JPH03216194A - Preparation of saccharide amino acid ester - Google Patents

Abstract

PURPOSE:To prepare a saccharide ester at a low cost by treating a mixture of an amino acid ester and a saccharide (alcohol) with a microorganism or enzyme capable of performing the conversion reaction of the ester compound. CONSTITUTION:(B) A mixture of (i) an amino acid ester such as L-alanine methyl ester and (ii) a saccharide or an alcohol thereof is treated with (A) a microorganism (e.g. Rhodotorula.lactosa IFO 1423 strain) or enzyme having an ability for the conversion reaction of the ester compound to provide a saccharide amino acid ester.

Description

Detailed Description of the Invention (Field of Industrial Application) The present invention is directed to the production of a compound (hereinafter referred to as sugar amino acid ester) in which a sugar or sugar alcohol and an amino acid form an ester bond in their respective hydroxyl and carboxyl groups. The present invention relates to a method for producing an amino acid ester, which comprises allowing a microorganism or an enzyme capable of carrying out a transfer reaction of an ester compound to act on a mixture of an amino acid ester compound and a sugar or sugar alcohol.

(Prior art) Conventionally, L-asbarthyl-L has been used as a low-calorie sweetener.
~ Peptides such as phenylalanine methyl ester, glycyrrhizin, glycosides such as stevioside, and sugar alcohols and saccharides that are said to be low in calories, including maltitol, are known.

The method for producing ester compounds of sugar and amino acids was disclosed in Japanese Patent Application Laid-Open No. 1983-1989.
-188037, JP 60-188039, JP 61-124354, JP 61-1
77963, etc., but all of these are based on organic chemical synthesis methods, and methods using microorganisms or enzymes are not known.

(Problems to be Solved by the Invention) A conventionally known method for producing an ester compound of sugar and an amino acid is a method of introducing an amino group into glucose whose 1-position is methylated. It is not possible to obtain unmethylated compounds.

Furthermore, since this method is a chemical synthesis method, it requires heating, organic solvents, expensive catalysts, etc., and thus has to be extremely expensive in terms of manufacturing equipment and manufacturing costs.

(Means for Solving the Problems) In order to produce a substance in which the 1st position of the sugar is not methylated, and to produce it with simpler equipment, the present inventors have developed a method for combining sugar and amino acids using microorganisms or enzymes. As a result of extensive research into methods for producing ester compounds, the present invention was completed.

That is, the present invention provides a method for producing a sugar amino acid ester, which is characterized by allowing a microorganism or an enzyme capable of carrying out a transfer reaction of an ester compound to act on a mixture of an amino acid ester compound and a sugar or sugar alcohol. . According to the present invention, an amino acid residue of an amino acid ester compound is transferred to a sugar or sugar alcohol using a microorganism or an enzyme capable of carrying out a transfer reaction of an ester compound, and the carboxyl group of the amino acid is transferred to the hydroxyl group of the sugar or sugar alcohol. A compound formed by an ester bond is obtained.

Examples of amino acid ester compounds include L-alanine methyl ester, L-alanine ethyl ester, chrysine methyl ester, glycine ethyl ester, L-valine methyl ester, L-valine ethyl ester, L-aspartic acid methyl ester, and L-threonine methyl. Ester, L-proline methyl ester or their hydrochloride salts, etc. can be used. Further, as the sugar or sugar alcohol, glucose, fructose, sorbose, sucrose, sorbitol, maltitol, glycerin, erythritol, dextrin, etc. can be used.

Examples of microorganisms or enzymes that have the ability to carry out transfer reactions of ester compounds include Rhodotorul.
a) Yeast or Neurosbora belonging to the genus
Filamentous fungi belonging to the genus Pora) or enzymes extracted from them can be used. In addition, some strains that produce sugar amino acid esters have been found among these strains treated with mutagenic agents or naturally occurring mutants, and these mutants can also be used in the present invention.

A typical strain is Rhodotorula lactosa (Rhodotorula lactosa).
otorula lactosa) IP0 1423
strain or Neurospora cytophylla (Neurosp)
ora sitophila) IF0 6070
Stocks are one example.

In culturing the fungi used in the present invention, conventional fungal culture methods are generally used. As a culture medium, carbon source,
Any medium can be used as long as it contains an appropriate amount of nitrogen source, inorganic substances, etc. and allows the bacteria used to grow well.

As the culture method, liquid shaking culture is preferred. The grown bacterial cells are collected by centrifugation, filtration, etc., and thoroughly washed with an appropriate buffer or sterile water.

The obtained microbial cells are crushed by vacuum expansion, grinding with glass beads, or other techniques commonly used for disrupting fungal cells to obtain a bacterial cell extract.

Furthermore, the bacterial cell extract obtained in this manner is purified by a method commonly used for protein purification, such as gel filtration or ion exchange, and used as an enzyme.

The thus obtained bacterial cells, bacterial cell extracts, or enzymes are added to a substrate solution containing a sugar or sugar alcohol serving as a receptor and an amino acid ester compound, and the mixture is stirred while stirring.
When kept at 0 to 40°C, it usually forms in a few hours, and 2 to 40°C.
A sufficient amount of the target substance accumulates in the reaction solution in 3 days. After the reaction is completed, the target product is purified and isolated from the reaction solution.

For isolating and purifying the sugar amino acid ester from the reaction solution, a conventional method for purifying natural organic compounds is used. For example, first, the reaction product is separated into a reaction solution and bacterial cells by centrifugation, filtration, or the like. After diluting this reaction solution to an appropriate ratio, it is purified using a known separation and purification method such as adsorption chromatography using a normal phase filter, silica gel, etc., to obtain a sugar amino acid ester. The purity of the sugar amino acid ester thus obtained can be further increased by repeating the same operation or by combining other purification methods.

(Example) Examples are shown below.

Example 1 The bacterial species is Rhodotorula lactosa (Rhodotorula lactosa).
ula Iactosa) using IFO 1423)
Ta. One platinum loopful of the strain was inoculated into a medium food with the following composition in a 500 d Erlenmeyer flask with a flange, and heated at 27°C.
The cells were cultured with shaking for 3 days and used as seed culture.

Medium composition: Yeast extract 3g/! , malt extract 3g/L peptone 5g/2, glucose 10g/β 10 Inl of seed culture was brought into contact with a medium of the same composition in a 52-volume Erlenmeyer flask, and cultured with shaking at 27°C for 3 days. This culture solution was heated at 18.00 O r. p. m. The cells were centrifuged for 20 minutes, the supernatant liquid was discarded, and the cells were washed twice with sterile water to obtain stationary bacterial cells.

Add 2 g of quiescent bacterial cells to 8 ml of the substrate solution shown below,
The reaction was carried out at 30°C for 48 hours.

Substrate solution: sugar (glucose, fructose, sorbose, sucrose, sorbitol or maltitol) 8.8 g. ．． L-alanine methyl ester hydrochloride 2 g, 0.2 M citric acid buffer (pH 5.4) 5 In 1 The obtained reaction solution was diluted 2 times, and filter paper for chromatography manufactured by Advantech Toyo Co., Ltd. was used. 50 (40x40cm)
When spotted, developed with butanol:acetic acid:water (2:l:1), and colored with ninhydrin, L-
Compared to the Rf value of alanine methyl ester of 0.83 and the Rf value of L-alanine of 0.57, when each receptor was used, new spots were observed to appear at the Rf values shown in Table 1 below. Ta.

Table 1 Also, when ester coloring was performed as shown below,
Each spot showed a positive reaction.

Ester coloring: Solution A: 1,44N hydroxylamine hydrochloride Methanol solution B: 3.5N sodium hydroxide Methanol solution C: 0.64N ferric chloride Methanol solutions A and B
Mix the solutions at a ratio of 1:1 and remove the precipitate to obtain Solution D. Mix liquid C and concentrated hydrochloric acid at a ratio of 4:1 to obtain liquid E. After spraying Solution D, heat at 80°C for 10 minutes and spray Solution E. At this time, the ester becomes purple and is confirmed.

Furthermore, sugar amino acid esters corresponding to the sugar compounds of each receptor were prepared by cutting out from the relevant portion of the chromatogram. The amounts shown in Table 2 were obtained for each sugar amino acid ester.

Table 2 The obtained sugar amino acid esters were subjected to alkaline hydrolysis by the following method.

Alkaline hydrolysis: Dissolve in commercially available ammonia water to a concentration of 1% and reflux for 30 minutes.

The obtained hydrolyzate! After condensation to dryness, it was dissolved in distilled water to a concentration of 1% and subjected to G chromatography in the same manner as shown above. The presence of L-alanine was detected by ninhydrin coloring, and the reducing sugar was detected by silver nitrate coloring. Liquid chromatography under the following conditions revealed the presence of Tang alcohol. In addition, for the sample before hydrolysis,
These were not included.

Liquid chromatography column: Tosoh Amide 80 4.6mmX
25cm Solvent: Acetonitrile/Water - 80/20 Flow rate: 1. 0/min Detector: RI In addition, 1% aqueous solutions of each compound were prepared and the sweetness of each was examined. As a result, the results shown in Table 3 below were obtained.

Table 3 Example 2 The bacterial species is Rhodotorula lactosa.
rula lactosa) IFO 1423 was used. One platinum loopful of the strain was inoculated into 100 d of a medium having the following composition in a 500 volume Erlenmeyer flask with a flange, and cultured with shaking at 27°C for 3 days to prepare a seed culture.

Medium composition: yeast extract 3 g/l, malt extract 3 g/1
．． , peptone 5 g/j2, glucose Log/1 10 d of the seed culture was inoculated into 5 × 1 liter medium of the same composition in a 5°C Erlenmeyer flask, and cultured with shaking at 27°C for 3 days. This culture solution was heated at 18.00 O r. p. m. The cells were centrifuged for 20 minutes, the supernatant liquid was discarded, and the cells were washed twice with sterile water to obtain stationary bacterial cells.

200g of stationary bacterial cells in Otake Seisakusho's French Brake 7! ．．
Cells were disrupted using Type 5501-MF at 1500 kg/cj and 4°C. The obtained cell disruption solution was heated to 18.00 ml.
Or. p. m. Centrifuge for 20 minutes at 2011d! using a Sartorius collodion bag. The enzyme solution was concentrated to obtain a crude enzyme solution.

Add the crude enzyme solution 2 to the substrate solution 8 shown below and mix for 30 minutes.
The reaction was carried out at °C for 48 hours.

Substrate? Volume: tM (gnorecose, fructose, sorbose, sucrose, sorbitol or maltitol) 8.8 g, L-alanine methyl ester hydrochloride 2 g, 0.21 citrate buffer (pH 5.4) 5 In1
The obtained reaction solution was diluted 2 times, and filter paper for chromatography manufactured by Advantenok Toyo Co., Ltd. was used. 50 (40X40cm
) and putanol:acetic acid:water (2:1:1).
When developed and colored with ninhydrin, L
- In contrast to the Rf value of 0.83 for alanine methyl ester and the Rf value of 0.57 for L-alanine, when each receptor was used, the appearance of new spots was observed at the same Rf values as in Table 1. . Furthermore, when ester coloring was carried out in the same manner as in Example 1, each spot showed a positive reaction.

Furthermore, sugar amino acid esters corresponding to the sugar compounds of each receptor were prepared by cutting out from the relevant portion of the chromatogram. The amounts shown in Table 4 were obtained for each sugar amino acid ester.

Table 4 Each of the obtained components was subjected to alkaline hydrolysis in the same manner as in Example 1. The obtained hydrolyzate was concentrated to dryness, then dissolved in distilled water to a concentration of 1%, and chromatography was performed in the same manner as shown above. Liquid chromatography under the same conditions as in Example 1 revealed the presence of reducing sugars and sugar alcohols.

Moreover, these were not contained in the sample before hydrolysis.

In addition, 1% aqueous solutions of each compound were prepared and the sweetness of each was examined. As a result, the results shown in Table 5 below were obtained.

Table 5 Example 3 The bacterial species used in Example 1 was Neurospora sitophila.
) Example 1 was carried out in the same manner as in Example 1, except that IFO 6070 was used, and the same results were obtained.

Example 4 The same procedure as in Example 1 was carried out except that the amino acid ester compound used in the substrate solution in Example 1 was changed to L-valine methyl ester hydrochloride or L-aspartic acid methyl ester hydrochloride. An ester compound of amino acid and sugar was obtained.

(Effects of the Invention) The present invention provides a method for producing an ester compound of sugar and amino acid using a microorganism or an enzyme. The ester compounds of sugar and amino acids obtained in this way are less likely to be contaminated with substances harmful to the human body than those obtained by organic chemical synthesis, and are suitable for use as foods. Furthermore, by using this method, the reaction can proceed under milder conditions than organic chemical synthesis methods, and equipment costs can be reduced.

Claims (1)

[Claims] 1. A method for producing a sugar amino acid ester, which comprises causing a microorganism or an enzyme capable of carrying out a transfer reaction of an ester compound to act on a mixture of an amino acid ester compound and a sugar or sugar alcohol.