JP3101640B2 - Pectin degrading enzyme - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a pectin-degrading enzyme capable of decomposing pectin into low-molecular-weight pectin having a molecular weight of 20,000 to 80,000. In particular, the present invention relates to one in which the degrading enzyme is an endo-type polygalacturonase.

[0002]

BACKGROUND OF THE INVENTION Dietary fiber is defined as an indigestible component in food that cannot be digested by human digestive enzymes. Not only plant cell wall components such as cellulose, lignin, and pectin but also widely used chitin and chitosan. Contains digested organic matter. In recent years, they have been shown to have various effects such as a blood cholesterol lowering effect, including an effect of improving bowel movement, and also play an important role in preventing adult diseases.

[0003] Among these dietary fibers, pectic substances such as pectin and pectic acid have a strong activity as dietary fiber, and have various effects such as improvement of bowel movement, suppression of elevation of blood cholesterol level, suppression of formation of gallstones, and suppression of hypertension. The effect has been reported. Conventionally, pectin has been used as a stabilizer in the food industry for jams, fruit jellies, drink yogurt, lactic acid bacteria drinks, etc.
Because of the above-mentioned effects, it is also expected to be added to foods and drinks as food fiber.

[0004] Pectic substances are present in the form of a complex called protopectin by binding to cellulose in immature fruits or plants, and are particularly contained in large amounts in citrus fruits, apples, and karin. This protopectin is insoluble, but is hydrolyzed to yield soluble pectin or pectic acid upon fruit ripening.

[0005] Among them, pectin is a polysaccharide having a molecular weight of 200,000 or more and containing galacturonan, which is a polymer of galacturonic acid, as a main component and containing trace amounts of rhamnose, arabinose, xylose, and galactose.

However, generally, pectin has the properties of low solubility, high viscosity, and strong gelling ability. Therefore, despite its various effects as described above, pectin can be added to foods and drinks only in small amounts due to its properties, and can be contained in foods and drinks in an amount that can be expected to have an activity as a dietary fiber. It was difficult.

[0007] Therefore, in order to solve this problem, a low-molecular-weight pectin having low viscosity and high solubility and maintaining the physiological activity as a fiber is required.

[0008]

SUMMARY OF THE INVENTION In view of the above circumstances, an object of the present invention is to provide a pectin-degrading enzyme capable of producing low-molecular-weight pectin.

[0009]

Means for Solving the Problems The present inventors have reduced the molecular weight of pectin with a pectin degrading enzyme,
It is thought that pectin with low viscosity and high solubility can be obtained,
A number of pectin-degrading enzymes were studied. As a result, the present inventors have found that, when endo-type polygalacturonase is allowed to act on pectin, the molecular weight of pectin is reduced only to about 20,000 due to the decomposition even when the pectin is allowed to act to the limit. It has also been found that by controlling the reaction conditions, a low-molecular-weight pectin having a molecular weight of about 20 to 80,000 can be obtained.

Therefore, the object of the present invention can be solved by using the following means.

That is, this means is a pectin degrading enzyme which degrades pectin and pectic acid having the following properties (1) to (6).

(1) The pectin-degrading enzyme is an endo-type polygalacturonase produced from Saccharomyces.

(2) The optimum pH when reacted at 35 ° C. for 20 minutes is 4.0.

(3) The stable pH range when heated at 35 ° C. for 60 minutes is from 4.0 to 8.0.

(4) The optimum temperature at the time of reaction at pH 5.0 is 45 ° C.

(5) When heated for 60 minutes under the condition of pH 5.0, the enzyme activity is stable up to 45 ° C.

(6) The molecular weight is 38,000.

The present inventors have studied various microorganisms that produce pectin-degrading enzymes, and as a result, have found that yeast belonging to the genus Saccharomyces (Saccharomyces bayanus)
charomyces bayanus) and JTF-4) were found to produce the endo-type polygalacturonase for the first time.

The yeast was deposited on July 9, 1992 with the Research Institute of Microbial Industry and Technology of the National Institute of Advanced Industrial Science and Technology.
No. 916).

Hereinafter, the present invention will be described in detail.

In general, endo-type polygalacturonase is present in microorganisms, higher plants and the like, but many steps are required to purify the enzyme therefrom. That is, the culture supernatant obtained by removing the cells from the culture solution of microorganisms is subjected to ammonium sulfate precipitation to salt out only the protein, which is separated by charge using an ion exchanger, and further separated by molecular weight by gel filtration. Purification is performed by a general enzyme purification step.

Since the endo-type polygalacturonase of the present invention obtained from the above JTF-4 is an extracellular enzyme secreted outside the cells, its culture supernatant can be directly used as a crude enzyme solution. it can. Usually, a culture supernatant can be obtained by seed-culturing the above yeast on an agar medium, further subjecting the yeast to main culture to perform large-scale culture, and centrifuging the obtained culture to remove bacterial cells. . Thus, the culture supernatant obtained from the yeast is preferably used because it can be directly used for the enzymatic reaction, thereby simplifying the enzyme purification step.

The simple treatment, such as dialysis, ultrafiltration, ion exchange, or gel filtration, applied to the culture supernatant can make the liquid color more transparent in the reaction using the present enzyme. preferable.

The physicochemical properties of the endo-type polygalacturonase (hereinafter referred to as JTFP-4) of the present invention will be described below.

JTFP-4, which is an enzyme of the present invention, acts on pectin and pectic acid to hydrolyze them, and has the following properties.

(1) Substrate Specificity The JTFP-4 of the present invention degrades pectin and pectic acid, but does not degrade soluble starch, dextrin and xylan.

(2) Optimum pH JTFP-4 of the present invention has an optimum pH around pH 4.

(3) Stable pH Range JTFP-4 of the present invention is stable at pH 4 to 8.

(4) Enzyme activity The enzyme activity of JTFP-4 of the present invention is 33.9 units / mg protein.

(1 unit: in the hydrolysis of pectic acid, the reducing group of the hydrolyzate is 1 μmol / minute at 35 ° C.)
(Amount of generated enzyme) (5) Optimum temperature JTFP-4 of the present invention has an optimum temperature around 45 ° C.

(6) Stable temperature JTFP-4 of the present invention is stable up to 45 ° C.

(7) Effects of Metal Ions and Inhibitors JTFP-4 of the present invention is inhibited 69% by barium chloride, but not by magnesium sulfate. Also, E
74% inhibited by DTA.

(8) Molecular Weight The molecular weight of JTFP-4 of the present invention is 38,000.

(9) Amino acid composition JTFP-4 of the present invention contains glutamine and glutamic acid most in the molecule (130 residues per molecule).

The case where the enzyme of the present invention is allowed to act on pectin will be described below.

When the endo-type polygalacturonase (JTFP-4) of the present invention is allowed to act on pectin,
Either a purified product, a culture supernatant (crude enzyme solution) or a processed product thereof may be used.

The low-molecular-weight pectin can be obtained by reacting the purified product, culture supernatant, or the processed product obtained as described above with a suspension of pectin in a buffer such as acetic acid. .

This enzymatic decomposition reaction has a reaction time of 12 to 4 hours.
It is preferable to perform the reaction for 8 hours under the condition that the quantitative ratio of the yeast culture supernatant to 1 part by weight of pectin is 5 to 20 parts by weight. In addition, the reaction temperature and pH, the reaction proceeds sufficiently,
The temperature and pH at which the endo-type polygalacturonase is not inactivated, that is, 30 to 50 ° C and pH 4.0 to 8.0 are preferable.

Even if this reaction is carried out at the decomposition limit, the decomposition of pectin stops at a molecular weight of about 20,000. However, by controlling the reaction conditions such as the above reaction time, the pectin has an arbitrary molecular weight in the range of about 20,000 to 80,000. Low molecular pectin can be obtained.

The low-molecular-weight pectin of the present invention may have a molecular weight of about 20,000 to about 80,000, but from the viewpoint of maintaining physiological activity as a dietary fiber and easy addition to foods and drinks, has a molecular weight of about 50,000 to about 70,000. It is preferred to have. Most preferred is a low molecular weight pectin having a molecular weight on the order of 60,000.

The decomposed product of pectin thus obtained may be used after drying as it is, or may be further treated.

In the case of performing further treatment, the pectin degradation product is subjected to dialysis, ultrafiltration, etc. to remove galacturonic acid and its oligosaccharides in the degradation product, and acetic acid used as a buffer during the enzyme reaction. After subjecting to a purification step, the generated decomposed product is powdered by a precipitation step with an organic solvent such as ethanol or acetone, or a drying step such as freeze-drying or spray-drying, and is used.

The low molecular weight pectin obtained by the above method has a molecular weight between existing polysaccharides such as pectin and agarose as food additives and oligosaccharides such as maltooligosaccharides and fructooligosaccharides. In addition, this low-molecular-weight pectin has a considerably lower viscosity than the original pectin, and has an effect of improving bowel movement, which is one of the physiological activities of dietary fiber, despite its high solubility.

On the other hand, the low-molecular-weight pectin of the present invention has the above-mentioned properties, so that it has been impossible in the past to maintain the physiological activity as a dietary fiber.
01 to 50% by weight, preferably 0.1 to 5% by weight can be contained in various foods and drinks such as juice, candy, bread, jam and the like.

[0045]

The present invention will be described in more detail with reference to the following examples.

Example 1 Culture method of JTF-4 Saccharomyces bayanus (JTF-4) was sucrose 2%
In potato decoction agar slant medium (pH 5.0) containing
The cells were cultured at 8 ° C. for 3 days. After that, one platinum loop of the cells grown on the slant medium was taken, and 50 ml of a liquid medium (5% glucose, 0.2% ammonium phosphate, 0.1% potassium phosphate 0.1%, magnesium sulfate) was placed in a 200 ml Erlenmeyer flask. 0.1% and yeast extract 0.4%, pH
5.0), and cultured at 28 ° C. for 3 days.
This culture was inoculated into a 3 liter Erlenmeyer flask containing 1 liter of medium of the same composition, and further cultured at 28 ° C. for 3 days. After completion of the culture, the culture was incubated at 8,000 rpm for 10 minutes.
After centrifugation for minutes, the cells were removed to obtain a culture supernatant.

Example 2 Preparation method of JTFP-4 and measurement of molecular weight The culture supernatant obtained in Example 1 was filtered using a Millipore filter (pore size: 0.45 μm) to completely remove the cells. Next, the culture filtrate was added to a 0.02 M acetate buffer (pH
Dialysis was performed overnight at 5.0 ° C. in 5.0). About 600 ml of the dialyzed culture supernatant is applied to an ion exchange column (S-Sepharos
e) and eluted by density gradient method using saline. Active fractions were collected and subjected to gel filtration column chromatography (Sephadex G-75) using a 0.02 M acetate buffer as an eluent. This chromatogram is
One high activity peak was shown. The fractions having a high activity were collected, dialyzed overnight against distilled water at 5 ° C., and then concentrated to 5 ml by gel filtration. Culture supernatant 6
About 1 mg of purified enzyme was obtained as a protein from 00 ml.

The enzymatic activity (1 unit) was quantified by measuring the amount of reducing group produced in the hydrolyzate formed by the enzymatic reaction using the Somogi-Nelson method. That is, one unit is the amount of the enzyme that generates 1 μmol of the reducing group of the hydrolyzate per minute at 35 ° C. (the amount of the reducing group generated is converted as the amount of galacturonic acid). As a result of the measurement, the enzyme activity of the present invention was 33.9 units / mg protein.

When SDS polyacrylamide gel electrophoresis was performed using the sample, a single band was detected.

Example 3 The following experiment was conducted to examine the properties of the enzyme of the present invention.

(1) Substrate Specificity In order to examine the substrate specificity of the present enzyme, the reactivity with the substrates shown in Table 1 below was examined.

Each substrate was added to 0.2 M acetate buffer (pH 5.0) so that the final concentration was 0.2%. 0.1 ml of the enzyme solution is added to 0.15 ml of these solutions,
Somogi-Nelson method by reacting at 35 ° C. for 20 minutes,
That is, the presence or absence of substrate decomposing activity was assayed by measuring the amount of reducing terminals generated by enzymatic decomposition. Table 1 shows the decomposition activity for each substrate. In Table 1, ○ indicates that the enzyme was decomposed by the enzyme, and X indicates that the enzyme was not decomposed.

[Table 1] As can be seen from Table 1, this enzyme degrades pectin and pectic acid, but does not degrade soluble starch, dextrin and xylan.

(2) Optimum pH In 0.23 ml of McIlvaine buffer containing pectic acid and having a pH of 2 to 7 so that the final concentration becomes 0.2%,
0.02 ml of the enzyme solution was added, reacted at 35 ° C. for 20 minutes, and the activity was measured by the Somogi-Nelson method. Enzyme activity values were expressed as relative activities with the maximum activity value taken as 100%.
As shown in FIG. 1, relative activity determined by the Somogi-Nelson method was plotted against pH to determine the optimum pH. As can be seen from FIG. 1, the optimum pH of the present enzyme was around 4.

(3) Stable pH range The buffer solution is a 0.2 M McIlvain buffer (pH
3-7) and a phosphate buffer (pH 7-10) were used.

0.125 ml of each buffer solution of pH 3 to 10
Was added thereto and treated at 35 ° C. for 1 hour. Then, 0.5 M acetate buffer (pH 5.0) was added to 0.1 mL.
15 ml was added to adjust the pH to 5.0. To this solution was added pectic acid to a final concentration of 0.2% and 35
The reaction was carried out at 20 ° C for 20 minutes, and the activity was measured by the Somogi-Nelson method. Enzyme activity values were expressed as relative activities with the maximum activity value taken as 100%. As shown in FIG. 2, the relative activity determined by the Somogi-Nelson method was plotted against pH to determine a stable pH region. As can be seen from FIG. 2, the enzyme was stable at pH 4-8.

(4) Optimum temperature The enzyme was added to 0.23 ml of a 0.2 M McIlvain buffer (pH 5.0) containing 0.2% pectic acid, and the temperature of
Then, the mixture was reacted at a temperature of 20 to 80 ° C. for 5 minutes, and the activity was measured by the Somogi-Nelson method. Enzyme activity values were expressed as relative activities with the maximum activity value taken as 100%.
As shown in FIG. 3, the relative activity determined by the Somogi-Nelson method was plotted against the temperature to determine the optimum temperature. As can be seen from FIG. 3, the present enzyme has an optimum temperature around 45 ° C.

(5) Stable temperature range 0.5 M McIlvaine buffer (pH 5.0)
Add 0.02 ml of this enzyme to 13 ml, and add 20 to 65
Heat treatment was performed at a temperature of 60 ° C. for 60 minutes. After cooling on ice, 0.1 ml of a 0.5% aqueous solution of pectic acid was added to each treatment solution, reacted at 35 ° C. for 20 minutes, and the activity was measured by the Somogi-Nelson method. Enzyme activity values were expressed as relative activities with the maximum activity value taken as 100%. As shown in FIG. 3, the relative activity determined by the Somogi-Nelson method was plotted against temperature to determine a stable temperature range. As can be seen from FIG. 4, the relative activity of this enzyme was 73% up to 45 ° C., but decreased to about 20% at 65 ° C. From this result, it was determined that the stable temperature range of the present enzyme was up to 45 ° C.

(6) Effects of Metal Ions and Inhibitors The effects of metal ions and inhibitors on the present enzyme were examined.

[0060] The purified enzyme solution containing 0.002 ml
To 15 ml of a 0.2 M acetate buffer (pH 5.0), various metal ions and inhibitors shown in Table 2 below were added to a concentration of 1 mM. After allowing each solution to react at 35 ° C. for 5 minutes, 0.1 ml of a 0.5% aqueous solution of pectic acid was added, followed by 3 minutes.
The reaction was carried out at 5 ° C. for 20 minutes, and the inhibition rate was measured by the Somogi-Nelson method. The results are shown in Table 2. The inhibition rate is a relative value based on the case where no metal or inhibitor is added (0%).

[0060]

[Table 2] As is clear from the table, barium ion (barium chloride) inhibited 69%, but magnesium ion (magnesium sulfate) did not inhibit it at all.

(7) Molecular Weight The molecular weight of the present enzyme obtained in Example 2 was measured by SDS polyacrylamide electrophoresis to be 38,000.
Met.

(8) Composition of Amino Acid The enzyme of the present invention was hydrolyzed with 6 M hydrochloric acid at 105 ° C. for 24 hours. This was analyzed using an amino acid analyzer (Hitachi, 83
5) and the amount of constituent amino acids was measured. 3 measurements
The amino acid composition was calculated by repeating the procedure twice and calculating the ratio of each amino acid. Table 3 shows the results.

[0063]

[Table 3] As can be seen from the table, glutamine and glutamic acid were the most abundant, followed by serine.

Example 4 (1) Preparation of low-molecular-weight pectin from JTF-4 culture supernatant 100 g of lemon pectin (Wako Pure Chemical Industries, Ltd.) was added to 0.025.
M acetate buffer (pH 4.8) was suspended in 4 liters of the suspension, and the culture supernatant (JTFP) of JTF-4 produced in Example 1 was added thereto.
1 liter), and reacted at 40 ° C. for 24 hours. The obtained reaction solution was concentrated at 60 ° C. using a rotary evaporator, dialyzed overnight against 100 times the amount of deionized water of the sample solution, and further freeze-dried to obtain a low-molecular-weight pectin having a molecular weight of about 66,000. 70.4 g were obtained.

(2) Effect of improving bowel movement SD male rats of 4 weeks of age were bred on a solid feed (Oriental yeast solid feed MF) for 4 days, and divided into four groups of 5 rats each. Thereafter, each group was fed a feed having the composition shown in Table 4 below and a solid feed, and bred for 9 days.
Day stool was collected. The results obtained are shown in Table 5 below. On the basis of the softness of the stool of the solid feed, the cured food was defined as-and the softened one was evaluated as +.

[0066]

[Table 4]

[Table 5] From the results shown in Table 5, it was found that low-molecular-weight pectin produced by the enzyme of the present invention had a stool softening effect similarly to pectin, and was effective in improving bowel movement.

[0067]

Since the enzyme obtained in the present invention is an extracellular enzyme secreted outside the body, the culture supernatant thereof can be used as it is as a crude enzyme solution and can be directly used for the enzyme reaction. Therefore, there is an advantage that the enzyme purification step can be simplified. In addition, the enzyme of the present invention can easily decompose pectin into low-molecular-weight pectin, and the obtained low-molecular-weight pectin has low viscosity, high solubility, and physiological activity of dietary fiber such as improved bowel movement. , It can be easily added to foods and drinks to the extent that physiological activity as dietary fiber can be added.

[Brief description of the drawings]

FIG. 1 is a diagram showing the relationship between pH and relative activity for determining the optimum pH of the present enzyme.

FIG. 2 is a diagram showing the relationship between pH and relative activity for determining a stable pH region of the present enzyme.

FIG. 3 is a diagram showing a relationship between a temperature for determining an optimum temperature of the present enzyme and a relative activity.

FIG. 4 is a diagram showing the relationship between temperature and relative activity for determining the stable temperature range of the present enzyme.

──────────────────────────────────────────────────続 き Continuation of the front page (51) Int.Cl. ⁷ identification code FI C12R 1:85) (58) Investigated field (Int.Cl. ⁷ , DB name) C12N 9/00-9/99 C12N 1 / 00-1/38 BIOSIS (DIALOG) MEDLINE (STN) WPI (DIALOG)

Claims (1)

(57) [Claims] 1. A pectin-degrading enzyme that degrades pectin and pectic acid, wherein (1) the pectin-degrading enzyme comprises:
(2) that the optimum pH is 4.0 when reacted at 35 ° C. for 20 minutes; (3) that the end-type polygalacturonase is produced from Saccharomyces;
(4) the optimum temperature is 45 ° C. when the reaction is carried out at pH 5.0, and (5) the pH is 5 when the reaction is carried out at pH 5.0. A pectin-degrading enzyme comprising: when heated for 60 minutes under the condition of 0.0, the enzyme activity is stable up to 45 ° C; and (6) the molecular weight is 38,000.