JPH0362393B2 - - Google Patents

Description

[Detailed description of the invention]

[Technical field] The present invention relates to a levorotatory optically active form of mandelic acid [(R)-levorotatory compound which is useful as a raw material or synthetic intermediate for pharmaceuticals such as penicillin and cephalosporin antibiotics or sympathomimetic drugs such as ephedrin. mandelic acid] (hereinafter simply referred to as levorotatory mandelic acid)
The present invention relates to an industrially advantageous method for producing , by a continuous reaction in which enzymes necessary for the synthesis are repeatedly used. [Prior Art] Known methods for producing levorotatory mandelic acid include optical resolution using fractional crystallization of racemic form, optical resolution using chromatography, and asymmetric synthesis using organic chemistry. This method has disadvantages such as complicated operations, low yield, and low optical purity of the product. On the other hand, in order to obtain levorotatory mandelic acid, a method of asymmetric reduction of benzoylformic acid using microbial cells (Japanese Patent Application Laid-open No. 198096/1983) is also known.
According to this method, although the drawbacks of the above-mentioned known methods are eliminated, a decrease in activity due to autolysis by microbial proteases is unavoidable, and there are also problems of product contamination and loss of purity due to intracellular components and culture medium components. etc. remain. The present inventors considered synthesis using enzymes extracted from microorganisms without using microbial cells, and found that enzymes extracted from microorganisms belonging to the genus Streptococcus are suitable for the purpose, and have actually applied this enzyme to produce levorotatory mandelic acid. However, this method simply uses enzymes in a reaction tank, and has an economical problem in that the expensive enzymes are discarded for each batch reaction. [Purpose] As a result of intensive research aimed at the repeated use of enzymes, the present inventors discovered that the above enzyme and reduced nicotinamide adenine dinucleotide (NADH), which is a reducing agent necessary for the reaction, could be used on the spot. The present inventors have discovered that levorotatory mandelic acid can be produced continuously for more than one week by using one other enzyme required for regeneration in an ultrafilter, and have completed the present invention. [Configuration] That is, according to the present invention, enzyme A having the ability to reduce benzoylformate derived from a bacterium of the genus Streptococcus
and formate dehydrogenase are put into an ultrafilter, and a substrate solution containing benzoylformic acid and formic acid is passed therethrough to carry out ultrafiltration while carrying out an enzyme reaction. A continuous manufacturing method is provided. Next, the enzymatic reaction in the present invention will be shown. In this reaction, electrons from formic acid are transferred to NAD (oxidized nicotinamide adenine dinucleotide) by the action of formate dehydratase, resulting in reduced form of NAD (oxidized nicotinamide adenine dinucleotide).
It becomes NAD [NADH]. As a result of stereoselective transfer of electrons from NADH to benzoylformic acid by enzyme A, asymmetric reduction occurs and levorotatory mandelic acid is produced. At the same time, NADH returns to its original NAD. The above cycle continues to rotate as long as mandelic acid is removed and the raw materials formic acid and benzoylformic acid are supplied. In the present invention, an ultrafiltration membrane is used to extract only mandelic acid from the reaction solution. Ultrafiltration membranes allow low-molecular compounds to pass through, but not macromolecules. When the reaction solution of the above formula is ultrafiltered, enzyme A and formate dehydrogenase remain in the reaction solution without being filtered, and only the product mandelic acid is taken out. By the way, as can be seen from the above formula, NAD is a reaction element that can be used repeatedly if it is retained within the system. Since NAD is originally a low-molecular compound, it will pass through the ultrafiltration membrane as it is. In order to prevent this passage, for example, it may be bonded to a polymer compound. As is clear from the above description, the following four material elements constitute the present invention. (1) Enzyme A with benzoylformate reducing ability, (2) formate dehydrogenase, (3) ultrafiltration membrane and device, and NAD
(4) NAD
A polymer substance that combines First, enzyme A (1) is prepared by destroying and extracting the cells of a bacterium belonging to the genus Streptococcus.
Bacterial strains used for this purpose include, for example, Streptococcus faecalis
faecalis). As the medium and culture conditions, any medium may be used as long as it allows for good growth of the bacterial cells and the desired enzyme activity is high, such as culturing with shaking at 30°C for 15 to 25 hours using tomato youth medium. Methods include: Conventional methods such as ultrasonication can be used to destroy the collected bacterial cells, and affinity chromatography, ion exchange chromatography, etc. can be used to purify the target enzyme solubilized in this way. Any normal method may be used. This purification does not necessarily need to be carried out to the extent that the target enzyme is isolated as a single protein. Usually, low-molecular components derived from bacterial cells, polysaccharides, nucleic acids, proteases, etc.
Excluding interfering enzymes such as NADH oxidase, enzymes with increased specific activity of about 100 U/mg are sufficient. For this purpose, for example, affinity chromatography using a dye-binding resin is effective. However, the present invention is not limited in any way by these descriptions. The properties of this enzyme A are as follows. (1) Activity measurement method: 0.1m phosphate buffer (PH7.5) 2.5
ml, 0.2ml of 83mM benzoylformic acid (sodium salt) aqueous solution, and 0.05ml of 10mg/ml NADH aqueous solution
Prepare the substrate solution by mixing and keep warm at 30℃.
Add and mix 20μ of Enzyme A solution, and measure the change in absorbance over time at 340 nm to determine the initial velocity. Measurement is performed using water instead of the benzoylformic acid aqueous solution as a blank, and the rate of change in absorbance at that time is subtracted from the previous value. As an initial rate, the amount of enzyme that changes 1 μmol of NADH in 1 minute is defined as 1 unit (U). (2) Specific activity of purified enzyme: 91U/mg protein (3) Molecular weight: 7200± as measured using gel filtration method
2000. When measured using SDS/polyacrylamide gel electrophoresis, it was 3400±2000. Therefore, this enzyme A is considered to be a dimeric protein consisting of two subunits with a molecular weight of around 34,000. (4) Isoelectric point: 4.9~5.0. (5) Optimal pH: When using benzoylformic acid as a substrate
PH4.5. In the reverse reaction, when (−)-mandelic acid is used as a substrate and NAD ⁺ is used as a coenzyme, the PH
9.2. (6) Effect of PH on stability: 70% residual activity when heated at 55℃ for 1 hour using various buffer solutions
The pH range exceeding 6.2 to 5.5. Under the same conditions, it is completely inactivated at pH 7.2 or higher and pH 4 or lower. Therefore, this enzyme A is stable under slightly acidic conditions of pH 6.5 to 5.0. (7) Stability against heating: 0.1M phosphate buffer containing 2mM mercaptoethanol (PH6.0)
The residual activity after heating for 1 hour at 55℃
35%, 50% at 50°C, and 80% at 45°C. Next, when 1% bovine serum albumin was coexisted as a stabilizer, the results (residual activity) were 70% at 55℃, 50%
It was 87% at ℃ and 96% at 45℃. (8) Stability under conditions that mirror the actual conditions for producing (-)-mandelic acid: 0.1 m benzoylformate, 1 mM NAD, 2 mM 2-mercaptoethanol, 1% bovine serum albumin, potassium sorbate (preservative) ) The residual activity of enzyme A when dissolved in 15mM phosphate buffer (PH6.3) containing 0.02% and kept at 30°C was 73 on the 5th day.
%, 52% on day 10 and 42% on day 19. (9) Stability during refrigerated storage: 1% at 4°C in 15mM phosphate buffer (PH6.3) containing 2mM mercaptoethanol and 3.2M ammonium sulfate.
It remained almost inactive for several months. (10) Michaelis constant: 0.1M phosphate buffer (PH
7.5) at 30℃, 3.3mM for benzoylformic acid and 35μ for NADH.
It was m. (11) Substrate specificity: NADPH as a coenzyme
Not a substitute for NADH. This enzyme A reduces various α-keto acids in addition to benzoylformic acid. Typical examples are as follows (the numbers in parentheses are % of the reaction rate when used at a concentration of 10mM, based on the value of benzoylformic acid as 100).
): α-ketobutyric acid (3), α-ketovaleric acid (23), α-ketocaproic acid (24), α-
Ketoisovaleric acid (67), α-ketoisocaproic acid (132), α-keto-β-methylvaleric acid (63), phenylpyruvic acid (60), 3-
Brompyruvate (152), and 3-chloropyruvate (17). Among α-keto acids, pyruvic acid,
It showed no activity against oxaloacetic acid, α-ketoglutaric acid, etc. (12) Stereoselectivity of reaction: with benzoylformic acid
Mandelic acid produced using NADH as a substrate was extracted and its optical rotation was measured, and its optical purity was measured as a diastereomeric derivative by gas chromatography. % optical purity. On the other hand, when the activity was measured using NAD ⁺ and (-)-mandelic acid or (+)-mandelic acid (manufactured by Aldrich) as a reverse reaction, (-)-mandelic acid was completely oxidized, but
(+)-Mandelic acid reacted only slightly. (13) Inhibitor: The activity of this enzyme A is inhibited by some heavy metal ions. Typical examples are listed below (in parentheses are the residual activity measured using 0.1M Tris/HCl buffer pH 7.5, expressed as a percentage with the value without metal ions added as 100), and metal ion concentration):
Cu ²⁺ (9%, 1mM), Zn ²⁺ (0%, 1mM),
Ni ²⁺ (55%, 1mM), Mg ⁺ (6%, 0.1mM),
Hg ²⁺ (31%, 0.1mM), Cd ²⁺ (2%, 1mM).
In addition, this enzyme A was completely inhibited by 0.5mM p -chloromercury benzoate, and 1
It is also inhibited by 60% by mM N -ethylmaleimide. As described above, this enzyme A is novel in that it has the ability to reduce benzoylformate, has high specific activity as an enzyme, is not easily inactivated under practical conditions, and is similar to levorotatory Mandel. It is an enzyme with excellent properties such as producing acid with 100% optical purity. Next, the formate dehydrogenase (2) is as stable as or more stable than the enzyme A having the benzoylformate reducing ability of (1), and the pH range in which it performs catalytic action is within the pH range used by enzyme A. Any item may be used as long as it overlaps with . For example, commercially available formate dehydrogenase from the Candida boidinii bacterium can be cited. In addition
In addition to formate dehydrogenase, alcohol dehydrogenase and glucose dehydrogenase can be expected to be used as enzymes for NADH regeneration. However, from the viewpoint of equilibrium constant and ease of removal of byproducts (carbon dioxide in the case of formate dehydrogenase and gluconolactone in the case of glucose dehydrogenase), formate dehydrogenase is the most It is considered to be effective. The ultrafiltration membrane (3) allows mandelic acid to pass through completely, but has pores with a pore size large enough to completely block the two enzymes mentioned above, and is stable under actual usage conditions. Any material can be used as long as it does not have the disadvantageous property of adsorbing enzymes or polymeric substances. For example, Amicon's PM-10 membrane and
Examples include PM-30 membrane. As the ultrafiltration device, any device may be used as long as it can be easily used by installing the above-mentioned membrane. However, it must be equipped with an input port for the PH electrode, a supply port for the acid/alkaline solution for pH adjustment, a supply port for the substrate liquid, an outlet for carbon dioxide gas, and a stirrer. A control device (PH stat) is also required to keep the pH constant. Next, as a polymer substance that binds NAD (4), it has high activity against formate dehydrogenase when it is in the oxidized form (NAD type), and has high activity against enzyme A when it is in the reduced form (NADH type). It may have any structure. Of course, it is water-soluble and has a molecular weight (approximately 10,000 or more) that does not pass through the ultrafiltration membrane used. As such, NAD is chemically modified to form a derivative having a polymerizable functional group (for example, N ⁶ -[2-[ N -[2-[ N
-(2-methacrylamidoethyl)carbamoyl]
Examples include those obtained by copolymerizing ethyl [carbamoyl] ethyl]-NAD) with acrylamide or N , N -dimethylacrylamide through a radical reaction. These are easily synthesized by known methods (Agric.Biol.Chem. vol. 45, p. 2277 (1981);
Patent No. 1226768). Finally, the conditions for carrying out the reaction will be explained. First, a buffer solution is selected, and any buffer solution that is normally used around neutrality may be used, such as a phosphate buffer solution or a Tris/hydrochloric acid buffer solution. The concentration of the buffer solution may be appropriately selected within the range of several mM to 200-300 mM. Concentrations higher than this are acceptable. PH is an appropriate value between 4 and 8. Which value to choose depends on the two enzymes and
It is determined by considering the stability of NAD and NADH and the PH region in which the two enzymes exhibit activity. The optimum pH of enzyme A having the ability to reduce benzoylformate used in the present invention is
The pH is around 4.5, and the most stable against heating is 5.8 to 6.0. Furthermore, among formate dehydrogenases, the optimum pH for the one derived from Candida boidinii bacteria is 8, and the stable pH range is around 7. These facts and
Considering that NAD is unstable in alkaline conditions and NADH is unstable in acidic conditions, it is concluded that it is practical to set the pH to 6.5 to 7.5 in the case of the above enzyme combination. It is desirable to add about 0.1 to 2 mM of 2-mercaptoethanol or dithiothreitol to this buffer as an enzyme stabilizer. In addition, if a salt such as ammonium sulfate has a stabilizing effect, it may also be added. Enzyme A and
The formate dehydrogenase of Candida boidinii is significantly stabilized by the presence of 1-2 m ammonium sulfate (60°C at pH 6.5).
When heated for 1 hour, both enzymes are completely inactivated in the absence of ammonium sulfate, but with 1M ammonium sulfate, enzyme A is completely stable, and formate dehydrogenase also maintains 50% activity). Highly concentrated ammonium sulfate can be expected to have a preservative effect during enzymatic reactions and a salting-out effect during organic solvent extraction of mandelic acid. A substrate solution is prepared by dissolving benzoylformic acid and formic acid in the form of an appropriate salt such as sodium salt or potassium salt in these buffer solutions. If NAD is to be used up, it is also added to the substrate solution.
In the case of benzoylformic acid, its concentration is the Michaelis constant for enzyme A (3.3mM at 30℃, pH 7.5).
It is practical to set the concentration to about 10 times (about 30mM or 0.5%) to about 100 times (about 300mM or 5%). Of course, it may be above or below this range. The concentration of formic acid should be in excess of the concentration of benzoylformic acid, but about twice that is sufficient.
The concentration of NAD is NAD and NADH at the pH used.
The Michaelis constant (30
It is sufficient to set the concentration to about 10 to 100 times that of 94 μm at ℃ and PH6.5). Of course, the rotation speed (turnover number) should be much lower than this.
It is also acceptable to improve the A certain amount of this substrate solution is put into an ultrafilter, and both enzymes and
If necessary, NAD bound to a polymer is added to initiate the reaction. In this case, of course, use the substrate solution.
No NAD added. The amount of enzyme used may be appropriately determined depending on the reaction rate required for the reactor. The concentration of NAD bound to a polymer is set depending on the concentration when free NAD is used by adding it to the substrate solution, but it is better to set it high to take into account the decrease due to decomposition. The reaction temperature is preferably around 30°C. The reaction solution is constantly stirred. Ultrafiltration is carried out by suction or pressurization at the same time as the reaction starts or after a predetermined conversion rate is reached. At the same time, the substrate liquid is supplied at a rate equal to the filtration rate to keep the volume of the reaction liquid in the ultrafilter constant. In addition, the specified pH is determined by the PH stat.
Try to keep it. When the reaction is carried out in neutral to slightly acidic conditions, for example, when sodium formate is used as the substrate,
NaHCO ₃ generated by the reaction of formate dehydrogenase decomposes, CO ₂ gas escapes, and NaOH is left behind, resulting in a gradual increase in PH. To suppress this, it is necessary to add acid. Suitable acids for this purpose include formic acid and acetic acid. The rate of ultrafiltration and feed rate of substrate liquid are adjusted to achieve the desired conversion. Naturally, increasing the flow rate will decrease the conversion rate. To isolate the levorotatory mandelic acid product from the ultrafiltrate, conventional methods such as organic solvent extraction may be applied. For example, if the reaction solution is made acidic with a pH of 2 to 1 using dilute hydrochloric acid or dilute sulfuric acid, then if necessary, salt such as common salt is dissolved to saturation concentration, and then extracted with ethyl acetate or ether. The mandelic acid contained therein is recovered almost quantitatively. Separate the organic layer, distill off the solvent, dissolve the residue in hot benzene, etc., if necessary, perform hot filtration after treating with activated carbon, etc., and cool the filtrate to obtain crystals of levorotatory mandelic acid. . [Example] Next, the present invention will be described in more detail with reference to Examples. Example 1 N6- [ ^2- [ N- [2-[ N- (2-methacrylamidoethyl)carbamoyl]ethyl]carbamoyl]ethyl]- synthesized by a known method (J.Biochem., 95109 (1984)) NAD, acrylamide
A known method using a combination of K ₂ S ₂ O ₈ and NaHSO ₃ as an initiator (Agric.Biol.Chem., 452277
(1981)), purified by ultrafiltration and dialysis, and freeze-dried to obtain a white solid of polyacrylamide-bonded NAD (hereinafter abbreviated as PA-NAD). When analyzed using the method described in the above literature, the content of coenzyme-active NAD was found to be
It was 48.8 μmol/g, and the average molecular weight was 56,000. Streptococcus faecalis IFO 12964 was cultured with aeration and stirring in a medium containing tomato youth, malt extract, and CoSO ₄ . After culturing at 30° C. for 24 hours, the bacteria were collected, and the cells were subjected to ultrasonication to extract enzyme A having the ability to reduce benzoylformate. This was purified by affinity chromatography using a column packed with Matrex Red A resin to obtain a sample with a specific activity of 621 U/mg protein. Candida Boideini
boidinii) formate dehydrogenase was purchased from Boehringer Mannheim Yamanouchi. A bioreactor as shown in the drawing was assembled using Amicon's PM-30 membrane and a Model 52 ultrafilter. 0.1M in 0.1M phosphoric acid drying solution
A substrate solution containing sodium benzoylformate, 0.2M sodium formate, 2mM 2-mercaptoethanol, 1M ammonium sulfate, and 0.03% potassium sorbate was prepared, and the pH was adjusted to 6.5. This is substrate solution B in the figure.
It is. Take a portion of the substrate solution and add bovine serum albumin.
0.2%, enzyme A 100U/ml, dihydrogenase
10U/ml and PA-NAD as the concentration of NAD
The mixture was dissolved to a concentration of mM, and finally, a new substrate solution was added to bring the total volume to 30 ml, and the mixture was poured into an ultrafilter (reaction solution A in the figure). In the PH stat reservoir,
Contains 5M formic acid aqueous solution D. Ultrafilter is 30
Immersed in a constant temperature bath at ℃. While stirring the reaction solution A with a magnetic stirrer, ultrafiltration is performed by suctioning it at a rate of 9 ml/hr with pump _P2 , and at the same time
At _P1 , substrate solution B was supplied at a rate of 9 ml/hr. PH
will rise, so keep it at 6.5
5M formic acid D was added using a Stat5. The generated CO ₂ gas was discharged through a filter 4.
At 120 hours, 30 μmol of PA-NAD was added as NAD. Filtrate C from _P2 was pooled separately for each day. The pH of each aqueous solution is 6NHCl.
< 2, and then extracted with the same amount, half amount, and 1/3 amount of ethyl acetate as the aqueous layer while saturating with sodium chloride. The ethyl acetate layer and the aqueous layer were combined, concentrated, and then crystallized from boiling benzene to obtain plate crystals of levorotatory mandelic acid. The yield, melting point, and specific rotation are shown in Table 1.
The decrease in flow rate appears to be due to membrane clogging. The yield was calculated based on the volume of the filtrate and the amount of benzoylformic acid contained therein. The melting point and optical rotation data were consistent with literature values for optically pure levorotatory mandelic acid. It was found that the decrease in yield from day 8 was due to inactivation of both enzymes. From the results in this table, it is clear that levorotatory mandelic acid can be continuously produced in this bioreactor over a period of one week or more.

〔effect〕

According to the present invention, it is possible to continuously produce optically pure levorotatory mandelic acid while repeatedly using the enzyme and, if necessary, the coenzyme NAD without releasing it to the outside of the reactor. Economical use of NAD can be achieved.

[Brief explanation of drawings]

The drawing is an explanatory diagram of an example of a bioreactor used in carrying out the present invention. 1...Ultrafiltration membrane, 2...Magnetic stirrer, 3...PH electrode, 4...Sterilization filter, 5
...PH stat, 6...Thermostat (30℃), P ₁ , P ₂
...Peristaltic pump, A...Reaction solution (enzyme and PA-NAD), B...Substrate solution (formic acid and benzoylformic acid), C...Ultrafiltrate (levorotatory mandelic acid), D...5M formic acid.

Claims (1)

[Claims] 1. A solution of enzyme A, which has the ability to reduce benzoylformate derived from Streptococcus bacteria, and formate dehydrogenase is placed in an ultrafilter, and a substrate solution containing benzoylformate and formic acid is passed through to carry out an enzyme reaction while performing ultrafiltration. 1. A method for continuously producing a levorotatory optically active form of mandelic acid.