JPH08256769A - Immobilized ultrathermostable beta-galactosidase - Google Patents

Abstract

PURPOSE: To obtain an immobilized enzyme by supporting an ultrathermostable β-galactosidase derived from Pyrococcus furiosus on a carrier, excellent in durability, hardly eluting the enzyme, capable of sterilizing various germs at a high temperature, useful for processing a food and producing a saccharide compound. CONSTITUTION: Ultrathermostable β-galactosidase collectable from Pyrococcus furiosus DSM3,638, etc., is supported on an immobilizing carrier (insoluble carrier) stable to heat and physical shocks to give a high-temperature bioreactor. The immobilization can be carried out by a method for subjecting a carboxyl group, etc., of the enzyme to covalent bond formation with a functional group (amino group, etc.) introduced to the immobilizing carrier.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an immobilized super thermostable β-galactosidase useful in the fields of sugar engineering and the food industry.

[0002]

2. Description of the Related Art β-galactosidase
ase, EC 3.2.1.23.) is an enzyme that decomposes β-galactoside and is found in animals, plants, and microorganisms. In particular, in bacteria, Streptococcus lacti (Streptococcus lacti), including Escherichia coli, is used.
s), Bacillus subtillis,
Streptococcus thermophilus (Streptococcus
thermophilus), sulfolobus solfataricus (Su
lfolobus solfataricus). This β-galactosidase is used for the production of low-lactose milk by utilizing its ability to hydrolyze lactose into galactose and glucose. It is also used to produce galactose or glucose from lactose in whey, which is produced in large quantities during cheese production. Further, in recent years, various production of sugar compounds such as oligosaccharides and complex sugars by imparting sugars by utilizing the transglycosylation reaction of β-galactosidase has been carried out (JP-A-6-25).
275, 6-38785).

When soluble β-galactosidase is used in food processing by a batch reaction, it is necessary to carry out operations such as inactivation of enzyme after the reaction and removal of enzyme protein.
In addition, when it is impossible to remove the enzyme protein as in the production of low-lactose milk, deterioration of flavor becomes a problem. Super thermostable β-galactosidase which is active at high temperature and has high thermostability [European Journal of Biochemistry, No. 2
13: 305-312 (1993)], the inactivation of the enzyme after the reaction is a problem because the enzyme is stable. As a means for solving these problems, the use of immobilized β-galactosidase in which β-galactosidase is retained on a certain carrier is required. For example, when β-galactosidase derived from the moderate thermophilic anaerobic bacterium NA10 strain is immobilized on a ceramic carrier, it is stable at 65 ° C. for up to 100 hours, but the activity is about 5 at 65 ° C. for 400 hours.
Drop to 0% [Applied Microbiology
And Biotechnology (Applied Microbiology a
nd Biotechnology), 40, 618-621 (1994)].

[0004]

As described above, in the processing of foods at high temperature and the production of sugar compounds, they can be used repeatedly and even for a long period of time due to economical problems, and there is no elution of enzymes, and microorganisms do not elute. There is a need for an immobilized β-galactosidase that can be used in a short time high temperature sterilization process to prevent contamination. An object of the present invention is to provide an immobilized hyperthermophilic β-galactosidase that can be used repeatedly and for a long time, does not elute an enzyme, and can be used in a sterilization process at a high temperature of 80 ° C. or higher. .

[0005]

Means for Solving the Problems The present invention will be described in brief. The present invention relates to an immobilized super thermostable β-galactosidase, which comprises a carrier containing the super thermostable β-galactosidase derived from Pyrococcus furiosus. It is characterized by

The present inventors paid attention to the super thermostable β-galactosidase derived from Pyrococcus furiosus and attempted to immobilize the enzyme. Furthermore, the present inventors have examined the properties of the super thermostable β-galactosidase that was successfully immobilized, and surprisingly, the immobilized super thermostable β-galactosidase has stable activity at 85 ° C. for at least 240 hours. And that the reaction is possible without elution of the enzyme, its biochemical properties were determined, and the present invention was completed.

The present invention will be described in more detail below. The β-galactosidase used in the present invention is not particularly limited as long as it is a β-galactosidase excellent in heat resistance, and for example, β-galactosidase derived from a strain belonging to the genus Pyrococcus can be used. For example, from Pyrococcus furiosus DSM3638 to JP-A-6-189
Super thermostable β-galactosidase that can be collected according to the method described in Japanese Patent No. 756 can be used.
For example, from a transformant in which a recombinant plasmid containing the hyperthermostable β-galactosidase gene derived from Pyrococcus furiosus DSM3638 is introduced, there is disclosed in
No. 253855, or Japanese Patent Application No. 6-15435.
Ultra thermostable β-galactosidase that can be collected according to the method described in No. 6 can be used. The transformant described in Japanese Patent Application No. 6-154356 is named and displayed as Escherichia coli JM109 / pTG2ES-105, and is designated as FERM BP-5023 at the Institute of Biotechnology, Ministry of International Trade and Industry. Has been deposited as.

The immobilization carrier (insoluble carrier) used in the present invention is not particularly limited as long as it is an immobilization carrier which is stable to heat and physical shock, and for example, a commercially available carrier can be used. . For example, commercially available porous chemically synthesized polymer POROS media [manufactured by Perseptive Biosystems] can be used. In addition, for example, control pore glass (manufactured by Control Pore Glass Co., Ltd.) made of commercially available glass and having a stable and large surface area can be used.

The immobilization method used in the present invention is as follows.
The method is not particularly limited as long as it is a method that is stable to heat and physical shock, but for example, an amino group, a carboxyl group, and a sulfhydryl group of an enzyme protein are added to functional groups such as an epoxy group, an amino group, and an aldehyde group that are introduced into an immobilized carrier. A method of covalently bonding under chemically mild conditions can be used.

Below, Pyrococcus furiosus DSM
The hyperthermostable β-galactosidase derived from 3638 will be described as an example of immobilized ultraheat-resistant β-galactosidase retained on an immobilization carrier. Pyrococcus furiosus DSM363 described in Japanese Patent Application No. 6-154356
A recombinant plasmid-introduced transformant containing a super thermostable β-galactosidase gene derived from Escherichia coli 8, Esch
erichia coli JM109 / pTG2ES-105 (FERM BP-5
023) under normal culture conditions, for example, L-broth medium containing 0.01% ampicillin (1% tryptone, 0.5%).
% Yeast extract, 1% NaCl, pH 7.2) at 37 ° C
The super thermostable β-galactosidase is accumulated in the culture by culturing at. When purifying ultra-thermostable β-galactosidase from the culture, for example, after collecting the bacterial cells, the bacterial cells are disrupted by ultrasonic treatment, and the supernatant obtained by centrifugation is, for example, gel filtration chromatography, The enzyme can be purified by combining methods such as ion exchange chromatography and hydrophobic chromatography. In particular, when purifying super thermostable β-galactosidase, it is effective to perform heat treatment before and after ultrasonication, because denatured contaminating proteins can be easily purified and purification can be performed. Further, it is purified to a single size by SDS-polyacrylamide gel electrophoresis and used for immobilization. For example, the purified enzyme solution is diluted or concentrated according to the required amount, and used for immobilization as a super thermostable β-galactosidase solution. Further, for example, the purified enzyme solution is freeze-dried according to a conventional method to prepare a freeze-dried preparation of super thermostable β-galactosidase. Next, the freeze-dried preparation is dissolved in, for example, sterilized water, a buffer or the like according to the required amount to prepare a super thermostable β-galactosidase solution and used for immobilization.

The super thermostable β-galactosidase solution,
The required amount of the immobilization carrier is added and the immobilization reaction is performed. After completion of the reaction, the absorbance (280 nm) of the reaction supernatant is measured, and it is confirmed that the enzyme protein of super thermostable β-galactosidase is retained on the immobilized carrier and the protein concentration of the reaction supernatant is lowered. Then, the immobilized carrier sufficiently provided with the super thermostable β-galactosidase is washed with a buffer solution or the like, and collected by performing filter filtration or the like. This is recovered,
The insolubilized super thermostable β-galactosidase is referred to as immobilized super thermostable β-galactosidase.

The biochemical properties of the immobilized hyperthermophilic β-galactosidase are as follows. (1) Enzyme activity measurement method The immobilized super thermostable β-galactosidase thus obtained was added to 500
50 mM citrate buffer containing mM lactose (p
H5.1) Suspended in 2.0 ml, stirred well, and allowed to react at 85 ° C. for 2 minutes under shaking conditions (130 rpm). Next, the reaction is stopped by filtering with a filter to separate the immobilized enzyme. After completion of the reaction, the glucose concentration produced in the supernatant of the reaction solution is measured using Glucose Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.). The immobilized enzyme activity is expressed as the enzyme activity per 1 ml of the immobilized enzyme and the enzyme activity per 1 mg of the enzyme protein used for immobilization. One enzyme unit (unit) of the enzyme activity is the amount of enzyme that produces 1 μmol of D-glucose per minute at 85 ° C. As the immobilization carrier, epoxy group-introduced Poros Media Self Pack 20EP (manufactured by Perceptive Biosystems), aldehyde group-introduced Poros Media Self Pack 20AL (Perceptive Biosystems), and amino group-introduced Poros Media. Table 1 shows the results when using Media Self Pack 20NH (manufactured by Perceptive Biosystems), EP4612 (manufactured by Organo), and FP4611 (manufactured by Organo). As shown in Table 1, the super thermostable β-galactosidase retained on the immobilized carrier shows high activity.

[0013]

[Table 1]

(2) Thermostability The thermostability of the immobilized super thermostable β-galactosidase is, for example, heat treated at 85 ° C. for 24 hours, and the residual activity after the treatment is shown by the enzyme activity shown in (1) above. Confirm by measuring with the measuring method. The results are shown in Table 2. As shown in Table 2, any immobilized carrier prepared using the immobilized super thermostable β-galactosidase has a residual activity of 80% or more after heat treatment for 24 hours.

[0015]

[Table 2] Table 2 Thermal stability of β-galactosidase immobilized with superheat resistance ───────────────────────────────── ──── Immobilized carrier Enzyme amount Residual activity rate (%) after heat treatment at 85 ℃ for 24 hours ──────────────────────────── ────────── I Porosmedia EP 2mg 121.0 III AL 2mg 96.2 V NH 2mg 83.8 VII FP4612 1mg 93.3 VIII FE4611 1mg 97.6 ────────── ────────────────────────────

(3) Confirmation of Durability and Eluted Enzyme The durability of immobilized super thermostable β-galactosidase and the eluted enzyme eluted from the immobilized carrier are confirmed. For example, as an immobilization carrier, Poros Media Self Pack 20E
In the case of immobilized ultra-thermostable β-galactosidase using P (manufactured by Perceptive Biosystems), the residual activity and the amount of eluted enzyme of immobilized ultra-thermostable β-galactosidase were measured at 85 ° C. for 240 hours with time. The results are shown in FIG. That is, FIG. 1 is a diagram showing the residual activity of immobilized hyperthermophilic β-galactosidase by heat treatment and the amount of eluted enzyme. Regarding the residual activity on the vertical axis, the immobilized ultrathermostable β-galactose before treatment (0 hour) is shown. The residual activity rate (%, open circles) is shown when the galactosidase activity is 100%. The amount of eluted enzyme was 10 mM o-nitrophenyl-β-based on 100 μl of the supernatant of the immobilized ultra-thermostable β-galactosidase solution filtered through the filter after the above treatment.
After adding 2.0 ml of 50 mM citrate buffer (pH 5.1) containing D-galactopyranoside and reacting at 85 ° C for 3 minutes, the amount of o-nitrophenol produced was measured by the absorbance at 410 nm. The enzyme amount obtained by comparing the value obtained by performing the same with the value obtained by similarly measuring super thermostable β-galactosidase (10 μg / ml) as a standard substance was the amount of enzyme to be eluted (μg / ml,
It is shown as a black square mark). As shown in FIG. 1, the immobilized hyperthermostable β-galactosidase has a residual activity rate of 80% or more even after treatment at 85 ° C. for 240 hours, and no elution of the enzyme retained on the immobilized carrier is observed. .

The immobilized ultra-thermostable β-galactosidase obtained as described above has no activity decrease and enzyme elution at 85 ° C., and can be used for a long time. It can be used in the sterilization process at a high temperature of 80 ° C. or higher, and is useful as a high temperature bioreactor in the food industry and sugar engineering fields.

[0018]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto.

Example 1 (1) Production of super thermostable β-galactosidase A recombinant plasmid containing the super thermostable β-galactosidase gene derived from Pyrococcus furiosus DSM3638 described in Japanese Patent Application No. 6-154356 was used. Introduced transformant, Escherichia coli JM109 / pTG2ES-
105 (FERMBP-5023) was added to L-broth medium containing 0.01% ampicillin (1% polypeptone, 0.
The cells were suspended in 5 ml of 5% yeast extract, 1% NaCl, pH 7.2) and shake-cultured at 37 ° C for 16 hours. Furthermore,
The culture was suspended in 1.2 liters of medium under the same conditions,
Shaking culture was performed at 16 ° C. for 16 hours. The bacterial cells recovered by centrifugation of the culture were suspended in 20 mM phosphate buffer (pH 7.0) containing 1 mM EDTA, and heat-treated at 100 ° C. for 10 minutes. Subsequently, ultrasonic treatment was carried out, heat treatment was further carried out at 100 ° C. for 10 minutes, and the supernatant after centrifugation was taken to obtain a crude enzyme solution. Further, the crude enzyme solution was added to DEAE-Sepharose (Pharmacia), Butyl Toyopearl (Toyo Soda),
Chromatography was performed in the order of Sephadex G-25 (manufactured by Pharmacia), and purification was performed by SDS polyacrylamide gel electrophoresis until it became single. The ultra thermostable β-galactosidase purified in this way is 20
2.5 mg / ml in mM phosphate buffer (pH 7.2)
Was prepared so that the enzyme concentration would be the same, and freeze-dried to obtain a freeze-dried preparation of ultra-thermostable β-galactosidase.

(2) Preparation of various immobilized super thermostable β-galactosidase The freeze-dried preparation of the super thermostable β-galactosidase obtained in (1) was dissolved in sterilized water to 10 mg / ml, and the enzyme was added. Solution I was used as a preparation of various immobilized ultra-heat-resistant β-galactosidase described below.

(2-1) The above enzyme solution I (0.2 ml) was added to 2.8 ml of 0.1 M borate buffer (pH 9.0) containing 2 M sodium sulfate, and the total amount was 3 ml (enzyme amount 2 mg). And To the solution, 0.2 g of Poros Media Self-Pack Poros 20EP (manufactured by Perceptive Biosystems) in which an epoxy group was introduced was added, stirred well, and then shaken (130 rpm).
The reaction was carried out at room temperature for 15 hours. After completion of the reaction, filtration with a filter was performed to recover the immobilized enzyme. Next, the immobilized enzyme was suspended in 3 ml of 0.1 M Tris-HCl buffer (pH 8.3) and reacted at room temperature for 2 hours under shaking conditions (130 rpm) to block excess epoxy groups. After completion of the reaction, filtration with a filter was performed to recover the immobilized enzyme. Further, the recovered immobilized enzyme was filtered on a filter to give 5
Washing was performed with 20 ml of 0 mM citrate buffer (pH 5.1). The immobilized superheat-resistant β-obtained in this way
Galactosidase was added to 50 mM citrate buffer (pH 5.
1) Suspended in 5.0 ml to give an immobilized super thermostable β-galactosidase I solution.

(2-2) The above enzyme solution I (0.4 ml) was added to 2.6 ml of 0.1 M borate buffer (pH 9.0) containing 2 M sodium sulfate, and the total amount was 3 ml (enzyme amount 4 mg). And Immobilized super thermostable β-galactosidase was prepared from the solution in the same manner as in (2-1) to obtain an immobilized super thermostable β-galactosidase II solution.

(2-3) The above enzyme solution I (0.2 ml) was added to 2.8 ml of 0.1 M phosphate buffer (pH 6.5) containing 2 M sodium sulfate, and the total amount was 3 ml (enzyme amount 2 mg). And To the solution, 0.2 g of an aldehyde group-introduced Poros Media Self Pack Poros 20AL (manufactured by Perceptive Biosystems) was added, and after thorough stirring, under shaking conditions (130 rpm),
The reaction was carried out at room temperature for 15 hours. After completion of the reaction, filtration with a filter was performed to recover the immobilized enzyme. Next, the immobilized enzyme was added to 0.1M containing 0.1M sodium borohydride.
The suspension was suspended in 3 ml of a phosphate buffer (pH 6.5) and reacted at room temperature for 2 hours under shaking conditions (130 rpm). As a result, the Schiff base formed between the primary amine of the enzyme protein produced by the above reaction and the aldehyde group of the poros media can be reduced to form a stable covalent bond. After completion of the reaction, filtration with a filter was performed to recover the immobilized enzyme. Further, the recovered immobilized enzyme was suspended in 3 ml of 0.1 M Tris-HCl buffer (pH 8.3) on a filter, and allowed to react at room temperature for 2 hours under shaking conditions (130 rpm) to remove excess aldehyde groups. Blocked. After completion of the reaction, filtration with a filter was performed to recover the immobilized enzyme. Furthermore, the immobilized enzyme recovered was filtered,
Washing was performed with 20 ml of 50 mM citrate buffer (pH 5.1). The immobilized superheat resistance β thus obtained
Galactosidase in 50 mM citrate buffer (pH
5.1) Suspended in 5.0 ml to give an immobilized super thermostable β-galactosidase 1II solution.

(2-4) The above enzyme solution I (0.4 ml) was added to 2.6 ml of 0.1 M phosphate buffer (pH 6.5) containing 2 M sodium sulfate, and the total amount was 3 ml (enzyme amount 4 mg). And Immobilized super thermostable β-galactosidase was prepared from the solution in the same manner as in (2-3) to give an immobilized super thermostable β-galactosidase IV solution.

(2-5) To 2.8 ml of 0.1M phosphate buffer (pH 6.0) containing 2M sodium sulfate and 12 mg of 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride. The enzyme solution I (0.2
ml) was added to make the total amount 3 ml (enzyme amount 2 mg). To the solution, 0.2 g of amino group-introduced Poros Media Self-Pack Poros 20NH (manufactured by Perceptive Biosystems) was added, and after thorough stirring, a reaction was carried out at room temperature for 15 hours under shaking conditions (130 rpm). After completion of the reaction, filtration with a filter was performed to recover the immobilized enzyme. Further, the recovered immobilized enzyme was filtered on a filter to give 5
Washing was performed with 20 ml of 0 mM citrate buffer (pH 5.1). The immobilized superheat-resistant β-obtained in this way
Galactosidase was added to 50 mM citrate buffer (pH 5.
1) Suspended in 5.0 ml to obtain an immobilized super thermostable β-galactosidase V solution.

(2-6) To 2.6 ml of 0.1 M phosphate buffer (pH 6.0) containing 12 mg of 2 M sodium sulfate and 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride. The enzyme solution I (0.4
ml) was added to make the total amount 3 ml (enzyme amount 4 mg). The solution was treated with the same method as in (2-5) to immobilize superheat-resistant β-
Galactosidase was prepared and used as an immobilized super thermostable β-galactosidase VI solution.

(2-7) 2M sodium sulfate and 0.4
% 1-Ethyl-3- (3-dimethylaminopropyl)-
The enzyme solution I (0.1 ml) was added to 2.9 ml of 0.1 M phosphate buffer (pH 6.0) containing carbodiimide hydrochloride.
ml) was added to make the total amount 3 ml (enzyme amount 1 mg). To the solution, 0.5 g of amino group-introduced FP4612 (manufactured by Organo) was added, and after thoroughly stirring, the mixture was reacted under shaking conditions (130 rpm) at room temperature for 20 hours. After completion of the reaction, filtration with a filter was performed to recover the immobilized enzyme.
Further, the recovered immobilized enzyme was washed on the filter with 20 ml of 50 mM citrate buffer (pH 5.1). The immobilized super thermostable β-galactosidase thus obtained was treated with 50 mM citrate buffer (pH 5.1).
It was suspended in 5.0 ml to prepare an immobilized ultra-heat resistant β-galactosidase VII solution.

(2-8) 2M sodium sulfate and 0.4
% 1-Ethyl-3- (3-dimethylaminopropyl)-
The enzyme solution I (0.1 ml) was added to 2.9 ml of 0.1 M phosphate buffer (pH 6.0) containing carbodiimide hydrochloride.
ml) was added to make the total amount 3 ml (enzyme amount 1 mg). To this solution, 0.5 g of FE4611 having an amino group introduced (manufactured by Organo) was added, and after thoroughly stirring, the mixture was reacted for 20 hours at room temperature under shaking conditions (130 rpm). After completion of the reaction, filtration with a filter was performed to recover the immobilized enzyme.
Further, the recovered immobilized enzyme was washed on the filter with 20 ml of 50 mM citrate buffer (pH 5.1). The immobilized super thermostable β-galactosidase thus obtained was treated with 50 mM citrate buffer (pH 5.1).
It was suspended in 5.0 ml to prepare an immobilized ultra-heat resistant β-galactosidase VIII solution.

(3) Method for measuring enzyme activity 0.5 ml of the immobilized super thermostable β-galactosidase I to VIII solutions prepared in (2-1) to (2-8) above were filtered and obtained. Fixed super heat resistance β
-Galactosidase was suspended in 2.0 ml of 50 mM citrate buffer (pH 5.1) containing 500 mM lactose, thoroughly stirred, and then shaken (130 rpm).
The reaction was carried out at 85 ° C. for 2 minutes below. Next, the reaction was stopped by filtering with a filter to separate the immobilized enzyme. After the completion of the reaction, the glucose concentration produced in the supernatant of the reaction solution was measured using Glucose Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.). Immobilized enzyme activity is 1 ml of immobilized enzyme
The enzyme activity per 1 unit and the enzyme activity per 1 mg of the enzyme protein used for immobilization were shown. One enzyme unit of enzyme activity is 1 μmol D per minute at 85 ° C.
-The amount of enzyme that produces glucose. Above (2-
Table 1 shows the results obtained by using the immobilized hyperthermophilic β-galactosidase I to VIII solutions prepared in 1) to (2-8).
As shown in Table 1, super heat resistance β retained on the immobilization carrier
-Galactosidase showed high activity.

(4) Thermal Stability The above (2-1), (2-3), (2-5) and (2-
7), the thermal stability of the immobilized super thermostable β-galactosidase I, III, V, VII, and VIII solutions prepared in (2-8) was evaluated by treating the residual activity after the heat treatment at 85 ° C. for 24 hours. It was confirmed by measuring with the enzyme activity measuring method described in (3) above. The results are shown in Table 2. As shown in Table 2, no decrease in activity was observed by heat treatment for 24 hours in any of the immobilized hyperthermostable β-galactosidases.

(5) Confirmation of Durability and Eluted Enzyme The durability and immobilized carrier of the immobilized ultra-thermostable β-galactosidase prepared by using the immobilized ultra-thermostable β-galactosidase I solution prepared in (2-1) The more eluted enzyme was confirmed. At 85 ° C., the residual activity of immobilized hyperthermophilic β-galactosidase and the amount of eluted enzyme were measured over time for 240 hours. The result is shown in FIG. That is,
FIG. 1 is a diagram showing the residual activity of immobilized hyperthermophilic β-galactosidase by heat treatment and the amount of eluted enzyme. The residual activity on the vertical axis is the immobilized ultrathermophilic β before treatment (0 hour).
-Percentage of residual activity (%, open circles) when galactosidase activity is 100%. Regarding the amount of the enzyme to be eluted, 100 μl of the supernatant of the immobilized ultra-thermostable β-galactosidase solution I (0.5 ml) filtered through the filter after the above treatment
In a 50 mM citrate buffer (pH: 10 mM o-nitrophenyl-β-D-galactopyranoside).
5.1) After adding 2.0 ml and reacting at 85 ° C. for 3 minutes, the value obtained by measuring the amount of o-nitrophenol produced by the absorbance at 410 nm and the super heat resistance β- as a standard substance Galactosidase (10 μg / m
The amount of enzyme obtained by comparing 1) with the value measured in the same manner is shown as the amount of eluted enzyme (μg / ml, black square mark). As shown in FIG. 1, the immobilized hyperthermophilic β-galactosidase had a residual activity rate of 80% or more even after the treatment at 85 ° C. for 240 hours, and no elution of the enzyme retained on the immobilized carrier was observed. It was

[0032]

INDUSTRIAL APPLICABILITY According to the present invention, an immobilized super thermostable β-galactosidase useful in the food industry and sugar engineering field is provided. By using the immobilized super thermostable β-galactosidase as a high temperature bioreactor, It is stable, can be used repeatedly, and can be used for a long time.

[Brief description of drawings]

FIG. 1 is a diagram showing the residual activity rate and the amount of eluted enzyme of immobilized hyperthermophilic β-galactosidase of the present invention.

 ─────────────────────────────────────────────────── ─── Continuation of front page (72) Inventor Susumu Tsunazawa 3-4-1, Seta, Otsu City, Shiga Prefecture, Central Research Laboratory, Mina Shuzo Co., Ltd. (72) Inventor Ikuno Kato, 3-4 Seta, Otsu City, Shiga Prefecture No. 1 in the Central Research Laboratory of Tao Shuzo Co., Ltd.

Claims (1)

[Claims] 1. An immobilized super thermostable β-galactosidase, which is characterized in that the super thermostable β-galactosidase derived from Pyrococcus furiosus is held on a carrier.