JP2683048B2 - Plant-derived 2-oxoaldehyde reductase - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention metabolizes 3-deoxyglucosone, which is an intermediate product of Maillard reaction by protein and sugar, so-called browning reaction, and further is a cell metabolite. However, the present invention relates to a plant-derived 2-osisoaldehyde reductase that inactivates methylglyoxal, which is said to exhibit strong cytotoxicity.

The browning protein is less susceptible to protease action, has a lower digestion and absorption rate, and is inferior in nutritional value. In addition, since proteins such as hemoglobin, crystallin, and collagen in the living body are polymerized, insolubilized, fluorescently generated, and colored by the Maillard reaction, it is inferred that they are related to aging and adult diseases.

On the other hand, methylglyoxal is known to have a mutagenic effect in addition to inhibiting synthesis of proteins, RNA and DNA and modifying DNA, and the presence of methylglyoxal is extremely disadvantageous for cell growth.

The plant-derived 2-oxoaldehyde reductase of the present invention is a novel enzyme that inactivates these highly reactive 2-oxoaldehydes, suppresses the progress of browning reaction in foods and living bodies, and eliminates cytotoxic effects.

[Conventional technology]

The Maillard reaction between protein and sugar, the so-called browning reaction,
The N-terminal amino group of the protein and the ε-amino group of the lysine residue are modified into a form that undergoes Amadori rearrangement via a Schiff base, and then this is decomposed to give 2-oxoaldehyde,
In particular, 3-deoxyglucosone is generated, which is a series of reactions that secondarily reacts with lysine, arginine, and tryptophan residues to form a crosslink, which causes polymerization, fluorescence generation, and browning.

In foods, not only the essential amino acids lysine and tryptophan are lost due to the Maillase reaction, but they are less susceptible to the action of protease due to polymerization, and the digestion and absorption rate is reduced.

On the other hand, in the living body, this Maillard reaction proceeds, and proteins with slow turnover, such as collagen, turn brown due to the Maillase reaction, undergo hardening when polymerized, are associated with delayed metabolism and aging or adult diseases, or turnover. The relationship between the opacification of crystallin in the eye lens, which is a special protein that does not occur, and cataract due to diabetes has been discussed. In addition, proteins modified and polymerized in vivo are less likely to be metabolized, and when deposited on the blood wall, they may cause arteriosclerosis. Since the Maillard reaction product generated in the living body is considered to be associated with the aging and the development of adult diseases, it is an inconvenient one for the living body and needs to be eliminated.

As an enzyme that metabolizes 3-deoxyglucosone, which is an important intermediate of this Maillard reaction, it has a carbonyl group and an aldehyde group in the molecule like 3-deoxyglucosone, and is a highly reactive cell growth inhibitor. 2-Oxoaldehyde dehydrogenase and methylglyoxal reductase which metabolize methylglyoxal are already known.

There are several enzyme systems involved in the degradation of methylglyoxal [protein nucleic acid enzyme 31 (11) 1010-1021],
A pathway for conversion to lactate by glyoxalase consisting of coenzyme glutathione, a pathway for conversion to lactate via lactaldehyde by methylglyoxal reductase, and a pathway for direct conversion to pyruvate by methylglyoxal dehydrogenase have been clarified. There is. These enzymes are
It is found in many microorganisms and animal brain, heart, liver, spleen, kidney, etc. The glyoxalase system has also been found in plants. However, an enzyme that converts to acetol in response to methylglucoxal has not yet been reported.

[Problems to be solved by the invention]

As described above, 3-deoxyglucosone is a major intermediate in the Maillard reaction, and methylglyoxal is known as a cell growth inhibitor.

The present invention acts on these 2-oxoaldehydes,
The object is to provide a plant-derived 2-oxoaldehyde reductase, which is a novel enzyme that has not been reported at all until now by reducing an aldehyde group and inactivating these 2-oxoaldehydes.

[Means for solving the problem]

The present inventors have never searched for 2-
As a result of earnest research for a plant-derived 2-oxoaldehyde reductase that inactivates oxoaldehyde, as a result, a crude extract obtained by adding a buffer solution to fresh edible plants and grinding the target 2- The present invention was completed by finding the existence of oxoaldehyde reductase. That is,
The present invention is a plant-derived 2-oxoaldehyde reductase that inactivates 3-deoxyglucosone and further inactivates methylglyoxal.

The 3-deoxyglucosone is a major intermediate in the protein-sugar Maillard reaction, and methylglyoxal is a compound involved in cell metabolism and proliferation.

 Hereinafter, the present invention will be described in detail.

The 2-oxoaldehyde reductase of the present invention (hereinafter,
The enzyme of the present invention) means, for example, roots, stems, leaves or flowers of plants such as turnips, celery, lettuce, honeywort, bok choy, green onions, spinach, chrysanthemum, chive, parsley, broccoli, and edible chrysanthemums, for example, as follows. Can be obtained.

To the roots, stems, leaves, or flowers of the above-mentioned plants, an appropriate amount (double amount) of a buffer solution, for example, a phosphate buffer solution containing β-mercaptoethanol is added, ground or homogenized, and filtered by an appropriate means such as filtration, The liquid fraction is separated by centrifugation or the like, or the liquid fraction is dialyzed against the same buffer to obtain a crude extract.

The crude extract is subjected to ammonium sulfate saturation, dialysis, column treatment, for example, DEAE-cellulose, Sephadex G-.
A purified enzyme showing a single band by SDS-polyacrylamide gel electrophoresis (PAGE) can be obtained by appropriately combining and treating 100, hydroxyapatite (Hydroxylapatite) and the like column treatment.

The physicochemical properties of the enzyme of the present invention are as follows.

Action It acts on 2-oxoaldehydes such as 3-deoxyglucosone and methylgluoxal to reduce aldehyde groups.

Substrate specificity It is active not only on 2-oxoaldehydes such as 3-deoxyglucosone, methylglyoxal and phenylglyoxal, but also on aldehydes such as glyceraldehyde, but not on ketones such as diacetyl and dihydroxyacetone. .

Optimum pH and stable pH Optimum pH 7-7.5 Stable pH 6-8 Inhibition, activation and stabilization Completely inhibited by 0.1 mM of PCMB (p-chloromercury-benzoic acid).

 Stabilized by SH reducing agent.

 Requires only NADPH as a coenzyme.

Molecular weight 67K by gel filtration method, 35K by SDS-polyacrylamide gel electrophoresis.

Purification method Purification is carried out by appropriately combining ammonium sulfate saturation, dialysis, column treatment such as DEAE-cellulose, Sephadex G-100 and hydroxyapatite. Specific examples of purification are as described in Example 2 below.

Optimal temperature 30 ℃ Deactivation condition by pH, temperature, etc. Stable for 10 minutes at pH 5 to 9, 5 minutes at pH 3 and 10 minutes at pH 10. (Relatively unstable) Titer determination method Incubate with 4 mM 3-deoxyglucosone and 0.3 mM NADPH in 100 mM phosphate buffer (pH 7.4) at 30 ° C, and measure OD at 340 nm.

As a result of analyzing the reduction product by the enzyme of the present invention,
Acetol from methylglyoxal is 3
-Deoxyglucosone produced 3-deoxyfructose. Since no enzyme that acts on 2-oxoaldehyde and reduces the aldehyde group has been reported so far, the enzyme of the present invention is recognized as a novel enzyme.

〔The invention's effect〕

The enzyme of the present invention is a Maillard reaction between protein and sugar,
It acts on 2-oxoaldehydes such as 3-deoxyglucosone, which is an intermediate organism of so-called browning reaction, and methylglyoxal, which is known as a cell growth inhibitor, to inactivate these 2-oxoaldehydes, and foods and in vivo. Because it helps to suppress the progress of browning reaction and eliminates cytotoxic effects,
It is a very useful enzyme.

〔Example〕

 Hereinafter, examples of the present invention will be described.

Example 1 Leaves (7 types) and flowers (2) of the plants described in Table 1 below.
20 mM phosphate buffer (pH 7.2) containing double amount of 10 mM β-mercaptoethanol (MCE) was added to 75 g of each of
After homogenizing for 3 minutes, centrifugation was performed at 12,000 G to obtain a supernatant, which was dialyzed against the same buffer as above (dialysis inner solution or outer solution) to obtain a crude enzyme extract (CE).

The metabolic activity of CE with respect to 3-deoxyglucosone (3-GD) and methylglycol oxal (MG) was measured in the presence of coenzymes NADPH, NADH, NADP and NAD in 100 mM phosphate buffer (p
Table 1 shows the results of measuring the amount of change in coenzyme when CE was allowed to act on each substrate at 30 ° C. in H7.4) for 5 to 10 minutes by the change in absorbance at 340 nm. The numbers in the table are plant tissues.
The activity amount of the enzyme present per 1 g is shown.

NA for 3-GD and MG in all CEs from Table 1
DPH- or NADH-dependent reductase activity is observed.

However, CE of all plants was NA against 3-GD and MG.
The DP- or NAD-dependent 2-oxoaldehyde dehydrogenase activity previously reported in the liver was not observed at all.

Example 2 30 ml of a fraction of 167 ml of CE of parsley obtained from leaves of parsley as described in Example 1 saturated with 45 to 70% ammonium sulfate and precipitated.
Was dialyzed against 20 mM phosphate buffer containing 10 mM β-mercaptoethanol, and the dialyzed solution was equilibrated with the above-mentioned buffer DE.
Applied to AE-cellulose column. After the non-adsorbed protein was eluted, oxygen was eluted by the salt concentration gradient method. The obtained active fraction was concentrated using an ultraperitoneal membrane, and then applied to a Sephadex G-100 column. Next, the active fraction eluted from this column was dialyzed against the above buffer solution, and the dialyzed solution was further concentrated. The concentrate was applied to a hydroxyapatite column, and the enzyme was eluted by a concentration gradient method of potassium phosphate. Thus, 0.5 mg of the purified enzyme of the present invention showing a single band on SDS-polyacrylamide gel electrophoresis (PAGE) was obtained.

When this enzyme was incubated with 10 mM phosphate buffer (pH 7.4) at 15 ° C and 15 mM substrate shown in Table 2 below and 0.3 mM NADPH at 30 ° C, the activity value of 3-deoxyglucosone was set to 100%. The relative activity value of each substrate of
It is as shown in Table 2.

Claims (1)

(57) [Claims] 1. A plant-derived substance having the following physicochemical properties:
Oxoaldehyde reductase. Action: It acts on 2-oxoaldehyde to reduce the aldehyde group. Substrate specificity: Not only 2-oxoaldehyde but also glyceraldehyde, but not ketone. Optimum pH and stable pH: The optimum pH is 7-7.5 and the stable pH is 6-8. Inhibition, activation and stabilization: Completely inhibited with 0.1 mM p-chloromercury-benzoic acid. Stabilized by SH reducing agent. Requires only NADPH as a coenzyme. Molecular weight: 67K by gel filtration, SDS-polyacrylamide gel electrophoresis
It is 35K. Purification method: Purify by appropriately combining ammonium sulfate saturation, dialysis and column treatment. Optimum temperature of action: 30 ° C. Conditions for deactivation due to pH, temperature, etc .: Stable for 10 minutes at pH 5-9. It is inactivated in 5 minutes at pH 3 and 10 minutes at pH 10. Method for measuring titer: Incubate with 4 mM 3-deoxyglucosone and 0.3 mM NADPH in 100 mM phosphate buffer (pH 7.4) at 30 ° C. and measure OD at 340 nM.