JPH1042868A - Activation method of immobilized papain - Google Patents

Abstract

(57) [Summary] [PROBLEMS] When using a fixed papain to batch-degrade a polypeptide such as a protein or a partially degraded product of a protein without using a drug not permitted to be used in foods And a method for activating the enzyme activity of immobilized papain. SOLUTION: 0.1 to 5.0% by weight of immobilized papain is used.
Better than untreated by treating in a mixed aqueous solution containing 0.07 to 3.45% by weight of L-cysteine or 0.5 to 7.5% by weight of L-cystine. The present invention relates to a method for activating immobilized papain capable of obtaining a high activity and a long-lasting activity.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to the industrial use of immobilized papain in which papain is immobilized on a carrier used in the field of food production such as production of functional food materials such as seasonings and partially decomposed products of milk protein. The present invention relates to an activation method which is highly safe and can express the activity of immobilized papain extremely effectively.

[0002]

2. Description of the Related Art Papain is a plant-derived proteolytic enzyme derived from papaya and is widely used as a food enzyme for the purpose of producing seasonings, producing functional materials, and the like.

The degrading enzymes are classified as cysteine proteases having one cysteine as an active center. The sulfhydryl group in cysteine has high reactivity and is easily oxidized by trace amounts of metal ions, oxidizing agents and the like. It is known to be deactivated. For this reason, a method of reacting a chelating agent such as ethylenediaminetetraacetic acid (hereinafter referred to as EDTA) and a cysteine having a sulfhydryl group with a substrate in a state where the cysteine coexists in the reaction substrate is described in "Method in Entology", Vol. 19, pp. 226-244. (“Metho
ds in enzymology ”, published by Academic press).

[0004] The applicant of the present invention has disclosed a method of immobilizing an enzyme on a chitosan-based carrier for immobilizing an enzyme, treating the enzyme with a mixed solution of EDTA and cysteine hydrochloride, and activating the same (Japanese Patent Application No. Hei 7-277046). Disclosed. Also, “Journal of fermentation and bioengineering”
gineering "78,3,241-245 (1994)) describes the use of immobilized papain immobilized on latex microparticles based on the same idea and adding EDTA and cysteine to the substrate to perform a continuous decomposition reaction. However, there is a problem in food production because EDTA is not approved for use.

[0005]

SUMMARY OF THE INVENTION When immobilized papain used for food production is subjected to batch decomposition reaction of polypeptides such as proteins and partially decomposed products of proteins, EDTA which is not permitted to be used for food is not permitted. The present invention provides a method for activating the enzyme activity of immobilized papain without using the same.

[0006]

SUMMARY OF THE INVENTION According to the present invention, immobilized papain is prepared by adding 0.1 to 5.0% by weight of metaphosphoric acid to 0.07 to 5.0% by weight.
3.45% by weight of L-cysteine or 0.5 to 7.5
The present invention relates to a method for activating immobilized papain, in which a treatment in a mixed aqueous solution containing L-cystine in an amount of more than 1% by weight gives a better activity and a longer-lasting activity than that of an untreated product.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The immobilized papain of the present invention is obtained by immobilizing papain, which is the above-mentioned protease, on a carrier. In the immobilized papain, the carrier for immobilizing papain is synthetic. Polymer ion exchanger,
Examples thereof include polysaccharide ion exchangers and inorganic carriers such as silica gel and glass, but are not particularly limited. Applicant filed Japanese Patent Publication No. 1-16420, Japanese Examined Patent Publication No. 63
The particulate porous chitosan and the crosslinked particulate porous chitosan obtained by the method disclosed in JP-A-54285 and the particulate porous chitosan disclosed in Japanese Patent Application No. 7-277046 are dispersed in an aqueous solution of carrageenan, and then polyfunctional. In the case of immobilized papain obtained by immobilizing papain using chitosan such as an enzyme immobilization carrier obtained by reacting a reagent as a carrier, a particularly excellent effect is exhibited.

The metaphosphoric acid used in the present invention is a linear polyphosphoric acid having an average degree of polymerization of 3 and hexametaphosphoric acid.
It is preferable to use sodium salt of linear polyphosphoric acid and hexametaphosphoric acid having an average degree of polymerization of 3 from the viewpoint of solubility. It is particularly preferable to use the hydrochloride of L-cysteine used in the present invention from the viewpoint of solubility. The activation treatment is carried out by adding immobilized papain to a mixed aqueous solution containing metaphosphoric acid and L-cysteine or L-cystine, and stirring the mixture.
A single treatment with either cysteine or L-cystine has no effect.

The concentration of metaphosphoric acid in the treatment solution is 0.1% by weight, more preferably 0.5% by weight or more from the viewpoint of the effect of activation, but at a high concentration, the solubility decreases and the viscosity increases. From the viewpoint of occurrence, it is used at 5.0% by weight, more preferably at 2.0% by weight or less. The concentration of L-cysteine used as a mixture is preferably 0.07% by weight, more preferably 0.14% by weight or more from the viewpoint of the effect of activation. However, at a high concentration, the solubility is reduced to 3.45%. % By weight, more preferably 1.38% by weight or less.

[0010] The concentration of L-cystine in the treatment solution is as follows:
It is preferably 0.5% by weight, more preferably 1.5% by weight or more from the viewpoint of the effect of activation, but at a high concentration, 7.5% by weight, more preferably 5.0% by weight because solubility is reduced. % Or less is preferable.

The activation treatment time and the volume ratio between the carrier and the mixed aqueous solution are 2 to 12 hours, 1: 2 to 2 in view of the operability.
1:10 is preferable, and the treatment temperature is preferably room temperature or lower from the viewpoint of not lowering the activity. After the activation treatment, the resultant is sufficiently washed with pure water to obtain highly active immobilized papain. Even if the activation treatment is performed before using immobilized papain with immobilized papain, this immobilized papain can be activated when reused after use in batch decomposition reaction It is.

[0012]

EXAMPLES The present invention will be described below with reference to examples, but the present invention is not limited to these ranges. The proteolytic activity of the immobilized papain was measured by the following method.

<< Method of Measuring Activity of Immobilized Papain >> (1) 100 g of casein was dissolved in 1,000 ml of 0.05 M Tris-HCl buffer (pH 8.0) by heating in a hot water bath. Make a solution. (2) 50 ml of a substrate solution is added to 1 ml of immobilized papain having papain immobilized on a carrier for immobilization, followed by stirring at 37 ° C. for 10 minutes. (3) After completion of the reaction, 1 ml of the supernatant is collected, and 3 ml of a 5% aqueous solution of trichloroacetic acid is added. (4) Centrifugation is performed at 3,500 rpm for 15 minutes, and the absorbance (c) at 280 nm of the supernatant is measured with a spectrophotometer (manufactured by Beckman KK, model: DU-640) in a quartz cell having a 1 cm optical path length. . At the time of measurement, dilution is performed so that the measured value does not exceed 0.5 (dilution ratio). (5) As a blank, a non-enzyme-fixed immobilization carrier was used in place of immobilized papain, and the same operation was performed as described above.
The absorbance (d) at 80 nm is measured. The dilution ratio at this time is the same as that in (4). (6) The activity of immobilized papain is calculated by the following equation. Activity of immobilized papain (U / (ml · carrier)) = 0.4 ×
(Cd) x dilution ratio

Example 1 210 g of chitosan having a degree of deacetylation of 79% and an average molecular weight of 58,000 was dissolved in 2,790 g of a 3.5% acetic acid aqueous solution. The chitosan acidic aqueous solution was mixed with 7% sodium hydroxide, 20% ethanol, 73%
% Chitosan after solidifying and regenerating chitosan into a granular porous material, and thoroughly washing with water until neutral, and an average particle size of 1%.
2,995 ml (wet) of regenerated granular porous chitosan having a thickness of 2 mm were obtained.

Next, 500 ml of the regenerated granular porous chitosan is
2% by weight kappa carrageenan aqueous solution (Hercules ・
Japan Co., Ltd., product name GENUVISCO Type CSW-2) 1,
After stirring for 24 hours, the aqueous solution of kappa carrageenan was removed by filtration, and 500 ml of 5.6% by weight epichlorohydrin and 120 ml of 5N-KOH were added, and the mixture was stirred and reacted at 70 ° C. for 2 hours. After completion of the reaction, the resultant was sufficiently washed with water to obtain a carrier for enzyme immobilization.

[0016] 100 ml of the obtained enzyme-immobilizing carrier was added to 1%
It was added to 500 ml of papain aqueous solution and stirred at room temperature for 2 hours. After completion of the stirring, the mixture was sufficiently washed with water to obtain 100 ml of immobilized papain.

Next, 1% of the immobilized papain was added with 1.0% by weight of linear sodium polyphosphate (manufactured by Wako Pure Chemical Industries, Ltd.) as an activator, and L-cysteine hydrochloride (Wako Pure Chemical Industries, Ltd.) was added thereto. 5 ml of Yakuhin Kogyo Co., Ltd. or 5 ml of L-cystine (Wako Junyaku Kogyo Co., Ltd.) was mixed at different concentrations as shown in Table 1, and stirred at 10 ° C. for 10 hours to perform an activation treatment. After that, it was sufficiently washed with water. After washing with water, the results of measuring the proteolytic activity of the immobilized papain are shown in Table 1.

[0018]

[Table 1]

As shown in Table 1, when the concentration of the linear sodium polyphosphate in the activator is 1.0% by weight, the L-
Cysteine hydrochloride concentration of 0.2-2.0% by weight, ie
L-cysteine conversion concentration is 0.14 to 1.38% by weight,
Alternatively, high activity is obtained when the concentration of L-cystine mixed with linear sodium polyphosphate is 1.5 to 5.0% by weight, and high activity value with respect to immobilized papain which has not been activated. it is obvious.

Example 2 1 ml of immobilized papain obtained in the same manner as in Example 1 was added to 0.5% by weight of L-cysteine hydrochloride.
After changing the concentration as shown in Table 2 and mixing with 5 ml of sodium hexametaphosphate (manufactured by Wako Pure Chemical Industries, Ltd.), stirring at 10 ° C. for 10 hours, and performing an activation treatment,
Washed thoroughly with water. After washing with water, the results of measuring the proteolytic activity of the immobilized papain are shown in Table 2.

[0021]

[Table 2]

According to Table 2, when the concentration of L-cysteine hydrochloride in the activator is 0.5% by weight, that is, when the concentration in terms of L-cysteine is 0.35% by weight, linear sodium polyphosphate mixed therewith or High activity is obtained when the concentration of sodium hexametaphosphate is 0.5 to 2.0% by weight,
It is evident that it has a high activity value against immobilized untreated papain.

Example 3 1 ml of immobilized papain obtained in the same manner as in Example 1 was added to 4.0% by weight of L-cystine, and 5 ml of sodium linear polyphosphate or 5 ml of sodium hexametaphosphate was added thereto. After changing the concentration as shown in Table 3, mixing and stirring at 10 ° C. for 10 hours to perform an activation treatment, the mixture was sufficiently washed with water. After washing with water, the results of measuring the proteolytic activity of the immobilized papain are shown in Table 3.

[0024]

[Table 3]

According to Table 3, when the concentration of L-cystine in the activator is 4.0% by weight, the concentration of sodium linear polyphosphate or sodium hexametaphosphate mixed with this is 0.5 to 2.0% by weight. It is clear that a high activity is obtained at the time of and that the compound has a high activity value with respect to the untreated activation.

Example 4 1 ml of immobilized papain obtained in the same manner as in Example 1 was collected, and 1.0% by weight of linear sodium polyphosphate and 0.5% by weight were added before the batch decomposition reaction of casein. % L-cysteine hydrochloride mixed aqueous solution 5
The mixture is put into a ml, activated at 5 ° C. for 10 hours, washed with water, and then subjected to a decomposition reaction. This was repeated five times to perform an activation treatment and a batch decomposition reaction (A). Similarly, 1 ml of immobilized papain was collected and placed in 5 ml of a mixed aqueous solution containing 1.0% by weight of sodium linear polyphosphate and 4.0% by weight of L-cystine at 5 ° C. for 10 hours before the batch decomposition reaction of casein. After activation treatment and washing with water, a decomposition reaction was performed, and this was repeated five times to perform a batch decomposition reaction (B). The batch decomposition reaction was repeated 5 times without performing the activation treatment before the batch decomposition reaction (C).
Table 4 shows the results obtained by measuring the proteolytic activity in each of the repeated batch decomposition reactions (A), (B), and (C).

[0027]

[Table 4]

From Table 4, it is apparent that the activation treatment before the reaction maintains a high proteolytic activity in the repeated batch decomposition reaction.

Example 5 Water contained in 600 ml of regenerated granular porous chitosan obtained in the same manner as in Example 1 was sufficiently removed with dimethylformamide. 500 ml of dimethylformamide and 500 ml of the regenerated granular porous chitosan
5.2 g of hexamethylene dissocyanate was added and reacted at room temperature for 2 hours. After removing the reaction residual liquid, the resultant was washed with dimethylformamide and then thoroughly washed with pure water to obtain an isocyanate crosslinked regenerated granular porous chitosan carrier.

As the carrier, the isocyanate cross-linked regenerated granular porous chitosan, a commercially available carrier X (Diaion HP-50, trade name, manufactured by Nippon Rensui Co., Ltd.), and a carrier Y (trade name, manufactured by Nippon Rensui Co., Ltd.) Name Diaion WA-30), carrier Z
(Amberlite IRA-9 manufactured by Organo Corporation)
3) 15 ml each of 2.5% glutaraldehyde aqueous solution 3
0 ml, and reacted at room temperature for 2 hours. After completion of the reaction, the mixture was washed with water, added to 5 ml of a 1% aqueous papain solution, and stirred at room temperature for 2 hours. After completion of the stirring, the mixture was sufficiently washed with water to obtain 15 ml of immobilized papain.

Next, 1 ml of each immobilized papain was collected and placed in 5 ml of a mixed aqueous solution containing 1.0% by weight of sodium linear polyphosphate and 0.5% by weight of L-cysteine hydrochloride, and activated at 5 ° C. for 10 hours. Treated and washed with water. Similarly, 1 ml of each immobilized papain was collected and not treated with the activator. The proteolytic activity of each of these immobilized papains was measured, and the results are shown in Table 5.

[0032]

[Table 5]

From Table 5, it can be seen that activation of the immobilized papain by the method of the present invention shows a higher proteolytic activity than that of the unactivated papain even if the carrier on which papain is immobilized is different. It is.

Example 6 1 ml of each immobilized papain obtained in the same manner as in Example 5 was collected, and a mixed aqueous solution containing 1.0% linear sodium polyphosphate and 4.0% by weight L-cystine was obtained. The mixture was placed in 5 ml, activated at 5 ° C. for 10 hours, and washed with water. Similarly, 1 ml of each immobilized papain was collected and not treated with the activator. The proteolytic activity of each of these immobilized papains was measured, and the results are shown in Table 6.

[0035]

[Table 6]

From Table 6, it is apparent that the activation treatment of the immobilized papain by the method of the present invention shows a higher proteolytic activity than that of the unactivated treatment even if the carrier on which papain is immobilized is different. .

According to the method of the present invention, when performing a batch decomposition reaction of a polypeptide using immobilized papain, 0.1-5.0% by weight of metaphosphoric acid and 0.1% by weight of metaphosphoric acid are added before the decomposition reaction. 07
-3.45% by weight of L-cysteine or 0.5-7.
By performing the activation treatment in a mixed aqueous solution containing 5% by weight of L-cystine, the activity value is remarkably increased, the reaction can be effectively performed, and the activation is performed even after use. The effect that the activity value can be increased again can be obtained. Furthermore, metaphosphoric acid, L-cysteine, and L-cystine are substances that are permitted to be used in foods, so that the method of the present invention can be used for the production of foods, which is industrially advantageous.

Continued on the front page (72) Inventor Yoshitaka Tamura 5-1-83 Higashihara, Zama City, Kanagawa Prefecture Morinaga Dairy Co., Ltd. (72) Inventor Hiroshi Miyagawa 5-1-83 Higashihara, Zama City, Kanagawa Prefecture Morinaga Milk Products Co., Ltd. Nutrition Science Laboratory (72) Inventor Hiroshi Ochi 5-1-83 Higashihara, Zama City, Kanagawa Prefecture Morinaga Milk Industry Nutrition Science Laboratory Co., Ltd.

Claims (1)

[Claims] 1. An immobilized papain containing 0.1 to 5.0% by weight.
Activation of immobilized papain, characterized in that the treatment is carried out in a mixed aqueous solution containing 0.07 to 3.45% by weight of L-cysteine or 0.5 to 7.5% by weight of L-cystine. Method.