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Novel Taste-Modifying Polypeptide Nas, Dna Thereof and Use Thereof

a technology of taste-modifying polypeptides and dna, which is applied in the direction of peptides, plant/algae/fungi/lichens ingredients, and peptides, etc., can solve the problems of insufficient taste-modifying functions of such reagents, disadvantages in laborious work and production costs, and inability to use such reagents in industrial production, etc., to achieve excellent taste-modifying activity and protein supply efficiency

Inactive Publication Date: 2008-12-11
MITSUKAN GROUP CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0043]In accordance with the invention, a novel dimeric protein neoculin with an excellent taste-modifying activity and in a heterodimer structure different from that of curculin is provided. Using the protein, a novel taste-modifying composition practically applicable to foods and the like is provided.
[0044]In accordance with the invention, further, the amino acid sequence of a subunit constituting the protein is provided. Thus, the protein can be provided by an appropriate synthetic method following the amino acid sequence.
[0045]In accordance with the invention, the DNA of the gene encoding the protein is provided. Selecting appropriate hosts, specifically koji molds, and using genetic engineering technology, the protein can be provided efficiently.

Problems solved by technology

However, the taste-modifying functions thereof are not sufficient enough to be added into foods.
The requirement of the use of such reagent is disadvantageous in terms of laborious works and production cost.
Additionally, such use of the reagent is not suitable for industrial production from the standpoint of safety profile when the produced neoculin is used for foods.

Method used

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  • Novel Taste-Modifying Polypeptide Nas, Dna Thereof and Use Thereof
  • Novel Taste-Modifying Polypeptide Nas, Dna Thereof and Use Thereof
  • Novel Taste-Modifying Polypeptide Nas, Dna Thereof and Use Thereof

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0123]A fruit of Curculigo latifolia was purified by the following procedures to obtain the novel protein with the taste-modifying activity.

(1) Preparation of Crude Extract Solution

[0124]40 liters of pure water were added to about 1 kg of the freeze-dried fruit of Curculigo latifolia (freeze-dried fruit powder in Table 1) for homogenization for 15 minutes. Then, centrifugation at 6,000 rpm for 20 minutes was done to discard the supernatant (the supernatant had no taste-modifying activity). The procedure described above was repeated twice, to obtain the residual precipitate.

[0125]Then, 20 liters of 0.05N sulfuric acid were added to the residual precipitate, for homogenization for 10 minutes. Then, centrifugation at 6,000 rpm for 20 minutes was done to recover the supernatant. The procedure described above was repeated twice. The resulting precipitate had no taste-modifying activity.

[0126]Then, 2 liters of 1N sodium hydroxide were added to the extract solution for neutralization, to o...

example 2

[0134]Neoculin obtained in Example 1 was purified further by the following procedures, to make the analysis of the individual subunits constituting neoculin.

(1) Purification on HiTrap SP Sepharose Fast Flow Column

[0135]100 mg of the neoculin powder obtained in Example 1 was dissolved in 20 ml of buffer A (50 mM Tris-HCl buffer, pH 7.5 containing 8M urea and 30 mM DTT). Then, the whole volume was applied to HiTrap SP Sepharose Fast Flow column (manufactured by Amersham Biosciences; a 1.6 cm diameter×2.5 cm) equilibrated with the buffer A. Continuously, the column was washed with 50 ml of the buffer A. Continuously, elution was done with 50 ml of the buffer A containing 1M NaCl, to obtain a purified NBS fraction. Furthermore, 70 ml of the wash fraction was dialyzed against ion exchange water to a volume of 100 ml.

(2) Second Purification on HiTrap SP Sepharose Fast Flow Column

[0136]To 100 mg of the wash fraction obtained above in (1) was added 8 M urea, 30 mM DTT and 50 mM acetate buff...

example 3

[0139]The amino acid sequence of the NAS fraction constituting neoculin as obtained in Example 2 was analyzed by the following procedures.

(1) Analysis of N-Terminal Amino Acid Sequence

[0140]70 μg of the purified NAS powder obtained in Example 2(1) to (3) was subjected to two-dimensional electrophoresis. The gel after electrophoresis was overlaid on a polyvinylidene difluoride (PVDF) membrane, where an electric current passed vertically for transfer. The PVDF membrane after the transfer was stained with SYPRO Ruby protein blot stain (manufactured by Molecular Probes), from which bands were excised out by a general method for the analysis of the N-terminal amino acid sequence with an amino acid sequencer (HP G1005A Protein Sequencing System). In other words, phenylisothiocyanate (PITC) is allowed to react with a free amino residue at the N terminus to prepare a phenylthiocarbamyl derivative (PTC amino acid), which is then released with trifluoroacetic acid in the form of anilinothiazo...

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Abstract

It is an object of the invention to find a substance with a better taste-modifying function and determine the structure of the taste-modifying substance, as well as to elucidate the structure thereof at a gene level and determine the primary structure of the substance and obtain the gene encoding the substance. Additionally, it is an object of the invention to provide a novel taste-modifying composition characteristically containing the taste-modifying substance.Specifically, the invention provides the polypeptide shown below in (A) or (B), a protein dimer neoculin comprising the polypeptide NAS and the polypeptide NBS and having a taste-modifying activity:(A) a polypeptide comprising an amino acid sequence shown in SEQ ID NO.2 in the sequence listing;(B) a polypeptide comprising an amino acid sequence with the substitution, deletion, insertion, addition or inversion of one or several amino acids in the amino acid sequence shown in SEQ ID NO.2 in the sequence listing, which polypeptide can form the neoculin dimer having a taste-modifying activity together with the polypeptide NBS.

Description

TECHNICAL FIELD[0001]The present invention relates to a novel taste-modifying polypeptide NAS, the DNA thereof and the use thereof. More specifically, the invention relates to the polypeptide NAS, the gene thereof, the dimeric protein neoculin containing the polypeptide and having a taste-modifying activity, and a taste-modifying composition containing neoculin.BACKGROUND OF THE INVENTION[0002]Curculigo latifolia is a plant spontaneously growing in west Malaysia and a southern part of Thailand, which is classified into Liliaceae. It is said that the curculin isoform (referred to as curculin hereinafter) contained in the plant is useful as a taste-modifying substance giving sweet taste when the isoform is eaten before drinking water or eating a sour substance.[0003]Conventionally known subunits constituting curculin include for example curculin A and curculin B.[0004]The entire amino acid sequence of curculin A has been determined (see for example patent literature 1). The entire ami...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07K14/415C12P21/02C12N15/29C12N15/80C12N15/63C12N1/15A23L27/30C07K14/47
CPCA23L1/2361C07K14/47A23L27/31A61P1/00A61P1/02A61P25/02A61P43/00
Inventor ABE, KEIKOASAKURA, TOMIKOSORIMACHI, HIROYUKIUENOYAMA, TAZUKONAKAJIMA, KENICHIROKITAMOTO, KATSUHIKOMARUYAMA, JUNICHIKISHI, MIKIYA
Owner MITSUKAN GROUP CORP
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