JPH044874A - Fructosylamino acid hydrolase, production and utilization thereof - Google Patents

Abstract

NEW MATERIAL:An enzyme, capable of acting on fructosyllysine and/or fructosylalanine and having the following enzyme chemical properties. Action; hydrolyzing fructoamino acid and producing saccharides and amino acids. Optimum pH and stable pH range; optimum pH is pH 7.5 and stable pH is pH 6-8. Action temperature range; 20-70 deg.C. Optimum temperature range; about 30 deg.C. USE:Capable of preventing foods from browning and lysine from being lost and effective in treating the foods. PREPARATION:A microorganism (preferably FERM P-11408), belonging to the genus Penicillium and capable of producing fructosylamino acid hydrolase is cultured in a culture medium and the resultant enzyme is collected from the obtained culture.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to an enzyme, and more specifically, a novel fructosyl amino acid degrading enzyme that acts on fructosyl lysine or fructosyl alanine, and a method for producing the same. ,
Regarding its use and producing bacteria.

[Conventional technology]

The food browning reaction (Maillard reaction) is a complex non-enzymatic reaction that occurs between reducing sugars and proteins and amino acids in foods. The Maillard reaction is known to cause browning and changes in aroma components in foods, especially with lysine, an essential amino acid that has two amino groups in one molecule, resulting in loss of lysine. In the Maillard reaction, a carbonyl group of a saccharide such as glucose and an amino group of an amino acid reversibly generate a Schiff base, which then irreversibly undergoes amatri rearrangement to generate a ketoamine called an amatri compound.

Therefore, in order to prevent the Maillard reaction in food,
There have been attempts to utilize enzymatic decomposition of Maillard reaction products. Fructosyl amino acid oxidase produced by Corynebacterium (Japanese Unexamined Patent Publication No. 61-268178) is known as an enzyme that decomposes the fructosyl amino acid produced in the initial stage of the Maillard reaction. It has poor reactivity with compounds and cannot be put to practical use. Additionally, there is a statement that fructosyl amino acid oxidase produced by Aspergillus acts on amatoli compounds bound to the ε-amino group of lysine (Journal of the Japanese Society of Agricultural Chemistry, Vol. 64, No. 3, 1990 Conference Abstracts, p. 579). )
, only has degrading activity against ε-fructosylaminocapric acid, and no degrading activity against fructosyl lysine has been observed.

The present invention relates to a fructosyl amino acid degrading enzyme derived from a Penicillium genus bacterium. It is also unknown that bacteria produce such enzymes, and the present invention is a novel invention that was previously unknown.

[Problem that the invention is trying to solve]

The object of the present invention is to search for a fructosyl amino acid degrading enzyme that acts on fructosyl lysine and/or fructosyl alanine, to produce the enzyme, and to use the enzyme to prevent food browning and lysine loss. be.

[Means to solve the problem]

In order to search for bacteria that produce an enzyme that degrades fructosylurisine, we screened for bacteria that grow in a medium with fructosylurisine as the sole nitrogen source.

As a result, microorganisms belonging to the genus Penicillium were discovered in the soil in front of the Faculty of Agriculture, Tokyo University of Agriculture and Technology, Saiwai-cho, Fuchu City, Tokyo. This bacterial strain was named Penicillium sp. 5335 and was deposited at the Institute of Fine Technology. (Feikoken Bibori No. 11408) The reason why this strain was identified as Penicillium and the enzymatic chemical properties of the new enzyme collected from this strain will be described below.

(1) Identification of producing bacteria of the present invention "Isolation, cultivation and identification of molds" (translated by Yoshitoshi Uda and Tetsuo Muroi, Ishiyaku Publishing Co., Ltd. 1983), "Food Fungi Handbook" (Yotoshi Uda and Matsuda) As a result of examining the mycological properties of this strain, it was identified as a Penicillium genus based on the following grounds.

1) Does not form ascocyst generation.

2) Venicilla formation was observed.

3) The conidial type is phialoid and does not form ring patterns.

4) Conidia are formed from the phialide tips and are chained.

5) Fearide is plate-shaped and does not have a tapered tip.

(2) Production and separation and purification of the enzyme of the present invention The enzyme of the present invention can be obtained by culturing Penicillium sp. 5335 bacteria in a medium;
It can be obtained by separating it from the culture. For culturing this strain, conventional methods for culturing microorganisms, especially filamentous fungi, are appropriately used, and culture is carried out under aerobic conditions in the range of 25 to 37°C, such as shaking culture or aerated agitation culture, using a commonly used liquid medium. Bye.

As the medium used in the present invention, a synthetic medium or a natural medium containing a carbon source, a nitrogen source, an inorganic salt, and other nutritional components can be used as appropriate. As a carbon source, glucose,
Fructose, glycerin, starch, sucrose, etc. are used. As the nitrogen source, peptone, casein decomposition product, defatted soybean, yeast extract, malt extract, broth, cornstarch liquor, etc. can be used. As the inorganic salt, salts such as chlorides, phosphates, and sulfates of potassium, sodium, calcium, magnesium, manganese, cobalt, iron, etc. are used as appropriate.

Particularly in the present invention, when fructosyl amino acids are added to the medium, the target enzyme is efficiently produced and accumulated. As the fructosyl amino acid, fructosyl lysine, fructosyl alanine, hydroxyacetonitrile glycine, and other fructosyl amino acids are appropriately used. Fructosyl amino acids can be freely used as commercially available products, but they do not necessarily have to be pure products. Crude products such as those from which the solvent has been removed or those obtained by dissolving and suspending the same in water can also be freely used.

The enzyme is extracted from the culture. When extracting from bacterial cells, the bacterial cells are extracted directly from the bacterial cells using water and/or an organic solvent, or the bacterial cells are crushed using enzymes, ultrasound, mechanical crushing, etc., and then extracted with an extraction solvent. When extracting from a culture solution, the culture solution is concentrated under reduced pressure and subjected to ultrafiltration treatment as required, and then extracted with an extraction solvent. In this way, an enzyme-containing solution can be obtained.

The enzyme-containing solution can be used as it is or after being concentrated as a crude enzyme, but if desired,
Purification can be carried out using conventional methods for isolation and purification of enzymes, either alone or in combination.

For example, purified enzymes can be obtained by using known purification systems such as various chromatography, dialysis, and recrystallization.

(3) Enzyme chemical properties of the enzyme of the present invention The cells were frozen at -80°C, mixed with the same weight of quartz sand in a mortar, and ground to crush the cells.
Add 7mM phosphate buffer (containing 0.1mM ethylenediaminetetraacetic acid, 05mM phenylmethanesulfonyl fluoride), extract, and dilute the extract with the same buffer for 2 hours.
Dialyzed for 4 hours, centrifuged at 12,000G for 30 minutes at 4°C,
The protein content of the supernatant was 1. Adjust so that it becomes B,
An enzyme having the following characteristics was produced as a crude enzyme solution.

1) Action This enzyme decomposes fructosyl amino acids to produce sugars and amino acids.

2) Substrate specificity Degrading fructosyl lysine and/or fructosyl alanine.

3) Optimum pH and stable P)l range It acts between pH 5 and 8, with an optimum pH of around 87.5.

(The activity is 0.06 using sifructosyl lysine as a substrate.
The reaction was carried out at 30°C for 10 minutes in a 7M phosphate buffer.

Fructosyl lysine was prepared by the method of Huinot et al. J, He1v, Chim, Acta52.1
488-1495) It is also stable in the pH range of 6 to 8. (Activity is measured at pH 7 after being left at 30℃ for 10 minutes at each pH.)
The degrading activity of sifructosyllysine was measured. ) Figure 1 shows the optimum pH and pH stability.

4) Working temperature range and optimum temperature range The working temperature range is 20°C to 70°C, and the optimum temperature range is around 30°C. (The activity was determined by reacting for 10 minutes in a 0.067M, pH 7.5 phosphate buffer using sifructosyllysine as a substrate.) The optimum temperature and temperature stability are shown in FIG.

5) Thermostability After being left in a phosphate buffer at pH 7.0 at 40°C for 10 minutes, 45% activity remains using sifructosyllysine as a substrate.

(4) Prevention of browning of foods using the present enzyme The enzyme according to the present invention has a strong effect and is very stable, so simply adding it to foods can prevent browning of foods.

This enzyme can widely prevent browning of various foods, including miso, soy sauce, mirin, dairy products, fruit juice, bread, biscuits,
It can be widely used as an additive to confectionery and other food and drink products. In this case, since one enzyme has a strong effect, browning can be sufficiently prevented by using a small amount, but since there is no off-taste and there is no problem in terms of safety, there is no problem in using a large amount.

As the present enzyme, not only a purified enzyme can be used, but also a crude enzyme solution can be sufficiently used, and the present strain itself can also be used.

〔Example〕

The present invention will be explained in more detail with reference to Examples.

Example 1 (a) Preparation of substrate - A methanol solution containing 1.0% lysine and 6.5% glucose was refluxed for 4 hours, and 0.5% of activated carbon was added.
Add 1% and stir for 1 hour. After evaporating methanol, it is dissolved in 100 mΩ of water to obtain a crude fructosyl lysine solution.

(b) Cultured bacterial strain (Penicjllium sp, 5335 FE
RM P-1]408), glucose 1%, dipotassium phosphate 0.1%, potassium chloride 0.05%, ferrous sulfate 7
Cultured for 1 week at 30°C on a slanted agar medium (10 mρ) consisting of 0.001% hydrate, 0.1% magnesium sulfate, 6.7% crude fructosyl lysine solution, and 1.5% agar to produce spores. let Create a spore suspension by suspending the spores in 1.0 mg of sterile water. Ten 500 mQ inverted flasks each with 100 mQ of medium (1% glucose, 0 dipotassium phosphate)
．． 1%, potassium chloride 0.05%, ferrous sulfate heptahydrate 0.001%, magnesium sulfate 0.1%, crude fructosyl lysine solution 16.7%), and spore suspension 2IIip were added. Culture with shaking at ℃ for 5 days. . The culture solution was filtered by suction to obtain 24 g of wet bacterial cells.

(c) Preparation of crude enzyme solution The bacterial cells are frozen at -80°C, mixed with the same weight of quartz sand in a mortar, and ground to crush the bacterial cells. A 67+nM phosphate buffer (containing 0.1mM ethylenediaminetetraacetic acid and 0.05mM phenylmethanesulfonyl fluoride) is added to the paste obtained by crushing the bacterial cells for extraction. The extract was dialyzed for 24 hours with the same buffer and
Centrifuge for 0 min at 4°C, and collect the supernatant at a concentration of 1 mg of protein.
This was adjusted to give a crude enzyme solution.

(d) Confirmation of decomposition activity of fructosylurisine Crude enzyme solution and 0.2 mg of crude enzyme solution heat-treated in boiling water for 0 minutes
, 0.1 mg of 50 mM difructosyllysine solution or Nt-fructosyllysine solution, 0.2 mg of 0.6
Mix with 7M, pH 7.0 phosphate buffer and incubate at 30°C for 1 hour.
The reaction was allowed to proceed for 5 hours. The reaction product was purified using high performance liquid chromatography at 80 mM as a mobile phase, pH 4,
36 ammonium acetate buffer at a flow rate of 1 mg per minute, 5CX-1151-N4.6 as analytical column.
X 150+nm (trade name, manufactured by Senshu Kagaku Co., Ltd.), and the detector was 5hodex RI 5E-31 (trade name, manufactured by Showa Denko Co., Ltd.) for analysis. the result,
In the crude enzyme solution, fructosyl lysine was decomposed and production of lysine or monofructosyl lysine was observed;
No decomposition of fructosyl lysine was observed in the heat-treated crude enzyme solution.

FIG. 3 shows a diagram of the high performance liquid chromatography and will explain it. Figure 3A shows a mixture of Nt-fructosyllysine and a crude enzyme solution reacted together, Figure 3B shows a mixture of Nt-fructosyllysine and a heat-treated crude enzyme solution reacted together, Figure 3 C is only the crude enzyme solution, the third diagram is the mixture of sifructosylurisine and the crude enzyme solution and reacted, the third diagram E is the mixture of sifructosylurisine and the heated crude enzyme solution, and the reaction is carried out. FIG. 3F shows only the heat-treated crude enzyme solution analyzed by high performance liquid chromatography.

From FIG. 3A, it is confirmed that this enzyme decomposes Nt-fructosyl lysine and lysine is produced. The third diagram confirms that this enzyme decomposes cyfructosyllysine to produce lysine, that is, this enzyme also decomposes fructosyllysine in which fructose is bound to either the α or E amino group of lysine. . Furthermore, from the results shown in Figure 3 B and D, the heat-treated crude enzyme lost its activity;
This suggests that this reaction is enzymatic.

(f) Confirmation of reduction in browning The color of the test solution immediately after mixing Nt-fructosyllysine and the crude enzyme is light brown, and the absorbance at 420 nm was measured by a spectrophotometer (manufactured by Hitachi, Ltd., model 320). When measured, it was 0.213, but the color of the test solution after 15 hours at 30°C was clear and colorless, and the absorbance at 420 nm was 0.054, indicating that this enzyme is useful for removing browning substances. I understand.

Example 2 According to the culture method of Example 1, 200 g of wet bacterial cells were obtained.

After freezing at -80℃, crush it by mixing it with the same weight of quartz sand.
+nM, pH 7.0 phosphate buffer, add 0.2n+g of protamine sulfate per 1mg of protein, centrifuge, and precipitate the supernatant with saturated ammonium sulfate.
The precipitate was dissolved in the above phosphate buffer, dialyzed against the same buffer,
A crude enzyme solution of 160 mQ was obtained.

It was prepared according to the method for preparing fructosylurisine.

Crude enzyme solution 0, 0.2 mg of 100 mM solution of fructosyl-α-alanine or fructosyl-β-alanine in 25+nQ, 60 mM, pH 7,0 phosphate buffer 0.
1 mg were mixed and reacted at 30°C for 24 hours. After the reaction, each sample was diluted 100 times with 0.02N hydrochloric acid and analyzed using an amino acid analyzer (Hitachi High Speed Amino Acid Analyzer Model 835-50, manufactured by Hitachi Seisakusho). Under these conditions, fructosyl-α-alanine and fructosyl-β-alanine completely disappeared and were not observed, and production of α-alanine or β-alanine, respectively, was observed.

Example 3 10 mQ of the crude enzyme solution prepared in Example 1(c) was added to 500 g of commercially available koji miso (raw materials: soybean, rice, salt, alcohol, natural seasoning, chemical seasoning) and mixed well.

As a control, 500 g of the same miso was added with 67+nM phosphate buffer (0,]mM ethylenediaminetetraacetic acid, 0.15mM
Contains phenylmethanesulfonyl fluoride. )10m
1 and mix well, put both in a sealed container, 30
When the samples were left in a constant temperature bath at ℃ for one week, no browning was observed in the sample to which the enzyme was added, but browning was observed in the control sample without the enzyme. In addition, it was observed that the product had no off-taste or odor due to the addition of the crude enzyme solution.

〔Effect of the invention〕

The present invention relates to a novel fructosyl amino acid degrading enzyme that acts on fructosyl lysine or fructosyl alanine, and contributes to the practical application of an enzyme-based method for preventing food browning.

[Brief explanation of drawings]

FIG. 1 is a graph illustrating the optimum pH and stable pH range of the enzyme according to the present invention, and FIG. 2 is a graph illustrating the optimum temperature and temperature stability of the enzyme according to the present invention. FIG. 3 is a confirmation diagram of fructosyl lysine degrading activity, and shows A-
F is a chromatogram in the following cases: A: NE-Fructosylurisine and a crude enzyme solution were mixed and reacted. B: NE-Fructosylurisine and a heat-treated crude enzyme solution were mixed and reacted. C: Crude enzyme solution only D: A mixture of difructosyllisine and a crude enzyme solution and the reaction E: A mixture of a heat-treated crude enzyme solution and a mixture of difructosylurisine and a reaction F: A heat-treated crude enzyme Liquid only 2nd 1st 1st L'do I (-・-) to "Ia Temperature (-△-) Temperature stability 岑Peak heightPeak heightPeak heightPeak heightPeak height peak height

Claims (5)

[Claims] (1) A fructosyl amino acid degrading enzyme that acts on fructosyl lysine and/or fructosyl alanine. (2) A method for producing a fructosyl amino acid degrading enzyme, which comprises culturing a fructosyl amino acid degrading enzyme-producing bacterium belonging to the genus Penicillium in a medium, and collecting the fructosyl amino acid degrading enzyme from the culture. (3) The fructosyl amino acid degrading enzyme-producing bacterium is Penicillium sp., which belongs to the genus Penicillium. 5335. A method for producing a fructosyl amino acid degrading enzyme. (4) Penicillium sp. 5335. (5) A method for preventing browning of foods, which comprises treating foods with the fructosyl amino acid degrading enzyme according to claim 1.