JPH01222783A - Production of immobilized biological catalyst - Google Patents

Abstract

PURPOSE:To obtain the title catalyst (enzyme) with enhanced stability, by immersing an activated fibrous protein carrier in a buffer solution containing a specified amount of a biological catalyst having functional group selected from phenolic group amino group and so on to make a reaction at room temperature. CONSTITUTION:Firstly, an inorganic acid e.g., a combination of dil. HCl with sodium nitrite is acted on an aromatic amino group-contg. fibrous protein carrier to prepare an activated fibrous protein carrier as a diagonium compound. Thence, this carrier is immersed in a buffer solution containing 1-3mg/ml of a biological catalyst having functional group selected from phenolic group, amino group and imidazole group and allowed to react for ca. 1-4hr to obtain the objective immobilized biological catalyst. Said biological catalyst is pref. aspartase, amylase or the like, whereas said buffer solution is pref. 0.05M sodium borate-hydrochloric acid buffer solution with the pH adjusted to 7.5-8.0.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for producing an immobilized biocatalyst in which a biocatalyst such as an enzyme, a coenzyme, or a microorganism is chemically bonded to a fibrous protein carrier.

[Prior art and problems] In recent years, various developments have been made to immobilize biocatalysts, especially enzymes, on carriers and use them as biosensors, bioreactors, etc. in the medical, environmental, and food industries. It is being said.

Among these developments, attempts have been made to immobilize enzymes on fibrous carriers, and are based on the idea of adding enzyme functions to fibers. Compatible fibers and products based on the idea of expressing enzyme functions in the form of fibers, such as immobilized enzymes that can be easily processed into cloth or paper, are being developed.

Conventionally, the fibrous carrier on which enzymes are immobilized is a fibrous protein selected from the group consisting of shaving waste, crushed cowhide, and silk buproin.

Enzyme immobilized on a carrier (Special Publication No. 60-990)
is known, and here immobilized enzymes are produced by the so-called azide method, in which an azide derivative is produced using carboxyl groups in a fibrous protein carrier and reacted with an enzyme.

This azide method is an excellent method for immobilizing various biocatalysts and microbial cells onto protein carriers, but the reaction temperature range is narrow and limited in the production of this azide derivative and the reaction of the azide derivative with the biocatalyst. Since the reaction is usually carried out under water cooling, temperature control has been difficult. Another problem is that fibrous protein carriers, particularly silk fibroin, have a small number of functional groups that can serve as reaction sites.

In order to solve these problems, the present inventor has conducted repeated research and found that in a method of introducing a diazo group into a fibrous protein carrier and reacting this with a biocatalyst, a biocatalyst is used under certain conditions. The present invention was completed based on the knowledge that the immobilized enzyme is very stable and the reaction proceeds over a wide range of temperatures around room temperature.

[Structure of the Invention] The present invention provides an activated fibrous protein carrier in which a fibrous protein carrier having an aromatic amino group is converted into a diazonium compound using an inorganic acid and sodium nitrite, and a fibrous protein carrier having a phenol group, an amino group, or an imidazole group. immersed in a buffer solution containing 1 to 3 mg/l of a biocatalyst having a functional group selected from
The present invention relates to a method for producing an immobilized biocatalyst, characterized in that the reaction is carried out at room temperature for up to 4 hours.

Fibrous protein carriers used in the present invention include silk fibers (silk fibroin, silk sericin), collagen, keratin, etc., but these fibrous protein carriers have aromatic amino groups in their structure, or An aromatic amino group must be derived within its structure. The induction of this aromatic amino group is, for example, a hydrogen atom at the ortho position of a phenol group in a tyrosine residue in a fibrous protein carrier. This is carried out by substituting a nitro group, and further reducing this nitro group to an amino group. Here, the hydrogen atom at the ortho position of the phenol group is replaced with a nitro group by, for example, acting on the fibrous protein carrier with a mixed acid of -a sulfuric acid and nitric acid, and the amino group of the nitro group is replaced. The reduction is carried out using a reducing agent such as sodium dithionite or lithium aluminum hydride.

This method is particularly effective because silk buproin has many tyrosine residues with phenol groups in its structure.

This fibrous protein carrier having aromatic amino groups is reacted with an inorganic acid such as dilute hydrochloric acid and sodium nitrite,
It becomes a diazonium compound, that is, an activated fibrous protein carrier.

The activated fibrous protein carrier thus obtained is immersed in a buffer solution containing 1 to 3 mg/+wl of a biocatalyst having a functional group selected from the group of phenol group, amino group, and imidazole group, and Allow to react at room temperature for ~4 hours.

In order for a diazonium compound to form a diazo bond through a coupling reaction, it is necessary for the force/combination component to have one of the following functional groups: a phenol group, an amino group, and an imidazole group. Those having a functional group selected from the group of phenol group, amino group, and imidazole group are used. As this biocatalyst, enzymes such as aspartase, amylase, and invertase, and coenzymes such as nicotinamide adenine dinucleotide, adenosine triphosphate, and coenzyme-A are preferably used. Alkaline phosphatase is preferred.

In addition, the buffer solution is used here to solidify and electrify the activated fibrous protein carrier of the biocatalyst, and the synthesis reaction of both for this immobilization and the stability of the biocatalyst are affected by pH. This is because the activity of the biocatalyst decreases significantly outside the appropriate pH range. For example, when alkaline phosphatase is used as a biocatalyst, the buffer solution is 0,0
It is preferable to use a 5M sodium borate-hydrochloric acid buffer, a 0.05M boric acid-hydrochloric acid buffer, or the like.

A biocatalyst is dissolved in advance in this buffer solution, and the concentration of the biocatalyst is 1-b. This is because if it is less than 1 m3/m1, the amount of biocatalyst immobilized will be small, so the activity will naturally be low, and 3n+g/l
This is because if the amount is exceeded, the biocatalyst becomes excessive and inhibits each other's reactions, resulting in a decrease in activity.

Note that the reaction time between the activated fibrous protein carrier and the biocatalyst in the buffer solution needs to be 1 to 4 hours.

Here, the reason why the reaction time is 4 hours or less is because the diazo group of the activated fibrous protein carrier of the present invention has a very high activity (
This is because it is a °1 functional group, in other words, an unstable functional group, and needs to be reacted quickly, and it is not preferable for the biocatalyst to exist in the reaction solution for too long. - On the other hand, the reason why the reaction time is 1 hour or more is because immobilization does not proceed sufficiently if the reaction time is less than 1 hour.

The immobilized biocatalyst thus obtained has the advantage that its activity is sufficiently maintained and the biocatalyst is excellent in stability.

It has particularly excellent thermal stability in the high temperature range, and maintains some activity even at a high temperature of 65°C, where unimmobilized biocatalysts and biocatalysts immobilized by other manufacturing methods are deactivated. can be maintained.

In addition, in the above reaction, the stability of the biocatalyst is guaranteed in a relatively wide range of mild reaction temperatures at room temperature, and there is no difficulty in temperature control such as conducting under water cooling as in the case of the azide method. Note that room temperature here is 1 to 35°C (JIS -
0050).

(Example 1) The silkworm surface was scoured with a 0.5% citric acid aqueous solution at 100° C. for 3 hours at a bath ratio of 50 f&, and then thoroughly washed with distilled water and dried to obtain silk fibroin.

This silk fibroin was mixed with concentrated sulfuric acid, concentrated nitric acid, and distilled water (1:
1:2 to 3) for several seconds to introduce a nitro group into the side chain of the tyrosine residue of silk buproin.

After thoroughly washing the nitrated silk fibroin with distilled water until the washing solution becomes neutral, the nitrated silk fibroin is washed with sodium dithionite in an amount of 20 times the molar amount based on the tyrosine residue of the silk fibroin, at a pH of 8 to 10. The mixture was reacted in a buffer at room temperature for about 1 hour to reduce the nitro group to an amino group.

Next, this aminated silk fibroin was thoroughly washed with distilled water and dried, then immersed in 1M hydrochloric acid under water cooling, and 0.5M sodium nitrite solution was gradually added dropwise to this while stirring for 1 hour. A diazo compound was obtained by reaction.

This diazo compound, activated silk fibroin, was mixed with distilled water and pH 7.5 (7) 0.05M? jI
m After washing with sodium buffer, 0.05 at pH 7.5 containing 2 mg/l alkaline phosphatase.
The alkaline phosphatase was immobilized on the silk fibroin by immersing it in M sodium borate buffer at a bath ratio of 1 tag and 7 cc and reacting at a temperature of 30° C. for 1 hour.

This immobilized alkaline phosphatase was treated with salt by immersing it in a 1M aqueous potassium chloride solution at 4°C for 3 hours, and was further reacted with a 0.5M glycine solution at 4°C for 1 hour.1. Free diazo groups were blocked.

The activity of the obtained immobilized alkaline phosphatase was determined by
An enzymatic reaction was carried out using p-nitrophenyl phosphate as a substrate, and the amount of p-nitrophenol produced was investigated by monitoring the absorption intensity at 400 nm using a spectrophotometer. H
Over a long period of time, the activity was almost the same as that of unimmobilized alkaline phosphatase, and its stability was excellent. In particular, at the operating temperature of 65°C, unimmobilized alkaline phosphatase is inactivated, while immobilized alkaline phosphatase has a relative activity of about 20 to 25% (relative activity when the activity at 25°C is taken as 100). It remained active and had excellent stability in a high temperature range.

In addition, when the reaction temperature was varied in the range of 4 to 30 °C in the above immobilization reaction, the activity was maintained at 80% or more at all temperatures, and there was no problem with stability.
In particular, this activity was higher as the temperature approached 30°C.

(Examples 2 to 3. Comparative Examples 1 to 2) Immobilized alkaline produced phosphatase.

The activity of the obtained immobilized alkaline phosphatase was investigated in the same manner as in Example 1, and the relative activity is shown in Table 1, setting the activity of Example 1 as 100.

As is clear from Table 1, the pH of the buffer solution is between 7.5 and 8.
Between 0 and 0, the relative activity is maintained at 88% or more and is stable, but when it deviates from these ranges, the activity decreases to 25%.
It can be seen that the temperature decreases significantly below % and becomes unstable.

[Margins below] Table 1: Tris is tris(hydroxy n+ethy
l) Abbreviation of amino n + ethane (2-7 mino 2-human 〇 N sime t 1.3) 00 ha 0 unc hion) (Examples 4 to 5. Comparative Example 3) Alkaline phosphatase and diazotized silk fibroin in Example 1 An immobilized biocatalyst was produced in exactly the same manner as in Example 1, except that the reaction time with each of the following was changed to the reaction time shown in Table 2.

The activity of the obtained immobilized alkaline phosphatase was investigated in the same manner as in Example 1, and the relative activity is shown in Table 2, setting the activity of Example 1 as 100.

As is clear from Table 2, when the reaction time is between 1 and 4 hours, the activity is maintained at 82.5% or more and is stable, but at (L5 hours) the activity is extremely low at 40% or less. ,
It turns out that it becomes unstable.

[Margins below] Table 2 (Examples 6 to 7) Completely the same as Example 1 except that the concentration of alkaline phosphatase in the buffer solution in Example 1 was replaced with each concentration shown in Table 3. An immobilized biocatalyst was produced in the same manner.

The activity of the obtained immobilized alkaline phosphatase was investigated in the same manner as in Example 1, and the relative activity is shown in Table 3, setting the activity of Example 1 as 100.

As is clear from Table 3, the biocatalyst concentration is 1 to 3 mg/
The activity was maintained at 50.8% or higher between +al and is considered to be within a usable range.

Table 3 [Effects of the Invention] As explained above, the method for producing an immobilized biocatalyst of the present invention is a novel method for immobilizing a biocatalyst on a fibrous protein carrier, and the reaction temperature is room temperature. Nearby, relatively wide and mild conditions W! The biocatalyst can be immobilized in a stable state where the activity of the biocatalyst is sufficiently maintained. Moreover, according to this method, an immobilized biocatalyst with excellent thermal stability in a high temperature range can be obtained.

In particular, when silk fibroin is used as a fibrous protein carrier, silk fibroin contains a relatively large amount of tyrosine (5 to 6%), so there are plenty of phenol groups in the side chains of tyrosine residues for introducing diazo groups. , it can be used as a carrier as is without any special surface treatment, and since only the tyrosine residue side chain is modified with the diazo group, it is not immobilized on other amino acid side chains or terminals. By controlling the reaction conditions, activity can be obtained depending on the intended use.

Furthermore, since the diazo group introduced by chemical modification causes intramolecular crosslinking at the same time as the biocatalyst immobilization reaction, the strength of the support after biocatalyst immobilization is improved.

Furthermore, M! The advantages of fibrous protein carriers, particularly silk fibroin, such as biocompatibility, are maintained, and these properties can also be effectively utilized.

Therefore, the present invention is an extremely useful method for producing immobilized biocatalysts suitable for biosensors, bioreactors, biocompatible materials, etc. with high productivity.

Claims (3)

[Claims] (1) An activated fibrous protein carrier in which a fibrous protein carrier having an aromatic amino group is converted into a diazonium compound using an inorganic acid and sodium nitrite has a functional group selected from the group of a phenol group, an amino group, and an imidazole group. immersed in a buffer solution containing 1-3 mg/ml of a biocatalyst with a
A method for producing an immobilized biocatalyst, which comprises reacting at room temperature for 4 hours. (2) The method for producing an immobilized biocatalyst according to claim 1, wherein the fibrous protein carrier is silk fibroin. (3) The method for producing an immobilized biocatalyst according to claim 1 or 2, wherein the biocatalyst is alkaline phosphatase.