JPH03218400A - Sweetness-inducing substance - Google Patents

Abstract

NEW MATERIAL:A high-purity miracullin having an amino acid sequence represented by the formula (CHO is sugar chain), etc., a sugar composition (molar ratio) of 3.0 glucosamine, 3.0 mannose, 2.1 fucose, 0.7 galactose and 1.0 xylose, about 25000 molecular weight and about pH9.0 isoelectric point. USE:A sweetener. PREPARATION:For example, water is added to the lyophilized sarcocarp of a miracle fruit and the mixture is homogenized and centrifuged to collect the resultant precipitate. To the collected precipitate, 0.5M sodium chloride solution is added and the resultant mixture is homogenized and centrifuged. The resultant supernatant is collected and ammonium sulfate is added to the collected extract to 40% saturation to deposit an active substance. The deposited active substance is separated and dissolved in a phosphate buffer solution and the resultant solution is subjected to ion-exchange chromatography, affinity chromatography and high-performance liquid chromatography in order for purification, thus obtaining the objective sweetness-inducing substance of the formula.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to miraculin, a novel sweetness-inducing substance isolated from natural miracle fruit.

[Conventional technology]

Miracle fruit (Richard) native to West Africa
lla Dulcifica) has the property that if you put it in your mouth and then taste something sour, it will taste sweet.

It is already known that this taste-altering active ingredient (taste modifier) of the miracle fruit is a glycoprotein (with a molecular weight of around 45,000). In addition, the proportion of sugar contained in this glycoprotein is approximately 10 to 15%, and the types of sugar are L-arabinose, L-rhamnose,
D-xylose, D-mannose, D-galactose,
It has been confirmed that it is D-glucose.

The mechanism behind the sweetness of this miracle fruit has not yet been elucidated, but the pulp of the miracle fruit is mixed with sour substances in advance and does not exhibit any sweetness even if it is left in the mouth for a certain period of time, so it is believed to be the active ingredient in the miracle fruit. Unlike conventional sweeteners, sweet taste inducers are thought to produce sweet taste through some kind of interaction between their active ingredients and sweet taste receptors in the epithelium of the tongue, rather than by converting sour taste itself into sweet taste.

For these reasons, miracle fruit sweetness inducers are expected to be a new type of sweetener, especially as a sweetener that is effective for people who have limited sugar intake.

[Problem to be solved by the invention]

The present inventors have previously provided a method for producing sweetness-inducing substances from miracle fruits with high purity and high yield without losing activity, but in the present invention, the primary structure of this sweetness-inducing substance has been further elucidated. The purpose of the present invention is to provide a novel high-purity miraculin with excellent sweetness.

[Means to solve the problem]

The novel high-purity miraculin of the present invention has the following physical and chemical properties: (1) The amino acid composition is as shown in Table 1.

(Margins below this page) Table 1 Amino acid composition Amino acid % *Numbers indicate the sum of logarithms of amino acid residues when the total residues are 100.

(2) I! Composition: Table 2 (3) Molecular weight: Approximately 25,000 (4) Isoelectric point: Approximately pH 9.0 Table 2 Sugar composition of Miraculin 8sp Ser Ala ProAsp Gly
Glu Lys Tyr Tyr Ile Val Gly Gly Gly Leu Eight sn Pro Val LeuLeu Arg
Thr Gly Pro Val Leu Arg Thr Vat Ser AlaAsp He Thr Asn Asp His Thr Thr Thr Val Vat Gln ThrAsp Arg
Pro Leu Pro Lys Glu AspAsp Le
u Asn TieArg Lys Glu V
al Ala Phe Phe ProVal Va
l Arg VatAsn Phe Ser
AlaAsp Old S Glu Asn Ser Thr Phe Met Arg Leu Asp Lys Tyr Asp
Glu Ser Thr GlyGly Asn
Pro Gly Pro Glu Thr11
e Ser Ser Trp Phe Lys Tie Glu Glu Phe CysGl.
y Ser Val Cys Gly Ser Cys Lys
Val Lys Cys GlyPhe The novel high-purity miraculin of the present invention can be obtained by washing the pulp of miracle fruit with water until the washings are no longer colored, then extracting with a sodium chloride solution, and then purifying this extract.

Specifically, water is added to the freeze-dried pulp of the miracle fruit, homogenized, and centrifuged. At this time, the supernatant shows a deep pink color. This supernatant has no activity to change sourness into sweetness. Add the same amount of water as the initial amount to the resulting sediment, homogenize it, and centrifuge. Repeat this operation until the supernatant becomes colorless. There is no activity in the supernatant when it becomes colorless.

Then extract using sodium chloride solution. That is,
A sodium chloride solution is added to the precipitate after the water washing operation, homogenized, and centrifuged.

For extraction near neutrality, the resulting extract exhibits high activity without loss of activity. The extract can be purified by salting out the extract with ammonium sulfate and then using a conventional chromatography method. For example, a precipitate obtained by salting out is subjected to CM-Sepharose ion exchange chromatography and further purified by affinity chromatography. The amino acid sequence of the obtained high-purity miraculin was determined by hydrolyzing the high-purity miraculin with enzymes (trypsin, chymotrypsin, lysyl endobeptidase), and then performing HPL using an ODS 120T column.
This was done by fractionating each peptide fragment with C.

Miraculin is a completely new type of sweet substance that can be applied to foods, medicines, etc. With this invention,
Its practical application will be further accelerated.

Reference Example (1) Water washing and extraction with sodium chloride solution Take 10 g of freeze-dried pulp of miracle fruit, add 40 cc of water, homogenize, and centrifuge (12.50 O r
pm, for 20 minutes). At this time, the supernatant had a deep pink color and had no activity to change sourness into sweetness.

Add 40 cc of water to this sediment, homogenize it, and centrifuge (12.50 rpm, 20 minutes). This supernatant had no taste-altering activity.

Next, 0.5M sodium chloride solution was added, homogenized, and centrifuged (12.50 rpm, 20 minutes).
did. The resulting curd was colorless and exhibited sweetness-inducing activity. The extraction operation using 40 cc of 0.5M sodium chloride solution was repeated three times, and the supernatants from the three times were combined.

(2) Salting out with ammonium sulfate Ammonium sulfate was added to the extract obtained in the above procedure to achieve 40% saturation to precipitate the active substance. The precipitate obtained by centrifugation (13.00 rpm, 20 minutes) was Dissolve in OIM phosphate buffer.

(3) CM-Sepharose ion exchange chromatography The solution prepared in (2) was placed on a CM-Sepharose column that had been sufficiently equilibrated with 0.01M phosphate buffer in advance. Then, first phosphate buffer (pH 6.8
) After sufficient elution with sodium chloride solution O~1.0M
Elution was performed using a linear concentration gradient elution method (flow rate 5cc/15m).
in, 1 fraction 5 cc, total eluate volume 400 cc). Protein was monitored by absorption at 280 nm. Figure 1 shows the results. Peak 0 in FIG. 1 is the sweet taste-inducing active substance.

(4) Affinity oral tomography The above peak 0 portion was concentrated to 10 cc by ultrafiltration, and the solvent was added to 0.0 cc containing 0.5 M sodium chloride. The solvent was replaced with OIM phosphate buffer (pH 6.8). This concentrate was processed through a ConA-Sepharose 4B column. First, 0.5M sodium chloride was added. Wash with OIM phosphate buffer (pH 6.8), then α-methyl-D-
Glucoside solution was eluted using a linear concentration gradient elution method of 0 to 0.15M (flow rate 3 cc/14 min, total eluate volume 20
0 cc). Protein was monitored by absorbance at 10280 nm. Figure 2 shows the results. Peak I showed activity and the yield was 35 mg.

(5) High performance liquid chromatography The purity of the active substance (peak ■ in Figure 2) was determined by high performance liquid chromatography (HPLC) using a reversed phase column.
Confirmed by.

The column used was TSK gel TMS-250.
Acetonitrile containing 0.5% trifluoroacetic acid (20~
70%) using a linear concentration gradient elution method and monitored by measuring absorbance at 210 nm.

As shown in FIG. 3, the results showed a sharp single peak, and it was confirmed that the purity was extremely high.

The molecular weight of this highly purified miraculin was 28,000 by SOS-PAGE. On the other hand, the sugar content is 14%
(Calculated from the phenol-sulfuric acid method and Lowry method.)
Therefore, when calculated from the amino acid primary structure of (P2), it is approximately 25,000. The composition of the sugar is shown in Table 2. Isoelectric point 11 pH is around 9.0. The amino acid composition is as shown in Table 1, and the amino acid sequence is as described above.

The results of the activity test of this highly purified miraculin are shown in FIG. From FIG. 8, the activity of high-purity miraculin whose amino acid sequence has been determined corresponds to 0.5M sucrose, while the conventional one corresponds to a maximum of 0.4M sucrose, so the miraculin of the present invention has a higher activity than the conventional one. It can be seen that it shows excellent activity.

〔Effect of the invention〕

This invention has made it possible to provide highly active and highly purified miraculin whose amino acid sequence has been determined.

Furthermore, this highly purified miraculin is useful as a source of structural genes when miraculin is produced in large quantities by genetic engineering techniques.

Example (1) Preparation of S-carpoxyamidomethylated miraculin 7 mg of high purity miraculin prepared according to Reference Example was added to 5 mfl of 0.4M I-Lys buffer 12 containing 6M guanidine hydrochloride, 2mM EDTA and 60mM dithiothreitol. dissolve in This solution was dissolved in nitrogen gas for 37
℃, incubated for 24 hours. Add 0.2 g of iodoacetamide to this solution and let stand at room temperature for 10 minutes.
Then, it was left standing in an ice water bath for 60 minutes. The resulting S-carboxyamidomethylated miraculin was treated with Sephadex G-
25 with 2M urea and 2mM EDTA.
Desalting was carried out using M sodium bicarbonate buffer (pH 8.0) as a solvent.

(2) Enzymatic cleavage of S-carboxyamidomethylated miraculin Glycine endopeptidase digestion of S-carboxyamidomethylated miraculin with 2 M urea and 2 mM EDTA
at 37°C in 50mM ammonium bicarbonate buffer containing
I went for 120 hours.

The protein concentration is lIIlg/rd and the enzyme:substrate ratio is 1:1.
00 (w/w). For the digestion reaction, add HCL and pt
It was stopped by setting the value to +2.0. No insoluble material was present in the digestate. In addition, chymotrypsin digestion of S-carpoxyamidomethylated miraculin was carried out at 37°C for 90 minutes using the same buffer, protein concentration, and enzyme:substrate as in the above digestion. Digestion reactions were stopped in the same manner as above. No insoluble material was formed in the digestate.

S-carpoxyamidomethylated miraculin was added to 50mM ammonium bicarbonate (containing 2M urea and 2mM EDT8).
V B (Staphylococcus aureus) in pH 7.8).
s V8) Digested with protease at 37°C for 3 hours.

The protein concentration was 1 mg/mfl, and the enzyme:substrate ratio was 1:
30w7w. A precipitate formed after digestion. Solid urea was added to the reaction mixture until the solution became clear.

(3) Separation of peptides Each peptide obtained by digestion with the three types of enzymes mentioned above was separated by HPLC (Toyo Soda PC 8000) using a TSK-003-120T (manufactured by Toyo Soda) column. Each peptide was eluted using a linear gradient elution method using acetonitrile containing 0.05% trifluoroacetic acid. Veptides were detected by absorption at 210 nm and each peak was collected.

(4) Amino acid analysis and determination of amino acid sequence 14 The amino acid composition of each peptide is determined by Waters Picot.
Determined by agsystem. That is, the peptide is H
Hydrolysis was carried out using CL steam at 110°C for 22 hours. The obtained amino acid was used as a phenylthiocarbamyl (PTC) derivative, and the TSK-OI]S-80TM column 0.4
llPLf using 6 x 15cm (manufactured by Toyo Soda)
Analyzed by l:.

PTC derivatives of amino acids were prepared in 50 lIIM phosphate buffer (
p117.0) in 3.0% acetonitrile for 20 minutes;
The column was then flushed with 40% acetonitrile for 5 minutes each at a flow rate of 1 d/min. Leaked PT
C-amino acids were detected by absorbance at 254 nm. Amino acid sequence was determined using 470A Applied Bio
System Protein Sequencer was used. That is, phenylthiohydantoin derivative (P
TH-amino acid) as TSK 10DS-120
Analysis was performed by +1 PLC using a T column (Toyo Soda).

The C-terminal amino acid sequence was determined using carboxypeptidase as described by Yambler. Dissolve 200 tt g of miraculin in 0.9 ml of 0.1M N-ethylmorpholine acetate buffer (pH 8.0). Add 1 μg of carboxypeptidase A to this solution.
is added and the reaction mixture is incubated at room temperature. Aliquots of the reaction solution are collected every 15, 30, 60, and 120 minutes. Trichloroacetic acid was added to these reaction solutions to precipitate proteins, which were removed by centrifugation, and the free amino acids in the supernatant were purified using Haters Picotag Sy.
It was analyzed by stem.

The amino acid sequence determined by the above method is as follows.

8sp Ser Ala ProAsp Gly
Glu Lys Tyr Tyr Ile Val Gly Gly Gly Gly Leu ^sn Pro Val LeuLeu Ar
g Thr Gly Pro Val Leu Arg Thr Val Ser AlaAsp Il
e Thr Asn Asp His Thr Thr Asp Arg Pro Leu Ala Phe
Phe Pro Glu AsnPro Lys
Glu Asp Val Val Arg Val
Ser ThrArg Leu Asp Lys
Tyr Asp Glu Ser Thr Gly16 Gln Tyr Phe Val Thr I
le Gly GIy Val LysGly A
sn Pro Gly Pro Glu Thr
Ile Ser SerTrp Phe Lys
Ile Glu Glu Phe Cys Gly
SerAsp Val Gly 11e Tyr 11e Asp Gin Lys Gly

[Brief explanation of drawings]

FIG. 1 shows the elution pattern of a sweet taste-inducing substance obtained from miracle fruit by washing with water, extraction, and salting out by CM-Sepharose ion exchange chromatography. FIG. 2 is an elution pattern of affinity chromatography in which the beak (■) portion of FIG. 1 was treated with a ConA-Sepharose 4B column. FIG. 3 is an elution pattern of high performance liquid chromatography of peak I in FIG. 2. FIG. 4 shows the IPLc separation of peptides obtained by S-carboxyamidomethylated miraculin and dylyl endopeptidase digestion. FIG. 5 shows HPLC of peptides obtained by chymotryptic digestion of S-carboxyamidomethylated miraculin. Figure 6 shows S-carboxyamidomethylated miraculin of Staphylococcus aureus V B (Staphy1o
Figure 3 shows HPLC separation obtained by protease digestion of C. coccus aureus V8). Figure 7 shows the amino acid sequence of miraculin. In FIG. 7, LEP and ch indicate the peptides from lysyl endopeptidase and chymotrypsin digestion, ■ indicates the peptides from ■8-protease digestion, and N indicates the amino acid sequence determined from the N-terminus, respectively. ←← indicates the sequence from the C-terminus. Furthermore, the relationship between alphabets and amino acids is as follows. G: G1y, A: A1a, S: Ser, T: T
hr, C: Cys, N: Asn, Q: GIn, L
:Leu, I:Ile, V:Val18 M:Met, F:Phe. Y: Tyr, W: T
rp,P:ProD:Asp. E:G1u,
H: His, K: Lys, R: 8rg
, FIG. 8 shows the activity of highly purified miraculin of the present invention.

Claims (1)

[Claims] 1. High purity miraculin having the following physical and chemical properties (1) Amino acid composition as shown in Table 1 (2) Sugar composition as shown in Table 2 (3) Molecular weight: approximately 25,000 (4) ) Isoelectric point: approximately pH 9.0 2. High purity miraculin of claim 1 having the following amino acid sequence [There is a gene sequence] [There is a gene sequence]