipases obtained by culturing the mutant strains can be obtained. The manufacturing method of is also included in the present invention.

As a typical example of the lipase-producing bacterium used in the present invention, Pseudomonas ( Pse
udomonas ) genus NCIMB10541 strain as a parent strain and Pseudomonas resistant strains such as Pseudomonas SD703 (Microtechnology Research Institute No. 13307) obtained by the above operation and NCIMB10541 strain as the parent strain and Pseudomonas mendocina SD70 obtained by the above operation
4 (Microtech Lab. No. 13357) and the like, which are resistant to cell wall synthesis inhibitors. NCIMB10541 is a British National Collection of Industrial and Marin
It is a type culture obtained from e Bacteria, Aberdeen, Scotland, and its mycological properties are based on the Vergi's Manual of Systematic Bacteriology (Bergers).
gey's Mannual of Systematic Bacteriology (1984)). The mycological properties of streptomycin-resistant strains such as Pseudomonas SD703, which are streptomycin-resistant strains obtained by mutation of this strain, are substantially the same as the parent strain except for streptomycin resistance and lipase productivity, and similarly cell wall synthesis. The mycological properties of the cell wall synthesis inhibitor resistant strain such as Pseudomonas mendocina SD704, which is an inhibitor resistant strain, are substantially the same as those of the parent strain except for the degree of cell wall synthesis inhibitor resistance and lipase productivity.

The antibiotics used in the present invention are
It is defined as a substance that inhibits the growth of microorganisms and other cells. The antibiotic used in the present invention is not particularly limited, and is usually classified as a cell wall synthesis inhibitor, a protein synthesis inhibitor, a nucleic acid synthesis inhibitor, or an antibiotic that impairs the cell membrane structure. If Examples of cell wall synthesis inhibitors include β-
Examples include lactam antibiotics, lipid cycle-acting antibiotics, and antibiotics that inhibit D-alanine uptake. Examples of β-lactam antibiotics include:
Examples of antibiotics acting on the lipid cycle such as carbenicillin, penicillin, and ampicillin include vancomycin, restetin, enramycin, macorbomycin, moenomycin, and the like. Examples of antibiotics that inhibit D-alanine uptake include D-alanine. Examples thereof include cycloserine, and examples of other cell wall synthesis inhibitors include tunicamycin. Examples of protein synthesis inhibitors include streptomycin, spectinomycin and the like. Examples of the nucleic acid synthesis inhibitor include rifamycin and the like.

The medium used in the lipase production method of the present invention may be any medium as long as the strain used can grow, for example, a carbon compound capable of assimilation as a carbon source or a medium containing the same. For example glucose,
A starch hydrolyzate such as starch or liquefied starch, molasses, glycerin, oils and fats, corn steep liquor, Tween-based surfactants and the like may be used as long as they can contain assimilable nitrogen compounds or those containing nitrogen compounds such as soybean flour. ,
Organic nitrogen sources such as meat extract, pharmaceutical media and corn stipe liquor, and inorganic nitrogen sources such as ammonium salt and nitrate are used. In addition to these, as the inorganic salts, phosphates such as commonly used monohydrogen phosphate, magnesium salts, calcium salts, manganese salts and the like can be appropriately added.

Cultivation of the antibiotic-resistant bacterium in the present invention is carried out under aerobic conditions, for example, by aeration stirring method or shaking culture method. The culturing temperature is in the range of 10 to 40 ° C., but especially 2
It is preferably carried out in the range of 0 to 37 ° C. PH during culture
The pH may be adjusted to 5 to 12 throughout the entire culture period, and the initial pH is preferably set to 7 to 9.5. The culture time is usually in the range of 8 to 100 hours, but the culture may be terminated when the amount of accumulated lipase reaches the maximum. From the viewpoint of economy, it is particularly preferable to set it in the range of 20 to 50 hours. The lipase productivity of antibiotic-resistant strains varies greatly depending on the medium composition and culture conditions, but under any culture conditions, lipase is produced 1.5 to 2 times or more as much as each parent strain.

After culturing in this manner, the lipase can be collected from the culture medium by separating and purifying it according to a conventional method for collecting lipase. That is, bacterial cells and medium solids can be separated from the culture solution by a known appropriate method such as filtration or centrifugation to obtain a supernatant or a filtrate. Concentration of these separated liquids or salting out method of precipitating enzyme by adding soluble salts without concentration, organic solvent precipitation method of precipitating enzyme or contaminants by adding hydrophilic organic solvent, ion exchange resin The lipase can be obtained in a desired form such as a solution or powder by using a single or a combination of separation or purification means such as adsorption / desorption method, membrane separation or gel filtration method, spray drying method without a stabilizing aid, and freeze drying method. You can

The activity of the lipase obtained in the present invention is measured by a known method, for example, an emulsification system measuring method using a triolein-polyvinyl alcohol (PVA) emulsion as a substrate. Specifically, a reaction solution consisting of 0.1 ml of enzyme solution, 0.4 ml of 200 mM Tris buffer (pH 9.0), and 0.5 ml of triolein emulsion was placed in a test tube with a stopper at 37 ° C.
10 minutes at room temperature, 0.2m of 1N hydrochloric acid as a stop solution
Stop the reaction with l. Triolein-PVA emulsion is a 2% aqueous solution of PVA (Poval PVA11
7 (trade name of Kuraray Co., Ltd.): Poval PVA205
(Kuraray Co., Ltd.) = 9: 1) To 15 ml, 1.5 g of triolein was added and homogenized at 18,000 rpm for 10 minutes while cooling with ice. Stop solution, n-hexane 2m
l, 2 ml of isopropyl alcohol and 1 ml of distilled water were added, and the mixture was vigorously stirred, allowed to stand, and the hexane layer was sampled.
C-FID method (Minagawa et. Al. , Lipids. 18 732 (198)
Quantify oleic acid in 3)). The unit of activity is 1 unit (U) of the amount of enzyme that produces 1 micromol of oleic acid per minute.

[0015]

EXAMPLES Next, reference will be made to Reference Examples and Examples for obtaining these antibiotic resistant strains, but these are merely examples and do not limit the present invention. The antibiotic resistant strains used in the present invention are It may be obtained by another method.

Reference Example 1 NCIMB10541 strain was applied to the following medium composition 1 containing streptomycin at various concentrations to determine the minimum growth inhibitory concentration of streptomycin. That is,
The above NCIMB was added to the medium 1 containing streptomycin.
A suspension of 10541 strain (bacterial concentration: 10 ⁸ cells / ml) was applied on one platinum loop and then left standing at 35 ° C for 1 day to visually observe the surface growth of the strain. The results are shown in Table 1.

Medium composition 1 Heptone 1.0% Yeast extract 0.5% Sodium chloride 0.5% Sodium carbonate 0.3% Agar 2.0% (The medium was sterilized at 121 ° C. for 20 minutes by an autoclave.)

[0018]

[Table 1]

As is clear from this result, NCIMB1
The minimum inhibitory concentration for strain 0541 was about 50 ppm streptomycin.

Reference Example 2 The NCIMB10541 strain was applied to the following medium composition 2 containing carbenicillin at various concentrations to determine the minimum inhibitory concentration of carbenicillin. That is, one platinum loop of the above NCIMB10541 strain suspension (bacterial concentration: 10 ⁸ cells / ml) was applied to the medium 2 supplemented with carbenicillin,
It was allowed to stand at 35 ° C for 1 day, and the surface growth of the strain was visually observed. The results are shown in Table 2.

Medium composition 2 Heptone 1.0% Yeast extract 0.5% Sodium chloride 0.5% Agar 2.0% (The medium was sterilized by an autoclave at 121 ° C. for 20 minutes.)

[0022]

[Table 2]

As is clear from this result, NCIMB1
The minimum inhibitory concentration for strain 0541 is about 1 carbenicillin.
It was 00 ppm.

Example 1 NCIMB10541 strain was cultivated in a plate medium of medium composition 1 at 35 ° C. for 16 hours, and one platinum loop was placed in a test tube with Molton stopper, and the agar-free liquid medium of the above medium composition 1 was used. 5 ml was inoculated. This was cultured for 5 hours and centrifuged, and the obtained cells were suspended in physiological saline to a final concentration of 100.
Add N-methyl-N'-nitro-N-nitrosoguanidine (NTG) solution so that the concentration becomes ppm, and add 10 minutes at 30 ° C.
Left unattended. Then, the cells were collected by centrifugation, washed with physiological saline, and shake-cultured at 35 ° C. for 16 hours in a liquid medium having the above-mentioned medium composition 1 except for agar. This culture solution was diluted and plated on a plate medium of medium composition 1 containing 200 ppm of streptomycin so that the number of viable cells was about 10 ⁸ per plate. Most of the bacterial colonies that had grown after being cultured at 35 ° C. for 1 day were streptomycin-resistant strains.

Example 2 The strain and parent strain obtained in Example 1 were placed in a test tube with a Molton stopper, and 2 ml of a liquid medium having the medium composition 3 shown below was inoculated with one platinum loop. After shaking culture at 35 ° C. for 18 hours, the culture solution was centrifuged, and the supernatant was subjected to the above-mentioned method.
The lipase activity was measured. As a result, the lipase highly producing resistant strains shown in Table 3 were obtained. About 60% of the obtained streptomycin-resistant strains had lipase productivity higher than that of the parent strain, and the productivity was about 1.5 to 2 times that of the parent strain. No. 31, which is the most productive strain in Table 3, was named Pseudomonas SD703, and was deposited with the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology as Microindustry Research Institute No. 13307.

Medium composition 3 Soy flour 2.0% Glucose 2.0% Ammonium sulfate 0.1% Urea 0.1% Yeast extract 0.5% Dipotassium monohydrogen phosphate 0.5% Magnesium sulfate heptahydrate 0.1% Sodium carbonate 0.3% (The medium was 121 Sterilization was performed at 20 ° C for 20 minutes.)

[0027]

[Table 3]

Example 3 Pseudomonas SD703 and the parent strain were each placed in a test tube with a Molton stopper, and a liquid medium 2 having the following medium composition 4 was used.
One platinum loop was inoculated into ml. After shaking culture at 35 ° C. for 18 hours, the culture solution was centrifuged and the supernatant was assayed for lipase activity by the method described above. The results shown in Table 4 were obtained.

Medium composition 4 Casamino acid 2.0% Glucose 2.0% Diammonium monohydrogen phosphate 0.1% Dipotassium monohydrogen phosphate 0.3% Yeast extract 0.5% Magnesium sulfate heptahydrate 0.1% Sodium carbonate 0.3% Tween80 0.7%

[0030]

[Table 4]

Example 4 NCIMB10541 strain was cultured in a plate medium having a medium composition 2 at 35 ° C. for 16 hours, and one platinum loop thereof was placed in a test tube equipped with a Molton stopper, and the agar-free liquid medium having the above medium composition 1 was used. 5 ml was inoculated. This was cultured for 5 hours and centrifuged, and the obtained cells were suspended in physiological saline to a final concentration of 100.
Add N-methyl-N'-nitro-N-nitrosoguanidine (NTG) solution so that the concentration becomes ppm, and add 10 minutes at 30 ° C.
Left unattended. Next, the cells were collected by centrifugation, washed with physiological saline, and shake-cultured in a liquid medium having the above medium composition 2 without agar at 35 ° C. for 16 hours. This culture solution was added to a plate medium of medium composition 2 containing 100 ppm of carbenicillin,
It was diluted and scattered so that the number of viable bacteria was about 10 ⁸ per plate. Most of the bacterial colonies that had grown by culturing at 35 ° C. for 1 day were carbenicillin-resistant strains.

Example 5 One platinum loop was inoculated with 2 ml of the liquid medium having the medium composition 3 containing the strain and parent strain obtained in Example 4 in a test tube with a Molton stopper.
After shaking culture at 35 ° C. for 18 hours, the culture solution was centrifuged and the supernatant was measured for lipase activity by the method described above. As a result, the lipase high-producing resistant strains shown in Table 5 were obtained. The productivity of many of the obtained carbenicillin-resistant strains was higher than that of the parent strain, and the productivity was about 2-2.5 times that of the parent strain. No. 15, which is the most productive strain in Table 5, was named Pseudomonas mendocina SD704, and was deposited with the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology as Micromachine Research Institute No. 13357.

[0033]

[Table 5]

Example 6 Pseudomonas mendocina SD704 and the parent strain were each inoculated with 1 platinum loop in 2 ml of a liquid medium having a medium composition 4 placed in a test tube with a Molton stopper. After shaking culture at 35 ° C. for 18 hours, the culture solution was centrifuged and the supernatant was assayed for lipase activity by the method described above.
The results shown in are obtained.

[0035]

[Table 6]

[0036]

INDUSTRIAL APPLICABILITY The antibiotic resistant strain according to the present invention is
Since it has a lipase productivity of 1.5 to 2.5 times or more, it is extremely advantageously used for industrial production of lipase.

Claims (10)

[Claims] 1. A method for producing lipase, which comprises culturing a lipase-producing bacterium having antibiotic resistance and collecting the lipase from the culture. 2. The method for producing lipase according to claim 1, wherein the lipase-producing bacterium having antibiotic resistance is a bacterium belonging to the genus Pseudomonas . 3. The method for producing lipase according to claim 1, wherein the antibiotic is a cell wall synthesis inhibitor. 4. The method for producing lipase according to claim 3, wherein the cell wall synthesis inhibitor is a β-lactam antibiotic. 5. The method for producing a lipase according to claim 4, wherein the β-lactam antibiotic is carbenicillin. 6. The method for producing lipase according to claim 1, wherein the antibiotic is streptomycin. 7. A lipase producing bacterium having antibiotic resistance is Pseudomonas SD703 (Microtechnological Research Institute No. 13307).
No.), the method for producing the lipase according to claim 1 or 2 or 6. 8. The method for producing a lipase according to claim 1, wherein the lipase-producing bacterium having antibiotic resistance is Pseudomonas mendocina SD704 (Ministry of Industrial Science and Technology No. 13357). 9. Pseudomonas SD703 (Microtechnology Research Institute No. 13307) and mutants thereof. 10. A Pseudomonas mendocina SD70.
4 (Microtech Lab. No. 13357) and its mutants.