JP5779368B2 - Polygalacturonase - Google Patents

Description

  The present invention relates to polygalacturonase exhibiting an endo-type degradation mode and use thereof.

Polygalacturonase is generally called pectinase, and is a hydrolase that degrades polygalacturonic acid (also called “pectinic acid”) and pectin. Such polygalacturonase is mainly used as an enzyme for the food industry because it has an optimum reaction pH of acid-weak acidity, but it has also been used as a detergent enzyme for clothing. (Patent Documents 1 to 3).
In the food industry, polygalacturonase that generally works efficiently in the low working pH region is required. However, when used in clothing detergents, textile processing, etc., surfactants, chelating agents It is necessary to be stable against Further, since the pectin is present as protopectin, which is an insoluble pectin, in the plant fiber, it preferably has an ability to decompose protopectin (protopectinase activity).

  On the other hand, oligogalacturonic acid produced by the action of polygalacturonase on pectin or pectic acid is said to have a bacteriostatic action against Escherichia coli and the like, and an action that induces a microbial defense reaction against plants. A method for more efficiently producing oligogalacturonic acid has also been studied (Patent Document 4).

Polygalacturonase has an exo-type and an endo-type of action. However, when used as a detergent enzyme, it is an endo-type because of its good reactivity with modified substrates such as methyl esterification. Are preferred. End types are mostly derived from molds and plants, for example, PEC-M1 (Sigma) having an optimum pH of 4 and a molecular weight of 36000, PEC- having an optimum pH of 5.5 and a molecular weight of 42000. Polygalacturonase derived from the filamentous fungus Acremonium cellulolyticus having an optimum pH of 4.5 and a molecular weight of 41,000 is known (Patent Document 5).
However, there are problems such as a narrow stable pH range at room temperature and low protopectinase activity.

JP-A-60-226599 Japanese Patent No. 20335580 International Publication No. 98/06809 Pamphlet JP-A-6-205687 JP 2001-61473 A

  The present invention shows a polygalacturonase that exhibits an endo-type degradation mode, has protopectinase activity, is stable in a wide pH range at room temperature, and is useful not only for the food industry but also as a laundry detergent enzyme, and a method for producing the same. And a method for producing mono- and oligogalacturonic acid using the enzyme.

  The present inventors screened enzymes produced by microorganisms in the soil. As a result, they have high protopectinase activity, are resistant to surfactants and chelating agents, and have a wide pH range from acidic to alkaline at room temperature. An endo-type polygalacturonase was found that is stable in

That is, the present invention relates to the following [1] to [9].
[1] A polygalacturonase having the following enzymatic properties.
(1) Action: acts on polygalacturonic acid, pectin and protopectin, and hydrolyzes α-1,4 bonds of polygalacturonic acid to produce oligogalacturonic acid.
(2) Optimal reaction pH: around pH 5 (acetate buffer)
(3) Optimal reaction temperature: about 50 ° C. (acetate buffer, pH 5)
(4) Molecular weight: about 24000 (gel filtration method)
[2] The polygalacturonase according to [1], which is derived from a genus Plectosphaerella.
[3] The polygalacturonase according to [2], wherein the genus Plectosphaerella is Plectosphaerella KSM-P57 (FERM P- 22057).
[4] The polygalacturonase according to [1] to [3], which further has one or more enzymatic properties selected from the following (5) and (6).
(5) pH stability: pH 2 to 12 (treatment at 40 ° C. for 60 minutes)
(6) Heat resistance: stable up to about 50 ° C (acetate buffer, pH 5, treated for 15 minutes)
[5] A Plectosphaerella fungus that produces the polygalacturonase of [1].
[6] The fungus according to [5], which is Plectosphaerella sp. KSM-P57; FERM P- 22057.
[7] The method for producing a polygalacturonase according to [1], wherein a Plectosphaerella fungus is cultured, and polygalacturonase is collected from the culture.
[8] The method for producing a polygalacturonase according to [7], wherein the genus Plectosphaerella is Plectosphaerella KSM-P57 (FERM P- 22057).
[9] A method for producing mono- and oligogalacturonic acid, comprising causing polygalacturonase of [1] to [4] to act on polygalacturonic acid or pectin.

  The polygalacturonase of the present invention acts endically on polygalacturonic acid and pectin, has an optimum reaction pH in the vicinity of pH 5 and is stable in a wide pH range at room temperature, and thus has a high degree of storage freedom. And since it exhibits excellent protopectinase activity and maintains its activity in the presence of a chelating agent and in the presence of a surfactant, it is useful not only for the food industry but also as a detergent enzyme for clothing and a fiber processing enzyme. Moreover, mono- and oligo-polygalacturonic acid can be produced by using the polygalacturonase of the present invention.

It is a figure which shows the influence of pH which acts on the polygalacturonase activity of this invention. It is a figure which shows the influence of the temperature which acts on the polygalacturonase activity of this invention. It is a figure which shows the influence of pH which acts on the polygalacturonase stability of this invention. It is a figure which shows the influence of the temperature which acts on the polygalacturonase stability of this invention. It is the figure which compared the protopectinase activity of the polygalacturonase of this invention with that of a commercially available polygalacturonase. M1: PEC-M1, M2: PEC-M2

The polygalacturonase of the present invention has the following enzymatic properties (1) to (4).
(1) Action:
It acts on polygalacturonic acid (pectinic acid), pectin and protopectin, and hydrolyzes α-1,4 bonds of polygalacturonic acid to produce oligogalacturonic acid.
For pectin, the reaction rate is about 106% for pectin with a degree of esterification of 30%, about 48% for pectin with a degree of esterification of 60%, and about 8% for a pectin with a degree of esterification of 90%. Disassemble this with.
It also has protopectinase activity that acts on protopectin to produce mono-, di- or tri-galacturonic acid.
(2) Optimal reaction pH:
The highest reaction rate is shown in pH 5.0 acetate buffer, and the activity is more than 50% of the maximum activity over a wide range of pH 4.5 to 6.5 (FIG. 1). Accordingly, the optimum reaction pH is 4.5 to 6, that is, around pH 5 (acetate buffer).
(3) Optimal reaction temperature:
When the enzyme reaction is performed in an acetate buffer (pH 5.0), the optimum reaction temperature is shown at about 40 to 50 ° C., that is, around 50 ° C. Moreover, the activity of 50% or more of the maximum activity is shown in the range of 30 ° C. to 60 ° C. (FIG. 2).
(4) Molecular weight:
Estimated molecular weight measured by gel filtration method (elution with TSK-GEL G2000SWXL equilibrated with acetate buffer solution (pH 5.0) containing sodium chloride at a flow rate of 1 mL / min) is 22000 to 26000, that is, about 24000. It is.

In more detail, the polygalacturonase of the present invention has the following properties (5) to (8).
(5) pH stability:
The residual activity after constant incubation at 40 ° C. for 60 minutes in each buffer solution (pH 1 to 13) is in the range of pH 2.0 to 12.0 when the residual activity in the acetate buffer solution (pH 5) is taken as 100%. Is 70% or more (FIG. 3).
That is, the pH stability is pH 2 to 12 (treatment at 40 ° C. for 60 minutes).
(6) Heat resistance:
When the temperature is kept at 10 ° C. to 80 ° C. for 15 minutes in 50 mM acetate buffer (pH 5.0), it is stable up to about 60 ° C. (FIG. 4).
(7) Effect of chelating agent:
It is hardly inhibited by the addition of chelating agents such as ethylenediaminetetraacetic acid (EDTA) and glycol etherdiaminetetraacetic acid (EGTA) (Table 1).

(8) Effect of surfactant:
It is stable even in the presence of a cationic surfactant (0.2% (w / v)) (Table 2).
For example, the activity of 124% in a liquid containing 0.2% polyoxyethylene (3) lauryl ether and 121% or more in a liquid containing 0.2% polyoxyethylene (8) lauryl ether is retained.

As described above, the polygalacturonase of the present invention is stable in a wide pH range at room temperature and has polygalacturonase activity, that is, polygalacturonic acid hydrolysis activity. The polygalacturonase activity is endo-type, and oligogalacturonic acid is generated from polygalacturonic acid.
Furthermore, since the polygalacturonase of the present invention acts on protopectin, which is an insoluble natural pectin, the production of pectin degradation products using insoluble pectin as a substrate, soil attached to protopectin on plant fibers, and ketchup It is effective in removing food spills and stains from foods with a high content of insoluble pectin such as jam.

The polygalacturonase of the present invention is produced by culturing a polygalacturonase-producing bacterium and collecting it from the culture.
Examples of the bacteria that produce the polygalacturonase of the present invention include fungi belonging to the genus Plectosphaerella, for example, Plectosphaerella KSM-P57 (Plectosphaerella sp. KSM-P57). The Plectosfalera KSM-P57 strain has the following morphological properties.

  The potato dextrose agar medium “Daigo” (Nippon Pharmaceutical, Tokyo) (PDA medium), 2% Malt Agar (MA medium), Bacto Oatmeal Agar (Becton Dickinson, MD, USA) (OA medium) or LcA (Miura medium) ) In (LcA medium), when cultured under conditions of culture temperature: 25 ° C. and culture period: 2 weeks, yellow to light-skinned wet colonies having a diameter of about 60 mm are formed. A phialide is formed on the tip part of the conidial pattern or is laterally formed, and the tip part of the phialide has a cylindrical coloret, and an oval to spindle-shaped 1-2 cell conidia is formed in a lump shape. The formation of sexual reproductive organs such as ascii is not observed.

Plectosphaerella KSM-P57 showed the highest homology with 100% homology to Plectosporium tabacinum from the results of homology search of 28S rDNA base sequence. Therefore, this strain is Plectosporium anamorph of Plectosporium cucumerina. It was considered a tabacinum. In February 2011, this strain was transferred to the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (Tsukuba Center Chuo 6th, 1-1-1 Tsukuba City, Ibaraki Prefecture, 305-8566). On the 1st, it was deposited as Plectosphaerella KSM-P57 (Plectosphaerella sp. KSM-P57; FERM P- 22057).

  In order to produce the polygalacturonase of the present invention using a polygalacturonase-producing bacterium such as Plectosfalera KSM-P57 strain, the strain is cultured in a medium containing an anabolic carbon source, a nitrogen source, and other essential nutrients. And incubating with shaking or aeration and agitation according to a conventional method. The pH of the medium is preferably adjusted to 7-9.

The collection and purification of polygalacturonase from the thus obtained culture can be performed according to a general method. That is, the cells are removed by centrifuging or filtering the culture, and the resulting culture supernatant is subjected to conventional means such as ammonium sulfate precipitation, solvent precipitation, ultrafiltration, various chromatography, lyophilization, spray drying and the like. The target enzyme can be concentrated. The enzyme solution or dry powder thus obtained can be used as it is, but can be further crystallized or granulated by a known method.
The detailed enzymological properties of polygalacturonase derived from the Plectosfalera KSM-P57 strain are described in Example 4.

Mono- and oligogalacturonic acids can be produced using pectin or polygalacturonic acid as a raw material using the polygalacturonase of the present invention.
Here, examples of the oligogalacturonic acid include those in which 2 to 8 galacturonic acids are bonded.
The enzyme reaction between the polygalacturonase of the present invention and the raw material is not particularly limited, and a known method can be adopted. For example, pectin or polygalacturonic acid is dissolved or suspended in water or a buffer solution. It can be carried out by allowing polygalacturonase to act on this, or binding polygalacturonase to an insoluble immobilization carrier and allowing pectin or polygalacturonic acid to flow down thereto.

  Here, the buffer used is preferably a buffer combined with acetic acid, hydrochloric acid, citric acid, phosphoric acid, trisaminomethane, bis-tris, glycine, sodium hydroxide, and the like. For example, acetate buffer (pH 3.5-5.5), bis-Tris buffer (pH 6.0-7.0), etc. are mentioned.

  The amount of polygalacturonase used is not limited as long as the effect is not impaired. It is.

  The reaction conditions are preferably the reaction at the optimum temperature and pH of polygalacturonase, for example, 25 to 60 ° C, preferably 35 to 55 ° C, pH 4.0 to 6.5, preferably Can be reacted in a pH range of pH 5.0 to 6.0.

  Although reaction time is not specifically limited unless the effect of this invention is impaired, 6 to 72 hours are preferable and 18 to 48 hours are more preferable.

The mono- and oligogalacturonic acids thus obtained can be used as they are, but can be further desalted, separated and purified by ion exchange chromatography, gel filtration or the like, if necessary.
In addition, the pectin or pectic acid which is a raw material can be prepared by extraction from a plant body or the like as long as it is plant-derived, or can be purchased.

Example 1 Screening for Polygalacturonase-Producing Bacteria Suspended soil from various parts of Japan in sterile water was applied to an agar plate medium having the following composition. Static culture was performed in a 30 ° C. incubator for 3 to 5 days. After growth of the fungus, 0.2% (w / v) polygalacturonic acid, 0.1% potassium hydrogen phosphate, 1% sodium chloride, 50 mM EDTA, 50 mM Tris-hydrochloride buffer (pH 8.0) 0.8 Soft agar consisting of% agar was overlaid and incubated at room temperature overnight. Pour a 1% cetyltrimethylammonium bromide solution on the layered soft agar, leave it for 1 hour at room temperature, select the ones where lysis spots associated with pectin degradation have been detected around the grown bacteria, and repeat single colonization Then, the ability to produce polygalacturonic acid-degrading enzyme was assayed. Many of the strains thus obtained mainly produced pectate lyase, and among them, the Plectosfalera KSM-P57 strain was obtained as a polygalacturonase producing bacterium. The strain was deposited as a Plectoshaerella sp. KSM-P57 (FERM P- 22057) to the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology.

Example 2 Production of polygalacturonase by the Plectosfalera KSM-P57 strain The culture of the Plectosphaerella KSM-P57 strain obtained by the above-described screening was performed by adding 50 mL of medium to a 500 mL Sakaguchi flask, and 30 ° C. Aerobic for 2 days. Medium composition is 0.5% (w / v) pectin, 2.0% polypeptone S, 0.5% yeast extract, 1.0% fish meat extract, 0.15% dipotassium monohydrogen phosphate, 0.02 % Magnesium sulfate heptahydrate and 0.005% manganese sulfate pentahydrate. The polygalacturonase activity produced in the culture solution was measured by adding 5 mM EDTA to completely deactivate the pectate lyase activity. By this measurement method, a production amount of 0.1 to 0.2 U / L was obtained per culture solution.

Example 3 Purification of Polygalacturonase The culture solution of Plectosphaerella KSM-P57 strain was centrifuged (8000 × g, 30 minutes, 4 ° C.) to obtain a supernatant (about 3 L). To the obtained supernatant, the phenylmethanesulfonyl fluoride solution was added to and stirred at a final concentration of 1 mM, and the calcium chloride solution was stirred at a final concentration of 50 mM, and then centrifuged again (8000 × g, 30 minutes, 4 ° C.). The obtained supernatant was concentrated and desalted by an ultrafiltration module (ACP1010: Asahi Kasei). The resulting concentrated solution (455 mL) was applied to a DEAE Toyopearl 650M column (5 × 7 cm: Tosoh) equilibrated with 20 mM Tris-HCl buffer (pH 7.0) containing 1 mM dithiothreitol. About 400 mL of equilibration buffer was used to wash and elute non-adsorbed protein. Endo-type polygalacturonase activity was eluted in the non-adsorbed part. The obtained non-adsorbed fraction was concentrated (21 mL) using an ultrafiltration module (YM-10: Millipore). Of these, 20 mL was replaced with 10 mM phosphate buffer (pH 6.0) using a P6-DG desalting column (2.5 cm × 30 cm: Bio-Rad), and SP Toyopearl equilibrated with the same buffer. The sample was applied to a 650M column column (1.5 cm × 13.5 cm: Tosoh). After washing with the equilibration buffer, the adsorbed protein was eluted by a concentration gradient method using 0 to 100 mM NaCL. Endo-type polygalacturonase activity is eluted at about 10 mM NaCl concentration, and the fractions are collected, concentrated by ultrafiltration, and paralleled with 10 mM acetate buffer (pH 5.0) in advance. Buffer replacement was performed with a column (BioRad). (4 mL, 11.9 U, 640 μg protein).

Example 4 Measurement of Enzymatic Properties Various enzymological properties of the polygalacturonase fraction obtained by the purification procedure of Example 3 were measured.
The polygalacturonase activity was measured as follows.
[Standard enzyme activity assay]
In a test tube, 0.2 mL of 0.5 M acetate buffer (pH 5.0), 0.2 mL of 1% (w / v) polygalacturonic acid (ICN biomedical; lot14482, pH 6.8 with sodium hydroxide solution) Adjustment), 0.5 mL of deionized water was added, and the temperature was kept constant at 40 ° C. for 5 minutes. To this was added 0.1 mL of an appropriately diluted enzyme solution (diluted with deionized water), allowed to react for 30 minutes, 1 mL of dinitrosalicylic acid reagent was added, and coloring of reducing sugar was performed in boiling water for 5 minutes. went. After quenching 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. In addition, after adding a dinitrosalicylic acid reagent to the reaction liquid processed without adding an enzyme liquid, the blank added the enzyme liquid and prepared the same color. One unit (1 U) of enzyme was defined as an amount that produces 1 μmol of reducing sugar equivalent to D-galacturonic acid per minute under the above reaction conditions.

(1) Substrate specificity Using a pectin (30, 60, 90%) having a different degree of esterification as a substrate instead of polygalacturonic acid, the reaction rate was examined by a standard activity measurement method. About 106% for pectin with 30% esterification (Sigma; lot118K0974), about 48% for pectin with 60% esterification (Sigma; lot069K0976), and 90% esterification (Sigma; lot18K1650) On the other hand, it had about 8% degradation activity. Next, a reaction was performed at 40 ° C. for 1 hour using one 15 cm basting yarn (gold bell mark) as a substrate (about 12 mg), and then the supernatant was analyzed by liquid chromatography. As a result, since mono-di-trigacturonic acid was detected as a reaction product, this enzyme was considered to have protopectinase activity that can act on protopectin contained in cotton fibers.

(2) Substrate degradation mode 0.05 U of enzyme was added to a reaction solution consisting of 100 mM Tris-HCl buffer (pH 5.0), 0.2% polygalacturonic acid, and 5 mM EDTA to make the total volume 0.1 mL. . The liquid reacted at 40 ° C. for 60 minutes was analyzed by liquid chromatography. As a result, a peak was also detected as a reaction product in di-trigalacturonic acid and elution time zones longer than that, and this enzyme was an endo-type polygalacturonase.

(3) Optimum reaction pH for polygalacturonase activity
Using each buffer solution (100 mM) of acetate buffer (pH 3.5 to 5.5), Bistris-HCl buffer (pH 6.0 to 7.0), and MOPS buffer (pH 7.0 to 8.5) As a result of investigating the optimum reaction pH, this enzyme showed the highest reaction rate in pH 5.0 acetate buffer, and showed an activity of 50% or more of the maximum activity in a wide range of pH 4.5 to 6.5. (FIG. 1).

(4) Optimum reaction temperature As a result of carrying out an enzyme reaction at a temperature of 10 ° C to 70 ° C in 100 mM acetate buffer (pH 5.0) and examining the optimum reaction temperature, this enzyme has an optimum reaction temperature around 50 ° C. The activity was 50% or more of the maximum activity in the range of 30 ° C. to 60 ° C. (FIG. 2).

(5) Stable pH range Potassium chloride-hydrochloric acid buffer (pH 1) Macclebain buffer (pH 2-3), acetic acid buffer (pH 4-5), bistris-hydrochloric acid buffer (pH 6), Tris-hydrochloric acid buffer ( This enzyme is added to each buffer solution (50 mM) of pH 7 to 8), glycine-sodium hydroxide buffer solution (pH 9 to 10), and potassium chloride-sodium hydroxide buffer solution (pH 12 to 13), and 40 ° C. for 60 minutes. As a result of measuring the residual activity after the constant temperature, the enzyme showed a residual activity of 70% or more in the range of pH 2.0 to 12.0, assuming the residual activity in acetate buffer (pH 5) as 100%. (FIG. 3).

(6) Heat resistance The enzyme was added to 50 mM acetate buffer (pH 5.0), and the residual activity was measured after incubation at 10 to 80 ° C. for 15 minutes. The enzyme is very stable up to 60 ° C. under these conditions (FIG. 4).

(7) Molecular weight Gel filtration method: The enzyme is placed on TSK-GEL G2000SWXL (7.8 × 30 mm) equilibrated with 50 mM acetate buffer (pH 5.0) containing 100 mM sodium chloride at a flow rate of 1 mL / min. Elution was performed. Using thyroglobulin (670000), γ-globulin (158000), ovalbumin (44000), myoglobin (17000), vitamin B12 (1350) as standard proteins, a calibration curve was prepared from the respective eluate amounts and molecular weights. When the molecular weight of the enzyme was determined, it was estimated to be about 24,000.

(9) Effects of various compounds The effects of various compounds on the activity of this enzyme were examined by adding each compound to the reaction system to a predetermined concentration and measuring the activity. Resistant to the described modifiers and chelating agents (Table 1).

(10) Effect of surfactant As a result of examining the reactivity of the enzyme in the reaction system in which various surfactants were added to 0.2% (w / v), in the presence of the cationic surfactant. The enzyme expressed about 120% of the activity compared to the subject (Table 2).

(11) Effect of metal salt 1 mM of various metal salts was added to the standard activity measurement conditions, and the effect on enzyme activity was examined. As a result, the enzyme was resistant to the metal compounds described (Table 3).

Example 5 Protopecnase Activity In the standard enzyme activity measurement method described above, instead of polygalacturonic acid, a 15 cm basting thread (gold bell mark) (about 12 mg) was used as a substrate at 30 ° C. for 24 hours. Thereafter, the color development of the reducing sugar in the supernatant was measured. Each enzyme was used at a concentration of 100 mU / mL as polygalacturonic acid degradation activity. As a result, it was found that this enzyme has about three times the protopectinase activity as compared with commercially available pectinases (PEC-M1 (Sigma), PEC-M2 (Sigma)) (FIG. 5).

Claims (5)

Polygalacturonase derived from Plectosphaerella sp. KSM-P57 (FERM P-22057) having the following enzymological properties.
(1) Action: acts on polygalacturonic acid, pectin and protopectin, and hydrolyzes α-1,4 bonds of polygalacturonic acid to produce oligogalacturonic acid.
(2) Optimal reaction pH: around pH 5 (acetate buffer)
(3) Optimal reaction temperature: about 50 ° C. (acetate buffer, pH 5)
(4) Molecular weight: about 24000 (gel filtration method) Furthermore, the following (5) and polygalacturonase of claim 1 Symbol mounting having one or more enzymatic properties selected from (6).
(5) pH stability: pH 2 to 12 (treatment at 40 ° C. for 60 minutes)
(6) Heat resistance: stable up to about 50 ° C (acetate buffer, pH 5, treated for 15 minutes) A fungus identified by Plectosphaerella sp. KSM-P57 (FERM P- 22057). The fungus specified by Plectosphaerella KSM-P57 (Plectosphaerella sp. KSM-P57; FERM P-22057) is cultured, and polygalacturonase is collected from the culture. A method for producing polygalacturonase. The polygalacturonic acid or pectin, preparation of mono- and oligogalacturonic acids, characterized in that the action of polygalacturonase mounting according to claim 1 or 2 SL.