JP2001178454A - Polygalacturonase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide polygalacturonase useful for detergents, textile treatment agents, etc. SOLUTION: The polygalacturonase is such as to have protopectinase activity, exhibiting optimally reactive pH near neutrality, being resistant to surfactant and chelating agents, and act even in alkaline region.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a polygalacturonase useful as a detergent, a fiber treatment agent and the like.

[0002]

2. Description of the Related Art Polygalacturonase (pectinase), pectate lyase, pectin lyase and the like are known as enzymes for decomposing pectic substances. These enzymes are used for clarification of citrus juice, improvement of squeezing rate of citrus juice, recovery of soluble components from fruit residue, peeling of orange peel, etc. In addition, pectin-degrading enzymes have been used for a long time for enzymatic scouring of vegetable fibers. In addition, there has been an attempt to use polygalacturonase (pectinase) as an enzyme for clothing detergents.
No. 0-226599, Japanese Patent Publication No. 6-39596, WO98 / 06809, and the like.

[0003]

SUMMARY OF THE INVENTION In the food industry,
Generally, a polygalacturonase that works efficiently in the low pH range is required.However, when used in clothing detergents, textile processing, etc., the enzyme must work in the neutral to alkaline range. , A surfactant, a chelating agent, and the like. Further, since pectic substances are present as insoluble pectin, protopectin in plant fiber, the ability to degrade protopectin is also required.

However, the only known alkaline polygalacturonase to date (JP-B-48-655)
No. 7) is an endo-type enzyme produced by an alkaliphilic Bacillus P-4-N strain and having an optimum reaction pH of about 10, but an enzyme having a low chelating agent resistance, which requires calcium ions in the reaction. is there. An enzyme derived from Fusarium oxysporum strain has an optimum reaction pH around 5 when polygalacturonic acid is used as a substrate. The reaction product is monogalacturonic acid, but S
Remarkably inhibited by DS, inhibited by metal ions such as calcium, magnesium, cobalt and zinc (Maceira et al., FEMS Microbiol. Lett., 154 , 37-43,
1997), not suitable as a detergent enzyme. Also, Selenomonas ruminantium
Derived enzymes show the optimum reaction pH around pH 7, but the working pH
It is characterized by a very narrow stable pH range, the optimum reaction temperature is around 40 ° C., and the reaction product is digalacturonic acid (Heinrichova et al., J. Appl. Bacterol., 66 , 1
69-174, 1989).

Accordingly, an object of the present invention is to provide a polygalacturonase which exhibits an optimum reaction pH near neutrality, is excellent in surfactants and chelating agents, has protopectinase activity, and acts even in an alkaline region. It is in.

[0006]

Means for Solving the Problems The inventors of the present invention screened an enzyme produced by a microorganism in soil and found that the enzyme exhibited an optimum reaction pH near neutrality and was resistant to surfactants and chelating agents. A polygalacturonase having a protopectinase activity that acts even in an alkaline region was found.

That is, the present invention provides a polygalacturonase having the following enzymatic properties, a bacterium producing the same, and a method for producing the same. (1) Action: Acts on polygalacturonic acid (pectic acid), pectin and protopectin, exo-hydrolyzes the α-1,4 bond of polygalacturonic acid to produce monogalacturonic acid. (2) Optimal reaction pH: around pH 7 (MOPS buffer, Tris-HCl buffer). (3) Optimal reaction temperature: about 60 ° C (Tris-HCl buffer,
pH 8.0). (4) pH stability: pH 8 to 11 (treated at 40 ° C for 30 minutes). (5) Heat resistance: about 50 ° C. (Tris-HCl buffer, pH 8.
0, 15 minutes). (6) Molecular weight: about 175000 (gel filtration method). (7) Isoelectric point: around pH 6.3 (isoelectric focusing). (8) Effect of metal ions: not inhibited by the addition of a chelating agent, but activated by calcium, strontium and manganese ions.

[0008] The polygalacturonase of the present invention can be produced, for example, by culturing a polygalacturonase-producing bacterium and collecting it from the culture. Examples of such a producing bacterium include a bacterium belonging to the genus Bacillus, for example, a Bacillus sp. KSM-P816 strain having the following mycological properties.

A Morphological properties (a) Cell shape and size: Bacillus (0.6-0.8 × 2.
(B) polymorphism: no (c) motility: yes (d) spores (size, shape, position, presence / absence of swelling): oval, 0.6-0.8 × 0 0.8 to 1.0 μm, semi-edge, no swelling (e) Gram stain: undefined (does not grow on CVT agar medium) (f) Acid-fast: negative (g) Growth on gravy agar medium: milky white, not Form regular colonies

B Physiological properties (a) Reduction of nitrate: + (b) Denitrification:-(c) MR test:-(d) VP test: + (e) Indole formation:-(f) Hydrogen sulfide Production:-(g) Starch hydrolysis: + (h) Gelatin hydrolysis: + (i) Casein hydrolysis: + (j) Use of citric acid: + (k) Use of inorganic nitrogen: + (l) Urease: -(M) oxidase:-(n) catalase: + (o) litmus milk: reduce indicator and convert to pecton (p) Growth temperature range: 12 to 59 ° C (q) Growth pH range: pH 6 to 10 (r ) Growth under anaerobic conditions: Growth (s) OF test:-(t) Gas production from glucose:-(u) Resistance to sodium chloride: 10% growth (v) Acid production from sugar: The following saccharides From the acid was observed. Ribose, galactose, xylose, arabinose, sucrose, glucose, mannitol,
Mannose, inositol, sorbitol, trehalose, lactose, glycerin, maltose, fructose, raffinose, salicin, rhamnose, soluble starch

As described above, Bacillus SP KSM-P8
Regarding the morphology and physiological properties of the 16 strains, see Bergey's Man
ual of Systematic Bacteriology "(Williams & Wilki
As a result of a comparative study in accordance with the description of B. licheniformis, the strain was considered to be a strain closely related to Bacillus licheniformis. However, its properties do not match those of known Bacillus licheniformis, and do not match the properties of other Bacillus bacteria. Therefore, this strain was sent to the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology Bacillus SP KS
Deposited as M-P816 (FERM P-17565).

In order to produce the polygalacturonase of the present invention using a polygalacturonase-producing bacterium such as Bacillus sp. KSM-P816, the strain must contain an assimilable carbon source, a nitrogen source and other essential nutrients. Inoculate the medium,
Shaking culture or aeration and stirring culture may be performed according to a conventional method.

The collection and purification of polygalacturonase from the obtained culture can be carried out according to a general method. That is, the cells can be removed from the culture by centrifugation or filtration, and the target enzyme can be concentrated from the obtained culture supernatant by a conventional method. The enzyme solution or the dry powder thus obtained can be used as it is, but can be further crystallized or granulated by a known method.

A polygalacturonase derived from Bacillus sp. KSM-P816, which is an example of the polygalacturonase of the present invention, has the following enzymatic properties.

[0015] The polygalacturonase activity was measured by the following method. [Standard enzyme activity measurement method] In a test tube, 0.2 mL of 0.5 M Tris-HCl buffer (pH 8.6), 0.2 mL of 1% (w /
v) polygalacturonic acid (ICN Biomedical; lo
t14482, adjusted to pH 6.8 with sodium hydroxide solution),
0.1 mL of 4 mM calcium chloride and 0.4 mL of deionized water were added, and the temperature was kept at 30 ° C. for 5 minutes. To this was added 0.1 mL of an appropriately diluted enzyme solution [diluted in 50 mM Tris-HCl buffer (pH 7.5) containing 1 mM calcium chloride], and allowed to react for 20 minutes. Then, 1 mL of dinitrosalicylic acid reagent was added. The reducing sugar was colored in boiling water for 5 minutes.
The mixture was quenched in ice water, 4 mL of deionized water was added, and the absorbance at 535 nm was measured to determine the amount of reducing sugar produced. A blank was prepared by adding a dinitrosalicylic acid reagent to a reaction solution treated without adding an enzyme solution, and then adding an enzyme solution to develop a color in the same manner. One unit (1 U) of the enzyme was an amount that produced 1 μmol of reducing sugar corresponding to D-galacturonic acid per minute under the above reaction conditions.

(1) Substrate Specificity Instead of polygalacturonic acid, pectins having different degrees of esterification were used as substrates, and the reaction rate was examined by a standard activity measurement method. Pectin with a degree of esterification of 28% (Sigma; lot74H
1092) has a degradation activity of about 60%, about 40% against pectin with 67% esterification degree (Sigma; lot74H1093), and about 30% against pectin with 93% esterification degree (Sigma; lot125H0123). Indicated. Next, 30
A commercially available detergent solution for clothing (pH 10.5) and an enzyme (0.2 U) were added using one cm basting thread (Kinuzuri) (about 25 mg), and the mixture was reacted at 30 ° C. for 1 hour. Reaction liquid (2mL)
Was centrifuged, and pectin released in the supernatant was quantified by the orcinol-hydrochloric acid method. As a result, about 15 μg of pectin was released under the above reaction conditions. This is considered to be due to the fact that this enzyme acts on protopectin on the surface of cotton fiber, and the activity of protopectinase belonging to type A (Sakai, Sakamoto, Textile Engineering, 45 , 301, 199).
2) was suggested.

(2) Decomposition mode of the substrate 0.05 U of the enzyme was added to a reaction solution comprising 50 mM Tris-HCl buffer (pH 7.5), 0.2% polygalacturonic acid and 0.2 mM calcium chloride, and To 0.25 mL. About 10 μL of the solution reacted at 30 ° C. for 30 minutes was spotted on a thin-layer chromatography plate (kiesel gel 60: Merck), and n-butanol: acetic acid: water = 5: 2: 3 (v / v).
Was developed in the solvent system. The reaction product was detected using an anisaldehyde-sulfuric acid solution.
The color was developed in an oven at 10 ° C. for 10 minutes. As a result, only monogalacturonic acid was detected as a reaction product, and this enzyme was determined to be an exo-type polygalacturonase.

(3) Optimal reaction pH MOPS buffer (pH 6-8), Tris-HCl buffer (pH
7-9), glycine-sodium hydroxide buffer (pH 8 ~
As a result of examining the optimum reaction pH using each buffer (100 mM) described in 11), this enzyme showed the highest reaction rate in MOPS buffer or Tris-HCl buffer at pH 7.0. Also,
It showed an activity of 70% or more of the maximum activity between pH 6 and 8 (FIG. 1).

(4) Optimal reaction temperature: 5 ° C. to 8 in 100 mM Tris-HCl buffer (pH 8.6)
The enzyme reaction was performed at each temperature of 0 ° C., and the optimum reaction temperature was examined. As a result, the enzyme showed an optimum reaction temperature around 60 ° C, and showed an activity of 60% or more of the maximum activity in the range of 50 ° C to 70 ° C. In addition, even when calcium chloride was not added to the reaction system, the optimum reaction temperature did not change and the reaction rate tended to be slightly lower than when calcium chloride was added (FIG. 2).

(5) Stable pH range McClubane's buffer (pH 2 to 8), glycine-sodium hydroxide buffer (pH 8 to 11.5), potassium chloride-sodium hydroxide buffer (pH 11 to 12.5) The enzyme was added to each of the above buffer solutions (50 mM), and the temperature was kept at 40 ° C. for 30 minutes, and then the residual activity was measured. As a result, assuming that the activity of this enzyme in a glycine-sodium hydroxide buffer solution (pH 9.0) was 100%, the enzyme was 80% in the pH range of 8 to 11.
% Or more of the remaining activity. When 1 mM calcium chloride was added in each treatment, the stability on the acidic side tended to increase. That is, it showed a residual activity of 80% or more of the maximum activity value in the pH range of 6 to 11 (FIG. 3).

(6) Heat resistance The enzyme was added to 50 mM Tris-hydrochloric acid buffer (pH 8.0), and the remaining activity was measured after being kept at each temperature of 5 ° C. to 80 ° C. for 15 minutes. The enzyme is kept at 50 ° C under these conditions.
Until very slowly deactivated above 60 ° C. (FIG. 4). When 1 mM calcium chloride is added,
It had no effect on the thermostability of the enzyme.

(7) Molecular weight (gel filtration method) 100 mM potassium chloride, 1 mM dithiothreitol and 1 mM
20 mM Tris-HCl buffer containing mM calcium chloride (pH
The enzyme was loaded on a Toyopearl HW55 column (1.5 × 65 cm) equilibrated in 7.0), and eluted at a flow rate of about 36 mL / h. Catalase (2
32000), aldolase (158000), bovine serum albumin (67,000), ovalbumin (4300)
0), a calibration curve was prepared from the respective eluate amounts and molecular weights, and the molecular weight of the enzyme was determined to be about 175,000.
It was estimated.

(8) Isoelectric point PAG-Plate (Pharmacia; pH 3.5 to 9.
Isoelectric focusing of this enzyme was performed using 5). After immersing the electrophoresed gel in a 10 mM phosphate buffer (pH 7.0), an agar plate containing polygalacturonic acid [1.0%
Polygalacturonic acid, 0.1% potassium monohydrogen phosphate,
1.0% sodium chloride, 50 mM Tris-HCl buffer (pH 7.5), 1.0% agar]. 37 ° C, 3
After incubating for 1 hour, the gel was removed and a 1% cetyltrimethylammonium bromide solution was poured. The isoelectric point of this enzyme was found to be around pH 6.3 from the protein mobility of the part where the lysis spot accompanying the activity occurred after about 10 minutes, the isoelectric point of the standard protein (Bio-Rad) and the calibration curve obtained from the mobility. It was decided to be.

(9) Influence of Various Compounds The influence of various compounds on the activity of the present enzyme was examined by adding each compound to the reaction system at a predetermined concentration and measuring the activity. As a result, the enzyme was inhibited by about 70% by N-bromosuccinimide (1 mM).
Other compounds, especially the chelating agents EDTA, EGTA
(5 mM each) did not inhibit (Table 1).

[0025]

[Table 1]

(10) Influence of Surfactant The enzyme activity was measured in a reaction system in which various surfactants were added to a concentration of 0.2% (w / v). as a result,
It was found that even in the presence of each surfactant at a high concentration of 0.2%, the present enzyme can express 70% or more of the activity as compared with the control (Table 2).

[0027]

[Table 2]

(11) Influence of metal salts Various metal salts were added at 1 mM under standard enzyme activity measurement conditions (without addition of calcium chloride), and the effects on the enzyme activities were examined. As a result, this enzyme was 117 to 14 compared to the control by calcium chloride, strontium chloride, and manganese chloride.
Activated with 2%. On the other hand, zinc chloride and nickel chloride inhibited about 90% and 50%, respectively (Table 3).

[0029]

[Table 3]

As described above, the polygalacturonase of the present invention has an optimum reaction pH of around pH 7, an optimum reaction temperature of around 60 ° C., is resistant to surfactants and chelating agents, and has an exo-type decomposition mode. Is a novel enzyme completely different from conventionally known polygalacturonase, particularly exopolygalacturonase, because it exhibits protopectinase activity belonging to the A type.

[0031]

[Embodiment 1] Screening of polygalacturonase-producing bacteria Suspension of soils from various parts of Japan in sterile water at 80 ° C, 20
The mixture was heat-treated for 1 minute and applied to an agar plate medium having the following composition. The culture was allowed to stand still for 3 to 5 days in a 30 ° C. incubator, and after cooling the bacteria, they were cooled in a refrigerator. Selection was made for those in which lysis spots associated with pectin degradation were detected around the grown bacteria,
Single colony formation was repeated, and the ability to produce polygalacturonic acid-degrading enzyme was assayed. Many strains thus obtained mainly produced pectate lyase. Among them, Bacillus sp. KSM-P816 strain was obtained as a polygalacturonase-producing strain. Screening medium composition Pectin (Sigma) 2.0% (w / v) Meat extract (Labemco) 1.5% Yeast extract (Difco) 0.5% Manganese sulfate 4-6 hydrate 0.005% Agar 1.5 % Tris-HCl buffer (pH 8.6) 50 mM (separate sterilization)

Embodiment 2 FIG. Bacillus sp KSM-
Production of polygalacturonase by strain P816 Culture of Bacillus sp. KSM-P816 strain obtained by the above screening was performed aerobically for 2 days at 30 ° C. by adding 50 mL of medium to a 500 mL Sakaguchi flask. The medium composition was 0.5% (w / v) pectin (Sigma), 1.5% polypeptone S (Nippon Pharmaceutical), yeast extract (Difco), 1.0% fish meat extract (Wako Pure Chemical),
0.1% potassium monohydrogen phosphate, 0.005% manganese sulfate 4-6 hydrate, 0.02% magnesium sulfate heptahydrate,
50 mM Tris-HCl buffer (pH 8.6; sterile separately). Under these conditions, the productivity of polygalacturonase was about 200 U / L.

Embodiment 3 FIG. Purification of polygalacturonase The culture solution of Bacillus sp. KSM-P816 was centrifuged (8000 × g, 15 minutes, 4 ° C.) to obtain a supernatant (2 L). This was put in a dialysis membrane, and polyethylene glycol 20000 (Wako Pure Chemical Industries) was sprinkled thereon to concentrate the internal solution. Furthermore, a module for ultrafiltration (AIP101
0: Asahi Kasei). 20 mM containing 1 mM calcium chloride and 1 mM dithiothreitol
After sufficient dialysis against a Tris-HCl buffer (pH 7.5), the obtained crude enzyme solution (145 mL) was equilibrated with the same buffer to obtain a DEAE Toyopearl 650M column (3.
5 × 23 cm: Tosoh). After washing and eluting the non-adsorbed protein using about 1000 mL of the equilibration buffer, the buffer containing 0 to 0.3 M sodium chloride (30
(0 mL each), and the adsorbed protein was eluted by a concentration gradient elution method. The polygalacturonase activity was eluted near the sodium chloride concentration of about 0.1M. On the other hand, the co-produced pectate lyase activity was eluted in the non-adsorbed fraction, so the polygalacturonase fraction was collected (80 m
L), and concentrated by ultrafiltration (YM3 membrane: Amicon) (8.5 mL, 1.0 U, 2.0 mg protein). The polygalacturonase fraction obtained by the above purification procedure exhibited the above-mentioned enzymatic properties.

[0034]

The polygalacturonase of the present invention has a pH of 7
It has an optimum reaction pH in the vicinity, acts even in the alkaline region, is stable to surfactants, has protopectinase activity, and is particularly stable to chelating agents. It is useful as an enzyme for use.

[Brief description of the drawings]

FIG. 1. Effect of pH on polygalacturonase activity of the present invention
FIG.

FIG. 2 is a graph showing the effect of temperature on the activity of the polygalacturonase of the present invention.

FIG. 3. Effect of polygalacturonase stability of the present invention
It is a figure which shows the influence of pH.

FIG. 4 is a graph showing the effect of temperature on the stability of polygalacturonase of the present invention.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12R 1:07) C12R 1:07) (72) Inventor Kazuhisa Sawada 2606 Kabane, Kaigamachi, Haga-gun, Tochigi Pref. Inside the company research institute (72) Inventor Yoko Kaito 2606 Kabane-cho, Akaga-cho, Haga-gun, Tochigi Prefecture Inside the Kao Co., Ltd. research institute (72) Inventor Toru Kobayashi 2606 Kabane-cho, Akaba-machi, Haga-gun, Tochigi pref. Person Shuji Kawai 2606 Akabane, Kaiga-cho, Haga-gun, Tochigi Prefecture F-term in Kao Corporation Research Laboratory 4B050 CC01 DD02 FF05E FF11E LL04 LL10 4B065 AA15X CA31 CA57 CA60

Claims (5)

[Claims] 1. A polygalacturonase having the following enzymatic properties: (1) Action: Acts on polygalacturonic acid (pectic acid), pectin and protopectin, exo-hydrolyzes the α-1,4 bond of polygalacturonic acid to produce monogalacturonic acid. (2) Optimal reaction pH: around pH 7 (MOPS buffer, Tris-HCl buffer). (3) Optimal reaction temperature: about 60 ° C (Tris-HCl buffer,
pH 8.0). (4) pH stability: pH 8 to 11 (treated at 40 ° C for 30 minutes). (5) Heat resistance: about 50 ° C. (Tris-HCl buffer, pH 8.
0, 15 minutes). (6) Molecular weight: about 175000 (gel filtration method). (7) Isoelectric point: around pH 6.3 (isoelectric focusing). (8) Effect of metal ions: not inhibited by the addition of a chelating agent, but activated by calcium, strontium and manganese ions. 2. A Bacillus sp. KSM-P816 (FER) that produces the polygalacturonase according to claim 1.
MP-17565). 3. A method for producing a polygalacturonase, which comprises culturing the microorganism producing the polygalacturonase according to claim 1 and collecting the polygalacturonase from the culture. 4. The method according to claim 3, wherein the microorganism is a bacterium belonging to the genus Bacillus. 5. The method according to claim 5, wherein the microorganism is Bacillus sp.
The production method according to claim 3 or 4, wherein the production method is P816 (FERMP-17565).