DE3523161A1 - IMPROVED TRANSAMINATION PROCESS FOR PRODUCING AMINO ACIDS - Google Patents

Description

The invention relates generally to an improved method for the production of L-amino acids (hereinafter referred to as

-IO denotes "amino acids") from their eL-keto acid precursors by biological transamination using aspartic acid as an amino group donor. The described The method is used in particular to improve the transamination rate and yield by increasing the rate of degradation of oxaloacetic acid, a by-product of the reaction. This achieves a significant increase in the reaction rate, so that essentially the entire preliminary stage is consumed in transamination. By eliminating the equilibrium pressure on the transamination and by converting a larger amount of the precursor, amino acid yields in excess of 90% can be achieved will. The reaction is catalyzed by adding metal ions to the transamination reaction system.

It is known that amino acid precursors can be enzymatically converted into their corresponding L-amino acids. For example, US Pat. No. 3,183,170 describes the transamination of phenylpyruvic acid in the presence of a multi-enzyme system obtained from various sources, including bacterial cells, dry cells, cell macerates or enzyme solutions. The US application 520 632 of August 5, 1983 describes the microbiological transamination of ⁰ ^ - -keto acids to amino acids in batch fermentations using conventional precursors in order to

35231S1

for example phenylpyruvic acid in L-phenylalanine, <? c-ketoisocaproic acid in L-leucine, «C-ketoisovaleric acid to convert to L-valine, etc.

The biotransformation of the eC-keto acid into the amino acid is an enzymatic transamination, which results in the exchange of the amino group of the amino group donor and the keto group of the precursor. It is generally accepted that enzymatic transamination is an equilibrium reaction. For example, Oishi states in " The Microbial Production of Amino Acids " (editor Yamada et al.) In Chapter 16 "Production from Precursor Keto Acids", pp. 440-446 (1972) that when using aminotransferases for a high product yield the

.j ₅ Use of a high concentration of the amino group donor is required. In US Pat. No. 3,183,170 it is reported that in a transamination reaction with L-glutamic acid as the amino group donor, the equilibrium is favorably shifted to the right by converting the cL- ketoglutaric acid formed during the transamination at the same rate by reductive amination in L -Glutamic acid is converted back in the same way as «c -ketoglutaric acid is formed.

In conventional transmissions there are a number of of compounds have been used as amino group donors. Oishi notes on pages 435-452 of the publication by Yamada et al. states that the best amino group donors are L-aspartic acid, L-leucine, L-isoleucine and 3Q are L-glutamic acid and that get better results when these amino acids are used in combination than when they are used individually. U.S. application 568,300 dated January 5, 1984 describes a process by means of which a microbiological transamination reaction can be brought to run completely continuously by using a

Solution uses approximately equimolar amounts of aspartic acid and the phenylalanine precursor, one precultures the microorganisms in the presence of the precursor and the biological catalyst used as dried cells and / or the loading of the system with the biological Catalyst increased.

Oxaloacetic acid (also known as oxosuccinic acid or keto succinic acid) forms a by-product of enzymatic transamination when aspartic acid is used as an amino group donor. Oxaloacetic acid has been investigated in a different context and it has been shown that it is broken down by various mechanisms. Bessmann reports in Arch. Biochem. 2Q, 418-21

(1950) "Preparation and Assay of Oxalacetic Acid" via the spontaneous degradation of oxaloacetic acid, which is catalyzed by a number of substances. Krebs reported in J. Biochem. 3_ £, 303-305 (1942) "The Effect of Inorganic Salts on the Ketone Decomposition of Oxalacetic Acid "states that a number of inorganic salts increase the rate of breakdown of oxaloacetic acid to increase pyruvic acid and carbon dioxide.

It has been found that the .Transamination of an amino acid precursor under the conditions described below into their corresponding amino acid if aspartic acid is chosen as the primary amino group donor and the precursor being ad-keto acid is significantly improved by catalyzing the breakdown of the transamination by-product oxaloacetic acid. The breakdown will catalyzed by adding certain multivalent metal ions or salts thereof to the reaction system. the ! ■ -Amino acid can be enriched and collected. By Such a breakdown of the oxaloacetic acid can shift the equilibrium of the system dramatically to the right

whereby the reaction is brought to essentially complete completion. The result of this metal ion catalysis is an increase in the transamination rate up to more than about 7.0 M / l / h and the amino acid yield up to about 90 to 100%.

One of the objects of the invention is to improve a biological transmutation response by reducing the rate of response and the overall amino acid yield based on both aspartic acid and precursor is dramatic increases. It is accordingly possible to adjust the relative amount of precursor necessary to achieve the desired product yield is required to lower.

The substantial increase in the rate of transamination increases the time required for the system to convert a given amount of prepress. In addition, the increased reaction rate significantly reduces the loss in product yield due to the degradation of the preliminary stage. Furthermore, this leads to reduced contamination with unconverted precursor, which makes purification and recovery of the product less expensive.

Another object of the invention is to provide a reaction in which the oL keto acid precursor need not be pure and which overcomes the inhibiting effect of impurities that may be present.

It is the overall object of the invention to provide those associated with the production of amino acids by transamination Significantly lower costs.

The method of the present invention provides a unique means of promoting the transamination of an amino acid precursor into the corresponding L-amino acid by adding

Aspartic acid used as an amino group donor and a suitable oC-keto acid as a precursor, a suitable intra- or extracellular transaminase selects and the transamination reaction coupled with the catalyzed degradation of the transamination by-product oxaloacetic acid under conditions which favor transaminase activity. The catalyzed Degradation is caused by adding multivalent metal ions or salts thereof to the reaction system and transamination is allowed to proceed significantly beyond equilibrium. The transamination can be carried out by mixing a solution containing the precursor oi-keto acid and aspartic acid with the brings intra- or extracellular transaminase into contact, i.e. with a cell or enzyme preparation which is able to mediate the transamination. The designation "Transaminase" is used according to the invention to denote an enzyme or an enzyme system that is capable is to mediate the previously described conversion of the precursor into the amino acid.

The microorganisms which have already proven to be suitable in such transamination processes include Pseudomonas pseudoalcaliqenes , Brevibacterium thiogenitalis , Pseudomonas aeruqinosa , Bacillus subtilis and

“ Escherichia coli , each of which mediates the transamination of a phenylalanine precursor to L-phenylalanine. The latter four bacterial species have also been shown to have activity for the formation of other amino acids by transamination with aspartic acid as a donor (shown for L-tyrosine (Example IV), L-leucine, L-isoleucine and L-valine). The group of microorganisms suitable according to the invention is of course very much broader. Organisms belonging to the genus Aerobacter , halophilic organisms and yeasts and also belonging to the genus Candida have proven to be suitable. It is assumed that

ORIGINAL INSPECTED '

any microorganism is suitable according to the invention, which is known to have transaminase activity for the production of amino acids and aspartic acid can use as an amino group donor. Mixed cultures of suitable strains can also be used.

The growth conditions should be selected based on the particular microorganisms used and this is within the knowledge and ability of a specialist

-jO in this area. Conditions that favor the rapid growth of healthy cells are preferred. For example, if Pseudomonas pseudoalcaligenes is used, the temperature is preferably kept at about 35 to about 39 C and the pH is kept at about 7.5. It is stirred and / or aerated to create an aerobic environment. Appropriate sources of energy and nutrients should be available.

The microorganisms are attracted to a predetermined cell density or growth phase. The exact The growth period and cell density is not critical, as the density can, if desired, be achieved by conventional methods such as Centrifugation or filtration after cultivation can be increased. A typical growing cycle of around 12 up to about 48 hours will generally provide sufficient cell count. The cells are then harvested and can be treated to permeabilize the cell membrane. This treatment can be carried out by drying, sonication, Incubation can be done with toluene or non-ionic surfactants. It may also be desirable to use the cells mobilize on a suitable substrate. Become viable microorganisms in the method according to the invention used, it is preferred that they are able to deliver the amino acid product into the medium to produce a to enable convenient and economical extraction. In the

Cell preparations described above are sufficient amounts of the transaminase in connection with the cells or the cell material present to carry out the reaction.

According to the invention at least about 2.0 to about 200.0 g cells (dry weight) per liter of substrate solution, preferably at least about 4.0 to about 80.0 g / l used. Regardless of the form in which the cells are actually

-IO borrowed to be used in the method according to the invention, The weight data for the cell catalyst are based on dry weight in the present case. With a lower catalyst content significant amounts of the precursor can be degraded before they enter the transamination process.

-j 5 The extent of the degradation caused by the preliminary stage The problem with conventional transamination processes varies depending on the oC -eto acid chosen as the precursor

■ and the reaction conditions. For example, the Degradation due to the presence of multivalent metal salts, as well as acidic pH conditions, ultraviolet light or the presence somewhat accelerated by oxygen.

Alternatively, a cell-free system can be used. It is an enzymatic transamination and this can take place in a solution containing the appropriate enzymes or transaminases. It is therefore possible an enzyme preparation, i.e. a crude extract or an isolated or purified enzyme for the transamination reaction to use. In another embodiment

3Q of the invention can the enzyme on a suitable substrate be immobilized.

The amino acid precursor is an o £ -ketocarboxylic acid or a salt of the same. For the method according to the invention

- 14 - 3523181

The selected eL-keto acid depends on the amino acid desired as the transamination product. For example, phenylpyruvic acid turns into L-phenylalanine, oe-ketoisocaproic acid into L-leucine, oL-ketoisovaleric acid into L-valine, pyruvic acid into L-alanine, ß-hydroxy- oL-ketobutyric acid into L-threonine, p-hydroxyphenylpyruvic acid into L-valine Indolepyruvic acid converted to L-tryptophan, ol ^ -Keto-ß-methylvaleric acid converted into L-isoleucine, <* _- ketohistidic acid (ß-imidazolylpyruvic acid) converted into L-histidine, etc. It is found that conventional amino acid precursors can be used in the process of the invention. The precursor can be purified or in unpurified form, for example as sodium, calcium, potassium or ammonium hydroxide hydrolyzate or as sulfuric acid precipi-

^ g fact exist.

In the preferred embodiment of the invention, a Substrate solution prepared, the aspartic acid and amino acid precursor in a molar ratio of about 1: 2 to about 2: 1 in a biocompatible buffer. It can any biocompatible solvent or buffer that does not interfere with the reaction and the pH can be used of the reaction system in the preferred range of about 6.5 to about 10.0. For example, about 0.1

P ₅ to 0.5 M phosphate buffer proved to be suitable. Alternatively, the pH can be controlled by adding a pH control agent to the system which adds base as needed. As the transamination process proceeds, there is a tendency for the pH to drop.

The amino group donor must be used in the inventive Transamination process be or must contain essentially aspartic acid. It was found, that the highest amino acid yields are achieved when aspartic acid is the only amino group donor in the

ORIGINAL INSPECTED

Reaction system is present. When using aspartic acid as an amino group donor, oxaloacetic acid is used as Formed by-product of transamination. The oxaloacetic acid is both spontaneously and catalytically under Formation of carbon dioxide and pyruvic acid are broken down.

It is of course permissible to have other amino group donors present in the system and these through to let the transaminase convert. However, the method according to the invention has no effect to the extent that the transamination reaction includes other amino group donors than aspartic acid, since in this respect no oxaloacetic acid occurs as a by-product that could be degraded.

To facilitate transamination, the presence of the Requires cofactor pyridoxal-5-phosphate (P-5-P). This cofactor forms a complex with the enzyme that is used transiently converted to pyridoxamine phosphate (PMP) and is recovered in the overall reaction. It can It may be desirable to add small amounts of P-5-P to accelerate transamination. For example, it can be beneficial to P-5-P in concentrations of about 0.1 / UM to be added, although small amounts of the cofactor are already due to the cell material added to the reaction system available.

In the transamination process according to the invention there are three potentially rate-limiting reactions: (1) the biological conversion of the precursor into the amino acid, (2) the breakdown of the cL -keto acid used as a precursor to worthless products and (3) the breakdown of the by-product oxaloacetic acid. The first reaction, namely the biological conversion, can easily be driven by increasing the loading of the transamination catalyst

wherein the catalyst is contained in the cell material. By driving the first reaction, the Decreased degradation of the precursor (the second reaction). Accordingly, the degradation of the oxaloacetic acid then becomes critical speed-limiting level. It turns out that there is an increase in the transmission catalyst available is only extremely advantageous if the rate of decomposition of the oxaloacetic acid is also achieved is increased.

The degradation of oxaloacetic acid is a first order reaction, i.e. the rate of degradation is proportional to the concentration of oxaloacetic acid in the system. At high concentrations there is faster degradation, but the rate is then decreases when the concentration is lowered by the degradation. The spontaneous rate of breakdown for a concentration of 0.1 M / l is about 6.7 χ 10 ~ M / l / h. The elimination of oxaloacetic acid from the reaction system through spontaneous degradation helps to allow the conversion of the precursor to the amino acid to take place completely.

In the early stages of biologically catalyzed transamination However, the oxaloacetic acid can decompose at a rate 5 to 10 times higher than the spontaneous rate or even be formed even more quickly if the transamination system allows it. Accordingly, there is also spontaneous Degradation has a clear tendency for oxaloacetic acid to accumulate in the system, especially in the early stages of the reaction, as the spontaneous rate of degradation is no longer rapid is enough to have the equilibrium effect on the transamination reaction to exclude.

The degradation of oxaloacetic acid can be caused by the presence of inorganic multivalent metal ions such as Al, Al,

₊ 2 ₊ 2 ₊ 2 ₊ 2 ₊ 2 ₊ 3, 2 ₊ 2, 2

Ni, Mn, Mg, Pb, Ag, Fe, Fe, Zn, Cr,

+ ³ + ² H- ³ + ² + ³

Cr, Co, Co, Pd, Au, etc. or salts thereof be catalyzed. The metal ions can, for example, be in soluble form as sulfates, sulfides or chlorides these metals are added. Alternatively, particles of metals such as alumina, lead or iron can be used as Catalyst are added. Solid metal particles are advantageous from a process point of view, since the catalyst is not lost with the product stream.

The multivalent metal ions which are suitable for catalysis of the oxaloacetic acid breakdown are to a certain extent also responsible for the breakdown of the d- keto acid used as a precursor. However, the rate of catalyzed oxaloacetic acid degradation is sufficiently high that the

relative amount in which the preliminary stage is broken down in the Overall reaction is insignificant. That is, the transamination process proceeds at such a rate continues that relatively few preliminary stages are dismantled

can. 20th

The choice of metal ions or their salts is specific to the system. For convenience and economic reasons it is generally preferred to select those already used in the transamination

th precursor or aspartic acid stream are present. The most preferred metal ions are magnesium, iron, Aluminum and zinc ions. Iron, aluminum and zinc generally provide higher rates and yields. Magnesium is something in spite of being preserved with it

lower speed and yield one of the preferred because of its solubility and low cost Metal catalysts.

3523131

Cost considerations can play a role, with silver and lead being more expensive and iron and nickel less expensive. Copper can and is believed to cause a coloration that is undesirable in certain applications it denatures the transaminase and forms chelates with the amino acids. Certain metal ions such as Copper and iron, can be cofactors or accelerators for other enzyme activities and form products that are eliminated Need to become. Calcium ions are in the process according to the invention because of their tendency to precipitate Preliminary stage from the solution is not desired. Manganese is toxic to humans and is not one of the preferred ones Choice because it creates problems in human contact with the process and waste fluids must be avoided. However, it has been shown that manganese salts are useful in the process of the invention are.

The metal ions increase the rate of degradation reaction by at least two orders of magnitude over the spontaneous rate of about 6.7 χ 10 M / l / h even at very low concentrations. For example, at high oxaloacetic acid concentrations of 0.1 M / l, the catalyzed degradation rate can be around 0.1 up to 2.0 M / l / h. At lower oxaloacetic acid concentrations of around 0.02 M / l, the catalyzed degradation rate about 0.01 to 0.2 M / l / h. This speed variation is due to variations in effectiveness and Concentration of the metal catalyst used and the temperature and pH of the reaction. In these Areas, the inhibition of the rate of transamination by the presence of oxaloacetic acid in the system is effective switched off. In the presence of these multivalent metal ions, volumetric transamination rates are up to about 10.0 M / l / h in the early stages of conversion and about 0.05 to 0.2 M / L / h total achieved with no evidence of oxaloacetic acid inhibition.

ORIGINAL INSPECTED

352316

The concentration of the metal ions used to catalyze the degradation is preferably influenced by the rate of transamination adapted to the rate at which oxaloacetic acid is formed by transition initiation. A demonstrable Catalysis of Qxalacetic acid degradation begins in the presence of

—4

at least about 1.0 10 M / l of the ions. At metal ion concentrations above about 1.0 χ 10 ~ M there is little or no significant increase in the rate of degradation. Of the The range is typically about 1.0 10 to about 1.0 10 ~ M / l. When determining the amount of salt that the If the system is to be added, it must be taken into account that the solubilities of the various salts vary. at the use of insoluble metals for the catalysis according to the invention, amounts of up to about 20 g / l can be used

Become 15.

The cell or enzyme preparation is brought into contact with the solution of aspartic acid and precursor in a suitable reaction vessel. The reaction conditions for the process of the present invention should be selected so as to increase the enzyme stability, and they are selected in accordance with the overall transamination reaction system. The temperature range may preferably be about 20 to about 50 ⁰ C, about 30 to about 40 ° C.

The pH can be about 6.5 to about 10.0, preferably about 7.5 to about 8.5. The pH adjustment can be made with any base that is compatible with the enzyme system is compatible, preferably with ammonium or potassium hydroxide, each of which has the effect of solubilizing the Amino acid precursor and aspartic acid supports. The reaction does not require ventilation, if not live, growing cells can be used, but the cells should be stirred or agitated a little over the substrate, about the interaction between biocatalyst and substrate

35 to maximize.

3523131

It is expected that the transamination reaction in less than 40 hours is complete, preferably in less than 4 hours if high biocatalyst concentrations be used. Another measure is the specific reaction rate, i.e. the moles formed Amino acid per gram dry cell weight per hour. In the method according to the invention, this value can be in the range from about 0.0001 to about 0.1, preferably from about 0.002 to about 0.05 moles of amino acid formed per gram of dry cell weight per hour. Usually, however, the specific reaction rate is about 0.0005 to about 0.01 moles Amino acid per gram dry cell weight per hour.

The breakdown products of oxaloacetic acid are carbon dioxide and pyruvic acid. The carbon dioxide escapes when the The reaction is carried out in an open vessel and the pH is close to neutral, or it can either be used for Disposal or collected for use in further proceedings. If the pH is above about 7.5, then the carbon dioxide remains dissolved in the liquid. It is believed that at least a very small fraction of the Pyruvic acid is converted to alanine by transamination or by decarboxylation of aspartic acid. The relative amount of alanine formed varies depending on the biocatalyst used in the transamination. Accordingly, both pyruvic acid and small amounts of alanine will be present, both of which can be however, can be easily disposed of by conventional separation techniques.

The improved biological transamination process of the present invention can produce extremely high amino acid yields both in terms of aspartic acid and in terms of the amino acid precursor deliver. That is, 90% or more of the aspartic acid in the reaction can be transaminated and

ORIGINAL INSPECTED

3523181

90% or more of the precursor can be converted to the amino acid. It also increases the rate of transamination significantly increased, which leads to both increased yields and shorter reaction times. After the reaction is complete, the amino acid product can be obtained by conventional methods will. Examples of typical processes for obtaining the desired product are ion exchange and / or fractionated Crystallization process. 10

The invention is illustrated below with the aid of examples.

The following abbreviations have been used in the description and examples:

DS - Developing Solution

OD - optical density

P-5-P - pyridoxal-5-phosphate

PPA - phenylpyruvic acid

ppm - parts per million

TYR - tyrosine

in the tables showing the results of the examples the specific reaction rate, the volumetric conversion rate and the yield were calculated as follows:

TNSPECTED

Specific reaction rate Volumetric turnover rate

_ Moles made grams of cells-hour

Mole formed liter - hour

yield

_ Moles formed moles of theoretical value

The theoretical value is calculated from the available moles of the precursor or aspartic acid.

Example I.

(The effect of manganese ions on transaminase activity)

A synthetic solution was prepared containing 17.4 g / l sodium phenylpyruvic acid (Na-PPA, monohydrate from Sigma Chemical Co.) and 11.09 g / l aspartic acid (from Sigma Chemical Co.) in a 0.1 M phosphate buffer 0.1 µM pyridoxal-5-phosphate (P-5-P) and its pH was adjusted to 7.5 with aqueous ammonia and sulfuric acid. Six 100 ml samples were prepared. 1.0 χ 10 M MnSO _{4 was} added to three samples; the remaining samples were used as controls. The solutions were filled into 150 ml jacketed mixing beakers, which were kept at 37 ° C. with careful stirring.

A preparation of dried cells was made as follows: Pseudomonas pseudoalcaligenes ATCC 12815 was grown in 14 liters of “Trypticase Soy Broth” for 24 hours at 35 ° C. The cells were harvested by centrifugation and dried in a vacuum oven at 37 ° C for 12 hours.

INSPECTED

ο ρ I ο Ί ο I

The dried cells became the test and control solutions added in amounts of 10/5 and 2 g per liter. The solutions were kept at about 37 ° C. with gentle stirring. Samples for analysis were after 5 and Taken 24 hours after the colorimetric ferric chloride Determination method on PPA (method is described below) and with HPLC on phenylalanine and Aspartic acid analyzed. The results are given in Table I. It turns out that in the case of the control samples, i.e. without the addition of the metal salt, the volumetric transamination rate despite the higher catalyst loading remained constant. The addition of MnSO. led to a dramatic increase compared to the control the volumetric and specific transamination rates

15 in addition to higher L-phenylalanine yields.

ORIGINAUIiSiSPECTED

Control (No Mn ⁺² )

Time:
Charge with catalyst 10 g / l

PPA (g / l)
ASP (g / l)
PHE (g / l)
ALA (g / 1)
Specific rate
Volumetric rate
Yield (PHE / PPA)
Yield (PHE / ASP)

Tabel I. (Addition of Mn 5 hours 10 g / l 5 g / l 2 g / i 4.70
3.77
8, 61 3.80
2.20
8.20 4.60
2.93
7.68 _f 00104
_f 0104
62%
62% , 002 ,
62%
62% 0046
, 01
57%
57%

24 10 g / l hours 2 g / l 0.70 5 g / l 0.50 2.28 0.80, 1.60 11.55 4.50 ^x 10.20 , 0003 10.55 , 0012 , 003 , 0005 , 0025 . 84% , 0027 75% 95% 76% 89% 120%

attempt

(1.0 χ ΙΟ " ³ Μ Mn ⁺² added)

Time:

Charge with catalyst 10 g / l

PPA (g / l) ASP (g / l) PHE (g / l) ALA (g / l)

Specific rate
Volumetric rate
Yield (PHE / PPA)
Yield (PHE / ASP)

0.30 2.84 12.69 0.62, 0015 . .015 93% 93%

ο hours 2 g / l 5 g / l 3.89 1.25 5.08 2.77 7.67 10.67 - 0.27 , 0046 , 002 6 , 01 , 013 57% 78% 57% 78%

1 - Obvious analytical error

24 hours 2 g / l CO I. 10 g / l 5 g / l 0.30 cn NJ
.N. 0.05 0.40 1.34 NJ) I. 0.69 1.00 12.16 CO 13.95 12.47 0.43 0.84 0.79 , 0015 , 00035 , 0006 , 003 , 0035 , 0031 88% 101% 90% 101% 106% 99.6%

Example II

(Effect of Magnesium Ions on Transamxnase Activity)

A synthetic solution was prepared containing 17.71 g / l Na-PPA (monohydrate, Sigma Chemical Co.) and 12.35 g / l aspartic acid (Sigma Chemical Co.) in 0.05 M phosphate buffer at 1.0 χ 10 M MgSO. and 0.1 µM P-5-P, the pH being adjusted to 7.5 with aqueous ammonia and sulfuric acid. Portions of 100 ml each were placed in five 150 ml jacketed mixing beakers, heated to 37 ° C. and carefully stirred. Pseudomonas pseudoalcaligenes ATCC 12815 dry cells were prepared as in Example I. The cells were added to the beakers in the following amounts, respectively: 15, 10, 5.0, 2.0 and 1.0 g / l. Samples were taken after one and 24 hours and analyzed as in Example I. The results, shown in Table II, show that when MgSO. no oxaloacetic acid repression of the transamination is observed and the volumetric reaction rates and the overall yields are increased.

Table II

(Addition of Mg)

Time: 15 g / l 1 hour 5 g / l ι g / i 15 g / l 24 hours 5 g / l ι g / l I. Loading with
Catalyst: 8.81 10 g / l 11.00 13.84 0 _f 00 10 g / l 0.15 0.95 σι PPA (g / l) * 9.44 9.76 10.13 11.75 0 _K 92 0.15 l _r 18 2 _t 08 I. ASP (g / l) 7.96 10.29 2 _f 99 0 _f 57 14.18 0.93 13.52 11 _T 47 PHE (g / l) - 4.60 - - 0.52 13.87 0.19 0.79 ALA (g / l) , 0032 - , 0036 _f 0035 , 00024 0.35 _f 00068 , 0029 Specific fate _f 048 , 0028 _r 018 , 0035 , 0036 , 00035 _T 0034 , 0029 Volumetric rate 56% , 028 21% 4% 100% , 0035 96% 81% Yield (PHE / PPA) 32% 98%

As phenylpyruvic acid

cn ho co

Example III

(Effect of the magnesium ion concentration on the

Transaminase activity) 5.

A synthetic solution was prepared containing 17.0 g / l Na-PPA (monohydrate, Sigma Chemical Co., sodium salt) and 11.0 g / l aspartic acid (Sigma Chemical Co.) in 0.1 M phosphor

-4
phate buffer containing 1.0 χ 10 M P-5-P, the pH value being adjusted to 7.5 with aqueous ammonia and sulfuric acid. 100 ml aliquots were poured into jacketed mixing beakers with varying amounts of magnesium sulfate

+2

brought so that the Mg concentrations given in Table III were reached. The solutions were kept at 37 ° C. with gentle stirring. Pseudomonas pseudoalcaligenes ATCC 12815 dry cells were prepared as in Example I. 0.5 g of dry cells was added to each solution. The samples were analyzed as in Example I after 0, 2 and 5 hours. The results, shown in Table III, show that increasing

+2
Mg ion concentrations with increasing transamination

rates and total yields correspond.

ORIGINAL INSPECTED

Table III

+2,

(Varying concentrations of Mg)

+2
Mg concentration:

(Moles per liter)

3 χ 10

-4

Ix 10 ³ 3 χ 10 " ³

1 χ

-2

3 χ

-2

0 hours

PPA (g / l) *
ASP (g / l)

17.20 13.00

16.70
13.20

16 _r 90 13.40

17.20 13.30

17.10 13.90

2 hours

PPA (g / l) *
ASP (g / l)

PHE. (G / l)
Specific rate

Volumetric rate

9.96 5.92 8.93 0054, 027 7 _; 82
3.00
11.73
_r 007
, 036

7.03

2.50

12.8 0

, 0078

.039

6.40 3.30 11.52, 007, 035

8.02 9.01 I. 3.4 4 3 45 to 11.07 11.60 OO , 0067 , 007 I. , 034 , 035

5 hours

PPA (g / l) *
ASP (g / l)
PHE (g / l)
Specific rate
Volumetric rate

As phenylpyruvic acid

6th , 50 2, 40 4% 2 , 70 1, 00 12th , 70 15, 20th r 003 , 0037 74 , 0% 80

2.21

1.00

15.40

_T 0037

92.0%

2.63

1.00

15.60

, 0038

92.0%

2.12 2.70 OJ 1.00 1.00 cn 16.00 15.90 ro , 0039 , 0039 OJ 93.0% 93 0%

cn

352316

Example IV

(Effect of various salts on transaminase activity)

A synthetic solution was prepared which contained 20.6 g / l Na-PPA (monohydrate, Sigma Chemical Co.) and 13.5 g / L aspartic acid (Sigma Chemical Co.) in 0.1 M phosphate buffer with 0.1 / UM P-5-P. 100 ml samples of this solution

-3

were prepared and each 1.0 χ 10 moles per liter one of the following salts added to each sample: magnesium sulfate, manganese sulfate, copper sulfate, calcium chloride, Aluminum sulfate and iron sulfate. One sample remained as Control without adding salts. The pH of each solution was adjusted to 8.5 and gently stirred. Dry

.15 cells from Pseudomonas pseudoalcaligenes ATCC 12815 were prepared as in Example I. 0.5 g of dry cells was added to the solution. The solutions were kept at 37 ° C. with careful stirring. Samples were taken after 0, 5 and 24 hours and analyzed as in Example I.

The results presented in Table IV show that

+2 +2 +3 +2
the Mg, Mn, Al and Fe salts to increase the

Transamination rate and yield are effective.

ORIGINAL INSPECTED

- "ί = 'ίΊ. r

Table IV

(Comparison of the different salts)

O hours

PPA (g / l) * ASP (g / l)

MgSO ₄

16 _r 30 13.10

MnSO,

16 _f 04 15.50

CuSO.

16.30 13.74

Al ₂ (SO ₄ I _{3 FeSO 4} control

16.55 12.74

15.90 14.34

16.40 14.32

5 hours

PFA (g / l) * ASP (g / l) PHE (g / l) Volumetric rate Specific rate

24 hours

PPA (g / l) * ASP (g / l) PHE (g / l) yield (PHE / PPA) yield (PHE / ASP)

8.33 6.33 9.00 8.10 4.47 5.47 9.00 16.80 8.44 8.59. 3.09 2.75 , 011 , 011 _Λ 0037 , 0033 , 002 f 0021 , 00075 , 00067 0.00 0.00 2.20 1.70 0.97 \\ yi 8.90 5.74 15.50 14.90 6.86 Γ
4.95 94.9% 93.0% 45.0% 30.0% 95.0% 78.0% 60.0% 36.0%

8.30 4.90 8 "85 , 011 , 0021

0 _r 00

1.68

14.95

90.0%

95.0%

9.30 6.93 7.10, 009, 0018

0.00

3.20

14.00

88.0%

80.0%

6.20 8.20 6.20, 007 _f 0017

0.00

4.10

13 _f 20

80.0%

74.0%

As phenylpyruvic acid CO

cn

NJ OJ

CD

3523-16

Example V

(Comparison of soluble and insoluble metals)

A synthetic solution was prepared containing 3 0.0 g / l Na-PPA (monohydrate, Sigma Chemical Co.) and 24.0 g / l aspartic acid (Sigma Chemical Co.) in 0.1 M phosphate buffer with 0.1 , µM P-5-P and 100 ppm Barquart MB-SO (TM, Lonza Inc.) as disinfectants. To 100 ml aliquots of the solution from the following substances were in each case: 1.0 M MgSO χ 10, χ 1.0 10 ^-2 M MgSO _4, 1.0 χ 10 ~ ² M Al ₂ (SO ₄ and J _3. 10 g / l aluminum oxide pellets (corresponding to a mesh size of about 0.42 mm). The pH value of the solutions was adjusted to 8.5 with aqueous ammonia and sulfuric acid. 5.0 g moist cells of Pseudomonas pseudoalcaligenes ATCC were added to each solution 12815 (72% moisture). The solutions were warmed to 35 ° C. and gently stirred. Samples were taken after 0, 1, 5 and 24 hours and analyzed as in Example I. The results given in Table V show that the The use of aluminum oxide pellets has about the same effect as that of in terms of increasing the volumetric reaction rate and the overall yield of the transamination

25 magnesium sulfate or aluminum sulfate.

ORIGINAL INSPECTED

Tabel

Metal catalyst:

0 hours

PPA ASP

(g / D * (g / l)

1 hour

PPA (g / l) * ASP (g / l) PHE (g / l) Volumetric rate Specific rate

5 hours

PPA (g / l) * ASP (g / l) PHE (g / l) Volumetric rate Specific rate

24 hours

PPA (g / l) * ASP (g / 1) PHE (g / l) yield (PHE / PPA)

1 χ

MgSO ₄

-3

23.54 23.20

16.52

18.49

7.18

, 044

, 0031

2.84

7th , 73 , 00 19th , 05 , 69 » 025 , 45 , 0016 .6% 0 4th 23 99

As phenylpyruvic acid

loxide
1 χ , 07 , 10 lpelle ^ ts
10 ~ ² sts , 07 , 22 MgSO ₄ , 31 , 74 23 , 73 024 24 , 18 .0017 15th 053 17th , 0038 9 2 r 6th 19th r

0.00

3 _T 47

22.79

98.9%

χ ΙΟ " ² Μ

(SO ₄ )

23.82
24.00

17th , 40 , 57 , 00 20th _r 20 , 73 , 80 6th , 40 , 05 , 20 r 038 023 .0% _r 0028 _r 0016 4th 0 7th 4th 19th 22nd f 93

10 g / l

Alumina pellets

23.83 24.03

15.17

16 _r 04

8.59

, 052

, 0037

2.07 6.02

21.95, 027

, 0019

0.00

3.46

22.75

95.5%

CO

cn

NJ

co

CJ)

3523 ^ 6

Example VI

(Manufacture of tyrosine)

A synthetic solution was prepared by adding 1/25 g of p-hydroxyphenylpyruvic acid (pH-PPA from Sigma Chemical Co.) in 50 ml of 0.1 M phosphate buffer along with 1.0 g of aspartic acid (Sigma Chemical Co.), 0.1 µM P-5-P

and dissolved 1.0 χ 10 M / l zinc sulfate. The pH of the

Ί ° solution was adjusted to 8.0. Cells of E-. coli ATCC 11303 were immobilized in Hypol polyurethane foam (TM, HFP 3000, WR Grace & Co.) by mixing 50.0 grams of a 75% moisture cell paste with 50.0 grams of Hypol prepolymer (TM). The mixture was allowed to cure (about 15 minutes) and the cured foam was cut into small pieces about 0.64 cm in diameter. A total of 4.0 g of the cured foam was added to the prepared pH-PPA solution. The solution was kept at 37 ° C. with careful stirring.

Samples were taken after 0, 3 and 18 hours and with Thin layer chromatography (TLC) and HPLC on tyrosine and Aspartic acid studied. After 3 hours the solution became cloudy due to the formation of tyrosine. To obtain the Product, the slurry and foam was washed with an equal volume of acetone and the foam with a Filtered off Buchner funnel. The solution, which contained about 1.2 g / L tyrosine in solution, was centrifuged at 3000 rpm. The amino acid precipitate formed was collected and dried. It became the total amount of 1.1 g Collected dry product and identified as tyrosine by TLC and HPLC. Using non-aqueous titration

3523 ¹ S "

a purity of the product of more than 90% was found. The results given in Table VI show that the method according to the invention for the fast Production of tyrosine with high yields is suitable.

• Babelle VI

Yield (ΤΥΚφΗ-ΡΡΑ) Yield (TYR / ASP)

(Manufacture of tyrosine)

Time: 0 hours

pH-PPA (g / l) 25.0 ASP (g / l) 18.5 TYR ¹ 0.0 TYR ² 0.0

3 hours

16.2 1.2

g / l tyrosine in solution at the time of sampling

g / l tyrosine as the total yield in the dried precipitate

hours

1.2

1.0

85%
80%

CO

cn

N> CO ... J ₁

CD

3523 ¹ G ¹

Colorimetric ferric chloride detection for phenylpyruvic acid, sodium salt

Principle : Phenylpyruvic acid (PPA) forms a green complex with iron (III) ions. The intensity of the green color is proportional to the amount of PPA present.

Reagents :

1. Developing solution (DS) 1000 ml: The following ingredients are mixed together and cooled to room temperature in an ice bath. The ingredients are placed in a 1 liter volumetric flask and topped up with deionized water. Ingredients : 0.5 g

FeCl ₃ .6H ₂ O, 20 ml glacial acetic acid, 600 ml dimethyl sulfoxide, 200 ml deionized water.

2. Na-PPA standard solution (10 g / l): 500 mg of Na-PPA.H ₃ O (Aldrich Chemical Co., purity 98 to 100%) are added to 40 ml of 0.1 M Tris / HCl buffer (pH 8.0) at 34 ° C. It is stirred until dissolved. The solution is placed in a 50 ml volumetric flask and made up with Tris / HCl buffer. The solution is stored in a cool place. Ingredients for 0.1 M Tris / HCl buffer : 900 ml of deionized water are added to 12.11 g of Tris, the pH is adjusted to 8.0 with HCl, the solution is placed in a volumetric flask and made up with deionized water 1000 ml filled up.

Calibration curve :

Na-PPA standard solution and 4.95 ml of developing solution are placed in glass spectrophotometer tubes. It is mixed on a vortex mixer. The tubes are allowed to stand for exactly 10 minutes (timing begins when the DS is added to the first tube). The optical density (OD) is read off at 640 nm. A Na-PPA.H ^ O standard calibration curve is drawn up, with the absorption being _{plotted on the vertical axis and the mg Na-PPA.H 2} O / ml DS on the horizontal axis.

Na-PPA standard 5, ul

15αι1 20, uI

25, ul

30, ul

mg Na-PPA per ml DS 0.01 0.02 0.03 0.04 0.05 0.06

Calculation ;

Na-PPA.H "O
(mg / ml)

or

PPA (g / l)

OD χ 100 x dilution Slope of the standard curve

OD 100 χ dilution Slope of the standard curve

164.4 204.16

Claims (33)

Claims 1st Process for controlling the transamination of an amino acid precursor into the corresponding L-amino acid, characterized in that one a) chooses aspartic acid as an amino group donor, b) the suitable aL-keto acid as an amino acid precursor chooses, c) a suitable intra- or extracellular transaminase chooses, d) the transamination reaction for the transaminase activity favorable conditions with the catalyzed. Degradation of the transamination by-product Oxaloacetic acid couples, the catalyzed degradation being brought about by adding multivalent metal ions or their salts to the reaction system, and e) allows transamination to proceed. 2. The method according to claim 1, characterized in that that multivalent metal ions from the group Al, Al ⁺⁴ , Mg ⁺² , Mn ⁺² , Ni ⁺² , Pb ⁺² , Ag ⁺² , Fe ⁺² , Fe ⁺³ , Zn ⁺² , Cr ⁺² , Cr ⁺³ , Co ⁺² , Co ⁺³ , Pd ⁺² and Au ⁺³ used. 3. The method according to claim 2, characterized in that the multivalent metal ions Al - or +4
Al ions are used. +2
that Mg ions are used as multivalent metal ions 4. The method according to claim 2, characterized in that that mar turns. 5. The method according to claim 2, characterized in that that mar turns. +2
that Zn ions are used as multivalent metal ions 6. The method according to claim 2, characterized in that + 2 that as multivalent metal ions Fe - or Fe ions are used. 7. The method according to claim 2, characterized in that that the multivalent metal ions in amounts of -4 at least about 1.0 χ 10 M / l used. 8. The method according to claim 7, characterized in that the ions are used in amounts of about 5.0 χ 10 to about 1.0 χ 10 ~ M / l. 9. The method according to claim 1, characterized in that the multivalent metal ions are used in the form of metal particles. ΙΟ. Process according to Claim 1, characterized in that the multivalent metal ions are used in the form of soluble salts. 11. The method according to claim 1, characterized in that that the degradation of the oxaloacetic acid formed as a product of the transamination at a rate of about 0.02 to about 2.0 M / l / h can take place. 12. The method according to claim 1, characterized in that that a volumetric transamination rate of about 0.05 to about 10.0 M / l / h is achieved. 13. The method according to claim 1 , characterized in that that you have the * <- keto acid precursor and the L-amino acid selects from the following pairs: phenylpyruvic acid for the production of L-phenylalanine, oL-ketoisocaproic acid for the production of L-leucine, «L-ketoisovaleric acid for the production of L-valine, pyruvic acid for the production of L-alanine, ß-hydroxy-cC-ketobutyric acid for the production of L-threonine, p-hydroxyphenylpyruvic acid for the production of L-tyrosine, indolepyruvic acid for the production of L-tryptophan, oL-ketohistidic acid for the production of L-histidine and oC-keto-ß-methylvaleric acid for Manufacture of L-isoleucine. 14. The method according to claim 13, characterized in that that L-phenylalanine is produced as the L-amino acid. 15. The method according to claim 1, characterized in that that steps (d) and (e) at a temperature of about 20 to about 50 C and a pH of about 6.5 to about 10.0. 16. The method according to claim 1 , characterized in that the molar ratio of aspartic acid to the amino acid precursor is about 1: 2 to about 2: 1. 17. The method according to claim 1, characterized in: that the transaminase used is present in connection with microorganisms from the group Pseudomonas, Brevibacterium, Bacillus , Aerobacter , Escherichia coli , Candida and halophilic microorganisms. 18. The method according to claim 17, characterized in that the microorganisms are used in concentrations of about 2.0 to about 80.0 g / l based on dry weight. 19. The method according to claim 1, characterized in that that one enriches and collects the L-amino acid. 20. Improved process for the production of L-amino acids by transaminating a <X - keto acid and an amino group donor to form an L-amino acid, wherein a <A. keto acid as a precursor and the amino group donor containing solution with a the cell or enzyme preparation which mediates transamination is brought into contact, characterized in that (a) chooses aspartic acid as the amino group donor, (b) catalysis of the degradation of the transamination by-product oxaloacetic acid by adding multivalent Brings about metal ions or their salts to the system and (c) enriches the L-amino acid. 21. The method according to claim 20, characterized in that multivalent metal ions from the group Al, Al ⁺⁴ , Mg ⁺² , Mn ⁺² , Ni ⁺² , Pb ⁺² , Ag ⁺² , Cu ⁺² , Fe ⁺² , Fe ⁺³ , Zn ⁺² , Cr ⁺² , Cr ⁺³ / Co ⁺² , Co ⁺³ , Pd ⁺² and Au ⁺³ are used. 22. The method according to claim 20, characterized in that the multivalent ions are used in amounts of at least about 1.0 χ 10 M / l. 23. The method according to claim 20, characterized in that that the volumetric transamination rate is about 0.05 to about 10.0 M / l / h. 24. The method according to claim 20, characterized in that that one collects the enriched L-amino acid. 25. The method according to claim 20, characterized in that that one uses phenylpyruvic acid as a precursor to manufacture used by L-phenylalanine. 26. Catalytic process for the degradation of oxaloacetic acid during its formation in the transamination of an et-keto acid to an L-amino acid using aspartic acid as an amino group donor, characterized in that multivalent metal ions or their salts are added to the transamination system. 27. The method according to claim 26, characterized in that that one metal ions from the group Al, Al, Mg , Mn ⁺² , Ni ⁺² , Pb ⁺² , Ag ⁺² , Cu ⁺² , Fe ⁺² , Fe ⁺³ , Zn ⁺² , Cr ⁺² , Cr ^{+ 3} , Co ⁺² , Co ⁺³ , Pd ⁺² and Au ⁺³ used. 28. The method according to claim 27, characterized in that
det. + +4
that one uses Al or Al ions as metal ions 29. The method according to claim 27, characterized in that -J. ² that one uses Mg ions. 30. The method according to claim 27, characterized in that that one uses Fe - or Fe ions. 31. The method according to claim 27, characterized in that that one uses Zn ions. 32. The method according to claim 27, characterized in that that the ions are in a concentration of at least - 4th
at least about 1.0 10 M / l used. 33. The method according to claim 27, characterized in that that the rate of degradation of the oxaloacetic acid is about 0.02 to about 2.0 M / l / h.