DE3427495A1 - METHOD FOR PRODUCING L-AMINO ACIDS FROM (ALPHA) KETO ACIDS - Google Patents

Description

The invention relates generally to the biological production of L-amino acids. In particular, it was found that an L-amino acid can be prepared from the corresponding a-keto acid as the starting compound can, by the a-keto acid of a culture in the active, logarithmic growth phase of supplies suitable microorganisms in a nutrient medium.

Numerous microorganisms are able to biologically convert a-keto acids into L-amino acids.

It is known that α-keto acids can be enzymatically converted into their corresponding L-amino acids. For example, US Pat. No. 2,749,279 describes a process in which α-ketoglutaric acid and ammonia are used treated with a biological enzyme-coenzyme catalyst. Suitable biological catalysts can be made from animal tissue, from fast-growing plants or from bacteria as autolysates or cell culture extracts be won. U.S. Patent 4,304,858 also describes the conversion of water-soluble α-ketocarboxylic acids into the corresponding amino acids in the presence a substrate-specific dehydrogenase, ammonium ions and nicotinamide adenine dinucleotide (NAD + / NADH). The conversion of α-keto acids into amino acids below Use of resting cell suspensions has been reported by Yamada et al., "The Production of Amino Acids", pp 440-446 (1972).

It was found according to the invention that the biological Conversion of an α-keto acid into the corresponding L-amino acid more effectively by an active one growing microorganism culture can be conveyed. Microorganisms that are capable of converting ot-keto acids into L-amino acids to be transferred are grown in a nutrient medium. The a-keto acid is used as the starting compound for the desired L-amino acid is supplied to the culture which is in the active, logarithmic growth phase. The transamination reaction is caused by one formed by the microorganisms of the growing culture coupled enzymatic system essentially driven to completion, resulting in continuous recycling of the amino group donor (L-glutamate) and the coenzyme (NADH or NADPH). The L-amino acid can be obtained from the culture medium.

It is primarily an object of the invention to provide an extremely effective method for biological transamination of cx-keto acids into their corresponding L-amino acids.

Further, the inventive method can be carried out with numerous microorganisms and for Her position serve numerous L-amino acids.

It is a further object of the invention to provide a method in which a single strain of microorganisms sufficient amounts of everything necessary for the conversion of a particular α-keto acid to the L-amino acid Forms enzymes.

A further object of the invention is for a substantial conversion of the α-keto acid into the L-amino acid to provide within a short response time.

Another object of the invention is to provide a process by means of which a high yield of L-amino acids is obtained with a low concentration of the amino group donor.
5

Bacterial growth can generally occur in three consecutive ways Phases are divided. The lag phase is a period of time during which the number of microorganisms in the culture increases little or not at all. In the logarithmic or exponential growth phase, the cell number increases exponentially as a function of time. Little or no cell growth or division occurs in the stationary phase.

It was found that the microbiological transamination an a-keto acid into the L-amino acid can be carried out with increased effectiveness if one the α-keto acid is added to a suitable microorganism culture in the logarithmic growth phase. the Batch fed fermentation according to the process of the invention provides high yields at high yields Conversion rates. The process makes from a coupled enzyme system present in the growing microorganisms Use to allow the reaction to proceed to completion. The L-amino acid product can be obtained from the Culture medium can be obtained.

The biological conversion of the α-keto acid into the L-amino acid is an enzymatic transamination reaction. The biological conversion is mediated by a coupled enzyme-coenzyme system, that a transaminase, a glutamate dehydrogenase, that Coenzyme NADP + / NADPH (or NAD + / NADH) and a dehydrogenase. Alternatively, the latter component can be added The enzyme system can be a hydrogenase for the return of the NADP + (or NAD +) to NADPH (or NADH).

3 * 27495

The coupled reaction system can be represented as follows:

(NADPH)

or

(NADH)

(tiyäroA

\ genase /

or -

/ De- \

I hydro-J \ genase /

(3)

(NADP ^) or (NAD +)

a-ketoglutarate

matdehydro
recovered

L-amino acid

Vaminase /

L-glutamate

ot-keto acid

(2)

(D

The desired transamination reaction (stage 1), α-keto acid to L-amino acid, is normally reversible and would only take place until equilibrium is established progress, thereby increasing the conversion efficiency into the Amino acid would be limited. By coupling the transamination reaction to other enzymatic reactions, Theoretically, the transamination can be allowed to run completely, but chemical degradation reactions can occur take place, which lower the actual yield.

The coupled enzymatic reaction referred to above as stage 2) leads to one of the transamination products, the a-ketoglutarate, back to L-glutamate. This step, a reductive amination, is carried out by glutamate dehydrogenase conveys and makes the present

'Required by ammonium ions. The L-glutamate available for the transamination of the a-keto acid into the L-amino acid is continuously replaced by this recycling step.
5

In stage 2) one of the coenzymes NADPH or NADH is used, which converts to NADP + or NAD + will. In a third enzymatic reaction, step 3), this NADP + (NAD +) is either by a Dehydrogenase reaction or a hydrogenase reaction converted back to NADPH (NADH). Accordingly, that too NADPH (NADH) required in step 2) is continuously replenished. The NH. + Required in stage 2) becomes provided by the nutrient medium.

The enzymatic reaction system itself is known. In the known applications of this system, however, the addition of three different microorganisms, cf. US-PS 3,183,170, or the addition of microorganisms, Amino group donors and cofactors, cf., Yamada et al., "The Microbial Production of Amino Acids, pp. 440-446 (1976), the reaction components of the three stages set out above. Furthermore, in this known method

25 resting cell suspensions were used.

According to the method of the invention, a single Microorganism strain used, which provides the entire coupled enzymatic reaction system. A KuI door suitable microorganisms in the logarithmic growth phase, with ammonium ions in the culture medium supplied, mediates all three stages in the biological conversion of the a-keto acids into their respective L-amino acids. In the method according to the invention is in step 3) of the coupled enzyme system of a

Dehydrogenase made use of to NADP + (NAD +) to be converted into NADPH (NADH).

The coupled enzymatic reaction system is present in actively growing microorganisms. This of course occurring system can now be used for an efficient conversion of a-keto acids into L-amino acids will. With the use of suitable microorganisms with the right substrate, the conversion is carried out with high yields carried out by the microorganisms themselves.

Very different types of microorganisms can be used in the method according to the invention. For example, strains of bacteria known as glutamic acid generators have been used with success. These include ^. !! "i ^ .äi.ifiliüül — i.lliP.S. ^ Ilii.äii ⁸ - (ATCC No. 31723), Br_e_y_i_b_ac_-

t_e_ir_ ^ u_m ^ ac ^ oJ ^ rjTie ^ t ^ m (ATCC No. 13655), §. £ .e_v_i_b_ac_t_e_r_i_ijm

ammon ^ ag_e_n_e_s (ATCC No. 13746), Brevibacterium glutamigenes (ATCC No. 13747, Br.ey ^ baci ^ r ^ ujTi f l_av_u [m (ATCC No.

13826) and Co £ y_ne_bacte_ £ ^ u_m il ^ ncu ^ e ^ (ATCC No. 13868)

a. The ATCC numbers give the deposit numbers , among which each of the above cultures are in the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 30852.

It is assumed that other glutamic acid generators are also suitable in the process according to the invention are .

It was also found that E__. c_oJjL HB101, the norma-

is not normally regarded as a glutamic acid generator, can be used in the process according to the invention can and thereby yields are obtained that match those with

correspond to the above-mentioned microorganisms. E__. c_o-

l_i_ is not a glutamic acid producer in that it is glutamic acid does not excrete into the medium. All

However, microorganisms form small intracellular quantities the substance. The enzyme system of interest according to the invention only requires catalytic amounts of glutamic acid to get the reaction going, and E__. c_o_-

l_i_ seems to have adequate amounts of glutamic acid form to expire stage 2) of the coupled system allow.

A wide variety of microorganisms are found to be involved in the biological conversion process of the present invention is usable. It is primarily necessary that the microorganisms are capable of producing a cx-keto acid resorb and convert into the corresponding L-amino acid. To the biological transformation with utmost To carry out effectiveness, the microorganisms should be able to participate in stages 1) to 3) To form enzymes of the coupled enzyme system in sufficient quantities to initiate the transamination reaction to catalyze essentially to completion beyond the equilibrium point. After all, they should Microorganisms preferably be able to release the L-amino acid into the culture medium in order to be convenient and to make economic extraction possible.

The selection of those used in the method of the invention α-Keto acid depends on the L-amino acid product desired. For example, phenylpyruvic acid is used to L-phenylalanine, a-ketoisocaproic acid to L-leucine, a-ketoisovaleric acid to L-valine, pyruvic acid to L-alanine, oxaloacetic acid to L-aspartic acid, ß-hydroxy-a-ketobutyric acid to L-threonine, p-oxyphenylpyruvic acid to L-tyrosine, indolepyruvic acid to L-tryptophan and a-keto-ß-methylvaleric acid to form L-isoleucine converted. It is obvious that the conventional Starting compounds for L-amino acids can be used in the method according to the invention.

According to a preferred embodiment, the α-keto acid added to the culture as an aqueous solution, as this form the pumping of the starting compound into the culture broth relieved. However, if desired, the α-keto acid can also be in other physical forms, such as Be solid or slurry.

It has been shown that the purity of the α-keto acid as the starting compound dramatically increases the L-amino acid yield influenced, at least in the case of the conversion of phenylpyruvate to phenylalanine (see. Example 5). Next to the fact that higher amounts of α-keto acids are available in the purified starting compound, it is assumed that the microorganisms are less sensitive to the purified α-keto acid .

The concentration of the aqueous a-keto acid solution can if this is the form in which it is added to the culture, vary considerably. For example, the inventive If a continuous fermentation method is used, the solution will be more dilute than for a batch fermentation. The upper concentration limit depends on the solubility of the α-keto acid in the desired solvent. Levels of about 1.0 to 200 g / l are believed to be suitable are, the concentration depending on the particular α-keto acid and on the aforementioned factors is. It is most convenient to dissolve the α-keto acid in water, but other solvents that compatible with the fermentation, e.g. additional growth medium, can be used if desired be used.

The growth medium should be chosen so that optimal growth conditions for the microorganisms used be created. Changing and adapting the media is within the capabilities of a person skilled in the art and need not be discussed in detail according to the invention will.

In addition to the basic nutritional requirements, should however, the medium contained a source of NH. +. The ammonium ions are used both by the microorganisms Increase in cell mass as well as for stage 2) des coupled enzymatic reaction system used. Accordingly, the ammonium ion level in the culture medium should be be controlled with regard to the desired amino acid production and cell mass. The setting of the (NH. +) level is also familiar to the person skilled in the art. For example, the ions can be conveniently added by adding of ammonium sulfate in a concentration of about 10 to about 50 g / l can be supplied to the nutrient medium.

Alternatively, urea, ammonium chloride, ammonium nitrate, Ammonium acetate, etc. can be used as (NH. +) Donors.

The biological conversion process of the invention requires a culture of actively growing microorganisms. This culture can be produced by Plot sterile nutrient or growth medium with an inoculum of the desired strain. The ones to inoculate The inoculum used in the culture medium is preferably left about 6 to 40 hours before use as Grow inoculum. The period of time during which the inoculum is grown prior to inoculation varies depending of the bacterial strain used. It is desirable that the inoculum be a healthy, growing cell mass contains that is sufficient to provide a good base for

the formation and growth of the culture for fermentation to build.

The size of the fermenter depends on the specific application of the method according to the invention. The expert it is common to adjust the quantities of medium and inoculum to the requirements of a specific reactor or adapt to the desired yield. The culture is allowed to grow in the fermenter until it adheres to the Has got used to the reactor environment and has developed into a cell mass that is sufficient to allow the addition of the α-keto acid to withstand. This is particularly important when an impure starting compound is used. The optical one Density can serve as a suitable measure of cell growth. An optical density ("O.D.", 640 nm) of about 10 generally indicates that the culture is ready for the addition of the α-keto acid. This matches with a growth time of about 6 to 40 hours, depending on the organism and the growth conditions. However, this can vary somewhat and should rather be viewed as a strict requirement left to the experience and discretion of the person carrying out the work.

The culture is grown at a temperature compatible with maximum growth of the bacterial strain used is. The general range is from room temperature up to about 37 C. For

and ^ .IlS.yi ^ .äS.i ^ E.iH. ^171, the preferred temperature is about 3O ⁰ C, for E _1- CoII about 37 ⁰ C. The pH of the culture should be from about 7 to eighth If this is necessary for the selected bacterial strain, it can be ventilated.

At this stage the reactor is charged with the desired α-keto acid. As indicated above, the α-keto acid is preferably in the form of an aqueous solution. The concentration of the α-keto acid in this aqueous solution can be from about 1 to about 20% by weight. The pH of the solution can be between about 7 and about 14. The pH value can be adjusted to lower values with glacial acetic acid or other biologically compatible acids (such as sulfuric acid or citric acid) or with carbon dioxide gas. The solution may (e.g., 15 minutes at 121 ⁰ C) by steam millipore filtration (for example, 0.22 μηη pore size) sterilized or other suitable means.

If necessary, a sterile glucose solution can be used together with or for continuous culture growth at about the same time as the a-keto acid in the ferments ^ be fed in. Excess glucose remaining in the culture broth at the end of fermentation can be however interfere with the production of the amino acid product. In such a case, the amount added to the culture should be used Glucose can be calculated on the amount that is consumed by the cell mass during fermentation.

The α-keto acid (and glucose if desired) is preferred made aseptically to avoid contamination of the reactor. The α-keto acid concentration in the culture broth of the reactor is in the range from about 20 to about 50 g / l for a batch charged Batch reactor. In the case of continuous fermentation, the concentration can be lower. However, it is desirable set the α-keto acid concentration as high as possible without disturbing the metabolism, to the consumption by the microorganisms during the

35 logarithmic growth phase to maximize.

The culture is then allowed to grow for an additional time, preferably about 20 to about 72 hours. This time, during which the α-keto acid is biologically converted into the L-amino acid, is preferably as short as possible, but allows maximum yields. According to the process according to the invention, the maximum expected yields can reach a level of 100 % within a period of time as short as 23 hours, as was shown on the basis of the preparation of L-phenylalanine according to Example 6. Following the growth phase, the L-amino acid product can be obtained from the culture broth by conventional methods.

The method according to the invention is illustrated below with the aid of examples.
* j

B_e_is_p_ie_i 1_

The following inoculum culture media were prepared

_{1 1}

Inoculation culture medium Components of growth medium

Glucose ² 100.00 g

Corn steep liquor

(corn steep liquor) 2.50 g

NZ amine B 2.50 g

1.00 g ^K 2 ^HP0 4 ¹ '°° 9

MgSO ₄ • 7H ₂ O 0.25 g

30.00 G 2.50 G 2.50 G 1.00 G 0.25 G 10.00 G 1.00 3
cm 1.00 cm

) _o S0 "50.00 g

"" ■ 3 3

Biotin stock solution 1.00 cm

4th
Thiamine HCl stock solution ₃

Stock solution 1.00 cm

20.90 g MOPS buffer ⁵ 20.9Og

1 - per liter of deionized water

2 - a 70% (w / v) solution of glucose stock solution

3 - 100 mg d-biotin per liter of deionized water
4-1.0g thiamine HCl per liter of deionized water

5 - MOPS = morpholinopropanesulfonic acid

Except for the glucose, all ingredients were combined and steam sterilized at 121 ° C. for 15 minutes. The glucose solution was separately sterilized according to the same procedure and then sterile to the 3rd
remaining medium given. Sore flasks (250 cm) with inoculation culture medium were used to cultivate two inoculation cultures of five bacterial strains forming glutamic acid. The strains and the optical densities for each of the strains after the inoculations were grown for a period of 8 hours are given below:

S; tamm optical density (640 nm)

Brevibacterium 3.9 + _ 0.1

thiogenitalis (ATCC 31723)

Brevibacterium 11.2 + 0.8

¹⁵ lactofenmentum (ATCC 13655)

Bnevibactenium 6.0 + _ 0.4

ammoniagenes (ATCC 13746)

Bnevibactenium 9.2 +0.2

glutamigenic (ATCC 13747)

Brevibacterium 13.3 +0.3

²⁰ flavum (ATCC 13826)

Conynebactenium 12.3 + 0.7

hencules (ATCC 13868)

The sterile growth medium wound in subsets

3 3

of 50 cm each in slotted 250 cm shake flasks. Each flask was inoculated with a single inoculation culture on a 1% v / v basis. It was both the Bnevibactenium- than 17 hours, even grow Conynebacterium cultures in shake flasks at an agitation rate of 300 rpm at 30 ⁰ C.

An aqueous solution of sodium pheny! Pyruvic acid sore (Na-PPA) used. The solution was an unpurified sodium hydroxide hydrolyzate of 5-benzylidene hydantoin. The production of the hydrolyzate of 5-Ben-

Cylidene hydantoin (Hamsphire Chemical Co.) was carried out by the following procedure: 2233 grams of deionized water and 223.3 grams of NaOH were combined in a 3 liter stainless steel beaker and heated to boiling. With continued boiling, 350 g of 5-benzylidene hydantoin were slowly added. The boiling process was continued at 101 to 103 ° C for 2 hours. During the reaction, the solution was mixed with an electric mixer. After the boiling was complete, the mixture was slowly cooled in an ice water bath. The final volume of this hydrolyzate was 1810 ml and had a Na · PPA'HpO concentration of 162 g / l, a pH of 13.51 and a clear amber color. A 1 M solution of the sodium phenylpyruvate (hydrolyzate) contained 1.2 M NaOH, 0.5 M sodium carbonate and 0.5 M urea. The pH of the solution was adjusted to 8 with 2N acetic acid and the solution was then sterilized through a millipore filter (0.22 [in pore size).

There was a separate glucose solution prepared (70 Gew./Vol.% In deionized water) and sterilized for 15 minutes at 121 ⁰ C. The glucose solution was given sterile in a volume ratio of 3: 7 to the sterile Na-PPA solution. A volume of the composite solution was added to each shake flask to give a sodium phenylpyruvate concentration of 10 g / l in the culture broth.

Each of the cultures was left in the shake flask for more Grow for 23 hours so that a total growth time of 40 hours was achieved. At the same time, medium samples were used removed and using a high pressure liquid chromatograph (HPLC) examined for L-phenylalanine. By millipores (0.22 μm pore size) filtered media samples were treated by the dansyl chloride method prior to injection into the HPLC.

In addition, the cell dryness was determined for each medium sample.

3
weight determined. A 10 cm sample of the medium from each flask was centrifuged at 10,000 rpm for 30 minutes. The moist cell paste was dried in an oven at 80 ° C. for 24 hours and then placed in a vacuum desiccator for 15 minutes. The dry cell sample was then weighed.

For each shake flask, the average L-phenylalanine production in 23 hours per g of dry cell mass calculated. The results obtained for each microorganism strain in two shake flasks each are shown in Table 1.

Microorganisms

ATCC No. 31723 ATCC No. 13655 ATCC No. 13746 ATCC No. 13747 ATCC No. 13826 ATCC No. 13868

Cell dry weight (g / l)

39 + 0.8

22.4 + 3.2 34.7 + 0.6

46.5 + 12.6 51.6 + 5.1 42.5 + 1.5

Table 1 mM (g / l) L-phenylalanine

40.9 +1.0 (6.8 +0.2)

25.7 + 4.5 (4.3 + 0.8)

28.2 + 0.3 (4.65 + 0.05)

29.4 + 1.5 (4.9 + 0.3)

41.2 + 7.9 (6.8 + 1.3)

38.4 + 2.1 (6.4 + 0.4)

1 = M L-phenylalanine formation / hour / g cell dry weight

%-Conversion average , 6 rate , 9 (Mole / mole) liche , 6 + 1 , 7 67 +1.5 45 , 3 + 1 , 2 42.3 + 7.5 49 + O , 6 46.2 + 0.5 . 35 , 4 + 9 , 2 48.2 +2.5 30th , 3 + 3 ,8th 67.6 + 12.9 34 + O 63.1 + 3.5 39

The following culture medium (Luria broth) was produced:

Components

2 Bacto tryptone

2 Bacto yeast extract

10 NaCl

MOPS buffer ³

Lot G 10.0 G 5, 0 G 5.0 G 20.9

1 - per liter of deionized water,
2 - available from Difco,

3 - MOPS = morpholinopropanesulfonic acid.

The ingredients were combined and heated at 121 ° C for 15 minutes long steam sterilized. The sterilized medium

3
was notched in increments of 50 cm each

3
250 cm shake flask given. Each shake flask was charged with a culture of §.s ^ 2 l ^e -.! ^R .i2 li5u- ^c ii ° * ~ "^ 1 ^ 1 ^ ^au the basis of 1% vol./vol inoculated.!...

The cultures in the flask were then added for 8 hours 37 C and a shaking frequency of 300 rpm as inoculation cultures at. At the end of this period the optical density for the double value shake flasks was at 640 nm 6.27 + 0.03. This inoculation culture was

~ 3

finally used to inoculate fresh 250 crn shake flasks and put on for 17 hours before adding the flasks were charged with the a-keto acid.

It became a sterile aqueous sodium phenylpyruvic acid-glucose solution produced according to example 1. This solution was at the end of the 17 hour growth phase (optical density = 10.3 + 1.3) according to Example 1 supplied to the test cultures in the shake flask in order to obtain a Sodium phenylpyruvic acid final concentration of Get 10 g / l in the broth. Samples were taken after an additional growth time of 23 hours and dry cell weights and L-phenylalanine concentrations -) 0 trations according to Example 1 determined. The results are shown in Table 2.

Table 2

Cell cam weight% conversion average / l) mM (g / l) L-phenylalanine (mol / mol) rate

19.4 + 0.3 31.8 + 5.8 (5.3 + 1.0) 52.2 + 9.5 71.3 + 13.8

ω. . > - μΜ L-phenylalanine formation / hour / g cell dry weight '.,' '

The following vaccination culture medium was produced:

Hysoy 3.0 g

! ..- • Isoleucine 1.0 g

MgSO ₄ · 7H ₂ O 0.4 g

(NH ₄ ) ₂ S0 ₄ 10.0 g

KH PO 3.0 g

2

Trace elements 1.0 cm

CaCO ,, 15, above

3

Biotin stock solution 1.0 cm

4th

- \ 5 thiamine HCl stock solution 1.0 cm

deionized water 700.0 cm

1 - available from Sheffield Co.

2 - the stock solution of the trace elements consisted of:

ZnSO ₄ · 7H ₂ O 8.8 g

FeSO ₄ · 7H ₂ O 10.0 g

CuSO ₄ · 5H ₂ O 0.06 g

Na ₂ B ₄ O ₇ · 10H ₂ O 0.088 g

Na ₂ Mo ₂ O ₄ · 2H ₂ O 0.053 g

MnSO ₄ · H ₂ O 7.5 g

CoCl ₂ • 6H ₂ O 0.12 g

CaCl ₅ 0.055 g

3 3Q deionized water 900.00 cm

The stock solution of the trace elements was adjusted to pH 2 with concentrated HpSO and deionized

3 th water up to a final volume of 1000 cm

admitted.

3 - 100 g D-biotin per liter of deionized water

4-1.0g thiamine HCl per liter of deionized water.

The medium described above was adjusted to pH 7.5 with 5 N NaOH and deionized water up to

added to a final volume of 900 cm. The medium was steam sterilized at 121 for 15 minutes. One Glucose stock solution (50 g / 100 cm deionized water) was separately steam sterilized at 121 ° C. for 15 minutes. After cooling, the sterile glucose solution was added sterile to the sterile medium until a

3
Medium end volume of 1000 cm was reached. The pH was

10 de adjusted to 7 with 5 N glacial acetic acid.

3
Partial amounts of 50 cm each of the inoculation culture medium were

3
placed in two 250 cm notched shake flasks.

The flasks were marked with § £ e y_ ^ b ac ^ e ^ i ^ m i.! Li ^o -§L ^e -! Li £. ™ i ^s _

ATCC No. 31723 and under for 8 hours at 30 ° C Shaking (300 rpm shaking frequency) is applied to form inoculum cultures. At the end of the 8 hour growth time, the optical density (640 nm) was approximately 10.0.

Growth medium was prepared in the same way as inoculation medium, with the difference that 50 g (NaH _A ) “SO. were used. Subsets of each

50 cm of the growth medium. were notched in two

3
250 cm shake flask given. Then the

Each flask was inoculated with a 2.5 cm sample of one of the inoculum cultures. After inoculation, the shake flask cultures were 33 hours
teln (300 rpm) attracted.

flask cultures for 33 hours at 30 ° C with shaking

A sterile glucose solution (70% w / v in deionized water) was prepared according to Example 1. It an aqueous, unpurified 17.5% w / w solution of sodium a-ketoisocaproic acid (density =

3
1.125 g / cm, pH = 13.5) was used. The solution was a sodium hydroxide hydrolyzate of isobutylidene hydantoin,

prepared according to the process of Example 1 using a sufficient amount of the isobutylidene hydantoin to form a 15% solution in water and NaOH before boiling. A 1 M solution of sodium α-ketoisocaproic acid (hydrolyzate) contained approximately 1.2 M NaOH, 0.5 M sodium carbonate and 0.5 M urea. The sodium-a-ketoisocapronsäurelösung was adjusted with concentrated acetic acid to pH 7 and steam sterilized for 15 minutes at 121 ⁰ C. Equal volumes of the sterile glucose solution and the sterile sodium a-ketoisocaproic acid solution were mixed in a sterile manner and the pH was again adjusted to 7 with glacial acetic acid.

Such volumes of the combined solution were added to the shake flask cultures that the final α-ketoisocaproic acid concentrations in the broth indicated in Table 3 were obtained. The cultures were allowed to grow for an additional 62 hours. Following this growth time, samples were taken, filtered through Millipores (0.22 μm pore size) in order to eliminate the cells, and analyzed for the L-leucine content using high pressure liquid chromatography (HPLC) according to Example 1. The results are shown in Table 3.

Sodium a-keto molars

1 2 2

■ isocaproic acid L-leucine formed yield

O.O312M (4.10 g / l) 0.025M (3.28 g / l) 80.0%

0.0354M (4.61 g / L) 0.037M (4.85 g / L) ⁴ 105.0 % ^A.

0.0624M (8.12 g / l) 0.058M (7.61 g / l) 93.7%

1 - added after 33 hours of growth

2 - after a further growth period of 62 hours

3 - molar yield g of L-leucine

=. x 100

g Na-α-ketoisocaproic acid

4 - the analytical error in HPLC determinations is approx

+ 10%; it is therefore obvious that this number should be replaced by a
analytical error comes about upwards.

The inoculation culture medium and the growth medium according to example 3 were used to B £ exib_a £ t ££ ^ u_m_th ^ o_g_e_n_i_- talis ATCC No. 31723 for the biological conversion of a-ketoisovaleric acid to attract L-valine. Inoculation cultures and shake flask test cultures were prepared using this strain by the procedure of Example 3 manufactured. An aqueous, unpurified 4.53% by weight solution of sodium a-ketoisovaleric acid was used. The solution was a sodium hydroxide hydrolyzate of isopropylidene hydantoin prepared according to Example 1,

3Q in which a sufficient amount of isopropylidenehyde

toins was used to form an approximately 6% solution in water and NaOH before boiling. One 1 M. Sodium α-ketoisovaleric acid solution contained approximately 1.2 M NaOH, 0.5 M sodium carbonate and 0.5 M urea.

The pH of the solution was 13.8 and was

sig set to 7.0. The solution was ^{steam-sterilized at 121 °} C. for 15 minutes.

A separate glucose solution (70% w / v in deionized water) was prepared and stored at 121 ° C Steam sterilized for 15 minutes. 70 parts of the sterile u-Ketoisovaleric acid solution became sterile to 30 Divide the sterile glucose solution given. In different stages of growth there were such volumes of these Mixture added to the shake flask test cultures that the final concentrations of sodium a-ketoisovaleric acid in the broth were either 10 or 15 g / l, as shown in Table 4. The total growth time was 40 hours for each shake flask test culture. At this point, samples were taken, filtered through millipores (0.22 μm pore size) and using analyzed by HPLC according to Example 1 with regard to the L-valine content.

in a control examination the same microorganisms were found and method used sodium a-ketoisovaleric acid but omitted from fermentation. The controls produced an average of 0.0205 M. (2.4 g / l) L-valine from glucose. This amount was found in the test results given in Table 4 not taken into account.

Table 4

N at r ium-a-keto-

time

isovaleric acid formed by adding L-'Valin

molar yield ⁴¹

0.0725M (10 g / l)

16 hours 0.0846 +, 008M

(9.90 + 0.90 g / l)

99%

O, O725M (10 g / l)

24 hours 0.0910 +, 003M

(10.65 + 0.35 g / D '

106

0.1087M (15 g / l)

16 hours 0.1004 + 005M

(11.75 + 0.55 g / l)

1 - after 40 hours of total growth time
2 molar yield g L-valine

78%

χ 100

g sodium a-ketoisovaleric acid

3 - the analytical error in HPLC determinations is approximately + 10.0; it is therefore obvious that this number came about through an analytical error upwards.

25th

30th

In this example, the biological conversion efficiency of Bll ^ i ^ ac ^ e £ ^ umtl2iogeini _L tali _: s ATCC 31723 was compared when a potassium hydroxide-5-benzylidene hydantoin hydrolyzate (potassium phenylpyruvic acid) or a 98% pure sodium phenylpyruvic acid was offered as a substrate . The α-keto acid solutions were prepared as follows:

Potassium phenylpyruvic acid: An unpurified potassium hydroxide hydrolyzate of 5-benzylidene hydantoin was obtained (obtained from Hampshire Chemical) according to Example 1 from

3M KOH produced per mole of 5-benzylidene hydantoin. Of the The pH of the hydrolyzate was adjusted to 8.0 with C0_ gas before it was added to the shake flasks.

Sodium phenylpyruvic acid: Solid sodium phenylpyruvate monohydrate (98%) obtained from Aldrich Chemical Company was dissolved in 0.6 M aqueous KOH to a concentration of 98 g / L (as sodium phenylpyruvate monohydrate (Na * ΡΡΑ · Η _? Ο)). The pH was adjusted to 8.0 with CO gas before adding to the shake flasks.

The inoculation and growth media used were prepared according to Example 1, with the difference that the MOPS buffer was replaced by 20 g / l CaCO2 and the glucose and other components of the medium were autoclaved together for 10 minutes at 110 ° C. (instead of the separate steam sterilization). The inoculation cultures were according to Example 1 using the strain § £. ^e - ^v _i ^ .2: 2.i. ^e . £ .i ^u . ^m ._i.! li ^o .ä ^e -Ilii.SS: i.i2. ^{AT (} ^ C 31723 made.

The inoculum cultures were used to inoculate the shake flasks according to Example 1. After an initial Individual shake flasks were filled with the phenylpyruvic acid solutions for the 17 hour growth period charged in quantities such that the final phenylpyruvate concentrations in the culture media was 76.22 mM (12.51 g / l as pheny! pyruvic acid). The cultures were left grow in the shake flask and the molar yields were after 23 hours (40 hours total growth time) and determined after 28 hours (45 hours total growth time). The HPLC determinations were according to Example 1 carried out. The results, shown in Table 5, show that the ability of

Liüü! ÜliP.S.f.n.ii. ^ i.ii ATCC 31723 for biological conversion

treatment of Phenylpyi-uvat to L-phenylalanine in the invention The process is not fully exploited when an impure output compound is used.

Table 5

Phenyl- ^

Pyruvate L-phenylalanine formation (rnM)

2 2

23 hours molar “28 hours molar

Yield yield

Potassium phe-

nylon pyruvate 35.96 + 0.85 47.2 + 1.1 49.52 + 12.7 65 +16.7

Sodium phe-

nylon pyruvate 67.80 + 0 89.0 + 0 69.19 + 1.2 90.8 + 1.6

1 - Double values of the shake flask examinations

2 - time since phenylpyruvate was added

3 - molar yield mM L-phenylalanine

χ 100

mM phenylpyruvate

Example 6

In this example sodium phenylpyruvic acid (Na * PPA) was used, which was precipitated from a sodium hydroxide hydrolyzate had been purified from 5-benzylidene hydantoin. The hydrolyzate was produced according to Example 1

30 have been presented.

Acetic acid precipitation was then carried out on the hydrolyzate carried out. 1500 ml of the hydrolyzate were in transferred to a 2000 cm glass beaker in an ice water bath. Glacial acetic acid (concentration 99.7%, 17.4 M) became

added slowly with stirring to lower the pH to 9. The temperature was not allowed to rise above 30 ° C. A precipitate began to separate out at about pH 12.95 and a pH of 9 was reached after 15 minutes.
5

The mixture was left covered at 23 ° C. for 1 hour and stand under a fume cupboard. The slurry was filtered through No. 4 Whatman paper without washing. The wet precipitate was in a for 18 hours Vacuum oven kept at 50C. The dry precipitate was passed through a sieve with a mesh size of 0.420 mm given. The end product was Na-PPA-H “0 with 79% purity, with the other ingredients acetate, Na +, small amounts of CO ~ and organic

15 trapped nitrogen.

The inoculum and growth culture media were prepared according to Example 5.

31723 was attracted in the inoculation medium according to Example 5

3 gen. The inoculum was to inoculate 250 cm

Shake flask used.

After an initial growth time of 18 hours, the precipitated Na-PPA-H ₂ O was dissolved in 0.3 M KOH (100

g / l as Na-PPA-H 0) in the shake flask, see above that the final concentrations shown in Table 6 in the broth were obtained. The cultures were left for grow another 23 hours. HPLC determinations of L-phenylalanine were carried out according to Example 1. The results are shown in Table 6. The results are approximate because the Liquid loss from evaporation from the shake flask was not measured.

Table 6

Trial number

Na »PPA» H ₀ O

1.73 + 0.12 3.06 +0 3.3 +0.08 5.71 +0 5.75 + 0.36 6.80 + 0.05 8.16 + 0.44 9.13 +0.36 9.45 +0 9.49 +0 12.91 + 0.04

! - Phenylalanine ^

1.8 + 0.09 3.69 + 0.02 3.57 + 0.19 6.32 + 0.19 6.18 + 0.11 6.05 + 0.05 7.4 + 0 6.99 + 0.14 8.35 + 0.15 7.82 + 0.12 10.05 + 0.95

Growth after 23 hours
(OD 640 nm) molar .
yield 34 + 4 41 + 2 100 40 + 2 100 24 + 1 100 27 + 1 100 23 + 0 100 43 + 2 88 43 + 0 90 27 + 0 76 36 + 7 88 24 + 1 82 37 + 5 78

- Double values of the shake flask tests

2 - colorimetrically determined PPA concentration in the medium (g / l)

- L-phenylalanine concentration (g / l) formed in 23 hours.

- Molar yield of M L-phenylalanine

M phenylpyruvate

χ 100.

Claims (18)

UEXKÜLL & STOLBERG PATENT LAWYERS BESELERSTRASSE 4 D 2000 HAMBURG 52 EUROPEAN PATENT ATTORNEYS DR. J. D FRHR from UEXKÜLL DR ULRICH GRAF STOLBERG DIPL ING. JÜRGEN SUCHANTKE DIPL-ING. ARNULF HUBER DR. ALLARD from KAMEKE W.R. Grace & Co. 1114 Avenue of the Americas New York, N.Y. 10036 V.St.A. (Prio .: 5th August 1983 US 520 632 0665 / UGS / VOE / wo) July 19 84 Process for the production of L-amino acids from α-keto acids Claims Process for the preparation of L-amino acids, characterized in that one a) selects microorganisms that are capable of producing a convert a-keto acid into the corresponding L-amino acid, b) attracts the microorganisms in a nutrient medium, c) supplies the desired α-keto acid to the microorganisms during the logarithmic growth phase and d) the α-keto acid from the growing microorganisms can be converted into the corresponding L-amino acid. 2. The method according to claim 1, characterized in that there is used microorganisms that are essentially contain a single strain of bacteria. 3. The method according to claim 1, characterized in that there is excreted glutamic acid as microorganisms Bacteria or! S ^ h ^ r ^ ch ^^ c ^ olä used. 4. The method according to claim 3, characterized in that the glutamic acid forming and excreted - \ 5 endogenous bacteria from the genera and Corvnebacterium selects. 5. The method according to claim 1, characterized in that the L-amino acid from the culture medium is 20 wins. 6. The method according to claim 1, characterized in that the growth phase according to step b) is about 6 to about 40 hours and the growth phase according to the study 25 fe d) is about 20 to about 72 hours. 7. The method according to claim 1, characterized in that a culture medium is used in step c), that the α-keto acid in amounts of about 0.5 to 30 contains about 5.0 w / v%. 8. The method according to claim 1, characterized in that the α-keto acid is used as an aqueous solution . 9. The method according to claim 8, characterized in that that the concentration of the aqueous solution is about 1.0 to about 20 w / v%. 10. The method according to claim 1, characterized in that that the α-keto acid is phenylpyruvic acid, α-ketoisocaproic acid, a-ketoisovaleric acid, Pyruvic acid, oxaloacetic acid, ß-hydroxy-a-ketobutyric acid, p-Oxyphenylpyruvic acid, indole-Ί0 Pyruvic acid or a-keto-ß-methylvaleric acid begins. 11. Process for the biological transamination of a-keto acids into their corresponding L-amino acid -j 5 ren using the coupled enzyme system a bacterial culture in the logarithmic growth phase, characterized in that that he a) a microorganism culture in the logarithmic growth phase which is capable of a-keto acids into their corresponding L-amino acids to convert, contacted in the presence of a nutrient medium with the desired a-keto acid and b) the transamination of the α-keto acid into the L-amino acid can be catalyzed by the coupled enzyme system of the growing culture. 12. The method according to claim 11, characterized in that the L-amino acid product of the transami nation reaction from the medium wins. 13. The method according to claim 11, characterized in that glutamic acid is formed as microorganisms ₃₅ dying and excreting bacteria or coli are used. 14. The method according to claim 13, characterized in that the glutamic acid-forming bacteria from the genera BH ^ vi ^ ä.Sif-lliy.OI ^unc '2.2. £ .y_ ⁿ .§.kä £ i ^e - ~ £ iu.m. selects.
5 15. The method according to claim 11, characterized in that the α-keto acid is phenylpyruvic acid, α-ketoisocaproic acid, α-ketoisovaleric acid, Pyruvic acid, oxaloacetic acid, ß-hydroxy-a-ketobutyric acid, p-oxyphenylpyruvic acid, indolepyruvic acid or a-keto-ß-methylvaleric acid is used. 16. The method according to claim 15, characterized in that the a-keto acid is used as an aqueous solution used. 17. The method according to claim 16, characterized in that the concentration of the aqueous solution is about 1 to about 20 % . 18. The method according to claim 11, characterized in that the culture in the logarithmic Contact with the α-keto acid for about 20 to about 72 hours during the growth phase.