JPH0331435B2 - - Google Patents

Description

[Detailed description of the invention]

INDUSTRIAL APPLICATION FIELD The present invention relates to a novel lipase, a method for producing the lipase, and a novel microorganism. BACKGROUND ART Lipase is used as a digestive agent, for decomposing fats and oils during soap production, and as an enzyme added to detergents. However, conventionally known lipases have the disadvantage that they have a very low rate of hydrolysis of castor oil. In other words, the majority of castor oil's constituent fatty acids are ricinoleic acid, which has an aquatic group, and it is known that the rate of hydrolysis by various lipases is lower than that of other fats and oils due to this hydroxyl group. . Therefore, the current situation is that the use of ricinoleic acid as a physiologically active substance, a chemical product, etc. and the effective use of castor oil are currently limited. Means for Solving the Problems In view of the above-mentioned current situation, the present inventors aimed to easily produce ricinoleic acid with a high hydrolysis rate of castor oil, and thus enable more effective use of castor oil. A soil screening was conducted using a medium containing castor oil as the sole carbon source. As a result, they discovered that strain f-B-24, a new bacterial strain belonging to the genus Alcaligenes, produces a new lipase that efficiently decomposes castor oil.Based on this, they conducted further research and completed the present invention. That is, the present invention uses castor oil with a molecular weight of about 30,000 (based on gel filtration), an optimal pH of around PH7.0, and an activity of 100% when olive oil is used as a substrate. Relative activity as substrate is 107
%, production of the lipase characterized by culturing a microorganism belonging to the genus Alcaligenes that produces the lipase in a medium, accumulating the lipase in the culture solution, and collecting it. method, and Alcaligenes sp.
It pertains to microorganisms that are In the present invention, the lipase titer is measured by the polyvinyl alcohol emulsification method of Yamada et al. (Journal of the Japanese Society of Agricultural Chemistry, Vol. 36, p. 860, 1962). i.e. 2% of polyvinyl alcohol
A reaction solution is prepared by mixing 5 ml of an olive oil emulsion obtained by adding 25 volumes of pharmacopoeial olive oil to 75 volumes of an aqueous solution and homogenizing for 3 minutes, 4 ml of a 0.1M phosphate buffer with a pH of 7.0, and 1 ml of an oxygen solution (test solution). This reaction solution was reacted at 37°C for 20 minutes, and immediately after the reaction was completed, a mixture of acetone and ethanol (1:1, volume ratio) was added.
The reaction was stopped by adding 20 ml, and the generated fatty acid was diluted with 0.05N using phenolphthalein as an indicator.
Titrate with NaOH. The titer of lipase activity of 1 unit (hereinafter referred to as U) indicates the amount of enzyme that liberates 1 micromole of fatty acid per minute. Also, 280n per ml
The number of units per m absorbance (U/ml/OD ₂₈₀ ) is
Define as specific activity. The characteristics of the novel lipase according to the present invention are as follows. (1) Action: Acts on the ester bonds of various oils and fats,
Produces fatty acids and glycerin. (2) Substrate specificity: 1g each of various animal and vegetable oils and fats
as a substrate, 9 ml of 0.1M phosphate buffer with pH 7.0
Add 0.5 ml (140 U) of enzyme solution in the presence of
The enzyme activity for various substrates was measured by hydrolysis rate when the reaction was carried out at 37℃ for 72 hours with shaking 120 times per minute, and the activity is expressed as relative activity with the activity when using olive oil as the substrate as 100%. and the first
As shown in the table.

[Table] From Table 1 above, it is clear that the present lipase acts well on various animal and vegetable oils and fats, and particularly acts well on castor oil, which has conventionally been difficult to degrade. Furthermore, the hydrolysis rate (absolute value), calculated from the saponification value and acid value of the oil and fat, is 82% for castor oil, 77% for olive oil, and 73% for coconut oil. For comparison, the substrate specificity of a commercially available lipase derived from R. nivenus (manufactured by Nagase Seikagaku Kogyo Co., Ltd., Lot. 6270113) was investigated in the same way, and it was found that when olive oil was used as a substrate, The relative activity using castor oil as a substrate was 52% when the activity was 100%. (3) Optimum pH: Around PH7.0. Figure 1 shows the relationship between lipase activity and pH when olive oil is used as a substrate. In Figure 1, the activity of PH3 to 6 is shown in Table 1.
The activity of pH 8.5 to 9 was measured using a phosphate buffer, and the activity of pH 8.5 to 9 was measured using a glycine buffer. (4) PH stability: When left at 4℃ for 22 hours, PH
The residual activity is 100% in the range of 9.0 to 10.0, and it is stable. After being left at 4°C for 22 hours, the relationship between the residual lipase activity when olive oil was used as a substrate and the pH at the time of standing is shown in Figure 2. In Figure 2, PH4~5
The treatment at pH 6 to 8 was carried out using a citrate buffer, the treatment at pH 6 to 8 was carried out using a phosphate buffer, and the treatment at pH 9 to 11 was carried out using a glycine buffer. (5) Optimum temperature: around 60℃. Figure 3 shows the relationship between lipase activity and temperature at pH 7.0 when olive oil is used as a substrate. (6) Temperature stability: At PH7.0, 100% activity remains when incubated at 35°C for 30 minutes;
When incubated for 30 minutes at 45°C, 76% activity remains, and when incubated at 45°C for 30 minutes, it is completely inactivated. Figure 4 shows the relationship between residual lipase activity and treatment temperature at pH 7.0 when olive oil is used as a substrate. (7) Inhibition and activation: This lipase inhibits Fe ³⁺ ,
Hg ²⁺ , EDTA (ethylenediaminetetraacetic acid),
PCMB (parachloromercuric benzoic acid)
etc. will be hindered. Various inorganic salts, EDTA or PCMB, PH6.9
The final concentration was adjusted to 1mM using 20mM Tris-HCl buffer, and the residual activity was measured after incubation at 37°C for 20 minutes.
The second value is a relative value with no additives as 100%.
Shown in the table.

[Table] In addition, this lipase is significantly inhibited by sodium dodecyl sulfate, sodium dodecylbenzene sulfate, etc., and activated by bile salts such as sodium cholate, polyoxyethylene sorbitan monooleate, etc. Various surfactants were prepared to a final concentration of 0.4% by weight using 20mM Tris-HCl buffer with pH 6.9, and the residual activity was measured after incubation at 37°C for 20 minutes. Relative values are shown in Table 3, with 100% being the case.

[Table] (8) Molecular weight: The molecular weight determined by gel filtration method is
It was about 30,000. Measurement is carried out using "Sephadex G"
-75'' (manufactured by Pharmacia). (9) Amino acid composition: After hydrolyzing this lipase at 110°C for 24 hours using 6N hydrochloric acid, the amount of each amino acid was measured using an automatic amino acid analyzer. Note that tryptophan was measured by ultraviolet absorption method. The results are shown in Table 4 below. The number of residues in one molecule was calculated based on the molecular weight of 30,000.

【table】

【table】

[Table] Table 5 below shows a comparison of the characteristics of the lipase of the present invention and conventional lipases derived from microorganisms.

[Table] * Effects of sodium deoxycholate.
In Table 5, Comparative Enzyme No. 1 and Tokukosho
Alcaligenes Meito PL- described in No. 58-36953
Lipase derived from No. 266 and Comparative Enzyme No. 3
Comparative Enzyme No. 4 is the lipase derived from Achromobacter Meito-AL-865 described in Japanese Patent Publication No. 49-32080, and Comparative Enzyme No. 4 is the lipase derived from Pseudomonas steutzeri described in Japanese Patent Publication No. 44-10754. enzyme
No. 5 is a lipase derived from Pseudomonas fratusi described in JP-A-48-88278, and comparative enzyme No. 6 is a lipase derived from Pseudomonas mephiteica varietus lipolyteica described in JP-A No. 50-25553. show. As is clear from Table 5, the lipase of the present invention is
It differs from other lipases in terms of molecular weight and is clearly distinguishable. In addition, it differs from lipases No. 4 and 5, whose molecular weights are not listed, in terms of optimum pH, and is clearly distinguishable. From the above points, the lipase of the present invention is different from conventionally known lipases and is recognized as a novel lipase. The lipase of the present invention can be obtained by culturing a microorganism belonging to the genus Alcaligenes that produces the lipase in a medium,
It can be suitably obtained by accumulating the lipase in a culture solution and collecting it. The present invention relates to a method for producing such a novel lipase and the microorganism. A preferred example of the above microorganisms is the f-B-24 strain, which is a new strain belonging to the genus Alcaligenes that was discovered in soil by the present inventor. The f-B-24 strain has been deposited with the National Institute of Microbial Technology, Agency of Industrial Science and Technology, and the accession number is FAIKEN BIKORI NO. 9715. The f-B-24 strain has the following mycological properties. (a) Morphology The shape of the bacteria grown on the broth agar medium is rod-shaped, and the size is 0.7 to 0.8 x 1.7 to 2.0 μm. It is motile. There are no polar flagella. Does not form spores. In addition, the Gram staining property is negative, and the acid fasting property is negative. (b) Growth status (1) Broth agar plate culture: Produces translucent, milky white colonies. The surface of the colony is smooth, the periphery is circular, the cross section is convex, and there is no pigment production. (2) Broth agar slant culture: Grows well. (3) Broth liquid culture: Grows well. The medium remains suspended even after standing for 1 hour, and there is no precipitate at the bottom. The surface is foamy. (4) Meat juice gelatin puncture culture: Growth is normal. Liquefaction is funnel-shaped. (5) Litmus milk: Slightly alkaline. Causes peptonization and liquefies. (c) Physiological properties In this section, + indicates positive or used, and - indicates negative or not used. (1) Nitrate reduction: + (2) Denitrification reaction: - (3) MR test: - (4) VP test: - (5) Indole production: - (6) Hydrogen sulfide production: - (7) Hydrolysis of starch: + (8) Use of citric acid Coser's medium: + Christensen's medium: + (9) Use of inorganic nitrogen sources Nitrate: + Ammonium salt: + (10) Urease test: + (11) Oxidase Test for: + (12) Hydrolysis of gelatin: + (13) Test for catalase: + (14) Growth range Grows in the pH range of 4.4 to 8.8. Also, 41℃
grow until. (15) Sodium chloride tolerant Grows in medium containing 2 wt% NaCl (16) O-F test: oxidative (17) Sugar utilization D-arabinose, D-xylose, D-
Glucose, D-mannose, D-fructose, D-galactose, D-sorbit,
Maltose, satucalose, lactose, trehalose, mannitol, glycerose,
Salicin, dextrin, cellulose, raffinose, inosine, cellobiose, starch, and α-methyl glucoside were all used, and acid production was observed in all of them. At this time, no gas generation was observed in any case. Based on the above-mentioned findings, classification is made according to Versey's identification criteria and the report by Iizuka et al. (Journal of General and Applied Microbiology, Vol. 9, pp. 73 and 83, 1963). It is recognized that it belongs to the genus Alcaligenes. As mentioned above, a strain that produces lipase similar to this strain is Alcaligenes Meito PL-266 (comparative strain No.
1), Achromobacter Meito-AL-865 (comparative strain No. 2) described in Tokuko No. 49-32080, Tokuko Akira
Pseudomonas steutzeri (comparative strain No. 3) described in No. 44-10754, Pseudomonas fratusi (comparative strain No. 4) described in JP-A-48-88278,
Pseudomonas mephiteica varietus lipolyteica (comparative strain No. 5) described in Japanese Patent Publication No. 50-25553 can be mentioned, but as is clear from the differences in the physiological properties of each strain shown in Table 6 below, This strain is clearly distinguished from these strains.

[Table] (Note) +: Positive. −: Negative.
As mentioned above, f-B belonging to the genus Alcaligenes
Strain -24 is clearly distinguished from known strains and is recognized as a new strain of the genus Alcaligenes. It goes without saying that the strain used in the present invention includes not only the f-B-24 strain itself, which belongs to the genus Alcaligenes, but also, for example, both natural and artificial mutant strains of this strain. Next, the culture of the lipase-producing bacteria of the present invention and the recovery of lipase will be described. Regarding the composition of the culture medium, any medium that is used by ordinary microorganisms and can be used by this bacterium can be used. Examples of the carbon source include glucose, maltose, starch, dextrin, etc., and examples of the nitrogen source include soybean flour, defatted soybean flour, peptone, meat extract, yeast extract, cornstap liquor, and the like. Additionally, inorganic salts such as potassium phosphate, magnesium phosphate, calcium phosphate, sodium chloride, magnesium sulfate, etc. are usually added to the medium. Further, as necessary, various organic substances, inorganic substances, antifoaming agents, etc. necessary for bacterial growth or enzyme production can be added to the medium. In particular, from the standpoint of inducing lipase production, olive oil, rapeseed oil, soybean oil,
It is preferable to add various fats and oils such as coconut oil, corn oil, and fish oil. As a preferred basal medium,
For example, a medium containing peptone, yeast extract, monopotassium phosphate, sodium chloride, and magnesium sulfate to which various oils and fats are added can be used. Cultivation is carried out by sterilizing a medium containing the above-mentioned components by a conventional method and inoculating the culture medium with the present bacterium. Cultivation is best carried out under aerobic conditions, and the culture temperature is usually between 20 and 40℃.
A temperature of about .degree. C. is suitable, and a temperature of about 28 to 30.degree. C. is particularly preferable.
The appropriate culture time is usually about 1 to 5 days.
Lipase activity usually reaches its maximum in about 40 to 60 hours. Separation and purification for recovering lipase secreted and accumulated in the culture solution can be carried out according to conventional methods.
For example, after the culture is completed, the bacterial cells are removed by centrifugation, the supernatant is salted out with a salt such as ammonium sulfate, and celite is added thereto. The precipitate is defatted with cold acetone, extracted with a buffer solution, and desalted by dialysis to obtain a crude enzyme. Furthermore, this crude enzyme can be purified using separation and purification means known in the art, such as ion exchangers, gelling agents, adsorbents, etc. as appropriate. The lipase of the present invention can be purified in this way, and the production of the enzyme will be explained in more detail in Examples below. Effects of the Invention According to the present invention, the remarkable effects of providing a novel lipase that efficiently decomposes castor oil, a method for easily producing lipase by culturing the lipase-producing microorganism, and the microorganism can be obtained. Moreover, this makes it possible to easily produce ricinoleic acid, which in turn enables a wide range of effective uses of castor oil. Examples Hereinafter, the present invention will be explained in more detail with reference to Examples. Example 1 2% by weight of olive oil, 4% by weight of peptone, 0.05% by weight of yeast extract, 0.1% by weight of monopotassium phosphate,
Magnesium sulfate 0.02% by weight and sodium chloride
Pour 100 ml of liquid medium containing 0.1% by weight into a 500 ml Sakaguchi flask, sterilize it at 110°C for 20 minutes, and then
Five cells of Alcaligenes f-B-24 strain (Feikoken Bacterial Serial No. 9715) precultured at 30°C for 16 hours were
ml was inoculated and cultured at 30°C in a reciprocating shaking incubator with an amplitude of 7 cm at 120 times per minute. Culture was stopped at 48 hours when lipase activity reached its peak. The lipase activity of this culture solution was 105 U/ml. Moreover, its specific activity was 6U/ml/ _OD280 . After the culture was completed, the culture solution was centrifuged at 12,000 rpm for 20 minutes to remove the bacterial cells and obtain a supernatant. This supernatant liquid
After saturated with 0.6 ammonium sulfate and adding Celite to it, the precipitate was degreased with cold acetone, and then 20 m
Extraction was performed with M Tris-HCl buffer (PH6.9).
The lipase activity of this crude enzyme solution was 455 U/ml. In addition, its specific activity is 114U/ml/OD ₂₈₀
It was hot. After dialysis of this crude enzyme solution, it was treated with DEAE-cellulose (manufactured by Braun) to obtain the enzyme contained in the non-adsorbed portion, and the lipase activity was
It was 198U/ml. In addition, its specific activity is
It was 221U/ml/OD ₂₈₀ . Furthermore, when dialyzed against 20mM Tris-HCl buffer (PH8.3), adsorbed on DEAE-Toyopearl 650M (manufactured by Tosoh Corporation), and eluted with a NaCl gradient (0→0.2M), at a salt concentration of 50mM, A single peak appeared, indicating activity. Lipase activity was 293 U/ml. In addition, its specific activity is 2437U/ml/OD ₂₈₀
It was hot. The obtained lipase showed a single band in disk electrophoresis using polyacrylamide gel and SDS-polyacrylamide gel. Example 2 In place of 2% by weight of olive oil in the medium of Example 1,
Culture was carried out in the same manner as in Example 1 using 1% by weight of rapeseed oil. The peak of lipase activity was reached at 56 hours. Lipase activity after 56 hours of culture was 104U/
It was hot in ml.

[Brief explanation of the drawing]

FIG. 1 is a graph showing the relationship between the activity of the lipase of the present invention and PH. Figure 2 shows the lipase of the present invention.
It is a graph showing PH stability. FIG. 3 is a graph showing the relationship between the activity of the lipase of the present invention and temperature. FIG. 4 is a graph showing the temperature stability of the lipase of the present invention. In each figure, □ indicates that the measurement was performed using a citrate buffer, ◯ indicates that a phosphate buffer was used, and ● indicates that the measurement was performed using a glycine buffer.

Claims (1)

[Scope of Claims] 1. Castor oil having a molecular weight of approximately 30,000 (by gel filtration method), an optimum pH of around PH7.0, and an activity of 100% when olive oil is used as a substrate. A novel lipase characterized by a relative activity of 107% when used as a substrate. 2. A method for producing the lipase according to claim 1, which comprises culturing a microorganism belonging to the genus Alcaligenes that produces the lipase in a medium, accumulating the lipase in the culture solution, and collecting the same. 3. The manufacturing method according to claim 2, wherein the microorganism is Alcaligenes f-B-24 (Feikoken Bacterial Serial No. 9715). 4. Alcaligenes f-B-24 having the ability to produce lipase according to claim 1 (Feikoken Bacterial Serial No. 9715)
Microorganisms that are.