JP2950865B2 - New aspartate racemase - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial application field) The present invention provides D-aspartic acid as an amino group donor in a D-transaminase reaction used for production of D-amino acids by racemizing L-aspartic acid. And a method for producing the same.

(Prior art and its problems) Aspartate racemase is described in Streptococcus faecalis ATCC97.
It is known that the presence of a single protein is not only confirmed in 90 strains but also alanine as well as aspartic acid (J. Biol. Chem. 247). (16) 5103 (2072)). Therefore, when considering that D-aspartic acid as an amino group donor in the D-transaminase reaction used for production of D-amino acids by the present enzyme is supplied by racemization of L-aspartic acid, the substrate specificity of the present enzyme is A big problem.

(Means for Solving the Problem) The present inventors sought a strain having an aspartate racemase-producing ability for the purpose of searching for an enzyme capable of racemizing aspartic acid, and intensively researched the strain including known conserved strains. As a result, Streptococcus genus and Lactobacillus genus were found to produce aspartate racemase, and Streptococcus thermophilus IAM10064 produced the most highly stable aspartate racemase among them, leading to the present invention.

The physicochemical properties of the aspartate racemase of the present invention will be described below.

(A) Action: From L-aspartic acid to D-aspartic acid and L-
Racemization reaction to produce aspartic acid, and D-
It catalyzes a racemization reaction that produces L-aspartic acid and D-aspartic acid from aspartic acid.

(B) Substrate specificity: The racemization reaction acts specifically on aspartic acid and hardly acts on other amino acids.

(C) Optimum pH: In the reaction measurement, at a temperature of 37 ° C., a 10 mM potassium phosphate buffer (pH 5.5 to 8) [FIG. 1], a 100 mM sodium pyrophosphate buffer (pH 7.9 to 9.0) ) [Fig. 1].

As a result, as shown in FIG. 1, the optimum pH is around pH 8 in the reaction at 37 ° C.

(D) Stable pH range For 0.1 ml of this enzyme solution, buffer
0.4 ml was added, the mixture was treated at 50 ° C. for 10 minutes, and cooled with ice to obtain a sample. The specific activity was determined for this. Buffer pH 4-6
Acetic acid-sodium acetate, 50 mM potassium phosphate for pH 6-8, and 50 mM Tris-hydrochloric acid for pH 8-9.

As a result, as shown in FIG. 2, the enzyme is stable up to around pH 6.5 to 8.0 when treated at 45 ° C. for 1 hour.

(E) Thermal stability: 0.1 ml of the enzyme solution was added to 50 mM potassium phosphate buffer (p
H7.0) Prepare 6 test tubes containing 0.4 ml, 25, 37, 4
Each was treated at a temperature of 5, 50, 55, or 60 ° C. for 60 minutes and cooled with ice to obtain a sample. Inactivity was determined for this.

As a result, as shown in FIG. 3, this enzyme is stable up to 50 ° C. in the treatment for 60 minutes, but almost no activity remains at 55 ° C.

(F) Molecular Weight The total molecular weight was measured by a gel filtration method using a column of Super Rose 12HR10 / 30 (Pharmacia).
The standard proteins include bovine serum albumin (molecular weight 67,000),
Ovalbumin (molecular weight 43,000), chymotrypsinogen A (molecular weight 25,000), ribonuclease (molecular weight 13,7
00) was used.

The molecular weight of the subunit was measured by SDS-slab electrophoresis. Standard proteins include phosphorylase b (molecular weight 94,000), bovine serum albumin (molecular weight 67,000), protein albumin (molecular weight 43,000), carbonic anhydrase (molecular weight 30,000), soybean trypsin inhibitor (molecular weight 20,000), α-lactalbumin (molecular weight 14,0
00) was used.

As a result, the total molecular weight is 60,000. In addition, the molecular weight of the subunit was 28,000 and there was one SDS protein band, indicating that the present enzyme has a dimer structure of the same subunit.

(G) Amino-terminal amino acid sequence The amino acid sequence was determined using 20 μg of this enzyme preparation in a gas phase sequencer by automatic Edman degradation. As a result, the following amino terminal amino acid sequence was confirmed (X represents an unknown amino acid).

The aspartate racemase of the present invention can be produced, for example, by culturing an aspartate racemase-producing bacterium belonging to the genus Streptococcus and collecting the aspartate racemase from the culture.

Microorganisms used in the method for producing aspartate racemase of the present invention are all strains belonging to the genus Streptococcus capable of producing aspartate racemase,
Includes mutants and variants. A preferred specific example is Streptococcus thermophilus, and the conserved bacteria belonging to this species include Streptococcus thermophilus IAM10064.

The culture of the aspartate racemase-producing bacterium for obtaining the aspartate racemase of the present invention can be performed in a usual lactic acid bacterium medium. For example, a medium containing peptone, yeast extract, meat extract, inorganic salts and the like is used.

The cultivation may be performed using either a solid medium or a liquid medium. However, in order to obtain a large amount of the target enzyme, it is desirable to perform cultivation using a liquid medium under anaerobic conditions such as static culture. The culture temperature may be within a temperature range in which the bacteria grow and aspartate racemase is produced, but is preferably 25 to 45 ° C. The culturing time may be selected as long as the enzyme activity is expressed, but is preferably 6 to 24 hours.
The pH of the culture is preferably 5 to 7. By this culture, most of the enzyme is accumulated in the cells.

After completion of the culture, the culture may be used as an enzyme source as it is, but usually, separation and purification are performed. As the purification method, an ordinary enzyme purification method can be used. For example, the cells are collected by centrifugation, and the cells are pulverized by a mechanical method such as ultrasonic treatment or dyno mill. Subsequently, a solid enzyme solution is obtained by removing solid matter such as cell debris by centrifugation or the like. Next, the crude enzyme solution is fractionated by an ammonium sulfate precipitation method, and a uniform enzyme preparation can be isolated by hydrophobic chromatography, ion exchange chromatography, gel filtration, or the like.

Next, measurement of the activity of the aspartate racemase of the present invention will be described.

D-aspartic acid generated from L-aspartic acid by aspartate racemase is oxidatively deaminated to oxaloacetic acid by D-amino acid oxidase, and the resulting oxaloacetic acid is reacted with 2,4-dinitrophenylhydrazine to form a hydrazone. At least, the reaction rate is determined by quantifying the amount of hydrazone produced. Incubate the reaction mixture as shown in Table 1 for 30-60 minutes at 37 ° C.
-Add dinitrophenyl hydrazone hydrochloride and add
Incubate at 37 ° C, add NaOH solution for another 30 minutes at 37 ° C
And the production of hydrazone is measured by absorbance at 435 nm.

The enzyme activity unit of this enzyme is 1 μmol / min D-
The amount of enzyme that produces aspartic acid is defined as 1 unit. The specific activity is represented by the number of units per 1 mg of the enzyme protein.

Next, a method for producing the aspartate racemase of the present invention will be described with reference to examples, but the present invention is not limited thereto.

(Example) Table 2 shows Streptococcus thermophilus IAM10064.
The resulting cells are cultured at 37 ° C. for 5 to 20 hours in a culture medium, disrupted by ultrasonic waves, and a cell-free extract is prepared by centrifugation or the like to obtain a crude enzyme solution.

The crude enzyme solution was first fractionated with ammonium sulfate, and then purified using column chromatography means of phenyl-Sepharose, Sephacryl S-300, DEAE-Sephacel, Sephacryl S-200, and Mono Q (Pharmacia, trade name). And
A single enzyme preparation can be obtained electrophoretically. Next, the total amount of protein in the enzyme solution obtained in each of the purification steps,
Total activity and specific activity were measured. Table 3 shows the results. Except for the final step, the total protein was measured by the Lowry method, and the final step was determined by the absorbance at 280 nm. The total activity was measured according to the above-mentioned activity measurement method.

[Brief description of the drawings]

FIG. 1 is a graph showing the relationship between the activity of the enzyme of the present invention and pH, FIG. 2 is a graph showing the pH stability of the present enzyme, and FIG. 3 is a graph showing the temperature stability of the present enzyme. It is a graph shown.

──────────────────────────────────────────────────続 き Continuation of the front page (56) References Biochemistry, 24 (6) (1985), p. 1333-1341 (58) Field surveyed (Int. Cl. ⁶ , DB name) C12N 9/00-9/99 BIOSIS (DIALOG) WPI (DIALOG)

Claims (3)

(57) [Claims] An aspartate racemase having the following physicochemical properties: (A) Action: catalyzes a racemization reaction for producing D-aspartic acid and L-aspartic acid from L-aspartic acid, and a racemization reaction for producing L-aspartic acid and D-aspartic acid from D-aspartic acid. . (B) Substrate specificity: The racemization reaction acts specifically on aspartic acid and hardly acts on other amino acids. (C) Optimum pH: around pH 8 at 37 ° C. (D) Stable pH range: No decrease in activity is seen at pH 6.5 to 8 at 45 ° C for 1 hour. (E) Thermal stability: No decrease in activity is observed at 60 ° C. for 60 minutes at pH 7. (F) Molecular weight: Total molecular weight about 60,000 (gel filtration method) Molecular weight of subunit 28,000 (2 in the same subunit)
(G) Amino-terminal amino acid sequence: 2. A method for producing aspartate racemase, comprising culturing an aspartate racemase-producing bacterium belonging to the genus Streptococcus, and collecting the aspartate racemase according to claim 1 from the culture. . 3. The method according to claim 2, wherein the aspartate racemase-producing bacterium belonging to the genus Streptococcus is Streptococcus thermophilus IAM10064.