JPS6013672B2 - Immobilized protease and its production method - Google Patents

Description

[Detailed description of the invention] The present invention relates to an immobilized protease and a method for producing the same.

Enzymes have high substrate specificity for various chemical reactions, mild reaction conditions, and high reaction efficiency, and are currently widely used in the food, pharmaceutical, and cosmetic industries as extremely useful catalysts. . However, since enzymes are generally water-soluble, they are difficult to recover after use, making them not only uneconomical but also difficult to separate from reaction products. Against this background, many proposals have been made to immobilize enzymes on various types of rods.

For example, methods such as immobilization of enzymes by covalent bonding or ionic bonding to glass bead carriers, methods of entrapping enzymes in polyacrylamide gel bodies, etc. have been widely studied, and some have been put into practical use. The same applies to proteases, but especially when using proteases in medicines, foods, chemical products, etc.
It is desired that the immobilization be performed on something with excellent thermal stability, pH stability, stability over time, etc., and that the obtained product should naturally have no adverse effects on the human body. However, when protease is immobilized using various chemical reactions, the enzyme may be deactivated or the crosslinking agent may degrade during storage or use, causing harm.

In addition, in the method of enclosing protease using polyacrylamide, enzymes can generally be immobilized stably, but the gel strength and activity retention of the enzyme are insufficient, and when used in medicines, foods, etc., toxicity may occur during storage. The disadvantage is that acrylamide monomers with high levels of oxidation are produced. Japanese Patent Application Publication No. 1973-
Publication No. 67785 describes a method for producing an insolubilized enzyme in which enzymes are immobilized on silk proteins, and fibers obtained by pulling silk threads taken out from silkworms immediately before spinning or silk threads immediately after being discharged from the mouth of silkworms. A method has been disclosed in which the fibers are passed through an enzyme solution, but in this method, the enzyme is immobilized on the fiber or film, so the enzyme content cannot be increased and the stability is also poor.

Furthermore, since it is in the form of a fiber or film, its uses are limited, and since it contains sericin, it is easily putrefied and lacks preservability. Furthermore, according to additional tests conducted by the present inventors, it was virtually impossible to immobilize protease on silk thread using this method. This is thought to be because the protease attached to the amorphous part degraded the silk protein and was washed out during the washing process, and no activity was expressed. Furthermore, Japanese Patent Application Laid-Open No. 52-57392 discloses that an enzyme is added to a fiproin solution and then mixed to form a membrane, and then this membrane is insolubilized to obtain an immobilized enzyme containing the enzyme in solid fibroin. Although it is described that it can be manufactured, not only is its use limited because it is the same membrane as the former, but also because when protease is immobilized using this method, proteolysis progresses from the time of addition to the time of membrane formation. It is not possible to obtain a decent sample of immobilized protease.

The present inventors completed the present invention as a result of intensive research aimed at improving conventional defects and obtaining an immobilized protease with excellent stability. The object of the present invention is to improve stability, especially thermal stability,
An object of the present invention is to provide an immobilized protease with excellent feed stability and stability over time.

Another object of the present invention is to provide a method for industrially easily and inexpensively producing an immobilized protease having excellent stability, particularly thermal stability, pH stability, and stability over time. The present invention is an immobilized protease containing 0.1 to 2% by weight of alkaline protease and/or neutral protease in fibroin. After mixing with an aqueous solution of protease and/or neutral protease, inorganic salt and/or
Alternatively, the method is characterized in that fiproin and the protease are precipitated by salt precipitation using an organic salt, and then the obtained precipitate is washed with water and then dried.

The immobilized protease of the present invention contains 0.1 to 2% by weight of the enzyme, preferably 1 to 15% by weight, particularly preferably 2 to 2% by weight.
Contains 10% by weight.

When the amount of enzyme is less than 0.1% by weight, the obtained immobilized enzyme has low enzymatic activity and is of little practical use. On the other hand, if the amount exceeds 2% by weight, the enzyme activity chamber becomes saturated, and the enzyme is likely to be eluted during use, and the cost efficiency is poor. The type of alkaline protease and/or neutral protease applied to the present invention is not particularly limited, and examples include tribucin, chymotrypsin, subtilisin BPN, Albergillus alkaline protease, plasmin, Streptococcus beptidase A, chymopapain, papiso, bromelain, ficin, etc. can be given.

Two or more types of these alkaline proteases and neutral proteases can also be used in combination. Fibro to be applied to the method of the present invention. The fiprone aqueous solution is obtained by scouring and removing sericin from silk raw materials such as raw silk, cocoon, raw silk calendar, kimo, bis, and boulet according to a conventional method, and then using an aqueous solution containing an alkali metal salt or an alkali metal salt capable of dissolving fiprone. Alternatively, it may be dissolved in Schwerer's reagent (copper-ammonia solution) or the like, or it may be further desalted by dialysis, and those obtained by desalting by dialysis are particularly preferred.

Examples of the alkali metal salts and alkali metal salts include Licl, LIBr, LINO3, Mmai12, MgB
r2, Mg(N03)2, ZM12, Zn(N03)2
In order to keep the solubility and molecular weight of fibroin as high as possible, Cac12 or Ca(NO
3) Use of 2 is preferred.

Further, the metal salt concentration is 5 to 8% by weight, preferably 20 to 8% by weight.
7% by weight, particularly preferably 40-6% by weight.
In order to further improve the solubility, it is preferable to add alcohols such as methyl alcohol, ethyl alcohol, propyl alcohol, etc. to the aqueous solution.

The timing of addition is preferably before or during the dissolution of silk, and the amount added is 20 to 6% by weight, preferably 2% by weight, based on the metal salt solution.
5-5% by weight. Fibroin dissolved in the above-mentioned aqueous solution may be used as it is as the fibroin aqueous solution, but it is preferable from the viewpoint of increasing the enzymatic activity of the immobilized protease or preventing the formation of water-insoluble salts during salt precipitation. Se.

The salts are removed using a dialysis membrane such as a fan membrane or a dialysis machine using hollow fibers. Huib. The concentration of the in aqueous solution is usually 2 to 2% by weight, preferably 3 to 2% by weight.
It is adjusted to 15% by weight, particularly preferably from 4 to 1% by weight. On the other hand, the enzyme concentration of the aqueous solution of enzymes such as alkaline protease and neutral protease is usually 0.5 to 3% by weight, preferably 1 to 2% by weight, particularly preferably 5 to 15% by weight.
Adjust to.

If an aqueous fibroin solution and an aqueous enzyme solution are mixed without any adjustment, the fibroin will be significantly decomposed within several seconds or at least several tens of seconds depending on the amount of enzyme, and it is virtually impossible to obtain an immobilized captured enzyme on an industrial scale.

Therefore, in order to stably obtain an immobilized enzyme, it is necessary to temporarily reduce the enzyme activity and suppress the decomposition of fiproin as much as possible. To do this, adjust the pH of the enzyme aqueous solution and the mixture of the enzyme aqueous solution and the fibroin aqueous solution to 3 to 5.
The pH should preferably be adjusted to between 3.5 and 5.0. At pH below 3, the protease is irreversibly inactivated in a short period of time, and the obtained immobilized protease does not express enzyme activity. Moreover, when the pH exceeds 6, fibroin is rapidly decomposed during mixing, making it virtually impossible to immobilize protease with fibroin. The fibroin aqueous solution is not particularly limited, but it is best to have a pH in the range of 3.0 to 5.5 without adjusting the pH when mixed with the enzyme aqueous solution. The same level is used. The pH of the enzyme aqueous solution may be selected from the range of 3.0 to 5.5, depending on the type of enzyme used, to ensure the most stable production. In addition, an enzyme aqueous solution or a mixture of an enzyme aqueous solution and a fibroin aqueous solution can be applied to a spot of 3.0 to 5.5.
If you adjust it and leave it for a long time, the fibroin will gel.
The enzyme gradually and irreversibly deactivates, so at least 2~
Preferably, the salt precipitation is carried out within 3 hours. Inorganic acids such as hydrochloric acid and sulfuric acid, organic acids such as acetic acid and citric acid, or aqueous buffer solutions of phosphoric acid, citric acid, and acetic acid are used to prepare fibroin aqueous solutions, enzyme solutions, and mixtures thereof. be able to. The fibroin aqueous solution and the enzyme aqueous solution are mixed with a fly group using a suitable fly-dressing device until both become uniform. In order to manufacture the immobilized enzyme more easily and stably during mixing, the solution temperature may be lowered, for example, from 0 to 1°. The obtained enzyme-containing fibroin aqueous solution is salted to precipitate the fibroin and enzyme using an inorganic salt and/or an organic salt.

Any type of inorganic salt and/or organic salt may be used as long as it can precipitate proteins, such as ammonium sulfate, sodium sulfate, magnesium sulfate, sodium chloride, potassium chloride, sodium nitrate, sodium phosphate, sodium acetate, sodium citrate, etc. Ammonium sulfate and sodium citrate are particularly preferred from the viewpoints of retaining enzyme activity, economy, and operability. The salting out precipitation method is carried out by adding the salt as a solid to an aqueous solution of enzyme-containing fibroin, or by mixing the salt in an aqueous salt solution.

The inorganic salt and/or organic salt may be used in an amount sufficient to precipitate fibroin and enzymes, but usually 25 to 25% of each salt is used.
Perform at 70% saturation by weight. Next, the salts are removed by washing with water, which is carried out two or more times, usually about four times.

If there is a possibility that fibroin may be decomposed by protease during washing, the pH of the first washing water may be adjusted to 3 to 6, preferably 3.5 to 5.0. After washing with water, the immobilized protease is obtained by drying. Drying can be carried out under normal pressure or reduced pressure at a temperature of 60 mm or less to prevent irreversible deactivation of the protease. The obtained dried and fixed protease can be shaped into powder, granules, etc. depending on the purpose. The immobilized enzyme obtained according to the present invention has a very high activity yield, maintains high activity even during continuous use in water, and hardly any enzyme is observed to leak out.

Furthermore, the immobilized protease of the present invention exhibits much higher stability than the original enzyme at high temperatures, in a wide pH range, or in various media.

Moreover, because of the advantage that it consists only of proteins that are harmless to living organisms,
It can be effectively used in fields such as food and cosmetics. The method of the present invention will be explained in detail below with reference to Examples. Example 1
Silk spun shoulder (boulet) lk9 Marcel soap 0.5
The mixture was immersed in a 30 wt % aqueous solution and mixed with a pestle at 80° C. for 1 hour to substantially completely remove sericin and oil, thoroughly washed with water, and then dried at 70 qo.

Next, 0.8k9 of the refined boulet was put into a kneader containing 4k9 of a 65% by weight calcium chloride aqueous solution and 1.6k9 of ethyl alcohol, and 80 to 85qo
Dissolved the tozusa for 1 hour. 80q to the resulting sticky solution
3.2 kg of warm water was added to dilute the mixture. The fiprone concentration of the solution was 85% by weight. Furthermore, a portion of the solution was subjected to a dialysis device using regenerated cellulose-based hollow fibers to obtain a fiprone dialysate. The fiprone concentration of the dialysate was 5.5% by weight. The solution and dialysate were diluted with 5N hydrochloric acid to obtain a fibroin aqueous solution.
On the other hand, as alkaline protease, Bioplase Conc (150000PUN') manufactured by Nagase Sangyo Co., Ltd. was dissolved in water so that the concentration of alkaline protease was 1% by weight, and then the enzyme aqueous solution was adjusted to pH 4.2 with 5N hydrochloric acid. I got it.

To 500 grams of the aqueous fibroin solution at a liquid temperature of 5° C., the aqueous enzyme solution in the amount shown in Table 1 was added and thoroughly mixed.

The value of the mixed solution was 4.3 to 4.5. Next, add 1 part of the mixture
The mixture was poured into the saturated ammonium sulfate aqueous solution and mixed to precipitate fiproin and alkaline protease. After the precipitate was separated in a furnace, it was repeatedly washed 5 times with 500 cc of water, then dried at 40 qo for 15 hours, and dried in a jet mill for 10
A powdered immobilized protease of ~60% was obtained. ○' Enzyme activity P Tosyl arginine methyl ester 0.033M aqueous solution (pH 8) with an appropriate amount of immobilization.

After adding o-tase and reacting at 30°C for 3 minutes, the methanol in the reaction mixture was determined using gas chromatography. The amount of methanol produced by immobilized protease (activity yield (%) = the amount of methanol produced corresponding to the initial addition amount).

Amount of methanol produced by thiase (100 times the amount of protease expressed by Takashi Nakaumi in the immobilized fermentation yarn) The amount of effluent protease was calculated from the measurement.

Immobilization rate (%) = added protease (- amount of effluent protease () .

Residual activity rate (%): Active lotus retention rate of heat-treated bell fists) The tube was packed, water was allowed to pass through the top at a rate of 2 min, and after continuous use for one week, the immobilized protease was taken out and the enzyme activity was measured.

Protease efflux rate (%) = Sansan XI.

〇A: Yield of immobilized protease activity (%) * B
: Flowing water treatment 〃 (%) {5- Protease content The protease content in the fixed protease was calculated using the following formula.

P.

Table 1 Table 1 Table 1 Protease content (%) = amount of protease added (x immobilization rate rainbow %2 yield of immobilized protease) %, the activity yield is low and the enzyme activity per unit weight of immobilized protease is extremely low.
It was difficult to use in practice.

Also, experiment no. [9} Or No. When the protease content exceeds 20% by weight, as in the comparative example, the activity yield is low, and the water efflux rate of the thermostable protease also deteriorates. In order to further increase the content, a large amount of protease must be used, but this is economically very disadvantageous because the immobilization rate drops rapidly. On the other hand, all of the immobilized proteases produced by the method of the present invention have high immobilization rates and high activity yields, and there is very little leakage of protease into water.Moreover, the heat-resistant activity residual rate of the original protease that is not immobilized is 47.5. % showed much higher stability. Example 2 5.3% by weight fibroin dialysate 1 obtained in the same manner as in Example 1 and bioplase/Conclo
50 wt % aqueous solutions were brought to 5°C in advance, and each was 2 wt %.
The pH values shown in Table 2 were set using an aqueous citric acid solution.

After leaving for about 5 minutes, both were mixed and stirred for about 3 minutes. Subsequently, this was added to a 66% by weight saturated aqueous ammonium sulfate solution at 5° C. while stirring. The resulting precipitate was collected in a furnace, washed with one portion of 0.01M citric acid buffer having a pH of 4.5, and further washed four times with one portion of water, and then air-dried. This was pulverized with a jet mill and the particle size was 10
~50 micron powder. For these powders, the yield, immobilization rate, activity yield, and protease efflux rate using the column filling water flow method were measured using the methods described in Example 1.

The results are shown in Table 2. Table 2 From Table 2, when the oxygen aqueous solution and the pH value when mixed with the fibroin aqueous solution are within the range of 3 to 5.5, both the yield and the immobilization rate are good, and the activity yield is high. We were able to obtain immobilized protease.

Furthermore, there is virtually no leakage of protease in distribution tests. However, experiment no. The pH of the enzyme solution as in (11)
If the value is low, the enzyme will be irreversibly inactivated and little activity yield will occur. Furthermore, the immobilization rate could not be determined because the enzyme was deactivated. Also, experiment no. When the pH value at the time of mixing is high as in (15), the decomposition of fibroin proceeds rapidly, and even when added to the ammonium sulfate aqueous solution, it becomes fine particles and becomes dispersed, resulting in a significantly low yield.

In addition, both the immobilization rate and the activity yield are low, making it impractical. Example 3 Immobilized protease was obtained using various inorganic salts or organic salts shown in Table 3, and by the same method as in Example 1, except that 500 hours of aqueous fiproin dialysis solution and 20 hours of aqueous protease solution were used.

Table 3 Although there were slight differences depending on the type, amount, or mixing method of the salt, the immobilized protease using any of the salts had excellent immobilization rates and enzyme activity.

Example 4 A dialyzed fibroin aqueous solution obtained in the same manner as in Example 1 was
．． Fibroin concentrations were adjusted to 0, 5.0, 10.0 and 15.0% by weight, and P was added with a citric acid aqueous solution of 2% by weight.
I set it to H4.8.

As alkaline protease and/or neutral protease, pioprase conc (manufactured by Nagase Sangyo Co., Ltd.), Neutrase (manufactured by Novo Corporation), Proteo Requifurase (manufactured by Ueda Chemical Industry Co., Ltd.), Type O Fungal Cmdefr
omA. The fourth test was performed using four types of or〆os (manufactured by Sigma).
It was dissolved in water at the concentration indicated in the table and made up to 4.3 with a 2% by weight aqueous citric acid solution. Thereafter, the mixture was mixed, salted out, washed with water, and dried in the same manner as in Example 1 to obtain an immobilized protease. In this case, the pH during mixing was 4.5 to 4.8. Enzyme activity was measured by the following method, different from Examples 1 to 3.
0. An appropriate amount of immobilized protease was added to a casein aqueous solution (pH 7) of 1% by weight, and the reaction was carried out at 30oC for 1M minutes.Then the furnace solution from which the immobilized protein was separated was colored with a phenol reagent and determined by the absorbance of 660. Calculated.

Table 4 1. 2. Use 500 ta of fibroin aqueous solution. Protease type, A: Piobrase conc., B: Neutrase, 0: Proteo cucuifase D: Type
11Fungal○rade frQhAoryzas
As shown in Table 4, various alkaline proteases and/or
All of the immobilized proteases obtained by the method of the present invention using neutral proteases had high activity yields, and very little leakage of protease into flowing water, resulting in stable immobilized proteases.

Example 5 Dialyzed fibroin aqueous solution (fibroin concentration 5.4% by weight) obtained in the same manner as in Example 1 lk9 and pioprase C
onc aqueous solution (bioplase concentration 10% by weight) and 2% by weight citric acid to feed 5.0 to 4.0% respectively.
Adjusted to.

In this case, the pH at the time of mixing was 4.9. Next, in the same manner as in Example 1, the precipitate was precipitated using a saturated ammonium sulfate aqueous solution and salt, and the precipitate was washed with water, air-dried, and then used in a jet mill to obtain 4 to 4 micronized immobilized protease. The activity yield of the immobilized protease was 22.6% using the method using p-tosylarginine methyl ester.

As a contrast to the immobilized protease, the original bioplase Conc was treated under the following environment, and its activities were measured and compared. [1] 2 days at 60°C in glycerin [2] 1 day in water (ion exchange water) 3 and 0 (3)
(Adjusted to pH 4 with acetic acid) 30qo for 1 day'4}
(Adjusted to PHil with sodium hydroxide) 30℃
■ 7 days at 60°C in a dryer [6] 7 days at 80°C Table 5 As shown in Table 5, the protease immobilized by the method of the present invention reverts back to the original rotease under any environment. In comparison, the activity was much more maintained, and the superiority of immobilization was obvious.

Claims (1)

[Scope of Claims] 1. An immobilized protease containing 0.1 to 20% by weight of alkaline protease and/or neutral protease in fibroin. 2. The immobilized protease according to claim 1, wherein the fibroin is in powder form. 3. The immobilized protease according to claim 1 or 2, which contains 1 to 15% by weight of alkaline protease and/or neutral protease. 4 After adjusting the pH to 3 to 5.5 by mixing an aqueous solution of fibroin and an aqueous solution of alkaline protease and/or neutral protease having a pH of 3 to 5.5, mix the fibroin and the above using an inorganic salt and/or an organic salt. A method for producing an immobilized protease containing 0.1 to 20% by weight of alkaline protease and/or neutral protease in fibroin, which comprises precipitating the protease by salt-folding, and then washing the obtained precipitate with water and drying it. . 5. The manufacturing method according to claim 4, wherein the pH of the protease aqueous solution and/or the liquid after mixing is 3.5 to 5. 6 The fibroin concentration of the fibroin aqueous solution is 2 to 20
The manufacturing method according to claim 4, wherein the amount is % by weight. 7 Fibroin aqueous solution is copper-ethylenediamine aqueous solution, copper hydroxide-ammonia aqueous solution, copper hydroxide-alkali-
The refined silk raw material was dissolved in at least one solvent selected from the group consisting of an aqueous glycerin solution, an aqueous lithium bromide solution, an aqueous solution of calcium, magnesium or zinc hydrochloride or nitrate or thiocyanate, and an aqueous sodium thiocyanate solution, and then dialyzed. The manufacturing method according to claim 4 or 6, which is 8. The production method according to claim 4, wherein the concentration of the protease in the aqueous solution of the protease is 0.5 to 30% by weight. 9. The manufacturing method according to claim 4, wherein the mixing is carried out at a liquid temperature of 0 to 15°C. 10. The production method according to claim 4, wherein the inorganic salt and/or organic salt is ammonium sulfate or sodium citrate. 11. The manufacturing method according to claim 4, wherein washing is performed with water having a pH of 5 to 6. 12. The manufacturing method according to claim 4, wherein the drying is carried out at 60° C. or lower and under normal pressure or reduced pressure.