JP2640958B2 - Hydroxamic acid hydrolase - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a hydroxamic acid hydrolase having amidase activity, a method for producing the same, and an application thereof.

[Conventional technology]

Hydroxamic acid has a strong affinity for metal ions, and can be an inhibitor of enzymes such as urease,
Recently, application to the pharmaceutical field has been attracting attention. The method for producing hydroxamic acid is carried out by reacting hydroxylamine with an acid chloride, an acid anhydride or an ester in an anhydrous alcohol in the presence of a base under heating (for example, Nihon Noka Kagaku, vol. 24, 291 (1949)). ].

The hydroxamic acid hydrolysis reaction has a wide range of applications such as recovery of metal ions from metal-hydroxamic acid complexes and desorption of hydroxamic acid functional groups as protecting groups for acyl groups and carboxyl groups. Conventionally, such a hydrolysis reaction of hydroxamic acid is usually carried out by a chemical method of heating under strongly basic or strongly acidic conditions similar to amides (for example, J. Chem. Soc. . (B) (1
971) 123].

On the other hand, the hydrolysis reaction of hydroxamic acid by the enzymatic method is based on the cell-free extraction of crushed extract of animal liver using the action of lipase [Biochim. Biopys. Acta. Liquid (J. Dairy Sci., Vol. 66, 23
37 (1983)]. There is also a report that acetohydroxamic acid is produced when acetamide and hydroxylamine are reacted in the presence of an amidase [Biochem. J., vol. 95, 24C (1965)]. By the way, amidase generally does not catalyze the hydrolysis reaction of hydroxamic acid, and no report has been made so far that hydroxamic acid serves as a substrate for the hydrolysis reaction of amidase.

[Problems to be solved by the invention]

As described above, a number of methods for chemically hydrolyzing hydroxamic acid have been proposed, but in such a method, the reaction conditions are severe, the color and smell are increased, and the separation of products and by-products is difficult. When complicated processing is required and industrial mass production and mass processing are considered, it cannot be put to practical use.

On the other hand, the enzymatic method, that is, the hydrolysis reaction of hydroxamic acid by lipase is possible under mild conditions,
Such a lipase has a problem in obtaining since it is derived from animals.

[Means for solving the problem]

In view of the above situation, the present inventors aimed at finding hydroxamic acid hydrolase from a culture of a microorganism and used the medium containing acetohydroxamic acid as a single nitrogen source to assimilate acetohydroxamic acid. When screening for microorganisms having the following, it was found that certain yeasts have the ability to assimilate hydroxamic acid, and that the enzymes collected from the yeasts are completely new types of enzymes having excellent hydroxamic oxidizing water decomposing ability. We have found and completed the present invention.

It is intended to provide a method for producing hydroxamic acid hydrolase having the following physicochemical properties, a method for producing hydroxamic acid having amidase activity, and a method for producing carboxylic acids and hydroxamic acid using the enzyme.

<Physicochemical properties> Action-Breaks CO-N bond and hydrolyzes hydroxamic acid.

Substrate specificity aliphatic hydroxamate, aromatic hydroxamate,
Hydrolyze amino acid hydroxamates and acid amides.

 Does not hydrolyze olive oil and tributyrin.

 Optimum pH range and stable pH range Optimum pH Approx. 8-9 Stable pH range Approx. 6.5-9.0 Optimum temperature range for action Approx. 10-40 ° C, preferably 25-35 ° C Deactivation conditions Deactivated at pH 10 or higher.

 Deactivated at temperatures above 45 ° C.

 Storage stability Stable at -20 ° C for more than 60 days.

Molecular weight 115,000 [HPLC (TSK G-3000 SW)] 65,500 [SDS-PAGE] subunit Protease activity (casein resolution), no lipase activity.

The hydroxamic acid hydrolase of the present invention is produced, for example, by culturing a hydroxamic acid hydrolase producing bacterium belonging to the genus Rhodotorula, and collecting a hydroxamic acid hydrolase having amidase activity from the culture.

The hydroxamic acid hydrolase-producing bacterium used in the present invention is a yeast belonging to the genus Rhodotorula, and is not particularly limited as long as it has a hydroxamic acid hydrolase-producing ability. Particularly, Rhodotorula grinis is used.
aminis) are preferred in terms of their productivity. Such microorganisms include IFO-0190, IFO-1422, ATCC16727, ATCC167
28, ATCC16729, ATCC16730, NRRL Y-2474, NRRL Y-
5791, CBS2826, CBS3043, CBS4698, CBS5016, CBS571
1, CBS5778 and the like include known microorganisms that can be obtained from various organizations, and further include strain Rhodotorula graminis KSM-18 isolated from soil by the present inventors.

The mycological properties of Rhodotorula graminis KSM-18 are as follows.

(1) Growth status in each medium MY liquid medium: good, proliferates by budding MY agar medium: good, proliferates by budding (2) Ascospore formation: none (3) Ejection spore formation …… None (4) Physiological properties Optimal growth conditions pH7 Temperature 28 ° C (preferably 25 ° C to 28 ° C) Growth range pH5 to pH8 Temperature 30 ° C or less Nitrate assimilation + Urea decomposition + Carotenoid generation + Remarkable Production of organic acids-Production of starch-like substances-(5) Carbon source assimilation D-arabinose + L-arabinose + D-ribose + D-xylose + D-glucose + fermentability-D-galactose + L-rhamnose -L-sorbose + maltose + sucrose + lactose-melibiose-cellobiose + trehalose + raffinose + melezitose-α-methyl-D-glucoside-al Chin (or esculin) + soluble starch - inulin - ethanol + D-Man'nitsuto + glycerin + DL-lactate - succinate + Citrate + or mycological properties "Yeast: Characteris-tics and
This strain is determined to belong to Rhodotorula dalaminis in accordance with the known mycological properties of Rhodotorula graminis described in "Idutification; Cambridge University Press". When the strain was searched on the basis of the above properties, the strain was a novel one that had not been known before, and was named Rhodotorla glaminis KSM-18. Deposited as Kenken No. 9928.

Next, the cultivation of the strain in the production of the hydroxamic acid hydrolase of the present invention will be described.

As the nutrient medium, any of a synthetic medium, a semi-synthetic medium, and a natural medium can be used as long as the medium appropriately contains assimilable carbon sources, nitrogen sources, inorganic substances, and the like.

The carbon source is not particularly limited as long as it produces hydroxamic acid hydrolase, but is preferably used alone or in combination with glucose, monosaccharides such as fructose, lactose, disaccharides such as maltose, and pyruvic acid. , Especially glucose, is particularly preferred. Its concentration is
0.5-5.0% by weight (hereinafter simply indicated as%), especially 1.0-3.0%
Is preferred.

As the nitrogen source, inorganic and organic nitrogen sources are used in a wide range. , Various amino acid mixtures, meat extract, yeast extract, NZ amine, hydroxyurea and the like are used alone or in combination. Among them, hydroxylamine is preferably used as an inorganic nitrogen source, and yeast extract, meat extract and the like are preferably used as an organic nitrogen source. Particularly when hydroxylamine is used, the production of the enzyme of the present invention is remarkably improved. The concentration of hydroxylamine is preferably 0.05 to 0.4%, particularly preferably about 0.2%.
The use of hydroxylamine as a nitrogen source is very specific and characteristic in general yeast culture.

By adding acetohydroxamic acid to the medium to a concentration of 0.1 to 0.5%, the production of the enzyme is remarkably improved. The pH of the medium is about 5.0 to 8.0, especially 6.5 to 7.5.
Is preferred.

As a culture method, a general yeast culture method is employed, but a liquid culture method, particularly a deep stirring culture method, is preferable. The cultivation is preferably performed under aerobic conditions at a temperature of 25 to 30 ° C, particularly 27 to 28 ° C. The cultivation time can be 2 to 10 days, but the amount of the produced enzyme is highest on the 3rd to 4th days.

In order to collect the enzyme of the present invention from the culture, for example, the cells may be disrupted by ultrasonic waves or the like and centrifuged. Further, if necessary, a fraction of 30 to 60% of ammonium sulfate fraction may be added to an anion exchange resin such as DEAE-Sephacel (Pharmacia), a hydrophobic resin such as phenylsepharose (Pharmacia), gel filtration once or each column. It can be purified by passing it twice or more to make it electrophoretic single.

It has the following physicochemical properties in addition to the above physicochemical properties.

<Physical and chemical properties> Method for measuring titer Enzyme is added to a 0.1 M potassium phosphate buffer (pH 7.0, 5.0 mg / ml acetohydroxamic acid) and reacted at 30 ° C. The hydroxylamine formed at this time is quantified by a colorimetric method using 8-hydroxyquinoline [Am. J. Bot., Vol. 41, 777 (1954)] reported by WEMagee et al. The amount of the enzyme that produces 1 μmol of hydroxylamine in one minute is defined as one unit.

From the above properties, the enzyme of the present invention has hydroxamic acid hydrolysis activity and amidase activity. Conventionally, among the amidases, an enzyme having hydroxamic acid hydrolysis activity has not been known, and the enzyme of the present invention is novel.

Carboxylic acids can be produced by reacting the enzyme of the present invention with hydroxamic acid.

The enzyme used in the hydrolysis reaction need not be a purified product, but may be a purified product of a part or a quiescent cell of a hydroxamic acid hydrolase-producing bacterium belonging to the genus Rhodotorula. Since the enzyme of the present invention has the ability to hydrolyze a wide range of hydroxamic acids, the hydroxamic acid to be used is not particularly limited, but the following general formula (I) [Wherein, R represents an aliphatic group or an aromatic group which may have a substituent, or an amino acid residue as RCO]. Further, preferred examples of the group R in the general formula (I) include an alkyl group or alkenyl group having 1 to 23 carbon atoms, an allyl group, and a heterocyclic residue such as a nicotinic acid residue. Arginine, glycine, bistidine, leucine, lysine, methionine, phenylalanine, serine, tryptophan, tyrosine, α-amino-n-butyric acid, α-aminoisobutyric acid, norleucine, norvaline and the like.

The hydrolysis reaction is preferably performed in a buffer having a pH of 6 to 10 at a temperature of 0 to 30 ° C. By such a reaction, various carboxylic acids corresponding to the hydroxamic acid used as the raw material are obtained.

Hydroxamic acid can be produced from hydroxylamine and carboxylic acids using the enzyme of the present invention. The enzyme of the present invention is a hydrolase, and this synthesis reaction is disadvantageous because the equilibrium is generally considered to be on the hydrolysis side in an aqueous reaction. However, when the target hydroxamic acid is hardly soluble in water, the hydroxamic acid is precipitated at the same time as the generation and goes out of the reaction system, so that the reaction from the carboxylic acids to the hydroxamic acid proceeds. For example, when a long-chain carboxylate is used as the carboxylic acid, the reaction proceeds easily because the corresponding long-chain hydroxamic acid is hardly soluble in water. Such a reaction is carried out under conditions in which the enzyme of the present invention is not inactivated, for example, 0 to 30 in a buffer solution of pH 6 to 10.
It is preferably carried out at a temperature of ° C.

〔The invention's effect〕

The enzyme of the present invention has excellent hydroxamic acid hydrolysis activity. Therefore, the hydrolysis and synthesis of hydroxamic acid, which has been conventionally performed by chemical means, is severe, and the separation treatment after the reaction is complicated by the generation of by-products. In the method using the enzyme of the present invention, the generation of by-products can be suppressed under mild conditions, a complicated separation operation is not required, and carboxylic acids can be advantageously produced. Hydroxamic acid can also be produced under mild conditions from carboxylic acids and hydroxylamine.

〔Example〕

 Next, the present invention will be described with reference to examples.

Example 1 30 g glucose, 5 g K ₂ HPO ₄ , acetohydroxamic acid 2
g, hydroxylamine hydrochloride _{2g, Nacl 1g, MgSO 4 ·} 7H 2
0.2 g of O and 0.05 g of yeast extract are dissolved in 1000 ml of tap water, adjusted to pH 7.0 with an aqueous sodium hydroxide solution, dispensed, and sterilized by an autoclave to obtain a liquid medium.

For the culture, 5 ml of the above medium was inoculated with 1 loopful of Rhodotorula graminis KSM-18 from the slant, and pre-cultured at 28 ° C for 2 days.
and shake culture at 28 ° C. for 3 days. As a result of measuring the degree of growth at OD ₆₁₀ , the culture reached 8.0 after 3 days of culture, and no significant proliferation was observed after 4 days.

The cells cultured for 3 days are centrifuged at 10,000 rpm (Hitachi, SCR20B) and sonicated (Kubota INSONATOR201)
M) for 15 minutes, and then centrifuged the supernatant (10000 rpm, 15 minutes).
Min) to obtain a crude enzyme solution.

The enzymatic activity was measured using a cell-free extracted crude enzyme solution, which is also shown for resting cells. As a result, 11.3 units of hydroxamic acid hydrolase were obtained from 500 ml of the culture solution.

Example 2 Under the same conditions as in Example 1, the productivity of hydroxamic acid hydrolase was examined by variously changing the nitrogen source. Table 1 shows the results.

Example 3 A solution of 5.0 mg (0.07 mmol) of acetohydroxamic acid in the presence of 0.025 units of a hydroxamic acid hydrolase (obtained in Example 1, using a cell-free extracted crude enzyme solution, the same applies hereinafter) was used to adjust pH.
It was dissolved in 1 ml of 8.0 and 0.1 M Tris-HCl buffer and shaken at 30 ° C. for 8 hours. After the reaction, 1.34 mg of hydroxylamine was detected by a hydroxylamine color reaction with 8-hydroxyquinoline, and 1.71 mg of acetic acid was detected by high performance liquid chromatography using an ODS reverse phase column. The decomposition rate was 61.0%.

Example 4 8.0 mg (0.08 mmol) of butylhydroxamic acid was added at pH 8.0 and 0.1 in the presence of 0.05 units of hydroxamic acid hydrolase.
M was dissolved in 1 ml of Tris-HCl buffer, and the mixture was shaken at 30 ° C. for 8 hours.
49 mg of hydroxylamine was detected. Decomposition rate is 58.0
%.

Examples 5 to 8 Various hydroxamic acids were hydrolyzed in the same manner as in Example 3 or 4. Table 2 shows the results. The second
The table also shows the results of Examples 3 and 4.

Example 9 695 mg (10 mmol) of hydroxylamine hydrochloride and 1.25 g (5 mmol) of sodium myristate were dispersed in 20 ml of a 0.1 M Tris-HCl buffer at pH 8.0 in the presence of 0.05 units of hydroxamic acid hydrolase, and dispersed at 30 ° C. for 48 hours. Reacted for hours. After the reaction, the mixture was acidified and separated with a chloroform / ethanol (95: 5) solvent using a silica gel (Wako gel C-200) column to obtain 60 mg (0.25 mmol) of myristic acid hydroxamate. The yield was 5%.

Example 10 In the same manner as in Example 3 or 4, substrate specificity was examined using various hydroxamic acids and acid amides. Table 3 shows the results.

Claims (6)

(57) [Claims] 1. A hydroxamic acid hydrolase having the following physicochemical properties. Action-Breaks CO-N bond and hydrolyzes hydroxamic acid. Substrate specificity Hydrolyzes aliphatic hydroxamates, aromatic hydroxamates, amino acid hydroxamates and acid amides. Does not hydrolyze olive oil and tributyrin. Optimum pH range and stable pH range Optimum pH Approx. 8-9 Stable pH range Approx. 6.5-9.0 Optimum temperature range for action Approx. 10-40 ° C, preferably 25-35 ° C Inactivation conditions Deactivated at temperatures above 45 ° C. Storage stability Stable at -20 ° C for more than 60 days. Molecular weight 115,000 [HPLC (TSK G-3000 SW)] 65,000 [SDS-PAGE] subunit No protease activity (casein resolution), no lipase activity. 2. A method for producing a hydroxamic acid hydrolase having amidase activity, which comprises culturing a hydroxamic acid hydrolase producing bacterium belonging to the genus Rhodotorula and collecting the hydroxamic acid hydrolase from the culture. . 3. The method for producing a hydroxamic acid hydrolase having an amidase activity according to claim 2, wherein the culturing is performed in a medium containing acetohydroxamic acid. 4. A method for producing carboxylic acids, wherein a hydroxamic acid hydrolase having an amidase activity produced by a hydroxamic acid hydrolase-producing bacterium belonging to the genus Rhodotorula is allowed to act on hydroxamic acid. 5. A method for producing a hydroxamic acid, comprising reacting a carboxylic acid with hydroxylamine in the presence of a hydroxamic acid hydrolase having an amidase activity produced by a hydroxamic acid hydrolase-producing bacterium belonging to the genus Rhodotorula. Manufacturing method. 6. Rhodotorula grinis (6)
aminis) A hydroxamic acid hydrolase-producing bacterium named KSM-18 and deposited as FERM P-9928.