JPH0670763A - New choline-decomposing enzyme - Google Patents

Abstract

PURPOSE:To provide a new choline-decomposing enzyme capable of being utilized for the measurement of the activity of choline esterase in vital body fluids in a clinical examination field. CONSTITUTION:The new choline-decomposing enzyme requires NAD(P) as a coenzyme, is activated with divalent iron ions, has the optimal pH at the pH of approximately 6.5, and has an action to decompose the choline into trimethylamine and glycolaldehyde under aerobic conditions.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel choline-degrading enzyme. More specifically, it relates to a novel choline-degrading enzyme derived from Candida tropicalis, and this enzyme can be used for measuring cholinesterase activity in biological fluids in the field of clinical examination.

[0002]

2. Description of the Related Art Studies on the degradation and metabolism of choline by microorganisms have been studied in detail in bacteria and filamentous fungi. That is, it has been revealed that choline is decomposed into glycine via betaine. Dehydrogenase and oxidase are known as enzymes involved in this metabolism.

That is, as an enzyme acting on choline, for example, choline dehydrogenase, Athrobacte genus of the genus Agrobacterium
r), enzymes derived from the genus Pseudomonas (Japanese Patent Publication No. 60-31470), and choline oxidase include genus Arthrobacter (Japanese Patent Publication 60-4716), genus Aspergillus (Japanese Patent Publication 60-10713). , Genus Corynebacterium (Japanese Patent Publication No. 60-10714, Japanese Patent Publication No. 53-81689), genus Penicillium (Japanese Patent Publication 61-20267), genus Streptomyces (Japanese Patent Publication No. 1-27714) , Alcaligenes (JP-A-54-17182), Brevibacte
The genus rium (JP-A-53-66492, JP-A-54-129187), the genus Sporobolomyces (JP-A-2-227072), and choline kinase are known.

Further, various methods for measuring cholinesterase activity in biological fluids using these enzymes have been reported. That is, by allowing cholinesterase in biological fluid to act on various synthetic substrates, each of the above enzymes is allowed to act on the produced choline, and choline is quantified to measure cholinesterase activity.

That is, as a method for measuring the activity of cholinesterase, a method using choline oxidase (JP-A-54-
136895), a method using choline kinase (JP-A-52-99)
888) and p-hydroxybenzoic acid hydroxylase [J. Biochemistry (J. Bio
chem.), 94, 11 (1983)].

In the field of clinical examination, a decrease in cholinesterase activity is observed in liver cirrhosis, chronic hepatitis, liver tumor, malignant obstructive jaundice, gastric cancer, sepsis, uterine myoma, liver cancer, organophosphorus poisoning and the like.

Generally, it is said that the reduction of liver function is directly expressed. Increased activity is also found in nephrotic syndrome. Thus, the relationship between each disease, particularly liver disease, and the serum cholinesterase activity value is very close and is one of the important items as a clinical test.

[0008]

However, the method using choline oxidase is susceptible to the interference of choline caused by the decomposition of phospholipids coexisting in serum, and
The method using p-hydroxybenzoic acid hydroxylase or choline kinase is not easily affected by interfering substances in serum,
There have been problems such as the need for complicated operations such as avoidance of side reactions and addition of a coupled enzyme system.

Therefore, a new measuring method has been desired, and for that reason, the development of a new degrading enzyme acting on choline has been desired.

[0010]

The present inventors have found that a strain belonging to the genus Candida decomposes choline into trimethylamine and glycolaldehyde under aerobic conditions. That is, by cultivating Candida tropicalis in a medium containing choline as a nitrogen source, choline is decomposed into trimethylamine and glycolaldehyde under aerobic conditions, and further oxidative demethylation reaction causes dimethylamine and methyl. Decomposes to ammonia via amine.

An enzyme that catalyzes the above reaction has not been known until now. The present inventors investigated this enzyme, including its assay method, and clarified its properties.

The main properties of the novel choline-degrading enzyme of the present invention are as described below. Action: It acts on choline under aerobic conditions and has the action of catalyzing the reaction described in the following reaction formula. (CH ₃ ) NCH ₂ CH ₂ OH + NAD (P) H → [choline] (CH ₃ ) ₃ NH + CHOCH ₂ OH + H ₂ O + NAD (P) [trimethylamine] [glycol aldehyde] coenzyme: NAD ( P) Request H. It is particularly specific to NADH. Activation: Activated with divalent iron ions. Optimum pH: around 6.5

That is, the present invention uses NAD as a coenzyme.
A novel choline-degrading enzyme that requires (P) H and decomposes choline into trimethylamine and ethylene glycol. More specifically, it requires NAD (P) H as a coenzyme and is activated by a divalent iron ion. Provided is a novel choline-degrading enzyme having an optimum pH of around 6.5 and having an action of decomposing choline into trimethylamine and ethylene glycol under aerobic conditions.

As the strain producing the choline-degrading enzyme of the present invention, for example, Candida tropicalis IFO-0
587.

In order to culture the choline-degrading enzyme producing strain of the present invention, a medium containing an appropriate carbon source and choline as a nitrogen source can be used. Various medium components can be added as needed.

The present invention will be described in detail below with reference to examples and test examples. The present invention is not limited to these.

[0017]

【Example】

Example 1 Candida tropicalis IFO-058
Cultivation of 7 and analysis of culture product Using a medium having the following composition, shaking culture was performed at 30 ° C. for 18 hours. Glucose 30 g Choline chloride 15 g K ₂ HPO ₄ 1 g KH ₂ PO ₄ 1 g NaCl 1 g MgSO ₄ .7H ₂ O 0.5 g Various minerals and vitamins ----------------------- −−−−−−−−−− pH 6.0 (1.0M HCl) 1000 ml

50 μl of 10 mM isopropylamine was added as an internal standard substance to 1 ml of the obtained culture solution, and the mixture was extracted with 1 ml of benzene and 1 ml of 65% potassium hydroxide. Amipak 141 was used as a packing material for this extract in a column (3 mm x 3 m).
(80/100 mesh), column temperature 80 ° C. constant temperature, carrier gas nitrogen (30 ml / min) were used for analysis by gas chromatography. The result is shown in FIG.

Example 2 Measurement of dehydrogenase activity, oxidase activity and hydroxylase activity of choline-degrading enzyme

By the measurement system as shown in Table 1 below,
The absorbance at 340 nm was measured to measure each activity.

[0021]

[Table 1]

In the table, "+" indicates addition as a reagent composition component. As the crude enzyme solution, the cells obtained in Example 1 were MES containing 5 mM dithiothreitol.
The solution was disrupted in a buffer solution, and the supernatant after centrifugation was subjected to gel filtration using Sephadex G-25 and the solution was concentrated by centrifugation.

As a result, dehydrogenase activity and oxidase activity were not recognized, and hydroxylase activity was recognized although it was low activity.

Example 3 Effect of buffer solution on choline-degrading activity As a result of using various buffer solutions shown in Table 2 in the system for measuring hydroxylase activity of Example 2, in the case of phosphate buffer solution and MES buffer solution Showed high activity.

[0025]

[Table 2]

Further, as a result of measuring the optimum pH using the MES buffer solution and the HEPES buffer solution, as shown in FIG.
Was about 6.5. When a phosphate buffer solution was used, the blank value increased during measurement by gas chromatography, so the MES buffer solution was used in the following studies.

Example 4 Effects of Various Additives The effects of glutathione, 2-ketoglutarate and divalent iron ions, and NADH and NADPH as coenzymes were investigated.
The results are shown in Table 3.

[0028]

[Table 3]

As a result, the activity was activated about 1.3 times by the divalent iron ion. Furthermore, this enzymatic reaction was considered to require NADH as a coenzyme.

Example 5 Examination of optimum concentration of iron ion The choline-degrading activity was measured by changing the iron ion concentration from 0 to 1.0 mM. As a result, as shown in FIG. 3, the activity was the highest at a low concentration of 0.1 to 0.2 mM, and the activity was about 3 times as high as that when the iron ion was not added.

From the above results, it was judged that the following conditions were optimum for the reagent composition for measuring choline-degrading activity. MES buffer solution (pH 6.5) 150 μmol Choline chloride 30 μmol NADH 9 μmol FeSO ₄ 0.3 μmol Enzyme solution 0.5 ml −−−−−−−−−−−−−−−−−−−−−−−−−−− 3.0 ml total reaction conditions: 30 ° C, 20 minutes shaking reaction (120 times / min)

Example 6 Effect of various stabilizers on stabilization of choline-degrading enzyme Choline-degrading activity was measured 12 hours after addition of various stabilizers at 5 ° C. The results are shown in Table 4.

[0033]

[Table 4]

As a result, glycerol, ethylene glycol and FAD were found to have a stabilizing effect.

Example 7 Confirmation of Reaction Product of Choline Degrading Enzyme After the reaction for measuring choline degrading enzyme, 10 mM of 3-
Methyl-2-benzothiazolone hydrazone (hereinafter MB
(TH) (0.1 ml), leave for 30 minutes, concentrate, and then 40 ℃
Dried in. 1 ml of purified water and 2 ml of chloroform were added, the chloroform layer was concentrated and dried at 40 ° C., and 0.1 ml of chloroform was added and dissolved. This solution was compared with the Rf value of the MBTH derivative of glycolaldehyde as a standard substance by thin layer chromatography. As a result, they showed the same Rf value.

[0036]

INDUSTRIAL APPLICABILITY The present invention provides a novel choline-degrading enzyme, which can be used for measuring cholinesterase activity in biological fluids in the field of clinical examination.

[Brief description of drawings]

1 shows a chart of gas chromatographic analysis of Candida tropicalis culture products.

FIG. 2 shows the optimum pH of choline-degrading activity of the present invention.

FIG. 3 shows the effect of iron ions on the choline-degrading enzyme of the present invention.

[Procedure amendment]

[Submission date] July 1, 1993

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0013

[Correction method] Change

[Correction content]

That is, the present invention uses NAD as a coenzyme.
Requires (P) H and converts choline to trimethylamine and gly
Novel choline degrading enzyme has an action of decomposing the call aldehydes, more particularly, as a coenzyme NAD (P) H
Is required and activated by divalent iron ions, and the optimum pH is 6.
Provided is a novel choline-degrading enzyme having an action of degrading choline into trimethylamine and glycolaldehyde under aerobic conditions.

Claims (1)

[Claims] 1. A novel choline-degrading enzyme which requires NAD (P) H as a coenzyme and has an action of degrading choline into trimethylamine and glycolaldehyde.