JP2000060544A - New alkali lipase and its production - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an alkali lipase having strong activity even in the presence of a surface active agent having high concentration under alkaline circumference. SOLUTION: This alkali lipase has the following physicochemical properties: (a) Action and substrate specificity: acting on glyceride such as olive oil and hydrolyzing the ester bond of the glyceride and hydrolyzing ester bond of various kinds of ester compounds to produce alcohols and acids; (b) Optimum pH: 10.0-11.0; (c) Molecular weight: about 63,000 daltons; (d) Inhibition: keeping >=70% relative activity even in the presence of various kinds of surface active agents having 1% concentration and not especially inhibited by PMSF.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel alkaline lipase, a method for producing the same, and an alkalophilic actinomycete capable of producing the alkaline lipase.

[0002]

2. Description of the Related Art Alkaline lipase is an enzyme that specifically hydrolyzes ester bonds of various lipids and various ester compounds in the alkaline region, and has recently been incorporated into laundry detergents and the like. It is known that lipase itself is widely produced by microorganisms such as bacteria, yeasts and filamentous fungi, but most of them have an optimum pH around neutrality and strongly inhibit their activity in the presence of a surfactant. To be done. Also,
There are few reports that alkaline lipases are produced even for alkalophilic bacteria, which has been actively researched in recent years (Horikoshi Horiki: Extreme microorganisms, p62-63, Kodansha), and alkaline alkase is produced for alkalophilic actinomycetes. It is completely unknown whether to do it. On the other hand, a lipase desirable as a lipase for formulating a detergent is one which maintains a sufficient activity even in a detergent solution under washing conditions. That is, the current state of detergents, etc.
Since the pH is in the alkaline region, alkaline lipase is the most suitable enzyme to be added to these. Further, it is desired to develop an alkaline lipase that maintains a sufficient activity even in the presence of various surfactants, which are the weak points of lipase.

[0003]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a novel alkaline lipase which has an optimum pH in the alkaline region and is resistant to various surfactants. Another object of the present invention is to provide a method for producing the alkaline lipase using a microorganism that produces the alkaline lipase.

[0004]

[Means for Solving the Problems] From the above viewpoints,
The present inventors have searched for microorganisms that produce alkaline lipase having a strong lipolytic activity in an alkaline environment, mainly on alkalophilic actinomycetes, and Japanese Patent Application No. 10-16344.
TOT, a novel alkaline protease-producing bacterium described in No. 1
The O-9805 strain (FERM BP-6359) was found to efficiently produce the desired novel alkaline lipase by aerobic culture, and the present invention has been completed. That is, the present invention provides an alkaline lipase having the following physicochemical properties. (a) Action and substrate specificity: It acts on glycerides such as olive oil to hydrolyze its ester bond. It also hydrolyzes the ester bonds of various ester compounds to produce alcohol and acid. (b) Optimum pH: pH is 10.0 to 11.0. (c) Stable pH: It is stable at pH 5.5 to 12.0 under the condition of 37 ° C. for 1 hour. (d) Optimum temperature: 70-75 ℃, 50% even at 100 ℃
The above relative activities are shown. (e) Stable temperature: It is stable up to 55 ° C under conditions of pH 10.0 and 10 minutes, but shows residual activity of 60% even at 70 ° C. (f) Molecular weight: About 63,000 daltons by SDS-electrophoresis. (g) Isoelectric point: pI 3.8-4.5 by isoelectric focusing. (h) Inhibition: Tween80, Span80 or SDS surfactant 1%
Even if it is allowed to act at a concentration, 70% or more of the activity is retained. 0.1% sodium hypochlorite did not inhibit at all,
Even at 0.5% concentration, about 90% of activity is retained. It is not inhibited by the enzyme inhibitors EDTA, DFP, PMSF, PCMB or iodoacetic acid in an aqueous system or a 10-20% ethanol aqueous solution. Further, the method for producing the novel alkaline lipase according to the present invention, a step of culturing a microorganism belonging to the alkalophilic Streptomyces genus and having the ability to produce the alkaline lipase, and the novel alkaline lipase from the culture obtained in the step And a step of separating the.

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION The alkaline lipase according to the present invention strongly hydrolyzes fat and ester compounds even in a strong alkaline environment of pH 10 or higher and in the presence of a high concentration of a surfactant. Therefore, it is easily mixed with detergents for clothes and tableware for the purpose of enhancing the cleaning effect, and it is easy for dirt containing fat such as bathtubs, bathtubs, bathroom drains, piping for circulating bathtubs, toilet bowls, and vanities to drain. It is extremely effective when added to a local detergent. Furthermore, the alkaline lipase can be used in a wide range of industrial fields including pharmaceuticals, reagents and the like. Further, the strain according to the present invention,
Since the above alkaline lipase is efficiently secreted out of the cells, it is advantageous in that it can be produced with high efficiency in a simple process. As the above-mentioned detergent composition, the above-mentioned alkaline lipase alone or known detergent components, for example, various surfactants, various builders (chelating agents, alkaline agents, etc.), anti-soil redeposition agents, bleaching agents, other than the above alkaline lipase These enzymes (other lipases, proteases, cellulases, etc.), fragrances, pigments and the like can be used alone or in combination of two or more. The form of the detergent composition or the like can be any form such as liquid, slurry, powder, granules and tablets. The detergent composition is used for various purposes such as clothes, dish washing, kitchen and the like, but it is preferably used as a dishwasher detergent and a kitchen detergent.

The novel alkaline lipase according to the present invention can be produced by using a microorganism. Particularly preferably,
The alkaline lipase according to the present invention is a Streptomyces sp. TOT described in Japanese Patent Application No. 10-163441 as a novel alkaline protease producing bacterium.
It is produced by strain O-9805 (FERM BP-6359). This strain is a mutant strain obtained by subculturing the TOTO-9305 strain (FERM P-13640). This strain is an alkalophilic actinomycete and has exactly the same mycological properties as the parent strain TOTO-9305, as shown in Tables 1 and 2 below. Since this strain is an alkalophilic microorganism and does not grow in a normal neutral medium or has a very poor growth, 0.5% Na ₂ was used when examining the mycological properties in Tables 1 and _2. An alkaline medium supplemented with CO ₃ was used. This strain
From the results of Table 1, the TOTO-9805 strain was morphologically actinomycete,
It has the characteristics of Streptomyces. Also, on the cell wall
This strain is classified as a strain belonging to the genus Streptomyces because it contains LL-diaminopimelic acid. The TOTO-9805 strain belongs to the white series due to the color of aerial mycelia and spores on various agar media, the spore surface is smooth, the spore chain is almost straight, and no melanin pigment is formed. And so on.

[0007]

[Table 1] Table 1 Various morphological characteristics of TOTO-9805 strain Physiological properties Assimilation of each carbon source

[0008]

[Table 2] Table 2 Growth status of each medium * 1 No melanin

This strain is different from the parent strain TOTO-9305 in that it produces a completely different protease. Therefore, this strain TOTO-9805 was judged to be a mutant strain of the parent strain TOTO-9305, and Streptomyces sp.
(Streptomyces sp. TOTO-9805). The strain has been deposited at the Institute of Biotechnology, Institute of Industrial Science and Technology under the deposit number (FERM BP-6359). Culture conditions Since the above strain is an alkalophilic actinomycete, it is necessary to culture it in an alkaline region. The method for making the medium alkaline is not particularly limited, and for example, sodium carbonate or sodium hydrogencarbonate may be added to a normal medium. As the carbon source, lipids such as olive oil, fatty acids such as stearic acid, sugars such as glucose, glycerin, soluble starch, and cellulose can be used. As a nitrogen source, nitrate,
Inorganic substances such as ammonium salts, urea, peptone, dried yeast, yeast extract, soybean powder, corn steep liquor, casein, meat extract, amino acids and the like are used. In addition to these carbon sources and nitrogen sources, various inorganic salts such as magnesium salts, potassium salts, sodium salts, and phosphates may be added as necessary. As an alkali source to be added to the medium, about 0.5 to 2.0% sodium carbonate, carbonate such as sodium bicarbonate or sodium hydroxide, ammonia can be used, and the pH of the medium is 8.0 to 11.0
The degree is desirable. Culture should be performed in such a medium at the culture temperature.
It is carried out at 20 to 40 ° C., preferably 25 to 37 ° C. for 2 to 5 days while aerobically stirring or shaking. The novel alkaline lipase according to the present invention is mainly secreted and accumulated in the culture medium under the above-mentioned culture conditions.

Collection of Enzyme In order to collect and purify the enzyme of the present invention from the above culture solution, known purification methods can be used alone or in combination. Since the present enzyme is mainly secreted outside the cells (in the culture solution), a crude enzyme solution can be easily obtained by removing the cells by filtration or centrifugation.
This crude enzyme is further purified by known purification methods such as salting out with ammonium sulfate; precipitation with an organic solvent such as methanol, ethanol or acetone; ultrafiltration; gel filtration chromatography; ion exchange chromatography; hydrophobic chromatography, affinity chromatography. Nite chromatography, other various chromatography, alone or in combination,
It can be purified. The preferred purification method is as follows. First, 60% saturated ammonium sulfate is added to the culture filtrate for salting out, and the obtained precipitate is dissolved in a buffer solution. Then
Ion exchange chromatography with DEAE-Toyopearl 650M (Tosoh Corporation), chromatography with hydroxyapatite (Wako Pure Chemical Industries), ion exchange chromatography with Mono-Q (FPLC, Pharmacia), Supe
By sequentially performing gel filtration with rdex 75 (FPLC, manufactured by Pharmacia), a purified enzyme uniform in SDS electrophoresis can be obtained.

Properties of the enzyme The properties of the alkaline lipase according to the present invention are as shown below. In addition, in the following, the activity measuring method means the following method. (Activity measuring method) The activity (esterase activity) measuring method using a synthetic substrate was adopted from the viewpoint of simplicity after confirming the lipase activity by the alkali titration method using olive oil which is the original substrate. That is, 2 ml of 2-butanol is added to 15 mg of para-nitrophenyl palmitic acid and dissolved. To this, deoxycholic acid and gum arabic were added to 0.
45m of 50 mM glycine buffer (pH 10.0) containing 1% and 0.2%
l was added to serve as a substrate. 0.1 ml of an appropriately diluted enzyme solution was added to 0.9 ml of the substrate, and the mixture was reacted at 37 ° C for 10 minutes. 0.2 M
After adding 0.5 ml of trichloroacetic acid to stop the reaction, 1
Color was developed by adding ml of 2 M sodium carbonate solution. This was filtered through a 0.2 μm filter, and the absorbance at 410 nm was measured. Enzyme titer: 1 U was defined as the amount of enzyme that liberates 1 μmol of para-nitrophenol in 1 minute.

(1) Action and Substrate Specificity It acts on glycerides such as olive oil to hydrolyze its ester bond. It also hydrolyzes the ester bonds of various ester compounds to produce alcohol and acid. (2) Optimum pH and stable pH Based on the above activity measurement method, the effect of pH on this enzyme was investigated. As a buffer solution, KCl / HCl buffer solution (pH 1.0
~ 2.0), glycine / HCl buffer (pH 2.5 to 3.5), acetate buffer (pH 4.0 to 5.5), phosphate buffer (pH 6.0 to 7.0), Tris hydrochloric acid buffer (pH 7.0 to 8.0) ), Borate buffer (pH8.0
~ 10.0), glycine / NaCl / NaOH buffer (pH 10.0-12.
0), KCl.NaOH buffer (pH 12.5-13.0) was used. Figure
1 shows the relative activity at each pH when the maximum activity is 100. From FIG. 1, it can be seen that the optimum pH of this enzyme is 10.0 to 11.0 at 37 ° C. Similarly, the pH stability of this enzyme is shown in FIG. Add 3 parts of this enzyme to the buffer at each pH.
After holding at 7 ° C for 1 hour, the activity was shown as the residual activity with the enzyme activity of untreated (left at 4 ° C for 1 hour) as 100.
From FIG. 2, the enzyme had a pH of 5.5 to 12.0 under the above treatment conditions.
It can be seen that it is stable in a very wide pH range up to.

(3) Optimum temperature and stable temperature The effect of temperature on the enzyme was examined according to the above-mentioned activity measuring method. FIG. 3 shows the relative activity at each temperature when the maximum activity is 100. From Fig. 3, the optimum temperature of this enzyme is 70.
It can be seen that the temperature is ~ 75 ° C. Furthermore, it was found that this enzyme shows a relative activity of 50% or more even at 100 ° C. Further, the present enzyme was added to 100 mM Tris-HCl buffer (pH 7.0) and kept under each condition in the range of 40 to 80 ° C for 10 minutes, and then the residual activity was measured. The results are shown in Fig. 4. From FIG. 4, it can be seen that this enzyme is stable up to 55 ° C. under the above conditions. Furthermore, this enzyme showed a residual activity of 60% or more even at 70 ° C. (4) Molecular weight The molecular weight of this enzyme was measured by SDS electrophoresis and found to be about 63,000 daltons. (5) Isoelectric point The isoelectric point of this enzyme was measured by the isoelectric focusing method, and the isoelectric point was about 3.8 to 4.5.

(6) Inhibition The influence of various metal ions on the activity was examined according to the above-mentioned activity measuring method. As a result, this enzyme was identified as Mg ²⁺ , Pb ²⁺ , Zn.
^It was moderately inhibited by the presence of ²⁺ ions. In addition, this enzyme was strongly inhibited by divalent and trivalent iron ions, like lipases other than this enzyme. On the other hand, Tween80 and Span8 are also inhibited by many lipases other than this enzyme.
With respect to 0 and SDS, 70% or more of the activity was maintained even at 1% concentration. In addition, a common enzyme inhibitor, EDTA
The effects of (ethylenediaminetetraacetic acid), iodoacetic acid, DFP (diisopropylfluorophosphate) and PMSF (phenylmethanesulphonylfluorite) on the activity of this enzyme were examined. 50 mM Tris-HCl buffer (pH
9.0), and ethanol was added to the same buffer solution to a concentration of 10% or 20% to dissolve in an ethanolic buffer solution, and the enzyme was added in an appropriate amount and treated at 37 ° C. for 5 minutes. Then, a fixed amount of the treated solution was sampled and its residual activity was measured according to the above-mentioned activity measuring method. As a result, this enzyme is ED in both aqueous and ethanol environments.
It was not inhibited by TA, iodoacetic acid, DFP, and PMSF. It is generally known that the active center of lipase is very similar to that of serine protease, and it is inhibited by PMSF especially in the alcoholic environment. On the other hand, since this enzyme was not inhibited by PMSF in any environment, it was revealed that this enzyme is an extremely specific alkaline lipase.

[0015]

The present invention will now be described in more detail with reference to the following examples, which should not be construed as limiting the invention thereto. Example 1 Preparation of Crude Enzyme Powder 100 ml of a preculture solution of Streptomyces sp. TOTO-9805 strain (37 ° C., shaking culture for 4 days) was mixed with stearic acid.
8%, corn steep liquor 0.5%, NaNO ₃ 0.1%, K ₂ HPO _{4
0.1%, MgSO 4 · 7H 2} O 0.05% and was added with separately sterilized N
Inoculate a small jar fermenter containing 4,500 ml of medium (pH 9.0) containing 1.0% of a ₂ CO _{3 and} incubate at 37 ° C for 4 days with aeration rate of 1 v / v / min and rotation speed of 200 rpm. went. After the culture was completed, the culture solution was filtered through Hyflo Super Cell to remove the cells. This gives about 3,900 crude enzyme solution of about 0.15 U / ml.
I got ml.

Example 2 Preparation of purified enzyme Streptomyces SP TOTO was placed in a small jar fermenter containing 10,000 ml of the same medium as in Example 1.
200 ml of preculture liquid of -9805 strain was inoculated. This is Example 1
After culturing in the same manner as above, the culture supernatant was centrifuged to 8,900 ml.
Got The lipase activity of this supernatant at pH 10.5 is 0.
It was 156 U / ml. Next, 60 mL of ammonium sulfate powder was added to this supernatant.
Add until it becomes% saturated, and leave it in a dark place at 5 ℃ for one day 8,000 r
The precipitate was collected by centrifugation at pm. The precipitate was dissolved in a small amount of distilled water and then filtered with a filter (Advantech Toyo, No. 1) to give a crude enzyme solution. The crude enzyme solution was desalted and buffer exchanged with PD10 (Pharmacia) which had been equilibrated with 20 mM Tris / HCl buffer (pH 8.0) in advance. DEAE-T equilibrated with this buffer
Adsorb the enzyme through an oyopearl 650M column and
Elution was performed with a M NaCl concentration gradient. The active fraction was desalted and buffer exchanged with PD10 equilibrated with 20 mM phosphate buffer (pH 8.0). Then, the active fraction was adsorbed by passing through a hydroxyapatite column equilibrated with the same buffer,
The adsorbed fraction was eluted by a concentration gradient method using a 20 to 200 mM phosphate buffer. The active fraction was replaced with 20 mM Tris-HCl buffer (pH 8.0) by PD10 and Mo equilibrated with the same buffer.
It was subjected to ion exchange chromatography with no-Q (FPLC). The adsorbed active fraction was eluted with a 0 to 1.0 M sodium chloride gradient method, further desalted with PD10, and then subjected to gel filtration with a Superdex 75 column (FPLC) equilibrated with the same buffer. The above series of purifications yielded 31 ml of purified alkaline lipase of about 1.70 U / ml. This fraction showed a single band and a single peak by SDS electrophoresis and gel filtration, respectively, and was confirmed to be homogeneous as an enzyme protein.

Formulation Example 1: Detergent for dishwasher / dryer 1 to 5 parts by weight of crude enzyme powder (1,000 U / g) of this enzyme, 10 to 30 parts by weight of sodium carbonate, 10 to 30 parts by weight of sodium sulfate, and sodium percarbonate 5-10 parts by weight, sodium citrate 1-5 parts by weight, polyoxyethylene alkyl ether (AE) 1-1
0 parts by weight-Effect-Cleaning of oil stains

Formulation Example 2: 1 to 5 parts by weight of kitchen detergent, crude enzyme powder (1,000 U / g), sodium straight chain alkylbenzene sulfonate (LA)
S) and sodium alkyl ether sulfate (A
ES) mixed surfactant 20 to 50 parts by weight, or up to 30 parts by weight up to 20 parts by weight, ethanol 1 to 5 parts by weight, carboxymethyl cellulose (CMC) 0.1 to 1.
0 parts by weight-Effect-Cleaning of oil stains

[Brief description of drawings]

FIG. 1 shows the optimum p of alkaline lipase according to the present invention.
It is a graph which shows H.

FIG. 2 shows stable p of alkaline lipase according to the present invention.
It is a graph which shows H.

FIG. 3 is a graph showing the optimum temperature of alkaline lipase according to the present invention.

FIG. 4 is a graph showing the stable temperature of alkaline lipase according to the present invention.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12R 1: 465) (72) Hideko Morimatsu 2-1-1 Nakajima, Kokurakita-ku, Kitakyushu, Fukuoka Ceramics Co., Ltd. (72) Inventor Yasuhiro Shimizu 4-19 Hie-cho, Kosai-cho, Koga-gun, Shiga Yamato Kasei Co., Ltd. (72) Inventor Atsushi Sugiyama 4-19 Hie-cho, Kosai-cho, Koga-gun, Shiga Yamato Kasei Incorporated (72) Inventor Akiyo Ueyama 4-19 Hie-cho, Kosai-cho, Koga-gun, Shiga Yamato Kasei Co., Ltd.

Claims (7)

[Claims] 1. An alkaline lipase having the following physicochemical properties. (a) Action and substrate specificity: acts on glyceride and hydrolyzes its ester bond. It also hydrolyzes the ester bonds of various ester compounds to produce alcohol and acid. (b) Optimum pH: The optimum pH of action is 10.0 to 11.0. (c) Stable pH: It is stable at pH 5.5 to 12.0 under the treatment condition of 37 ° C. for 1 hour. (d) Optimum temperature: The optimum action temperature is 70-75 ℃, 100 ℃
Also shows a relative activity of 50% or more. (e) Stable temperature: It is stable up to 55 ° C under the treatment condition of pH 10.0 for 10 minutes, but shows residual activity of 60% even at 70 ° C. (f) Molecular weight: About 63,000 daltons by SDS electrophoresis. (g) Isoelectric point: 3.8 to 4.5 by isoelectric focusing method. (h) Inhibition: Tween80, Span80 or SDS surfactant 1%
Even if it is allowed to act at a concentration, 70% or more of the activity is retained. 0.1% sodium hypochlorite did not inhibit at all,
Even at 0.5% concentration, about 90% of activity is retained. It is not inhibited by the enzyme inhibitors EDTA, DFP, PMSF, PCMB or iodoacetic acid in an aqueous system or a 10-20% ethanol aqueous solution. 2. The lipase according to claim 1, which is obtained from an alkalophilic actinomycete, Streptomyces. 3. Streptomyces sp.
omyces sp.) TOTO-9805 strain, The lipase according to claim 1. 4. A step of culturing a microorganism belonging to the alkalophilic Streptomyces genus and having an alkaline lipase-producing ability according to any one of claims 1 to 3, and a culture obtained in the step A method for producing alkaline lipase, which comprises the step of separating the alkaline lipase. 5. The production method according to claim 4, wherein the culture of the microorganism is carried out in an alkaline medium having a pH of 8.0 to 13.0. 6. The method for producing alkaline lipase according to claim 4, wherein the microorganism is Streptomyces sp. TOTO-9805 strain. 7. A detergent composition containing the alkaline lipase according to any one of claims 1 to 3.