JPH0143548B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to protopectin solubilizing enzymes.
Enzymes that decompose pectin substances are used in practical applications for clarifying fruit juice and crushing plant tissues. The present inventors conducted extensive studies to obtain a novel pectin-degrading enzyme, and as a result, they arrived at the present invention. The gist of the present invention resides in a protopectin solubilizing enzyme having the following physicochemical properties. (b) Action and substrate specificity Solubilizes protopectin. It acts particularly well on protopectin in plant tissues. It does not act on galactyuronic acid dimer, but decomposes trimer into dimer and monomer, and converts tetramer into trimer, monomer and two molecules of dimer.
The pentamer is decomposed into a trimer and a dimer. (b) Optimal PH 3.5 to 5.5 (c) Stable PH range 2 to 6.5 (d) Potency measurement method Add 0.5 ml of the enzyme solution of the test sample to 10 mg of protopectin and 0.02 M acetate buffer (PH 5.0). Add the mixture to make a total volume of 2.5 ml, react at 37°C for 30 minutes, filter, and measure the liberated pectin by a carbazole/sulfuric acid reaction. (E) Suitable temperature range for action: 45-55℃ (F) Molecular weight: Approximately 40,000 according to SDS-electrophoresis using bovine serum albumin, egg albumin, α-thymotrypsinogen A, myoglibin, and cytochrome C as internal standards, cytochrome C, and lysozyme. , α-thymotrypsinogen A, egg albumin, and Cephadex G using bovine serum ambumin as internal standards.
-75 gel (Sephadex is a gel filtration agent manufactured by Pharmacia Fine Chemical Co., Ltd., trademark) is about 30,000, and according to the sedimentation equilibrium method at 26,000 rpm it is about 26,800. (g) Sedimentation constant, isoelectric point The sedimentation constant measured by the sedimentation equilibrium method is
2.99S, and the isoelectric point (PH) measured by focal electrophoresis is 8.4-8.5. (h) Sugar content The sugar content determined by the phenol-sulfuric acid method was 3.2% by weight as pentose. (li) Inhibition Inhibited by Hg ⁺ or Ba ²⁺ . (N) Crystal form The crystal has a plate-like shape, but it is unstable and easily broken. (Le) Amino acid composition (molecular ratio) Lysine 19, Histidine 7 Arginine 4, Tryptophan 11 Aspartic acid 42, Threonine 22 Serine 33, Glutamic acid 20 Proline 7, Glycine 43 Alanine 18, Half cystine 1 Valine 20, Isoleucine 24 Leucine 13, Tyrosine 7 Phenylalanine 9, Carbohydrate 8 (as pentose) (W) Specific activity 3945 units/mg protein (W) Absorbance Absorbance E1% 1cm, 280nm is 11.93. (f) Purification method Obtained from Galactomyces reessii L strain belonging to Onygenaceae (tentative position). To separate and purify the protopectin-solubilizing enzyme from Galactomyces reesei L, culture filtrate was equilibrated with 0.02M acetate buffer (PH5.0) using CM-Sephadex C-50 (manufactured by Pharmacia Huain Chemical Co., Ltd.). gel filtration agent,
The enzyme is adsorbed onto a CM-Sephadex (Trademark) column, and the enzyme is eluted using a 0-0.4M salt gradient method. The active fractions are collected, concentrated, added to a CM-Sephadex C-50 column, and subjected to column chromatography to obtain a purified enzyme. The protopectin solubilizing enzyme of the present invention will be explained in more detail below. (b) Action and substrate specificity An enzyme that degrades and solubilizes protopectin.
It is a type of endo-polygalactylonase. Regarding its mode of action on galactyuronate oligomers, it does not act on galactyuronate dimers, but decomposes trimers into dimers and monomers, albeit at a very slow rate. In addition, the tetramer is decomposed into two forms: a trimer, a monomer, and two molecules of dimer. Furthermore, the pentamer is broken down into trimers and dimers. In addition, this enzyme acts well on protopectin in plant tissues. (b) Optimal PH The optimal PH of this enzyme is in the range of PH3.5 to 5.5. (c) Stable pH range The residual activity of this enzyme when treated at 50°C for 30 minutes is shown in Figure 1, and it is stable at pH 2 to 6.5. (d) Measurement method of titer Add 0.5 ml of the enzyme solution of the test sample to 10 mg of protopectin and 0.02 M acetate buffer (PH5.0) to make a total volume of 2.5 ml, react at 37°C for 30 minutes, and then The pectin released was determined by quantifying it by a carbazole-sulfuric acid reaction. One unit of oxygen activity equals 1 μmole of galactulonic acid per ml of reaction mixture in 30 minutes.
It is defined as the amount of enzyme that liberates pectin equivalent to . The substrate protopectin was obtained by collecting the albetic layer of the peel of Satsuma mandarin orange, pulverizing it finely, washing it with water repeatedly to completely remove the water-soluble pectin, and then freeze-drying it, and then adding 100 to 200 mesh pieces. The protopectin sample is prepared by pulverizing it into small pieces. (e) Suitable temperature range for action We conducted the reaction for 30 minutes at various temperatures under the condition of PH5.0, and investigated the effect of temperature on the activity.
It is a diagram. As is clear from Figure 2, 45 to 55
It has an optimum temperature at ℃. Furthermore, the residual activity of this enzyme when treated for 30 minutes at various temperatures under PH5.0 conditions is as shown in Figure 3, and is stable below 55°C. (F) Molecular weight: Approximately 40,000 according to SDS-electrophoresis using bovine serum albumin, egg albumin, α-thymotrypsinogen A, myoglibin, and cytochrome C as internal standards, cytochrome C, lysozyme, α-thymotrypsinogen A, egg albumin, and bovine Cephadex G with serum ambumin as internal standard
-75 gel (Sephadex is a gel filter agent manufactured by Pharmacia Fine Chemicals, trademark) is about 30,000, and according to the sedimentation equilibrium method at 26,000 rpm, it is about 268,000. (g) Sedimentation constant, isoelectric point The sedimentation constant measured by the sedimentation equilibrium method is
2.99S, and the isoelectric point (PH) measured by focal electrophoresis is 8.4-8.5. (h) Sugar content The sugar content determined by the phenol-sulfuric acid method was 3.2% by weight as pentose. (li) Inhibition Inhibited by Hg ⁺ or Ba ²⁺ . (v) Crystal form Crystallized by dialysis against ammonium sulfate solution. Although the crystal has a plate-like shape, it is unstable and easily broken. (Le) Amino acid composition (molecular ratio) Lysine 19, Histidine 7 Arginine 4, Tryptophan 11 Aspartic acid 42, Threonine 22 Serine 33, Glutamic acid 20 Proline 7, Glycine 43 Alanine 18, Half cystine 1 Valine 20, Isoleucine 24 Leucine 13, Tyrosine 7 Phenylalanine 9, Carbohydrate 8 (as pentose) (2) Specific activity The specific activity of protopectin solubilizing enzyme is 3945 units/mg protein. (W) Absorbance Absorbance of 1% solution in aqueous solution at 280 nm using a 1 cm thick cell E1% 1 cm, 280 nm is 11.93. (F) Purification method The enzyme of the present invention is derived from Galactomyces reesei (tentative position), which belongs to the family Onygenaceae.
reessii) obtained from L strain. Galactomyces reesei is described by Antony van Lebenhutsk, Journal of Microbiology and Therology, Vol. 25, pp. 458-464 (1959), Vol. 38, pp. 289-309 (1972), and Canadian Journal of Botany, Vol. 55. This is a known species described on pages 1701-1711 (1977). The mycological properties of the L strain were consistent with the descriptions in these literatures, so this strain was classified as Galactomyces reessii (van der Walt).
Redhead et Malloch]. The main mycological characteristics of this strain are listed below. (a) Growth status in each medium Wort medium (see Figure 4) After culturing at 25°C for 3 days, a fungal system with septa, asci and segmented conidia are formed. Width 2.5~8μm. Produces a dull, wrinkled, milky film and precipitate. Wort agar medium Growth is good; after 3 days at 27°C, the colonies are milky white and smooth, and later become slightly powdery. peripheral hairy, septated hyphae, ascus (usually
produces one ascospore) and segmented conidia. Does not form budding conidia. Potato glucose agar medium Growth is good. After 3 days at 27°C, hairy, wrinkled, milky white colonies are formed. Peripheral hairiness.
Forms septated hyphae, ascospores (usually producing one ascospore) and segmented conidia. Does not form budding conidia. (b) Formation of ascospores (see Figure 5) Ascospores are well formed by slide culture using the potato-glucose agar medium described above. Ascus formation is due to the fusion of gametes. Occurs on rather long, branched, irregular gametocyst hyphae. The gametocyst hyphae coalesce at the tip and then form two to two at the base.
It coils three times and is separated from the hyphae by a septum. Ascospores are oblate, wrinkled, thin, with an irregular outer membrane and one large oil body. Size: 6-9 μm x 5-7 μm (c) Conidial period (see Figure 6) Segmented conidia are produced, both ends are obtuse circles, and width is 2-2 μm.
7.5μm (d) Physiological properties Nitrate or nitrite assimilation: Negative Urea hydrolysis: Negative Ester production: Positive (apple aroma) NaCl tolerance: 4-6% Growth at 37℃: weakly positive or negative Fermentability: None Growth stimulation by vitamins: Pyridoxine and thiamine stimulate growth. Assimilation of carbon compounds glucose +, galactose + maltose, sucrose - trehalose, lactose - raffinose +, L-soribose + xylose +, arabinose - L-arabisose, ribose - L-rhamnose, ethanol - glycerin +, Erythritol - adonitol -, mannitol - sorbitol +, cellobiose - salicin -, glyconate - 2-ketogluconate +, D,L-lactate - succinate +, citrate - acetate -, inositol - inulin +, soluble starch - (e) Isolation source: Isolated from Japanese tangerine fruit.
The Galactomyces reese L strain has been deposited with the Institute of Microbiology, Agency of Industrial Science and Technology, and its microorganism accession number is Microbiological Research Institute No. 5726. Therefore, in order to separate and purify the protopectin solubilizing enzyme from Galactomyces reesei L., the culture solution was diluted with 0.02M acetate buffer (PH5.0).
It was added to a column of CM-Sephadex C-50 (a gelling agent manufactured by Pharmacia Fine Chemical Co., Ltd., CM-Sephadex is a trademark) equilibrated with 100% sodium chloride, and adsorbed on the column. Elute the enzyme. The active fractions are collected, concentrated, added to a CM-Sephadex C-50 column, and subjected to column chromatography to obtain a purified enzyme. Furthermore, a preferred purification method includes a method using affinity chromatography using a gel in which pectin is insolubilized. As a gel in which pectin is insolubilized, a gel in which a mixture of starch and pectin is immobilized is particularly preferable. For immobilization, mix soluble starch and pectin in a weight ratio of 2:1, for example, and dilute
What is necessary is to dissolve it in an aqueous NaOH solution and then add epichlorohydrin for reaction. Purification is carried out as follows. That is, the culture solution
Dialyzed against 0.01M acetate buffer pH 5.0, passed through a column of the above pectin-insolubilized gel equilibrated with the same buffer, washed the column, removed non-adsorbed proteins, and then adjusted to a saline concentration of 0 to 0.2. If the enzyme is eluted using a concentration gradient of M, the protopectin-solubilized enzyme will be eluted at a salt concentration of 0.1M. This method has the advantage of a high enzyme yield of approximately 90%. The protopectin solubilizing enzyme of the present invention as described above is different from known protopectin solubilizing enzymes. For example, it is a type of yeast. Trichosporon
Penicillatum (Trichosporon penicillatum)
The protopectin solubilizing enzyme obtained from the SNO-3 strain (described in Fermentation and Industry, Vol. 37, pp. 928-939, 1979; hereinafter referred to as S-enzyme) has a sedimentation constant, etc. They differ in electrical point, sugar content, specific activity, amino acid composition, etc. Furthermore, the protopectin solubilizing enzyme of the present invention is characterized in that it acts well on protopectin in plant tissues. Hereinafter, the present invention will be explained in more detail with reference to Examples. Example 1 (1) Cultivation of Galactomyces reesei L Galactomyces reesee L having the above properties, 0.5 g of glycose, caseic acid decomposition product (product manufactured by Nissui Pharmaceutical Co., Ltd. was used)
0.4g, KNO ₃ 0.11g, MgSO ₄・7H ₂ O, 0.05g,
The cells were cultured at 30° C. for 24 hours in a medium (PH 5.0) consisting of 0.01 g of CaCl ₂ , 50 μg of thiamine-HCl, 50 μg of pyridoxine-HCl, and 100 ml of water. Culture solution
20 contains 6450mg of protein and 735
×10 ³ units of protopectin solubilizing enzyme activity was observed. (2) Separation and purification of protopectin-solubilizing enzyme (a) Preparation of pectin-insolubilizing gel Dissolve 3 g of a 2:1 (weight ratio) mixture of soluble starch and pectin in a 3.5N NaOH aqueous solution and stir at 60°C for 30 minutes. did. Next, 15 ml of epichlorohydrin was added and the mixture was stirred at 60°C for 60 minutes, and then left at 60°C for 60 minutes. The resulting gel was crushed into 35 mesh pieces. This gel was thoroughly washed with water and used for purification. (b) Separation and purification The culture solution obtained in (1) was dialyzed against 0.01M acetate buffer PH5.0 and equilibrated with the same buffer.
It was passed through the column of pectin-insolubilized gel in (a). The column was washed to remove unadsorbed proteins by passing 0.01M acetate buffer (PH5.0) through the column, and then washed with 0.01M acetate buffer (PH5.0) with a concentration gradient of 0 to 0.2M NaCl. The enzyme was then eluted. Electrophoretically homogeneous protopectin solubilizing enzyme at a concentration of 0.1 M NaCl
% yield. The protein content is
150mg, protopectin solubilizing enzyme activity is 594
×10 ³ units, specific activity was 3945 units/mg protein. When the obtained enzyme solution was dialyzed against 50% ammonium sulfate 0.02M acetate buffer pH 5.5, plate-shaped crystals were obtained, but the crystals were unstable in shape and fragile. (c) Measurement of solubilizing effect on various pectins The effect of this enzyme was measured using mandarin orange, burdock, radish, watermelon, and carrot. That is, protopectin preparations were prepared from various plant tissues in the same manner as in the case of Satsuma mandarin, and this was mixed with 1 ml of 0.01M acetate buffer (PH5.0).
The suspension was suspended at a rate of 10 mg per ml, the enzyme unit obtained in (b) was added, and the mixture was reacted at 37°C for 30 minutes. For comparison, the aforementioned S-enzyme was also tested. The results are shown in Table 1. 【table】

[Brief explanation of drawings]

FIG. 1 is a curve showing the stable PH range of the protopectin solubilizing enzyme of the present invention, FIG. 2 is a curve showing its optimum temperature range, and FIG. 3 is a curve showing its stable temperature range. Figure 4 shows the Galactomyces leecii strain L, which is the source of the enzyme of the present invention, at 27°C.
Figure 5 is a diagram showing the various stages of asci formation of the strain; 1 is an initial ascus;
2 indicates a mature ascospore containing one ascospore, and 3 indicates an ascospore. FIG. 6 is a diagram showing segmented conidia of the strain.

Claims (1)

[Scope of Claims] 1. A protopectin solubilizing enzyme having the following physical and chemical properties. (b) Action and substrate specificity Solubilizes protopectin. It acts particularly well on protopectin in plant tissues. It does not act on galactyuronic acid dimer, but decomposes trimer into dimer and monomer, and converts tetramer into trimer, monomer and two molecules of dimer.
The pentamer is decomposed into a trimer and a dimer. (b) Optimal PH 3.5 to 5.5 (c) Stable PH range 2 to 6.5 (d) Potency measurement method Add 0.5 ml of the enzyme solution of the test sample to 10 mg of protopectin and 0.02 M acetate buffer (PH 5.0). Add the mixture to make a total volume of 2.5 ml, react at 37°C for 30 minutes, filter, and measure the liberated pectin by a carbazole/sulfuric acid reaction. (E) Suitable temperature range for action: 45-55℃ (F) Molecular weight: Approximately 40,000 according to SDS-electrophoresis using bovine serum albumin, egg albumin, α-thymotrypsinogen A, myoglibin, and cytochrome C as internal standards, cytochrome C, and lysozyme. , α-thymotrypsinogen A, ovalbumin, and Cephadex G using internal standards as bovine serum albumin.
-75 gel (Sephadex is a gel filtration agent manufactured by Pharmacia Fine Chemicals, trademark) is about 30,000, and according to the sedimentation equilibrium method at 26,000 rpm, it is about 26,800. (g) Sedimentation constant, isoelectric point The sedimentation constant measured by the sedimentation equilibrium method is
2.99S, and the isoelectric point (PH) measured by focal electrophoresis is 8.4-8.5. (h) Sugar content The sugar content determined by the phenol-sulfuric acid method was 3.2% by weight as pentose. (li) Inhibition Inhibited by Hg ⁺ or Ba ²⁺ . (N) Crystal form The crystal has a plate-like shape, but it is unstable and easily broken. (Le) Amino acid composition (molecular ratio) Lysine 19, Histidine 7 Arginine 4, Tryptophan 11 Aspartic acid 42, Threonine 22 Serine 33, Glutamic acid 20 Proline 7, Glycine 43 Alanine 18, Half cystine 1 Valine 20, Isoleucine 24 Leucine 13, Tyrosine 7 Phenylalanine 9, Carbohydrate 8 (as pentose) (W) Specific activity 3945 units/mg protein (W) Absorbance Absorbance E1% 1cm, 280nm is 11.93. (f) Purification method Obtained from Galactomyces reessii L strain belonging to Onygenaceae (tentative position). To separate and purify the protopectin-solubilizing enzyme from Galactomyces reesei L, culture filtrate was equilibrated with 0.02M acetate buffer (PH5.0) using CM-Sephadex C-50 (manufactured by Pharmacia Huain Chemical Co., Ltd.). gel filtration agent,
The enzyme is adsorbed onto a CM-Sephadex (Trademark) column, and the enzyme is eluted using a 0-0.4M salt gradient method. The active fractions are collected, concentrated, added to a CM-Sephadex C-50 column, and subjected to column chromatography to obtain a purified enzyme.