JP4382952B2 - Exopolygalacturonase - Google Patents

Description

【００２８】
（１０）キレート剤の影響
本酵素を１０mM ＥＤＴＡを含む５０mMトリス−塩酸緩衝液（ｐＨ７．０）中で３０℃、３０分間恒温した後、１０倍に希釈し残存活性を測定した。標準活性測定法において塩化カルシウムを添加した場合と無添加の場合においての残存活性を比較すると双方ともに活性が９０％以上残存することから本酵素は反応、安定性にカルシウムイオンを要求しないことが判った。
【００２９】
このように本発明のエキソポリガラクツロナーゼは、最適反応ｐＨを７付近に、最適反応温度を６０℃付近に有するモノガラクツロン酸生成型の新規な酵素である。
【００３０】
【実施例】
実施例１ エキソポリガラクツロナーゼ生産菌のスクリーニング
日本各地の土壌を滅菌水に懸濁したものを８０℃、２０分間熱処理し、下記の組成を有する寒天平板培地に塗布した。３０℃の培養器で３〜７日間静置培養し、菌の生育後、０．２％（ｗ／ｖ）ポリガラクツロン酸、０．１％リン酸１水素カリウム、１％塩化ナトリウム、０．２Ｍクエン酸３ナトリウム、５０mMトリス−塩酸緩衝液（ｐＨ７．０）、０．８％寒天から成る軟寒天を重層し、３７℃で１時間恒温した。コロニー周辺にポリガラクツロン酸の分解に伴う溶解斑が検出されたものについて選抜し、シングルコロニー化を繰り返し、ポリガラクツロン酸分解酵素の生産能を検定した。このようにして得られた多くの菌株は、主にペクチン酸リアーゼを生産したが、その中でポリガラクツロナーゼ生産菌としてバチルス エスピー ＫＳＭ−Ｐ４４３株を得た。
【００３１】
【表２】

【００３２】
実施例２ バチルス エスピー ＫＳＭ−Ｐ４４３株によるエキソポリガラクツロナーゼの生産
上述のスクリーニングにより得られたバチルス エスピー ＫＳＭ−Ｐ４４３株の培養は、５００mL容坂口フラスコに５０mLの培地を加え、３０℃、２日間好気的に行った。培地組成は、０．５％（ｗ／ｖ）ペクチン酸、２．０％ポリペプトンＳ、０．５％酵母エキス、１．０％魚肉エキス、０．１５％リン酸１水素カリウム、０．００５％硫酸マンガン、５０mMトリス−塩酸緩衝液（ｐＨ８）であった。本培養条件においてエキソポリガラクツロナーゼの生産性は約２３０Ｕ／Ｌであった。
【００３３】
実施例３ エキソポリガラクツロナーゼの精製
バチルス エスピー ＫＳＭ−Ｐ４４３株の培養液を遠心分離（９０００×ｇ、２０分間、４℃）し上清液（２．８Ｌ）を得た。これを限外濾過用モジュール（ＡＣＰ１３０００：旭化成）により濃縮、脱塩を行った（４９０mL）。得られた濃縮液は２mMβ−メルカプトエタノール、０．５mM塩化カルシウムを含む１０mMリン酸緩衝液（ｐＨ６．０）にて平衡化しておいたＣＭトヨパール６５０Ｍカラム（２．５×５０cm：東ソー）に添着した。約７５０mLの平衡化緩衝液を用いて非吸着タンパク質を洗浄溶出させたところ、エキソポリガラクツロナーゼ活性はこの部分に溶出された。活性画分を集め（６００mL）限外濾過（ＰＭ１０メンブレン：アミコン）により濃縮、脱塩を行った（１００mL）。この濃縮液を予め２mMβ−メルカプトエタノール、１mM塩化カルシウムを含む２５mMトリス−塩酸緩衝液（ｐＨ７．５）にて平衡化しておいたＳｕｐｅｒＱトヨパールカラム（２．５×３０cm：東ソー）に添着した。約４５０mLの平衡化緩衝液を用いて非吸着タンパク質を洗浄溶出させたところ、エキソポリガラクツロナーゼ活性はこの部分に溶出された。活性画分を集め（１００mL）、限外濾過（ＰＭ１０メンブレン）により濃縮、脱塩を行った（１０mL）。この濃縮液を脱イオン水にて１０倍に希釈し、予め２mMβ−メルカプトエタノール、１mM塩化カルシウムを含む１０mMトリス−塩酸緩衝液（ｐＨ９．０）にて平衡化しておいたＤＥＡＥ−バイオゲルＡカラム（２．５×１２cm：バイオラッド）へ添着した。平衡化緩衝液にて洗浄した後、０から０．１Ｍ塩化カリウムを含む同緩衝液（３００mLずつ）を用い吸着タンパク質を濃度勾配法にて溶出させた。エキソポリガラクツロナーゼ活性は約７５mMの塩化カリウム濃度付近で溶出され、その画分を集め、限外濾過により濃縮、希釈を繰り返した。これを予め２mMβ−メルカプトエタノール、１mM塩化カルシウムを含む１０mMリン酸緩衝液（ｐＨ６．０）にて平衡化しておいたＤＥＡＥ−バイオゲルＡカラム（２．５×１０．５cm：バイオラッド）へ添着した。平衡化緩衝液にて洗浄した後、０から０．２Ｍ塩化カリウムを含む同緩衝液（２００mLずつ）を用い吸着タンパク質を濃度勾配法にて溶出させた。エキソポリガラクツロナーゼ活性は約８０mMの塩化カリウム濃度付近で溶出され、その画分を集め、限外濾過により濃縮した（１．７５mL）。この濃縮液を予め０．１Ｍ塩化カリウム、２mMβ−メルカプトエタノール、１mM塩化カルシウムを含む１０mMトリス−塩酸緩衝液（ｐＨ７．５）にて平衡化しておいたバイオゲルＡ０５ｍカラム（１．５×７３cm：バイオラッド）へ載せゲル濾過を行った。得られた活性画分を集め限外濾過により濃縮した（１．５mL、３３．９Ｕ、１．０１mgタンパク質）。以上の操作により電気泳動的に均一なエキソポリガラクツロナーゼを活性収率約６％、比活性約１６５倍までに精製した。
上記精製操作により得られたエキソポリガラクツロナーゼ画分は、前述の酵素学的性質を示した。
【００３４】
【発明の効果】
本発明のエキソポリガラクツロナーゼは、ｐＨ７付近に最適反応ｐＨを有し、界面活性剤耐性、キレート剤耐性を有する新規酵素であり、ガラクツロン酸の調製あるいは衣料用他、洗浄剤用酵素として有用である。
【図面の簡単な説明】
【図１】本発明のエキソポリガラクツロナーゼ活性に及ぼすｐＨの影響を示す図である。
【図２】本発明のエキソポリガラクツロナーゼ活性に及ぼす温度の影響を示す図である。
【図３】本発明のエキソポリガラクツロナーゼ安定性に及ぼすｐＨの影響を示す図である。
【図４】本発明のエキソポリガラクツロナーゼ安定性に及ぼす温度の影響を示す図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to exopolygalacturonase useful for the preparation of galacturonic acid, detergents and the like.
[0002]
[Prior art]
Known enzymes that degrade pectin are polygalacturonase (pectinase), pectate lyase, pectin lyase, pectin esterase, and the like. In the food industry, these enzymes are used effectively and applied to clarification of fruit juice, wine, etc., improvement of squeezing rate of citrus juice, recovery of soluble components from fruit residues, and peeling of mandarin orange peel. In particular, in the food industry, polygalacturonase that works efficiently in a region where the working pH is low is generally used. In fact, the optimum reaction pH of conventionally known polygalacturonase is almost on the acidic side. Yes.
[0003]
On the other hand, there has been an attempt to use polygalacturonase as an enzyme for garment detergents to remove food spills and stains on food clothes with high pectin content such as jam, ketchup and juice. JP-A-60-226599, JP-B-6-39596, WO98 / 06809 and the like are disclosed.
[0004]
[Problems to be solved by the invention]
When using polygalacturonase for laundry detergents and other detergents, it is necessary that the enzyme acts in the neutral to alkaline region and is stable against surfactants that are components of detergents. It is.
In addition, exo-type galacturonase can be used to efficiently obtain galacturonic acid and digalacturonic acid from pectic acid. In this case, considering the solubility of pectic acid, exopolygallium works best in the vicinity of neutrality. Cutronase is desired.
[0005]
However, most known exopolygalacturonases have an optimum reaction pH of 6 or less. Under such circumstances, two types of exopolygalacturonase exhibiting a high optimum reaction pH are known. Among them, the enzyme derived from Selenomonas ruminantium has an optimum reaction pH of around 7, but is characterized by a very narrow working pH and stable pH range. The optimum reaction temperature is around 40 ° C. The product is digalacturonic acid (Heinrichova et al., J. Appl. Bacteriol., 66 , 169-174, 1989). On the other hand, an enzyme derived from a Fusarium oxysporum strain has an optimum reaction pH in the vicinity of 5 when polygalacturonic acid is used as a substrate. Although the reaction product is monogalacturonic acid, it is significantly inhibited by SDS, inhibited by metal ions such as calcium, magnesium, cobalt, zinc, etc. (Maceira et al., FEMS Microbiol. Lett., 154 , 37-43, 1997) Not suitable as a cleaning enzyme.
[0006]
Accordingly, an object of the present invention is to provide an exopolygalacturonase having an optimum reaction pH near neutrality and exhibiting resistance to chelating agents and surfactants.
[0007]
[Means for Solving the Problems]
The present inventors have found a microorganism that produces an exopolygalacturonase resistant to chelating agents and surfactants from among enzymes produced by microorganisms in soil, having an optimum reaction pH of pH 7, and completed the present invention. did.
[0008]
That is, this invention provides the exopolygalacturonase which has the following enzymological property, the microorganisms which produce it, and its manufacturing method.
(1) Action: Acts on polygalacturonic acid (pectinic acid) and pectin, and exohydrolyzes α-1,4 bonds of polygalacturonic acid to produce monogalacturonic acid.
(2) Optimal reaction pH: pH 7 (MOPS buffer, Tris-HCl buffer)
(3) Optimum reaction temperature: about 60 ° C. (Tris-HCl buffer, pH 7.0)
(4) pH stability: pH 7-12 (treatment at 40 ° C. for 60 minutes)
(5) Heat resistance: stable to about 55 ° C. (Tris-HCl buffer, pH 7.0, treated for 15 minutes)
(6) Molecular weight: about 45000 (SDS electrophoresis method)
(7) Isoelectric point: around pH 5.8 (isoelectric focusing method)
(8) Chelating agent: Not inhibited by EDTA (10 mM).
(9) Surfactant resistance: Stable to surfactant (0.1%).
[0009]
DETAILED DESCRIPTION OF THE INVENTION
The exopolygalacturonase of the present invention can be produced, for example, by culturing an exopolygalacturonase producing bacterium and collecting it from the culture solution. Examples of such producing bacteria include bacteria belonging to the genus Bacillus, for example, the KSM-P443 strain having the following mycological properties.
[0010]
A Morphological properties (a) Shape and size of cells: Neisseria gonorrhoeae (0.6-0.8 × 2.4-3.2 μm)
(B) Polymorphism: None (c) Mobility: Existence (d) Spores (size, shape, position, presence or absence of swelling): Ellipse, 0.6-0.8 × 0.8-1.0 μm , Near edge, no swelling)
(E) Gram staining: indefinite (does not grow on CVT agar medium and does not show viscosity by the potassium hydroxide method)
(F) Antiacidity: negative (g) Growth on broth agar medium (medium 1): Milky white, foliate colonies formed
B Physiological properties (a) Reduction of nitrate (medium 2): +
(B) Denitrification reaction (medium 2):-
(C) MR test (medium 3):-
(D) VP test (medium 3): +
(E) Indole production (medium 4):-
(F) Production of hydrogen sulfide (medium 5):-
(G) Starch hydrolysis (medium 6): +
(H) Gelatin hydrolysis (medium 7): +
(I) Casein hydrolysis (medium 8): +
(J) Use of citric acid (medium 9): +
(K) Use of inorganic nitrogen (medium 10): +
(L) Urease (medium 11):-
(M) Oxidase (medium 12): +
(N) Catalase: +
(O) Litmus milk (medium 13): produces acid and peptates (p) growth temperature range (medium 14): 12-59 ° C
(Q) Growth pH range (medium 15): pH 4-10
(R) Growth under anaerobic conditions (medium 16): Grows (s) OF test (medium 17):-
(T) Gas production from glucose (medium 18):-
(U) Resistance to sodium chloride (medium 19): grown at 10% (v) Acid production from sugar (medium 20): Acid production was observed from the following sugars. Galactose, ribose, xylose, arabinose, sucrose, glucose, mannitol, mannose, inositol, sorbitol, trehalose, lactose, glycerin, maltose, fructose, raffinose, salicin, soluble starch, melibiose, rhamnose
Medium 1: Nutrient agar (Difco)
Medium 2: Nutrient broth (Difco), 0.1% by weight potassium nitrate
Medium 3: Bactopeptone (Difco) 0.7% by weight, Potassium monohydrogen phosphate 0.5% by weight, Glucose (separately sterilized) 0.5% by weight
Medium 4: SIM medium (Nissui Pharmaceutical), Kobak reagent medium 5: TSI agar medium (Eiken Chemical)
Medium 6: Nutrient agar, 1.0% by weight soluble starch
Medium 7: Nutrient agar, 1.0% by weight of gelatin
Medium 8: Nutrient agar, casein 1.0 wt%
Medium 9: Simmons medium (Eiken Chemical)
Medium 10: 0.05% by weight of yeast extract, 0.1% by weight of sodium sulfate, 0.1% by weight of potassium dihydrogen phosphate, 1.0% by weight of glucose (separately sterilized), 0.25% by weight of sodium nitrate or chloride 0.16% by weight ammonium
Medium 11: Urea medium (Eiken Chemical)
Medium 12: cytochrome oxidase test filter paper (Nissui Pharmaceutical)
Medium 13: Bactritomas milk (Difco)
Medium 14: SCD medium (Nippon Pharmaceutical)
Medium 15: Sodium carbonate and hydrochloric acid are added to the nutrient broth after sterilization, and the pH is adjusted. Medium 16: Anaerobic Agar (Difco)
Medium 17: OF basal medium (Difco)
Medium 18: Nutrient broth, glucose (separately sterilized) 1.0% by weight
Medium 19: SCD medium, sodium chloride 2-10% by weight
Medium 20: ammonium monohydrogen phosphate 0.1% by weight, potassium chloride 0.02% by weight, magnesium sulfate heptahydrate 0.02% by weight, yeast extract 0.02% by weight, agar 1.5% by weight, bromocresol Purple 0.0006 wt%, sugar (separate sterilized) 1.0 wt%
[0013]
As described above, the morphology and physiological properties of the KSM-P443 strain were compared according to the description in “Bergey's Manual of Systematic Bacteriology” (Williams & Wilkins, 1984). It was thought that. However, since this property is not consistent with the known properties of Bacillus licheniformis, and is not consistent with other properties of other Bacillus bacteria, this strain is transferred to the Institute of Biotechnology, the Industrial Technology Institute as a new Bacillus bacterium. KSM-P443 Deposited as a strain (FERM-P17561).
[0014]
In order to produce exopolygalacturonase of the present invention using an exopolygalacturonase producing bacterium such as KSM-P443 strain, the strain is inoculated into a medium containing an assimilable carbon source, nitrogen source, and other essential nutrients. In addition, shaking culture or aeration and stirring culture may be performed according to a conventional method. The carbon source and nitrogen source to be used are not particularly limited and can be assimilated such as pectin, pectic acid, galactose, galacturonic acid, glucose, sucrose, maltose and the like. Examples of the nitrogen source include meat extract, fish extract, yeast extract, peptone, corn steep liquor and the like. In addition, phosphates, metal salts, and organic / inorganic trace nutrients can be added as appropriate. The pH of the medium may be adjusted using sodium carbonate or the like to a pH suitable for the enzyme production of the present invention.
[0015]
Collection and purification of exopolygalacturonase from the thus obtained culture solution can be performed according to a general method. That is, the cells can be removed from the culture solution by centrifugation or filtration, and the target enzyme can be concentrated from the obtained culture supernatant by conventional means such as salting out, solvent precipitation, ultraconcentration, etc. it can. An example of salting out is ammonium sulfate (30 to 90% saturated fraction), and an example of solvent precipitation is cold precipitation (50% or more), followed by centrifugation, desalting, and freezing. Dry powder or spray-dried powder can be obtained. As the desalting method, dialysis, gel filtration using Sephadex G-10 or the like, ultrafiltration, or the like is used. 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.
[0016]
The exopolygalacturonase derived from the Bacillus sp. KSM-P443 strain, which is an example of the exopolygalacturonase of the present invention, has the following properties. The enzyme activity was measured as follows.
[0017]
[Standard enzyme activity assay]
In a test tube, 0.2 mL of 0.5 M Tris-HCl buffer (pH 9.0), 0.2 mL of 1% (w / v) polygalacturonic acid (ICN biomedical; lot14482, pH 6.0 with sodium hydroxide solution). 8), 0.5 mL of deionized water was 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 [dilution was performed with 10 mM Tris-HCl buffer (pH 7.0)] and allowed to react for 20 minutes. Then, 1 mL of dinitrosalicylic acid reagent was added, and boiling water was added. The coloring of reducing sugar was performed for 5 minutes. 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 a reducing sugar equivalent to 1 μmol of galacturonic acid per minute under the above reaction conditions.
[0018]
(1) Substrate specificity The reaction rate was examined by a standard activity measurement method using pectin (28, 67, 93%) having a different esterification degree as a substrate instead of polygalacturonic acid. This enzyme is most reactive to polygalacturonic acid (ICN biomedical; lot14482), and when the same polygalacturonic acid (Sigma; lot112H3780, Fluka; lot53998) is used, the reaction rate is 82% of the maximum activity, respectively. About 70%. About 28% pectin (sigma; lot74H1092) having a degree of esterification of about 40% and about 4% of pectin having a degree of esterification of 67% (sigma; lot74H1093) showed a degradation activity of about 93%. (Sigma; lot125H0123) showed almost no degradation activity.
[0019]
(2) Decomposition mode of substrate 0.05 U of this enzyme was added to a reaction solution consisting of 50 mM Tris-HCl buffer (pH 7.0), 0.2% polygalacturonic acid, and 0.2 mM calcium chloride to bring the total amount to 0. To 25 mL. About 10 μL of the solution reacted at 30 ° C. for 30 minutes was spotted on a thin-layer chromatographic plate (kiesel gel 60: Merck), and using a developing solution of n-butanol: acetic acid: water = 5: 2: 3 (v / v) Deployed. Anisaldehyde-sulfuric acid solution was used for detection of the reaction product, and color was developed in a dryer at 100 ° C. for 10 minutes after spraying the plate. As a result, only monogalacturonic acid was detected as a reaction product, and this enzyme was determined to be an exo-type polygalacturonase.
[0020]
(3) Optimum reaction pH
As a result of examining the optimum reaction pH using each buffer solution (100 mM) of acetate buffer solution (pH 4 to 6), MOPS buffer solution (pH 6 to 8), and Tris-HCl buffer solution (pH 7 to 9.5), this enzyme Showed the highest reaction rate in Tris-HCl buffer at pH 7. Moreover, the activity of 50% or more of the maximum activity was shown between pH 6 and 8 (FIG. 1).
[0021]
(4) Optimum reaction temperature The enzyme reaction was performed at 30 ° C to 90 ° C in 100 mM Tris-HCl buffer (pH 7.0), and the optimum reaction temperature was examined. As a result, the enzyme showed an optimum reaction temperature around 60 ° C. (FIG. 2). Even when 1 mM calcium chloride was added, the optimum reaction temperature and reaction rate did not change at all.
[0022]
(5) Stable pH range Acetic acid buffer (pH 4-6), MOPS buffer (pH 6-8), Tris-HCl buffer (pH 7-9), Glycine-sodium hydroxide buffer (pH 8-11) and chloride Each buffer (50 mM) enzyme of potassium-sodium hydroxide buffer (pH 11 to 12.7) was added, and the residual activity was measured after incubation at 40 ° C. for 60 minutes. When the activity before the treatment was taken as 100%, the enzyme showed a residual activity of 80% or more in the pH range of 7-12 (FIG. 3). When 1 mM calcium chloride was added, the enzyme stability was not affected at all.
[0023]
(6) This enzyme was added to a thermostable 50 mM Tris-HCl buffer (pH 7.0), and the residual activity was measured after incubation at 30 ° C. to 70 ° C. for 15 minutes. The enzyme was stable up to 55 ° C. under these conditions (FIG. 4). The same stability was also obtained when calcium chloride (1 mM) was added.
[0024]
(7) Molecular weight (SDS electrophoresis method)
An SDS treatment solution was added to the enzyme, heat treatment was performed at 100 ° C. for 2 minutes, and then SDS electrophoresis was performed on a 12.5% acrylamide gel. Phosphorylase b (97400), serum albumin (67000), ovalbumin (43000), carbonic anhydrolase (30000), trypsin inhibitor (21500), α-lactalbumin (14500) are used as standard proteins, and their respective mobility and molecular weight. From this, a calibration curve was prepared and the molecular weight of this enzyme was determined, and it was estimated to be about 45000.
[0025]
(8) Isoelectric focusing of the enzyme was performed using isoelectric point PAG-Plate (Pharmacia; pH 3.5 to 9.5). After electrophoresis, the protein was stained with Coomassie Brilliant Blue G250. From the calibration curve obtained from the isoelectric point and mobility of the standard protein (BioRad), the isoelectric point of this enzyme was determined to be around pH 5.8.
[0026]
(9) Influence of surfactant Enzyme activity was measured in a reaction system in which various surfactants were added at 0.1% (w / v). As a result, it was found that even in the presence of each surfactant at a high concentration of 0.1%, this enzyme can express about 60% or more of the activity compared to the control (Table 1).
[0027]
[Table 1]

[0028]
(10) Influence of chelating agent The enzyme was incubated at 30 ° C. for 30 minutes in 50 mM Tris-HCl buffer (pH 7.0) containing 10 mM EDTA, diluted 10 times, and the residual activity was measured. Comparing the residual activity with and without calcium chloride in the standard activity measurement method, it is clear that the enzyme does not require calcium ions for reaction and stability because both activities remain at 90% or more. It was.
[0029]
Thus, the exopolygalacturonase of the present invention is a novel monogalacturonic acid-producing enzyme having an optimum reaction pH of around 7 and an optimum reaction temperature of around 60 ° C.
[0030]
【Example】
Example 1 Screening for Exopolygalacturonase-Producing Bacteria A suspension of soil in Japan in sterile water was heat-treated at 80 ° C. for 20 minutes and applied to an agar plate medium having the following composition. The culture was allowed to stand for 3-7 days in a 30 ° C. incubator. After the growth of the bacteria, 0.2% (w / v) polygalacturonic acid, 0.1% potassium monohydrogen phosphate, 1% sodium chloride, 0. Soft agar consisting of 2M trisodium citrate, 50 mM Tris-HCl buffer (pH 7.0) and 0.8% agar was overlaid and incubated at 37 ° C. for 1 hour. Those in which dissolution spots associated with the degradation of polygalacturonic acid were detected around the colony, and single colonization was repeated to test the ability to produce polygalacturonic acid-degrading enzyme. Many strains thus obtained mainly produced pectate lyase, among which Bacillus sp. KSM-P443 strain was obtained as a polygalacturonase producing bacterium.
[0031]
[Table 2]

[0032]
Example 2 Production of Exopolygalacturonase by Bacillus sp. KSM-P443 Strain The Bacillus sp. KSM-P443 strain obtained by the above-mentioned screening was performed by adding 50 mL of medium to a 500 mL Sakaguchi flask at 30 ° C. for 2 days. Went aerobically. Medium composition is 0.5% (w / v) pectic acid, 2.0% polypeptone S, 0.5% yeast extract, 1.0% fish meat extract, 0.15% potassium monohydrogen phosphate, 0.005 % Manganese sulfate, 50 mM Tris-HCl buffer (pH 8). Under the main culture conditions, the productivity of exopolygalacturonase was about 230 U / L.
[0033]
Example 3 Purification of Exopolygalacturonase The culture solution of Bacillus sp. KSM-P443 strain was centrifuged (9000 × g, 20 minutes, 4 ° C.) to obtain a supernatant (2.8 L). This was concentrated and desalted with an ultrafiltration module (ACP13000: Asahi Kasei) (490 mL). The resulting concentrated solution was attached to a CM Toyopearl 650M column (2.5 × 50 cm: Tosoh) equilibrated with 10 mM phosphate buffer (pH 6.0) containing 2 mM β-mercaptoethanol and 0.5 mM calcium chloride. did. When non-adsorbed protein was washed and eluted using about 750 mL of equilibration buffer, exopolygalacturonase activity was eluted in this portion. The active fractions were collected (600 mL) and concentrated and desalted by ultrafiltration (PM10 membrane: Amicon) (100 mL). This concentrated solution was applied to a SuperQ Toyopearl column (2.5 × 30 cm: Tosoh) equilibrated in advance with 25 mM Tris-HCl buffer (pH 7.5) containing 2 mM β-mercaptoethanol and 1 mM calcium chloride. When non-adsorbed protein was washed and eluted using about 450 mL of equilibration buffer, exopolygalacturonase activity was eluted in this portion. The active fractions were collected (100 mL), concentrated and desalted by ultrafiltration (PM10 membrane) (10 mL). This concentrated solution was diluted 10-fold with deionized water, and was previously equilibrated with 10 mM Tris-hydrochloric acid buffer (pH 9.0) containing 2 mM β-mercaptoethanol and 1 mM calcium chloride (pH 9.0). 2.5 × 12 cm: Bio-Rad). After washing with the equilibration buffer, the adsorbed protein was eluted by the concentration gradient method using the same buffer (300 mL each) containing 0 to 0.1 M potassium chloride. The exopolygalacturonase activity was eluted at a potassium chloride concentration of about 75 mM. The fractions were collected, concentrated and diluted repeatedly by ultrafiltration. This was applied to a DEAE-Biogel A column (2.5 × 10.5 cm: Bio-Rad) that had been equilibrated in advance with 10 mM phosphate buffer (pH 6.0) containing 2 mM β-mercaptoethanol and 1 mM calcium chloride. . After washing with the equilibration buffer, the adsorbed protein was eluted by the concentration gradient method using the same buffer (200 mL each) containing 0 to 0.2 M potassium chloride. Exopolygalacturonase activity eluted at around 80 mM potassium chloride concentration and the fractions were collected and concentrated by ultrafiltration (1.75 mL). This concentrated solution was equilibrated in advance with 10 mM Tris-HCl buffer (pH 7.5) containing 0.1 M potassium chloride, 2 mM β-mercaptoethanol, and 1 mM calcium chloride (1.5 × 73 cm: bio). Gel filtration. The resulting active fractions were collected and concentrated by ultrafiltration (1.5 mL, 33.9 U, 1.01 mg protein). By the above operation, electrophoretically uniform exopolygalacturonase was purified to an activity yield of about 6% and a specific activity of about 165 times.
The exopolygalacturonase fraction obtained by the above purification operation exhibited the aforementioned enzymatic properties.
[0034]
【The invention's effect】
The exopolygalacturonase of the present invention is a novel enzyme having an optimum reaction pH near pH 7 and having a surfactant resistance and a chelating agent resistance. It is.
[Brief description of the drawings]
FIG. 1 is a graph showing the influence of pH on exopolygalacturonase activity of the present invention.
FIG. 2 is a graph showing the influence of temperature on the exopolygalacturonase activity of the present invention.
FIG. 3 is a graph showing the influence of pH on the stability of exopolygalacturonase of the present invention.
FIG. 4 is a graph showing the influence of temperature on the stability of exopolygalacturonase of the present invention.

Claims (3)

Exopolygalacturonase derived from Bacillus sp. KSM-P443 (FERM P-17561) having the following enzymatic properties.
(1) Action: Acts on polygalacturonic acid (pectinic acid) and pectin, and exohydrolyzes α-1,4 bonds of polygalacturonic acid to produce monogalacturonic acid.
(2) Optimal reaction pH: pH 7 (MOPS buffer, Tris-HCl buffer)
(3) Optimum reaction temperature: about 60 ° C. (Tris-HCl buffer, pH 7.0)
(4) pH stability: pH 7-12 (treatment at 40 ° C. for 60 minutes)
(5) Heat resistance: stable to about 55 ° C. (Tris-HCl buffer, pH 7.0, treated for 15 minutes)
(6) Molecular weight: about 45000 (SDS electrophoresis method)
(7) Isoelectric point: around pH 5.8 (isoelectric focusing method)
(8) Chelating agent: Not inhibited by EDTA (10 mM).
(9) Surfactant resistance: Stable to surfactant (0.1%).   Bacillus sp. KSM-P443 (FERM P-17561) which produces the exopolygalacturonase according to claim 1. A method for producing exopolygalacturonase, comprising culturing the microorganism according to claim 2 and collecting the enzyme according to claim 1 from the culture.

Images