JPS6193948A - Sucrose sensor - Google Patents

Abstract

PURPOSE:To determine quantitatively and easily the sucrose (cane sugar) in a vital sample, culture liquid, etc. by passing a sample through a column immobilized with the alpha-glucosidase originating from beer enzyme, column immobilized with mutarose and beta-D-glucose detector in this order. CONSTITUTION:The sample injected through a sample injecting port 52 is passed together with an eluate 51 through the column 2 immobilized with the alpha- glucosidase originating from the bear enzyme and acting specifically with su crose ('-GLUCOSIDASe-Type VI(R)' of Sigma Inc., USA, etc.) and the enzyme column 3 immobilized with mutarose in this order. The sucrose in the sample is selectively hydrolyzed in the column 2 and is converted to alpha-D-glucose and beta-D-fructose then the alpha-D-glucose is converted to beta-D-glucose in the column 3. The beta-D-glucose is detected by the detector 4 and the sucrose concn. in the sample is calculated and recorded or displayed in an arithmetic display part 41. The quantitative determination of the sucrose is thus made possible even with the sample in which the other sugar contg. the beta-D-fructofuranosyl group is mixed without the need for a pretreatment, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION (a) Industrial Application Field The present invention relates to a sucrose sensor. More specifically, sucrose can be used to quantify sucrose in a sample using an immobilized enzyme, and the sucrose content in various samples such as natural products, biological samples, and culture solutions can be easily measured. Regarding sensors.

(b) Prior art Sucrose (sucrose) is composed of β-D-7 fuctofufunosyl-α-D-4'-copifusito bonds, as shown below, and upon hydrolysis, one molecule of β-ri-fructose and one It is known to produce the molecule α-D-glucose.

Recently, a method using Senna equipped with an immobilized enzyme column has been known as a method for quantifying the above-mentioned sucrose in a sample. This sensor enzymatically hydrolyzes sucrose into β-D-fructose and α-D by passing a sample solution through a column containing β-μfuctofufnosidase (inveμtase). - Generate glucose and pass it through a column on which mutalose is immobilized to convert α-D-glucose therein into β-D-glucose, which is then used as a β-D-glucose detector. (For example, a glucose meter that combines glucose okindase-immobilized sulfur and an oxygen electrode) is used to measure the amount of glucose, and based on this, the amount of sucrose in the sample is determined.

However, in the sensor using the β-fructofuranosidase-immobilized Kakara, β-fructofuranosidase originally has a β-D-fructofuranosyl group t.
-S woven to perform hydrolysis, in addition to the intended sucrose, it also acts on oligosaccharides with β-D-fufufunosyl bonds, and in the end, only under limited conditions, i.e., when fructofufunosyl bonds other than sucrose are used. The problem is that the amount of sucrose cannot be determined unless the group-containing saccharide is substantially absent.

(Purpose of the Invention This invention was made to solve the above problems, and it is possible to remove sucrose even in samples containing other β-D-hook tofurano V~ group-containing saccharides without performing any pretreatment such as separation. The purpose is to provide a sucrose senna that can easily detect and quantify only sucrose.

In the present invention, Nagisa et al. found that, in addition to the above-mentioned β-) Kutofufunosidase, there is an α-glucosidase that recognizes the α-D-Cutofufu Vfi/ group and performs hydrolysis. As a result of intensive research, we focused on α-glucosidase derived from brewer's yeast, which is said to act specifically on sucrose among other α-glucosidases. , this α-glucosidase can be immobilized on a carrier64), and even when immobilized, a specific hydrolysis effect on sucrose is expressed, and this immobilized α-glucosidase carrier, a mutarose-immobilized column, and The inventors have discovered that sucrose can be selectively quantified with high accuracy by combining a glucose detector, and have thus arrived at this invention.

(d) Structure of the invention Thus, according to the present invention, an immobilized enzyme column formed by immobilizing α-glucosidase derived from brewer's yeast, an immobilized enzyme column formed by immobilizing mutalose, and β-D-
A sucrose sensor is provided which is characterized in that it is equipped with a course detector in sequence.

In short, the present invention uses immobilized α-glucosidase derived from brewer's yeast instead of conventional immobilized β-fructofuranosidase to produce various β-D-phyctofuranosyl group-containing saccharides. The system is designed so that only sucrose can be quantified with high precision even in samples containing a mixture of sucrose and sucrose.

The α-glucosidase derived from brewer's yeast in the present invention is, for example, α-GL manufactured by Sigma (S-GMA) (USA).
It is easily available from UCOS under the name ASE-'117pe M.

The α-glucosidase immobilized enzyme column used in the present invention is a column that is filled with α-glucosidase immobilized on a particulate or membrane-like carrier by various known methods and is capable of passing a sample solution. Can be mentioned. Among these, as a carrier, a metal hydroxide-based gel, especially a particulate gel, produced by the hydrolysis of a metal alkoxyde or an organometallic chelate compound having oxygen as a ligand, is used. It is preferable to use the above immobilized α-glucosidase because it provides high activity and enables accurate quantification (as with sucrose). Metal 7 that is the raw material for the carrier in this case
As μcoxide, 5i (OCHs) 4.5i (OC
J, )4, Ti(OCsHy)4, V(002H5)3
, A#(OCsHy)s, Co(OC&)a, N1(O
CJs)z, Fe(QC,H,), etc., and among these, lower alkoxins are preferred. Further, as the organometallic chelate compound having oxygen as a ligand, metal chelate compounds of alkanediones or derivatives thereof are suitable, such as 2,4-pentanedione (acetylacetone), 3-phenylene/L'-2 , 4-pentanedione (3-phenylacetylacetone), 2.4-hexanedione, 2.4 (or 3,5)-hebutanedione, 2,
Examples include calcium, aluminum, titanium, zinc, iron or calylum chelate compounds of lower 7μ candiones such as 2,6,6-titrametheA/-3,5-heptanedione (dipivaloylmethane). Of course, these metal 7-koxides and organometallic chelate compounds may be used in combination.

Easily volatile O hydrophilic injection fusing solution (
For example, by adding an acid to a lower alcohol μ solution and leaving it to stand, hydrolysis proceeds to form a gel, and by drying and crushing this, a carrier can be obtained. .

The immobilization of α-glucosidase on such a carrier is usually carried out using a silane coupling agent such as γ-aminopropyltriethoxysilane, T-qualoproyltrimethoxysilane, vinyltriethoxysilane, etc. , γ-glycidoxyglobyltrimethoxysilane, N-β-(aminoethylμ)-γ-aminoprobyltrimethoxysilane, etc.) to inject the silane coupling agent into the gel body through the hydroxyl groups. After introduction and subsequent treatment with an aqueous solution of a bifunctional immobilizing agent (such as glycaldehyde), α-glucosidase is transferred to a silane cup by contacting with an aqueous solution of α-glucosidase derived from brewer's yeast. This can be carried out by immobilization by chemical bonding with a ring agent. However, in some cases, it is also possible to directly immobilize α-glucosidase without using a coupling agent, such as in the cyanogen bromide activation method. Note that the appropriate amount of immobilization at this time is 10 to 20 U/da carrier.

The immobilized mutarose enzyme column used in this invention may be a well-known one, but it is preferable to use a metal hydroxide-based gel as described above.

On the other hand, the β-D-glucose detector used in the present invention can be used in various ways, such as those capable of detecting and measuring the amount of β-D-glucose in at least four liquids. A method in which an enzyme column and an oxygen electrode are combined in a flow path to measure changes in the amount of dissolved oxygen before and after passing through the column to measure the amount of G/L/cose in a sample solution, using an oxygen electrode coated with a glucose oxidase immobilized membrane. A method using a glucose electrolyte, luminol and red blood salt or luminol and bistrichlorophenylene Iv oxalate ester A is added to the flow through a glucose oxidase-immobilized enzyme column.
/(2) A glucose detector such as a method in which a chemiluminescent agent is mixed and the amount of jig μ course is measured based on the amount of chemiluminescence can be applied.

(e) Examples The present invention will be explained in detail below with reference to examples shown in the drawings. Fig. 1 is a configuration explanatory diagram showing a sucrose sensor of the invention. In Fig. 1, the sucrose sensor (1) is
an α-glucosidase-immobilized enzyme Kakara (2) equipped with a supply path (5) for a buffer solution (51); and a mutarose-immobilized enzyme column (3) connected from the column (2) via a flow path (6). ) and a β-D-glucose detector (4) connected from the column (3) via a flow path ()). (41) is a calculation display unit, (52) is a sample introduction unit. The above a-glucondase-immobilized enzyme column (2) is
It was obtained as follows.

(Preparation of carrier) Tetraethoxysifun 5i (QC2Hs) 40.52 mo〃, Ethano-A/1.72 moμ, water 1.82 moμ, hydrochloric acid 0.028 mo〃, and heptathhydrotonic acid 0. 058~0.1
Mix and stir a 15 mol mixture (pH about 1) at room temperature for several minutes until it becomes homogeneous.

It was then heated in a water path at 80°C for 3 days and nights to evaporate the 7 μg of ethyl produced by the hydrolysis reaction, water, residual hydrochloric acid, and trace amounts of unreacted hydrofluoric acid, resulting in approximately 309 pores. A gel-like carrier was obtained.

(Aminoacrylation) The gel-like carrier obtained above was pulverized to 120/20
Obtained Q-metush beads.

5wt% γ-aminopropylμtriethoxykufun aqueous solution i Adjusted to pH 3.5 with 5N hydrochloric acid, and this solution 451+
52 of each of the above bead-like gelatinous materials were added to each of the 4 samples, and the pH was further adjusted to 3.5. This mixture was placed in a Yotsulov Fusco equipped with a stirrer, a thermometer, and a Dimroth, and the temperature was maintained at 75° C. with a water path, and the reaction was carried out for 3 hours while stirring.

After the reaction was completed, the beads were transferred to a suction membrane filter, unreacted γ-aminopropyltriethoxine was removed with distilled water in Step 14, and dried in a desiccator to obtain an aminoalkylated gum support. This Aminea ρkip
Store the gelled material in a desiccator.

(Immobilization of enzyme) The above aminoalkylated gel-like carrier (bead-like) was immersed in a phosphate buffer solution (pH 7.0) of bifunctional glutaraldehyde (2.5 wt%), and the pressure was reduced with an aspirator. Next, the mixture was allowed to react for about 30 minutes with stirring.

Subsequently, the reaction was continued for about 30 minutes under stirring at normal pressure. The reaction temperature was 30° C., and the mixture was thoroughly washed with a phosphate buffer solution of pH 7’ and dried. This process results in
A Schiff base having a 7μ dehyde group is introduced into the product.

100 m9 of the obtained gel-like carrier was
- Glucosidase-containing solution (Sigma, Lunar Phase VI; 40
α-glucosidase was immobilized by immersing it in 50M9 phosphate buffer (pH 7,0) solution (10m!') and shaking it slowly for 5 hours in a 30'C hot water bath to perform the immobilization reaction. The converted enzyme (in the form of beads) was obtained.

The α-glucosidase immobilized enzyme Kakara (2) was obtained by filling this immobilized enzyme in a glass cylindrical container (2, lJ2+' x 5.0 containers) equipped with a liquid inlet and a liquid outlet. Ta.

The mutarose-immobilized enzyme column (3) used was one in which mutarose 800 Ut' was immobilized on a gel carrier and packed in the same manner as described above. In addition, as the β-D-glucose detector (4), a high-speed linear glucose meter ((
Manufactured by Shimadzu Corporation; a method that detects the rate of decrease of dissolved enzyme in the liquid in the batch tank by the action of glucose oxidase, and detects the amount of glucose in the liquid based on this) As the equilibrium solution (51), a phosphate IIc solution was used.

In the nine sucrose sensor (1) constructed in this way, when a sample containing sucrose is introduced through the inlet (52), sucrose is first selectively hydrolyzed in the column (2) and α-D - converted into glucose and β-D-fructose, and then in column (3) α-D-glucose is converted into β-D-glucose, which is converted into β-D-fructose.
It is detected by the D-glucose detector (4), and the calculation display section (
In step 41), the sucrose concentration in the sample is determined based on the starting glucose amount.

FIG. 2 shows the results of investigating the relationship with the amount of glucose produced using samples containing various concentrations of sucrose.

In this way, a good linear relationship between the sucrose concentration and the amount of glucose produced was confirmed. Enzyme beads were prepared, and as a comparative example, immobilized enzyme beads containing 200 U of β-fructofuranosidase were similarly prepared, and their activity toward fructooligosaccharides was evaluated. In addition,
Fructooligosaccharides can be hydrolyzed to produce glucose and fuctose as described below.

In addition, experiments were conducted using a fructooligosaccharide aqueous solution (5, OW/V
%) Place the above immobilized enzyme beads in 50i1 and heat at 20°C.
The mixture was stirred with a lower stirrer, and the amount of glucose produced over time was measured in terms of glucose amount in the same manner as described above.

The results are shown in FIG. As described above, the glucosidase-immobilized enzyme used in the present invention has a lower glucose production rate than the β-fructofufunosidase-immobilized enzyme.
It was found that it is substantially inactive against fructooligosaccharides and selectively hydrolyzes sucrose. The amount of glucose produced after 3 hours is 4519/(Lg) for the glucosidase immobilized enzyme.
538■ for fructofuranosidase immobilized enzyme
/di, and it was also found that the former had no effect at all after 1 hour.

(F) Effects of the Invention As stated above, the sucrose sensor of the present invention allows for quantitative determination of sucrose without pretreatment such as separation, even in samples containing other β-D-7μ ctofufnosyl group-containing sugars. It is useful in various fields. It is particularly useful for determining the sucrose content in natural products containing oligosaccharides of β-D-fructofuranoside.

[Brief explanation of the drawing]

FIG. 1 is a configuration explanatory diagram showing one embodiment of the sucrose sensor of the present invention, FIG. 2 is a graph showing the sucrose responsiveness of the sensor of the same embodiment, and FIG. 3 is a
This is a graph showing the activity of glucosidase-immobilized enzymes on fructooligosaccharides together with comparative examples. (1)... Sucrose sensor, (2)... α-glucosidase immobilized enzyme column,
(3)... Mutarose-immobilized enzyme column, (4)...
- β-D-glucose detector. Agent: Patent attorney: Bedai Nogawa - ・53゛No. 1 Diagram 2: Exposed sucrose l friend ('/,)

Claims (1)

[Claims] (1) A sucrose characterized by sequentially comprising an immobilized enzyme column formed by immobilizing α-glucosidase derived from brewer's yeast, an immobilized enzyme column formed by immobilizing mutalose, and a β-D-glucose detector. sensor.