JPH01187085A - Novel lipase, production thereof and microorganisms producing same - Google Patents

Abstract

PURPOSE:To obtain the subject novel lipase capable of readily improving the degradation efficiency for e.g., castor oil, by making a culture of Alcaligenes microorganismus capable of producing lipase in a culture medium. CONSTITUTION:Alcaligenes f-B-24 (FERM-9715) strain capable of producing lipase is put to culture in a culture medium to accumulate lipase in the resultant culture solution. Thence, this lipase is collected and purified to obtain the objective lipase having the following characteristics: (1) molecular weight: ca. 30,000 (determined by the gel permeation method) (2) optimum pH: ca. 7.0 (3) optimum temperature for action: ca. 60 deg.C (4) substrate specificity: if the activity is assumed as 100% when olive oil is used as the substrate, the relative activity is 107% when castor oil is used as the substrate (5) inhibition and activation: inhibited by Fe<3+>, Hg<2+>, EDTA, etc., while activated by BaCl2, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to a novel lipase, a method for producing the lipase, and a novel microorganism.

Background of the Invention Lipases are used as digestive agents, for decomposing fats and oils in soap production, and as enzymes added to detergents.

However, conventionally known lipases have the disadvantage that the rate of hydrolysis of castor oil is very low. That is, castor oil has water a! as its constituent fatty acid! Most of the fat is ricinoleic acid, which has groups, and it is known that because of this hydroxyl group, the rate of hydrolysis by various lipases is lower than that of other fats and oils.

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 is currently limited.

Means for Solving the Problems In view of the current state of E2, the inventor of the present invention aimed to easily produce ricinoleic acid with a high hydrolysis rate of castor oil, and thereby 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, it was discovered that strain f-8-24, a new bacterial strain belonging to the genus Alcaligenes, produces a new lipase that efficiently decomposes castor oil.Based on this, further research led to the completion of the present invention.

That is, the present invention uses castor oil as a substrate, which has a molecular weight of about 30,000 (by gel filtration method), has an optimum pH around pH 7.0, and has an activity of 100% when olive oil is used as a substrate. A novel lipase characterized by having a relative activity of 107%, 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. The present invention relates to a method for producing the lipase, and a microorganism that belongs to the genus Alcaligenes and is characterized by having the ability to produce the lipase.

In this specification, the lipase titer refers to the polyvinyl alcohol emulsification method of Yamada et al. (Journal of the Japanese Society of Agricultural Chemistry, Vol. 36,
p. 860, 1962).

That is, 25 volumes of pharmacopoeia olive oil were added to 75 volumes of a 2% aqueous solution of polyvinyl alcohol, and homogenized for 3 minutes.
9 olive oil emulsion 51, pH 7; 0.1
M phosphate buffer 4-1 and enzyme solution (test solution) 11 are mixed to prepare a reaction solution. This reaction solution was reacted at 37°C for 20 minutes, and immediately after the reaction was completed, acetone-ethanol (1:
1. Volume ratio) Mixture 201 was added to stop the reaction, and the resulting fatty acid was measured at 0.00% using phenolphthalein as an indicator.
Titrate with 05N NaOH.

The potency of 1 unit (hereinafter referred to as U) of lipase activity is 1
It indicates the amount of enzyme that liberates 1 micromole of fatty acids per minute. Further, the number of units per absorbance of 280na+ per 1-1 (LJ/1f10D) is defined 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 to produce fatty acids and glycerin.

(2) Substrate specificity: Using 1 g of each of various animal and vegetable oils and fats as a substrate, add 0.5+el (140 U) of enzyme solution in the presence of 0.1 M phosphate buffer 9-: pH 7.0,
The enzyme activity for various substrates was measured by the hydrolysis rate when the reaction was carried out at 37°C for 72 hours at 120 cycles per minute.
Table 1 shows the relative activity based on 00%.

Table 1 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.

In addition, the hydrolysis rate (absolute value) is calculated from the saponification value and acid value of the oils and fats: castor oil is 82%, olive oil is 77%,
It is 73% coconut oil.

For comparison, commercially available Rhizopus nivenus (R, n1v
enus) derived lipase (manufactured by Nagase Seikagaku Kogyo ■,
Lot, 6270113) was investigated in the same way, and the activity when olive oil was used as a substrate was 10%.
The relative activity using castor oil as a substrate when it is 0% is 5
It was 2%.

(3) Optimum pH: around pH 7.0.

Figure 1 shows the relationship between lipase activity and pH when olive oil is used as a substrate. In FIG. 1, the activity at pH 3 to 6 was measured using a citrate buffer, the activity at pH 6 to 8 was measured using a phosphate buffer, and the activity at pH 8 and 5 to 9 was measured using a glycine buffer.

(4) pH stability: When left at 4℃ for 22 hours, the pH
The residual activity is 100% in the range of 9.0 to 10.0,
It is stable.

After standing at 4°C for 22 hours, the relationship between the residual lipase activity and the pH at the time of standing when olive oil was used as a substrate is shown in FIG. In Figure 2, citrate buffer was used for the treatment at pH 4-5, phosphate buffer was used for the treatment at pH 6-8, and phosphate buffer was used for the treatment at pH 9-1.
Treatments 1 were performed using glycine buffer, respectively.

(5) The optimum temperature is around 260°C.

FIG. 3 shows the relationship between lipase activity and temperature at pH 7.0 when olive oil is used as a substrate.

(6) Temperature stability: 30°C at 35°C at pH 7.0
100% activity remained after incubation for 40 min.
76% activity remains after incubation at 30 minutes at 45°C, and is completely inactivated at 45°C for 30 minutes.

FIG. 4 shows the relationship between residual lipase activity and treatment temperature at DH7.0 when olive oil is used as a substrate.

(1) Inhibition and activation: This lipase is activated by F03+, 2+ HQ, EDTA (ethylenediaminetetraacetic acid), PC
It is inhibited by MB (parachloromercuric benzoic acid), etc.

Various inorganic salts, EDTA or PCMB were adjusted to a final concentration of imH using 20 mH Tris-HCl buffer at pH 6.9.
After incubating at 37°C for 20 minutes, the residual activity was measured at 100% when no additive was added.
Table 2 shows relative values expressed as %.

Table 2 In addition, this lipase has been severely criticized by sodium dodecyl sulfate, sodium dodecylbenzene sulfate, etc.
It is also activated by bile salts such as sodium cholate, polyoxyethylene sorbitan monooleate, etc.

Various surfactants were prepared using 20-H Tris-salt 1lIII solution with DH6,9 so that the final concentration was 0.4%, and the residual activity after incubation at 37°C for 20 minutes was determined. The measurement results are shown in Table 3 as relative values, with the value of no additive being 100%.

Table 3 (8) Molecule ff1 = Molecular weight determined by gel or legal method is
It was about 30,000. The measurement was carried out using [Sephadex G-
75J (manufactured by Pharmacia).

(9) Amino acid composition: After hydrolyzing Honsubase with 6N hydrochloric acid at 110°C for 24 hours, each amino acid composition was analyzed using an automatic amino acid analyzer. l1ffi was measured. Note that tryptophan was measured by ultraviolet absorption method.

The results are shown in Table 4 below. In addition, the number of residues in one molecule is
This is calculated based on the fact that the molecule is 130,000.

Table 4 A comparison of the characteristics of the lipase of the present invention and conventional lipases derived from microorganisms is shown in Table 5 below.

In Table 5, comparative enzymes 1 and 2 are
Alcaligenes Meito PL-266 described in No. 6953
The comparative enzyme NQ3 is Achromobacter Meito described in Japanese Patent Publication No. 32080/1983.
The lipase derived from AL-865 was used, the comparative enzyme 11Q4 was the lipase derived from Pseudomonas steulaeri described in JP-A-44-10754, and the comparative enzyme NQ5 was the lipase derived from Pseudomonas fludge described in JP-A-48-88278. , the comparative enzyme Nα6 was obtained from the Special Publication Publication in 1984-25.
553 are shown, respectively.

As is clear from Table 5, the lipase of the present invention differs from other lipases in terms of molecular weight and is clearly distinguishable. Also,
Molecule 9 is different from undescribed lipases 8Q4 and 5 in terms of optimum pH and is clearly distinguishable. From the above points, the lipase of the present invention is recognized as a novel lipase, unlike conventionally known lipases.

The lipase of the present invention can be suitably obtained 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 the lipase. The present invention also relates to a method for producing such a novel lipase and the microorganism.

A preferred example of the microorganisms mentioned above is f-8, which is a new strain belonging to the genus Alcaligenes discovered by the present inventor in soil.
-24 strains can be mentioned.

The f-8-24 strain has been deposited with the National Institute of Microbial Technology, Agency of Industrial Science and Technology, and the accession number is FAIKEN BACKGROUND No. 9715.

The f-8-24 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. Also,
Durham staining is negative and acid fastness is negative.

(b) Growth status (1) Broth agar plate culture Two-handed, producing transparent, milky-white colonies. The surface of the colony is smooth, the periphery is circular,
The cross section is convex and no pigment is produced.

(2) Gravy agar slope ring! i: 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) Lidmus 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 knee (3) MR test knee (4) VP test knee (5) Indole formation knee (6) Hydrogen sulfide formation knee (7) Starch hydrolysis: + ( 8) Utilization of citric acid Coser's medium: ・← Christensen's medium: + (9) Utilization of inorganic nitrogen source Nitrate: + Ammonium salt: + (10) Urease test: + (11) Oxidase test 2 (12) Gelatin hydrolysis: + (13) Catalase test: + (14) Growth range: Grows in the pH range of 4, 4 to 8.8. It also grows up to 41°C.

(15) Grows in a medium containing sodium chloride resistant Na (16) 0-
F test: ionic (11) sugar utilization D-arabinose, D-xylose, D-glucose,
D-mannose, D-fructose, D-galactose, D-sorbitol, maltose, sucrose, lactose, trehalose, mannitol, glycerol, salicin, dextrin, cellulose, raffinose, inosine, cellobiose, starch and α-methyl glucoside. In both cases, acid production was observed. At this time, no gas generation was observed in any case.

Based on the above findings, we have established the identification criteria for Vaseeds and the report by Iizuka et al. (Journal of General and Applied Microbiology, Vol. 9, No. 73 and No. 8).
3, 1963), it is recognized that it belongs to the genus Alcaligenes.

As mentioned above, strains that produce lipase in the same way as this strain include Alcaligenes octasaccharide PL-266 (comparative strain 1) described in Japanese Patent Publication No. 58-36953 (comparative strain 1), Achromobacter Meito
AL-865 (comparative strain Nα2), Special Publication No. 1973
Pseudomonas steulaeri (comparative strain number 3) described in No. 754, Pseudomonas fludge (comparative strain Nn4) described in JP-A-48-88278, and JP-A No. 1983-
Pseudomonas mephiteica described in No. 25553
The present strain is clearly distinguished from these strains, as is clear from the differences in the physiological properties of each strain shown in Table 6 below.

As mentioned above, strain f-8-24, which belongs to the genus Alcaligenes, 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-8-24 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 used by ordinary microorganisms and available in the home country 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, various organic substances, inorganic substances, antifoaming agents, etc. necessary for bacterial growth or M element production can be added to the medium as necessary. In particular, from the point of view of guiding the production of lipase, olive oil,
It is preferable to add various oils and fats such as rapeseed oil, soybean oil, coconut oil, corn oil, and fish oil. Preferred basic media include, for example, a medium containing peptone, yeast extract, monopotassium phosphate, sodium chloride, and magnesium sulfate, to which various oils and fats are added.

Cultivation is carried out by sterilizing a medium containing the above-mentioned components by a conventional method and inoculating it with Honkan. Cultivation is preferably carried out under aerobic conditions, and the culture temperature is usually about 20 to 40°C.
Particularly preferred is about 28 to 30°C. Culture time is usually 1~
Approximately 5 days is appropriate, and lipase activity is usually 40~
It reaches its maximum in about 60 hours.

Separation and purification for recovering the 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 precipitated filtrate 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 appropriate separation and purification means known in the art, such as ion exchangers, gel filtration agents, adsorbents, etc. 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, a novel lipase that efficiently decomposes mustard oil, a method for easily producing lipase by culturing the lipase-producing microorganism, and the remarkable effect of providing 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 A liquid medium containing 2% by weight of olive oil, 4% by weight of peptone, 1% by weight of Fs Matrix Kiss 005fliff, 0.1% by weight of monopotassium phosphate, 0.02% by weight of magnesium sulfate, and 0.1% by weight of sodium chloride. 100-1 to 500
Cells of Alcaligenes sp. f-8-24 (Feikoken Bacterial Serial No. 9715) were placed in a 1-volume Sakaro flask, sterilized at 110°C for 20 minutes, and precultured at 30°C for 16 hours. 5-1 inoculation at 30℃, 120 times per minute, amplitude 7
The cells were cultured in a 0-reciprocating shaker incubator. Culture was stopped 48 hours after lipase activity reached its peak. The lipase activity of this culture solution was 105 U/al. Further, its specific activity was 6U/1f100230.

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 is 0.6
The mixture was saturated with ammonium sulfate, Celite was added thereto, the precipitated filtrate was defatted with cold acetone, and then extracted with 201M Tris-HCl buffer (pH 6,9). The lipase activity of this crude enzyme solution was 455 LI/ml. Moreover, its specific activity was 114 U/m110D28 (,).

After dialysis of this crude oxygen 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 198 U/ml. Further, its specific activity was 221 LJ/■110D28o. Furthermore, 201N Tris-HCl buffer (pH 8.3
), DEA [-Toyobar 650M (Tosoh ■
NaCl gradient (0→0.2M)
), a single peak appeared at a salt concentration of 50-H, indicating activity. Lipase activity is 2
It was 93U/-1.

Moreover, its specific activity is 2437 U/m11002B.

Met.

The obtained lipase was tested on polyacrylamide gel and 5D
In disk electrophoresis using S-polyacrylamide gel, all showed a single band.

Example 2 Culture was carried out in the same manner as in Example 1 except that 1% by weight of rapeseed oil was used in place of 2% by weight of olive oil in the medium of Example 1. The peak of lipase activity was at 56 hours. The lipase activity after 56 hours of culture was 104 U/i+l.

[Brief explanation of the drawing]

FIG. 1 is a graph showing the relationship between the activity and pH of the lipase of the present invention. FIG. 2 is a graph showing the pH stability of the lipase of the present invention. Figure '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, the mouth is citric acid! 11! i solution, O is phosphate buffer,
* indicates that the measurement was performed using the respective glycine buffer. (Above) Agent: Hideo Saegusa, Patent Attorney Figure 1: Lipase activity and pH-1f1 pH retention Figure 2: Lipase cr>pH9 Qualitative pH Figure 3: Lipase Sga1 staining. Viewing temperature: 1 (°C) Figure 4 Reparse's Mil, Degree foot surname SL R (''C)

Claims (5)

[Claims] (1) The molecular weight is approximately 30,000 (by gel filtration method), the optimum pH is around pH 7.0, and the activity when olive oil is used as a substrate is 100%, using castor oil as a substrate. A novel lipase characterized by a relative activity of 107%. (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 production method according to claim 2, wherein the microorganism is Alcaligenes f-B-24 (Feikoken Bacterial Serial No. 9715). (4) A microorganism belonging to the genus Alcaligenes and having the ability to produce the lipase according to claim 1. (5) Alcaligenes f-B-24
715). The microorganism according to claim 4.