JPH01225486A - Production of d-glyceric acid - Google Patents

Abstract

PURPOSE:To obtain the subject compound useful as a raw material for optical isomers from L-tartaric acid readily available as a by-product in producing wine at a low cost, by utilizing a biological L-tartaric acid metabolic system without passing through oxaloglycolic acid. CONSTITUTION:A biological L-tartaric metabolic system without passing through oxaloglycolic acid is utilized to afford D-glyceric acid from L-tartaric acid. A microorganism of the genus Pseudomonas, such as 51D1A strain (FERM P-9889), or microorganism of the genus Aerobacter, such as Aerobacter aerogenes ICR B00280, is preferably used.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Application] Optical isomers are attracting attention in the fields of medicine and agrochemicals, and D-glyceric acid is used as a raw material for organic synthesis of various optical isomers.

[Prior art]

In general, the method for producing D-glyceric acid is α.

D-glyceric acid is obtained by oxidizing and hydrolyzing β-dibromopropionic acid, or by racemic resolution of L-glyceric acid by chemical means or microbiological means.

The enzyme that converts L-tartaric acid to D-glyceric acid was discovered by Dougray et al. in the crude enzyme extract of Pseudomonas (Biochemical Journal).
m, J, ) vol. 89, p. 22, 1963],
Further studied in detail by Cohn et al., Pseudomonas putida and Pseudomonas acitoporans
L-tartrate dehydrogenase (an enzyme that catalyzes the oxidation of L-tartaric acid to oxaloglycolic acid) and oxaloglycolate reductase (an enzyme that catalyzes the oxidation of oxaloglycolic acid to D-
They discovered an enzyme that catalyzes the conversion to glyceric acid (NADH is regenerated to NAD+ during this reaction), and L-tartaric acid is converted to D-glyceric acid via oxaloglycolic acid through a coupled reaction between the two enzymes. [Journal of Biological Chemistry (J, Biol, Chem,) Vol. 243, 2]
465 pages, 1968].

[Problem that the invention seeks to solve]

Optical isomers can be obtained by synthesizing a racemate and then separating the racemates by chemical or biological means to obtain the desired optical isomer, or by using raw materials for the synthesis of the desired optical isomer. After converting it into an optical isomer, it is synthesized while maintaining optical activity (there are methods, but
Currently, the former method is used in most cases.

The reason why the latter is difficult to adopt is that optical isomers used as synthetic raw materials generally have the disadvantage of being expensive. In particular, monoglyceric acid, which is useful as a raw material for producing a number of optical isomers, is very expensive in current production methods, and therefore its use as a raw material for synthesis is extremely limited. This makes it possible to produce D-glyceric acid at a lower cost than conventional methods.

[Means for Solving the Problems and Their Effects] The present inventors have made repeated studies to supply D-glyceric acid as a raw material for the synthesis of optical isomers at low cost, and have developed a method for producing D-glycerin from L-tartaric acid in microorganisms. Focusing on the metabolic pathway leading to acid, L
- By isolating and identifying a microorganism that produces an enzyme system that catalyzes the conversion of tartaric acid to D-glyceric acid, and investigating various enzyme reaction conditions, L-tartaric acid can be efficiently converted to D-glyceric acid.
It was successfully converted biologically into glyceric acid. This reaction system was different from previously known metabolic systems, and its conversion efficiency was very good.

It should be noted that L-tartaric acid used as a raw material for this reaction is obtained in large quantities as a by-product of the wine manufacturing industry, making it even more practical. The present invention will be explained in detail below.

In order to efficiently produce D-glyceric acid from L-tartaric acid, microorganisms having the activity of converting L-tartaric acid to D-glyceric acid were screened. As the screening method, the method described below was adopted.

Potassium L-tartrate 1.0%, ammonium sulfate 0
．． Use a medium containing 3% dipotassium phosphate, 0.3% magnesium sulfate, O.I.% magnesium sulfate, and 0.02% yeast extract, and adjust the pH to 7.5 using 3N hydrochloric acid (
(hereinafter referred to as standard medium). 25 of each microorganism in this medium 5 relatives
The microorganisms obtained by shaking culture at °C for 2 to 3 days are transplanted to a 2% agar medium containing L-tartaric acid. Using a 5001d flask, the obtained microbial cells were mixed with the above medium 200ml.
Culture aerobically in Jd at 25° C. under shaking conditions until visual confirmation of cell growth. The cells are then centrifuged and washed twice with 0.85% sodium chloride solution.

Approximately Ig (wet weight) of the washed cells was suspended in 1 m of 0.1 M) Lis-HCl buffer (pH 7,5) and sonicated for 2 minutes at 4°C using Seiko Model 7040. . After further centrifugation at 40,000 x g for 20 minutes, L-tartrate dehydrogenase activity was measured using the supernatant, and D-glycerate production activity of cells that produce L-tartrate dehydrogenase activity was determined. It was measured. In addition, each enzyme activity was measured according to the method shown below.

(1) Measurement of L-tartrate dehydrogenase activity 100 umo
l Tris-HCl buffer (pH 7,5), 1 μmol magnesium chloride, 1 μmol NAD” and 50 μmol
Using a reaction solution containing potassium L-tartrate in 11111, the absorbance at a wavelength of 340 nm is measured at 30°C with the reaction starting when the sample solution is added. For measurement, a spectrophotometer UV-3000 manufactured by Shimadzu Corp. is used, and changes in absorbance are measured within a range that maintains linearity for at least 1 to 2 minutes.

(2) Measurement of D-glyceric acid production activity 100 μmo
1 of Tris-HCl buffer (pH 7,5), 1 μmol of magnesium chloride, 1 μmol of NAD” and 50
μmol of potassium L-tartrate II+! 1! After the sample solution was allowed to act on the reaction solution contained in the solution at 30°C for 1 hour, 0.2 parts of 3N hydrochloric acid was added to stop the reaction.

The amount of residual L-tartaric acid and the amount of D-glyceric acid produced were determined by adding HLC8030 manufactured by Toyo Soda to ULTRONP.
It was quantified by high performance liquid chromatography using S-80H (0.8X30aa, trade name, manufactured by Shinso Kako ■).

Q, 126% perchloric acid as eluent, 0.8 rai
The flow rate was 1/mf. The elution times of L-tartaric acid and D-glyceric acid are 9.2 minutes and 11.1 minutes, respectively.

In addition, the generated CO2 was measured using a gas chromatograph (CR-6A model) manufactured by Shimadzu Corporation, and the conditions were 60/8.
A stainless steel column (0.03 x 100 cm) packed with 0 mesh activated carbon (manufactured by Nishio Kogyo ■) was used, and the carrier gas (helium) was heated at 39II11;/mi.
n, at a temperature of 160°C. The retention time of CO□ is 1 minute.

The unit of enzyme activity was defined as 1 unit when 1 μmol of the product was produced per minute. In addition, specific activity is expressed as activity per unit of protein, and protein amount is determined by the method of Lowry et al. using bovine serum albumin as a standard [Journal of Biological Chemistry (J, Biol, Chem, ) 193].
Vol., p. 265, 1968].

As a result of screening a number of bacterial strains, 13 strains of microorganisms that produced L-tartrate dehydrogenase were obtained, and as shown in Table 1, the presence of D-glyceric acid production activity was observed in 8 of these strains. .

Margin table below - 1 Among these, the 5DIA strain with high D-glyceric acid production activity is described in the Manual of Clinical Microbiology ((Manual of Clinical Microbiology).
Microbiology), 4th edition, 1985]
Identified according to.

Cell morphology Bacillus cell size (
Width) 0.8-1.2u Cell size (length) 2.0-3.2um Motility
Presence or absence of flagella and epiphytic status
Polar monoflagellate Gram stain Negative Solubility in 3% KOH Positive Aminopeptidase Positive Sporulation
Negative oxidase production Negative catalase production Positive growth under anaerobic conditions Negative growth at 37°C
Positive Growth at 41°C Negative Pine Conchi Agar Medium Positive SS-Agar Medium Negative Cetrimide Agar Medium
Positive pigment formation Water-insoluble pigment production Yellow-orange water-soluble pigment production Negative fluorescent pigment production Negative biocyanin production Negative OF test glucose (aerobic) Positive glucose (anaerobic) Gas production from negative glucose Negative acid production (As
s) Glucose Positive fructose Positive xylose Positive 0NPC test Negative arginine dihydrolase Negative lysine decarboxylase
Negative indole formation Negative nitrate reduction Negative denitrification reaction
Negative phenylalanine deaminase Positive production of levan from sucrose Negative lecithinase Negative urease
Negative hydrolyzable starch Positive gelatin Negative casein Negative DNA Negative Tween 80 Negative Aesculin
Degradation of negative tyrosine Negative auxotrophy
Negative assimilable acetite Positive adivate Negative caprate Positive citrate Positive glycolate Negative levulinate Negative maleide Positive malonate Positive phenylacetite Negative L-arabinose Positive fructose Positive glucose Positive mannose Positive xylose Positive mannitol Positive gluconic acid Positive 2-ketogluconic acid Positive N-acetyl Glucosamine Negative L-serine Positive In addition, 2-hydroxy and 3-hydroxy fatty acids were confirmed, but cyclopropane fatty acids were not confirmed.

Based on the above results, this bacterial strain was identified as a Pseudomonas genus. This 5DIA strain has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, and its deposit number is FAIKEN BACKGROUND No. 9889. This strain was used in subsequent experimental examples.

Experimental Example 1 Comparison of carbon source D
- The effect of carbon source on the production of glyceric acid production activity was investigated.0 As shown in Table 2, when L-tartaric acid was used as the carbon source, the bacterial cell yield of strain 5DIA was highest, and the highest amount of D
- Glyceric acid producing activity was obtained.

Table 2 Experimental Example 2 Measurement of optimum reaction conditions ■ Measurement of optimum pii Optimal p when producing D-glyceric acid from monotartaric acid
H was around 7.5 (Figure 1). The reaction conditions were as described in Example 1, and the reaction was carried out at 30° C. for 1 hour. However, the buffer solutions used were acetate buffer, Tris-HCl buffer, and phosphate buffer, and the protein content of the crude enzyme extract in the reaction solution was 3.15 μ/d.

(2) Measurement of optimal temperature The optimal temperature for producing D-glyceric acid from monotartaric acid was 30°C for 3 hours of reaction (Figure 2). The reaction conditions were the reaction solution composition described in Example 1 at various temperatures (20, 30
, 40, 50 and 60°C) for 1 to 3 hours. However, the content of the crude enzyme solution in the reaction solution was 1.37 μ/d.

■Influence of metal salts Crude enzyme solution (contains 3.2■ as protein) and 100μ+1I
ol of Tris-HCl buffer (pi47.5) and 5μ
By incubating a solution containing mol of disodium ethylenediaminetetraacetate in a total volume of IFd at 30°C for 1 hour, the D-glyceric acid production activity was completely inactivated, and it was reactivated by M g '', M n '', and Co ''. activated (Table 3), and M g t- is 0, 2
At concentrations above mM, the effect on D-glyceric acid production was almost constant.゛Table-3 ■Influence of NAD+ NAD when producing D-glyceric acid from monotartaric acid
(Figure 3).

The reaction conditions were as follows with the reaction solution composition described in Example 1 at 30°C.
Reacted for 2.3 and 5 hours. However, the protein content of the crude enzyme extract in the reaction solution was 1.37 μ/d.

■Substrate specificity The substrate specificity of the crude enzyme extract of the 5DIA strain was investigated (Table-
4) The reaction conditions were a system containing 10 mM NADH when dihydroxyfumaric acid was used as a substrate, and a system containing 1 mM NADH in other cases.

Table 4 As shown above, the crude enzyme solution obtained from this strain acts on L- and meso-tartaric acid, but is considered an intermediate in the metabolic pathway from L-tartaric acid to D-glyceric acid. It had no effect on dihydroxyfumaric acid, a tautomer of oxaloglycolate. This indicates that L-tartaric acid is metabolized to D-glyceric acid by a novel enzyme system that is completely different from the enzyme system previously discovered by Jacopy et al. Such substrate specificity was confirmed in all strains shown in Table 1 other than the 5DIA strain.

Examples are given below, but the present invention is not limited thereto.

Example 1 Identification of the product generated from L-tartaric acid by the action of the crude enzyme extract of the 5DIA strain 100 mmol of dipotassium monotartrate;
Hydrochloric acid buffer (pH 7,5) 20 o1. Magnesium chloride 0.2 mmol, NAD” 2 mmol and 5
DIA strain crude enzyme extract 50d (938■ protein) 1
The solution contained in the suspension was incubated at 30°C for 24 hours. After the reaction, transfer the reaction solution to Amberlite IRA400.
(Product name) (OH-type) (5 x 21 cm, capacity 4
It was applied to a column of 18 yd), washed with water of 11, and eluted with IN-hydrochloric acid of 1f. After sufficiently concentrating both portions of hydrochloric acid in an evaporator, the method of Baer et al. [Journal of the American Chemical Society (J.
A+w, Chem.

Soc, Vol. 61, p. 2607, 1939], calcium glycerate was obtained. The obtained calcium glycerate gave NMR and IR spectra with the same pattern as commercially available DL-calcium glycerate.

The optical rotation [α]20 of the obtained calcium glycerate was +13.3 (the literature value of the optical rotation of D-glyceric acid is +12.9 to +15.5). The product was oxidized by a coupling reaction using glyoxylate reductase and diaphorase to give 87 nmol of formazan dye from 89 nmol of calcium glycerate. Based on the above results, the product was identified as D-glyceric acid. The optical yield under these conditions was 96%.

Example 2 Example 1 using 50d of crude enzyme extract obtained by culturing Aerobacter aerogenes ICRBOO280
As a result of operating in the same manner as above, the product was identified as D-glyceric acid, and the optical yield was 86%.

Example 3 The reaction process when D-glyceric acid was produced from 50 mH L-tartaric acid was measured. (Figure-4) 0.1M) Squirrel-
Approximately 1
L-tartaric acid was converted to D-glyceric acid with a yield of 0.00%.

In Figure 4, when 25.4 μmol of D-glyceric acid was produced, 23.3 μmol of CO□ was observed to be generated, indicating that the reaction proceeded stoichiometrically.

[Effects of the Invention] The present inventors screened a strain from nature that produces a novel enzyme system that converts L-tartaric acid to D-glyceric acid. In particular, by examining the reaction conditions using the 5DIA strain enzyme system, L-tartaric acid could be converted to D-glyceric acid with a yield of almost 100%, and its optical purity was also 96%.

[Brief explanation of the drawing]

Figure-1 shows the influence of pH on the production of D-glyceric acid, and Figure-2 shows the influence of temperature on the production of D-glyceric acid. The reaction results for 2 hours and -Δ- are the reaction results for 3 hours. Figure 3 shows the influence of NAD on D-glyceric acid production, -〇- is the reaction result for 1 hour, -
Maroichi is the reaction result for 2 hours, -Rho is the reaction result for 3 hours, and -.- is the reaction result for 5 hours. Figure-4 shows the reaction process when D-glyceric acid is produced from L-tartaric acid, -0- indicates the reduction process of L-tartaric acid, -
The row indicates the production process of D-glyceric acid.

Claims (2)

[Claims] (1) Biological L that does not go through oxaloglycolic acid
- A method for producing D-glyceric acid in which D-glyceric acid is obtained from L-tartaric acid using a tartaric acid metabolic system. (2) The method for producing D-glyceric acid according to claim (1), which uses a microorganism belonging to the genus Aerobacter or Pseudomonas.