JPS6043391A - Biological preparation of l-amino acid from alpha-keto acid - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention generally relates to the biological production of L-amino acids. The application was filed in August 1983 [of amino acids from alpha-keto acids! 1I3t+(prod
uction of L-A m1no A cid
s from Alpha-Keto Ac1dsJ (
Murray C., Fusee) discloses that L-amino acids can be prepared by supplying alpha-keto acids to cultures of suitable microorganisms growing actively and logarithmically in a nutrient medium. gi from the corresponding alpha-keto acid precursor
It has been disclosed that alpha-keto acids can be prepared by biologically converting them into monoamino acids.
There are many microorganisms that exist.

The optimal rate of alpha-keto acid supply can be directly related to the change in the l1l-1 value of the culture caused by the change in the growth rate of the culture. Addition of alpha-keto acids in this manner results in high conversion of the J:recursive precursor to monoamino acids. Furthermore, this method controls the culture medium and provides the microorganism with an optimal environment to continue the logarithmic growth phase.

It is known that alpha-keto acids can be enzymatically converted to their corresponding amino acids. For example, U.S. Pat.
No. 49279 discloses a method for treating alpha-ketoglutaric acid and ammonia with a biological enzyme-coenzyme catalyst. Suitable biological catalysts include autolysates from animal tissue, rapidly growing plants or bacteria.
ysate) or as an extract of cell culture.

Similarly, U.S. Pat.
The conversion of water-soluble alpha-keto carboxylic acids to the corresponding amino acids in the presence of H> is disclosed. Resting cells (
The conversion of alpha-keto acids to amino acids using a suspension was described by Yamada et al.
he production of a m1no
A aids.

440-46 (1972).

There are many microorganisms that can biologically convert alpha-keto acids to L-amino acids. Related application F usee
The patented method utilizes these microbial-based conjugated (formerly Goled) enzyme systems to induce transamination reactions that convert alpha-keto acids to L-amino acids. By utilizing an actively growing culture in this manner, the reaction is driven to substantial completion and the amino donor (glutamate) and coenzyme <NADPH or NADH> are continuously recycled. was discovered by Fusee. This amino acid can be recovered from the culture broth.

The Fusee method described above was improved by linking the alpha-keto acid supply rate to the rate of growth of the culture. It was thought that microorganisms produce various acids when they are in the active growth phase. The decrease in p1 value of culture broth acts as a natural constraint on cell proliferation and IIJII
If not treated, there will be negative effects on growth and metabolism. The improvement method disclosed herein consists of adding a basic solution of an alpha-keto acid in an amount sufficient to correct this naturally occurring decrease in 11H values. Thus, alpha-keto acid precursors are supplied to the culture at a rate approximately proportional to the growth rate. This rate is approximately proportional to the extent to which the coupled enzyme systems of the co-growing microorganisms are available for biological transformation. It has also been found that the upper pH range of the culture broth maintains an optimal environment for active metabolism.

Cultures grown by this improved method are alpha9- The timing and rate of monoketo acid supply is self-controlled.

A related object is to provide a method for maintaining optimal environmental conditions for microbial growth and metabolism.

Furthermore, it is an object of the present invention to eliminate a process step in biological transformation by using an alpha-keto acid solution to feed the culture without first having to neutralize it.

A related objective is to prevent possible loss of active ingredient by eliminating this neutralization step.

Furthermore, the methods of the invention are useful with a wide variety of microorganisms and are useful for the production of a wide variety of monoamino acids.

It is also an object to provide a method by which a single microbial strain can produce sufficient amounts of all the enzymes necessary to convert a given alpha-keto acid into a single amino acid.

Large amounts of alpha-keto acid L within a shorter reaction period
-Aim 10- to provide conversion to amino acids.

Furthermore, it is an object to provide a method that produces high yields of L-amino acids in the presence of low concentrations of amino donors.

Bacterial growth can generally be divided into three sequential phases. In the lag phase, the number of microorganisms in culture is almost
Or there is a period of no increase at all.

In the logarithmic or exponential growth phase, the number of cells increases exponentially over time. During stationary phase, little or no cell proliferation or division occurs.

It has been found that the biological conversion of alpha-keto acids to L-amino acids can be carried out more efficiently by adding the alpha-keto acids to suitable logarithmically growing microorganisms. This method has the advantage that the transamination reaction is completed by a coupled enzyme system present in the growing microorganism.

The present invention disclosed herein improves this biological amino group conversion method by adjusting the alpha-keto acid supply rate in direct response to the rate of acid production, which also corresponds to the growth rate of the culture. It is something to improve. This alpha-keto acid is supplied to the culture as a basic aqueous solution. This base is added to counteract the acid produced by the microorganism during the growth phase. As a result, the pl-1 value of the cultured broth remains substantially constant. This "demand feeding" method has the advantage of increasing the metabolic potential of the 18 nutrient solution since the culture is in the logarithmic growth phase. In addition, steady state DH values provide an optimal environment for enhanced bacterial growth and metabolism. When fermentation is complete, the amino acid product can be recovered from the culture broth.

ヱ32mυL The biological conversion of alpha-keto acids to L-amino acids is an enzymatic transamination reaction. This biological conversion involves aminotransferase, glutamate dehydrogenase, and coenzyme NA.
DP+/NΔDPH<or NAD+/NADH),
mediated by a coupled enzyme-coenzyme system consisting of dehydrogenase and dehydrogenase. The latter component of the enzyme system can also be a hydrogenase to recycle NADP+ (or NAD+) back to NADPH (or NAD+-1).

The conjugated reaction system can be represented by the following diagram: (NADPH> or (NAD+) (3) (2> (1) 13- Desired amino group conversion reaction [step (1)] The step from alpha-keto acids to L-amino acids is usually reversible and only equilibrated, thus limiting the efficiency of conversion to amino acids.By coupling this amino group conversion reaction with other enzymatic reactions, Although this amino group conversion is theoretically complete, chemical deterioration reactions exist, reducing the actual yield.

The coupled enzymatic reaction represented by step (2) above cycles some of the amino group conversion product alpha-ketoglutarate back to monoglutamate. This step, reductive amination, is mediated by glutamate dehydrogenase and requires the presence of ammonium ions. The monoglutamate obtained for the amino group conversion of the alpha-keto acid to the monoamino acid is continuously replaced by this cycle.

Step (2) is the coenzyme N A D P H or N A
D+-1, where this one is NADP
It is converted to 14- or NADH. The third enzymatic reaction, step (
In 3), this NADP+ (NADH) product is converted to N via either a dehydrogenase reaction or a hydrogenase reaction.
ADP+-1 (converted to NADI-11. Thus also the N A D P +-1 required for step (2)
(N A D +-1) is continuously replenished.

■NHI+ required by the culm (2) is supplied in the nutrient medium.

Enzyme reaction systems are known per se. However, before using this system, the above-mentioned US Pat. No. 3,183,170 (
The addition of three different microorganisms to supply the reaction components of the three steps described in Kitai et al.), or microorganisms;
Addition of amino donor and cofactor [Yamada et al., TheM 1crol)ial pro
Cluc mouth on of A m1no A cids.

440-46 (1976)]. Furthermore, these conventional methods use suspensions of resting cells.

The biological transamination method disclosed by Fusee uses a single microbial strain to provide an entirely coupled enzymatic reaction system. Logarithmically growing cultures of suitable microorganisms with ammonium ions in the culture medium mediate all three steps of the biological conversion of alpha-keto acids to their respective amino acids.

Step (3) in the coupled enzyme system of the Fusee method is to convert NADP+ (NADH) to NADPII (NADH
) using a dehydrogenase reaction.

Sentence i1 A coupled enzymatic reaction system exists in actively growing microorganisms. This naturally occurring system can be utilized for the efficient conversion of alpha-keto acids to single amino acids. By providing suitable microorganisms with suitable substrates, this transfer is carried out by the microorganisms themselves in high yields.

There is a wide variety of microorganism types suitable for use in this bioconversion process. For example, it is reported in a related application by Fusee that bacterial strains known as glutamate producers may be utilized. These include Brevibacterium ([3rf3Vil) a (iterillll)
) Species, Corynebacterium? (corVnebactar
ta ) species and E. coli LI B 101. The improved method of the present invention is based on Prehybacterium thiognitalis (th+-ogenitalis) (ATCCN).
o, 31723>, Brevibacterium lactofermentum (lactof-ermentum) (
ATCC No. 21420> and Prehyacterium flavum < AT CC No. 13
826) can be used (see Examples). ATCC No shown here.

are American Type C11ltllr respectively.
e Co11ection.

12301 Parklawn Drive, Roc
kville.

The designation number given to the culture by Marylanrl 30852, to which each of the above-mentioned cultures has been deposited. It is also anticipated that other microorganisms capable of carrying out basic biological transformations and which secrete significant amounts of acid during the logarithmic growth phase will be suitable for use in the improved process.

17- It is clear that there is a wide range of microorganisms useful for this biological transformation method. The first requirement is that the microorganism be able to absorb alpha-keto acids and convert them into the corresponding monoamino acids. In order to carry out this biological transformation most efficiently, microorganism-conjugated enzyme-based processes (1) to (
It should be possible to produce the enzyme contained in 3) in a sufficient amount to substantially complete the reaction and induce the transamination reaction beyond the equilibrium point. Finally, the microorganism should preferably be able to secrete L-amino acids into the culture medium so that they can be conveniently and economically recovered.

A second requirement is that the microorganism produce and secrete significant amounts of acid during logarithmic or exponential growth phases. Examples of the most commonly secreted acids include lactic acid, glutamic acid, and succinic acid, although secretion of other acids is also suitable for purposes of the present invention. The amount secreted, if not externally controlled by the addition of base, should be sufficient to lower the pH value of the culture broth to a range of about 4-6.

The alpha-keto acid chosen for use in the aqueous solution depends on which L-amino acid is the desired product. For example, phenylpyruvic acid converts to l-phenylalanine,
Alpha-ketoisocaproic acid converts to L-leucine, alpha-ketoisovaleric acid converts to L-valine, and pyruvic acid converts to [
-Alanine, oxaloacetic acid to L-aspartic acid,
β-Hydroxy-alphake: -acetic acid to L-threonine, 1)-oxyphenylbilhinic acid to 1-tyrosine, indolepyruvate to L-tria], alpha-keto-β-methylvaleric acid It is converted into isoleucine, etc. Thus, common amino acid precursors are used in the method of the invention.

The alpha-key I-acid used in the present invention is preferably a basic aqueous solution. For example, the alpha-key 1-acid may conveniently be dissolved in 0.1-0.6M aqueous potassium hydroxide. Mono-lithium hydroxide, ammonium hydroxide or urea can also be used, and any other base compatible with the growing culture and transamination reaction can be used. When using phenylpyruvic acid (PPA) as a precursor, it has been found that KOH is preferred since PPA is more soluble in K O+-1. It is anticipated that certain bases may be suitable for use with certain alpha-keto acids for solubilization purposes. The concentration can be varied to maintain the desired 11H value in the fermenter and the feed rate adjusted accordingly. However, typically the concentration of base will be between about 0.1% (w/v) to about 20% (w/v).

When using alpha-keto acids in the form of sodium or potassium hydrolysis products, it is usually not necessary to dissolve the alpha-keto acids in a base. For example, by boiling 5-penzylidenehytantoin (5-BH) with water and NaOH and then cooling in a water bath! 113
i! i (see Example 3) Sodium hydrolysis product of 5-BH (Na phenylbildate) is about 13.
This will result in a solution with a pH value of 5. Similarly, alphake 1--itsukaprone HNa hydrolyzed from isoptylidenehytantoin has an rll-
1, and alpha-ketoisovaleric acid hydrolyzed from isoprohylidenehydantoin has a value of ca.
It has an l11-1 value of 3.8. These compounds are suitable for use as l) H control agent in the method of the present invention.

It is preferable to add the alpha-keto acid (J) to the culture broth as an aqueous solution of the appropriate base, since it is in this state that the precursor is used for pumping into the culture broth. However, if desired The alpha-keto acid can be used as a slurry of a suitable base.

The concentration of the aqueous solution of alpha-keto acid can vary considerably when it is being fed to a culture. For example, when the present invention is used for continuous fermentation, the solution is more diluted than for batch fermentation. The upper limit for α21- may depend primarily on the dissolution of the alpha-keto acid in the desired solvent. About 1.0-200M for the specific alpha-keto acid and the concentration depending on the factors mentioned above.
It is anticipated that an amount of 1 may be suitable. The most convenient and economical solvent is water, but other solvents compatible with the system, such as culture media, can be used if desired.

Nutrient media should be chosen to provide optimal conditions for the microorganisms used. The selection, type and adjustment of media are well within the capabilities of the person skilled in the art and need not be described in detail here.

However, in addition to the basic necessary nutrients, this medium should contain sources of N HI+. This ammonium ion is used by the microorganism both for growing the cell body and for step (2) of the coupled enzyme reaction system. Thus, the amount of ammonium ions in the culture medium should be matched to the desired production of amino acids and cell bodies. The preparation of this NH4''I is well known to those skilled in the art.
~About 500. It may be convenient to supply ammonium sulfate by adding it to the nutrient medium at a concentration of 1 liter. In addition, urea, ammonium chloride,
Ammonium nitrate, ammonium acetate, etc. can be used.

Reaction Method The biological transformation of the present invention requires an actively growing culture of microorganisms. This culture is seeded with the desired strain (5e
ed) This culture can be prepared by inoculating the culture into a sterile nutrient solution or growth medium. Preferably, the seed culture used to inoculate the nutrient medium is allowed to grow for about 6 to 40 hours before being used as an inoculum. The amount of time allowed to grow the seed culture before inoculation will vary depending on the bacterial strain used. It is desirable that the seed culture consists of sufficiently healthy and growing cell bodies to form a good basis for the establishment and propagation of the fermentation culture.

The size of the fermentation reactor depends on the particular application of the process. It is within the knowledge of those skilled in the art to adapt the medium and seed culture volume to a particular reactor or desired yield.

The culture is grown in a fermentation reactor until it acclimatizes to the reactor environment and grows enough cell bodies to withstand the addition of alpha-keto acid.

This is particularly important when using precursors containing impurities. Optical density can be a convenient measure of cell growth. The optical temperature ([O9D,J) of approximately 10 (640n
m) generally indicates that the culture medium is ready for the addition of alpha-keto acids. This corresponds to a culture period of approximately 6 to 40 hours, depending on the organism and culture conditions.

However, this is somewhat variable and should not be seen as a strict requirement, but should be left to the experience and choice of the operator.

Cultivation is carried out at a temperature compatible with maximum growth of the bacterial strain used. A typical range would be from ambient temperature to about 37°C. For Corynebacteria and Brevibacterium the preferred temperature is about 30°C. ll l-11 of the culture medium before the drop in p)-1 value with the onset of logarithmic growth.
ii1 is preferably about 7-8. Aeration can be performed for selected bacterial strains as required.

During the lag phase (la(1) and logarithmic growth, many microorganisms,
For example, Prehybacterium and Corynebacterium species produce and secrete various acids.

These acids include glutamic acid, lactic acid, succinic acid and others. If not controlled by the addition of bases, the addition of these acids to the culture medium will result in a pH value of approximately 4.
It will drop to ~6. Bacterial growth and metabolic rates are significantly adversely affected by this range of reduced pl-1 values.

Conversely, when growth begins and reaches stationary phase, acid production by the microorganism decreases and almost ceases. As the population of cells dies, IJRW compounds such as amines are released into the fermentation broth. This uncontrolled release of bases causes the IIIH value to rise above about 8. High 1) H values are also harmful to the culture medium and inhibit metabolism and kill at least part of the remaining living cell population.

It is the actively growing and metabolizing cell culture that provides the coupled enzyme system that induces the transamination reaction of this method. It has been found that by feeding the alpha-keto acid precursor to the culture according to the needs of the IIH value, the precursor is provided to the microorganism at a rate that is substantially comparable to its ability to convert into L-amino acid products. Ta. As the culture enters the logarithmic growth phase and the pH value begins to decrease, the alpha ketone is preferably added at a rate that maintains the pH value at a predetermined value of about 6.5 to about 7.5 or higher. Supply a basic solution of the acid.

Additionally, it is desirable to establish an upper pH value in the range of about 7.5 to about 8. This will serve to maintain microbial health and metabolic live bamboo even after active growth has declined. This upper limit pl-
A value of 1 can be maintained by feeding the culture medium with any acid compatible with fermentation. Suitable acids include concentrated glacial acetic acid, sulfuric acid, citric acid, phosphoric acid, and the like. Any acid compatible with microbial growth or metabolism and transamination can be used.

Any suitable method can be used to continuously monitor the 11) value of the culture broth and add the base or acid necessary to control the IIH value within the range of the present invention. can. For convenience and accuracy, an automated pl-1 sensor or Preferably, an il controller is used. In this way, it is possible to respond immediately and appropriately to changes or fluctuations in the p l-1 value of the meat juice.

This equates to a virtually instantaneous response to changes in cell growth and metabolic rates.

Of course, the rate of base or acid feed depends on the 0 day value of the added solution and the 81r! I as well as the pl-1 requirement of the culture broth. Furthermore, it is conceivable to wait with continuous or slug feed of base or acid until the desired IlH value is reached. Approximately 1 to 3 for a fermenter with a capacity of 1700 m1
It has been found that a feed rate of ml/min is suitable, with the feed period optionally ranging from 1.0 seconds to about 10 minutes.

During the initial (induced) growth phase, ie after inoculating the seed culture, the lower pH limit can be controlled by adding any base compatible with the growing culture. During this initial period, the culture does not reach logarithmic growth phase in which alpha-keto acid precursors become active and convert to monoamino acids. During this induced growth phase, basic alpha-ketol solution can be used for pH control, but this is detrimental to the integrity of the culture, especially when using unpurified precursors.

In either case, the precursors are not fully utilized until logarithmic growth is reached. For this reason, it is preferred to use a suitable base during the initial growth phase, one compatible with the growing culture medium free of beaten precursors. Suitable bases include, for example, ammonium hydroxide, urea, sodium hydroxide, potassium hydroxide and ammoniacus.

The first mark starts at about 6-10 o'clock, 0. D, (640 nm) reaches about 10 and the IIIH value decreases to about 6.8-7°0, lower the pH by adding alpha-keto acids into the basic solution as shown in the previous section. - Further control the value. Although the feed rate during this phase of logarithmic growth (or acid production) is substantially determined by IIH requirements, it is desirable to maintain the alpha-keto concentration in the broth below about 15 M1.

Concentrations higher than this are detrimental to the growing culture, especially when using unpurified precursors.

As much precursor solution as possible is fed into the culture broth according to the pi-f value. The production of acid by bacteria increases the pf-1 value of the medium from about 6.8 to about 7.5, preferably about 7.
．． If it decreases to a value that is no longer sufficient to bring it below 2, the limit for the l) l-1 value can be raised in order to supply additional 29- precursor based on the pH requirement. For example, the lower rlH range can be raised to about 7.8.

After the feeding based on the pH requirement is completed, the remainder of the total volume of alpha-keto acid that would be added to the L-amic acid is fed to the gravy. The remainder of the alpha-keto acid is added for up to 24 hours or more, preferably 2 hours.
If fed over a period of ~8 hours, this is converted to a substantial amount of amino acids. For example, 80 (
]/I precursor solution 300-400 cc at 700C
If it is desired to add to a volume of meat broth of C, about 1/3 to 1/2 of the precursor can be supplied depending on the pal requirement during the logarithmic growth phase. Feed the remainder over the next 2-8 hours or more.

The upper limit value of pt-+ is set to a predetermined value in the range of about 7.5 to 8.0, preferably about 7.8.

As mentioned above, this range is also controlled for optimal preservation and metabolic activity of the culture medium. Regarding the lower limit p(-1 control), it is also preferable to provide an automatic p t-1 sensor (j゛-) for starting the addition of acid to the cultured meat juice.

Add 1+ acid at a rate sufficient to maintain the gravy +11-1 value below the specified upper limit. For example, the concentration of this acid is about 1%.
(weight/volume) to approximately 30% (weight/volume). However, particularly in the batch process, a concentrated solution of C is preferred, since a small amount is added to the total volume of the photofermented j8 product.

In the batch method, the culture solution is heated to a temperature ranging from about 20° to 7°C.
0. From the values, the 1-amino acid product can be recovered from the culture broth after reaching an apparent stationary growth phase. If a continuous process is used in the process of the invention, the product can be recovered continuously. L-amino acids can be recovered from cultured broth using conventional methods.

The following examples are presented for illustrative purposes only and are not intended to limit the invention described herein. In this example, the following approximately 8n are used: °C - cubic centimeters cc - grams hr - hours l - shoes 1 - lpm - liters per minute μ - microns M - moles 1n - minutes 1 - milliliters ram - millimeters mMo1 - millimoles N - normal nll1 - nanometers % - percent RPM - revolutions per minute vol - volume wt - ψ quantity Example 1 Seeds per l]Ga ionized water 11 as follows
' and growth medium were prepared: (30,., wa7.-]-2 Hand 100.0 0 2.5 g ~ Corn soaking liquid 2.5 g 12.5
(] NZ amine B22,5 (l B1, OOK2
HPO41,Og FO,25Q Ml]SO4・7f-1io 0.25
g 110.0 0 <NH4)! 804 40.
00 'g1.0 1111 Hithione storage solution n31.
Oml hl, Oml thiamine-1-I CI stock solution' 1.0 if f modified J-'--Qa CO3S
al-70% (wt/vol) glucose storage solution. h2
-8heffieltl Co, Ji3-Tax ion A 10oII 1g of d~Rubithion per 11 ionized water 4- Theory 1. Oo's Thiamine Kushi-l-I
C.I. I 5 - Added to seed medium for buffer purposes.

33- Combine the ingredients of 1 ply and heat to 110℃ for sterilization purposes.
Disinfect for 10 minutes. After disinfecting the medium with NaOH)
The lH value was adjusted to 7.4.

Prehybacterium thiogenitalis ArccNo.
, 31723 was inoculated. 300 ml before using this culture as inoculum for the acid tank. Shake with PM for 17 hours, then incubate at 30°C, volume 2+ (processing volume 11)
Equipment controlled, 4ultigen fermenters (N eW
BRILLNSWick CO,). Before use, one volume of 0.211 polyethylene glycol 2000 was added to the fermenter to prevent foaming. The fermenter was then extinguished at 110° C. for 10 minutes. Starting volume - 9 ports of 700 ml of growth medium were added to this fermentation 1. Seed [0, D, (64
0nm) = 22mm 501 were added. 1.0 per minute
3 while aerating by blowing air at 1.25 (LPM).
The fermentor was operated at 0° C. and 600 PPM.

34- Mild NH401-1 (
29%) solution was used to control the pH value of the fermenter by hand. During the first 4 hours of R, 1) l-1 values changed little or not at all; For the next 4 hours, both needles were maintained at a constant value by visual observation. N tl 401-1 as required
0 mouths were added, and the total amount of 0 mouths gained during this period was approximately 2. It was OCC.

After the first 8 hours, p t-11I11.11111M
odel 5997 (1-1orizon E col
ogy Co, ) and pl-1 electrode (200vn)
(1)] using I ngold > automatically.
I controlled hl+. 7 when the pH1ii of H in the culture is 6.8 or higher
．． A program was created to control the l)H value of this system so that it was 81 meters below. When the pH value of the medium decreases to 6.8, approximately 1-31/
Add the base for 1 minute (peristalt)
ic) program to send a signal to the pump pHiI'
I had given it to III in advance.

The base consisted of phenylpyruvin-11 salt in aqueous ammonium hydroxide. Purified phenylpyruvate (PPA) was dissolved in Na-PPA・H2O (98%
;MW204.16) as Aldrich Chem
ical Co. This was dissolved in 0.6M aqueous KOH and mixed with 98MI in a total volume of 3oocc.
Or a solution with a concentration of 0.144M was produced.

In this system, PPA aqueous solution is used as rlH control agent at about 8
Begins to be used during fermentation by an hour and ends at about 24 hours. This solution was automatically fed into the fermentor during a total of 175 CC logarithmic growth. The total Na-PPA.H2O fed during this period was 17.15o (84 mmol).

At this point, after 124 hours of fermentation - acid production by the microorganisms is no longer sufficient to reduce the I)H value of the medium to 6.8 or below. Therefore, 12.250 (6
The remaining 125 cc of aqueous PPA containing 0 mmol) was
It was pumped into the fermentor over a period of time (62,50 C/h). Photofermentation was allowed to continue for an additional 24 hours, for a total of 48 hours. During the last 17 hour period, 5NKOf-125cc was used as the base control agent.

Also, a second straining pump (Co1e ParIler)
By sending a signal to add thick glacial acetic acid to Co.), the DI-1111111 till machine was programmed to 1) prevent the l-1 value from moving above 7.8. A total of 15 Ca was added.

Glucose [70% (weight/passenger ship) stock solution] was added as necessary to prevent the glucose 111 degrees in the fermented meat juice from becoming zero. This was done to confirm that glucose was the carbon source rather than a precursor or product.

The final culture volume was 1.029 cc. The final calculated volumes were as follows: Initial medium 700
cc Wet material 50GO Water t9Na-PPA 300 cc Acetic acid 15 cc N+-1401-12cc 37- Glucose 100 cc 5N KOH25cc Subtotal -1,192cc Removed sample 7X10CC = 70 00 Total -1,
Evaporative loss over the 122 cc 48 hour fermentation period was approximately 8.3%.

L-phenylalanine in the fermented meat juice sample was analyzed using a high performance liquid chromatography (HPLC) system. Before injection into HPLC, danzyl chloride (D ansylc
hloride) and filtered with mysobore (0.
(22μ pore size) meat juice samples were derivatized. The results were as follows: 24 hours - 13,20(]/l 1-phenylalanine 28 n -17,06Q/ 1 31 〃 - 19,20 g/ 1 48 /J -18,69o/ 1 at 48 hours The low yield of was due to product decomposition.

38- Total of 1/14 mmol of Na-PPA・I''e 0(
29,40), a total of 115.93 mmol of L-phenylalanine (19,159) was produced during the fermentation. The molar yields were as follows: The culture medium, microbial strain and basic method of Example 1 were used with the following differences in volume and pl-1 control. After inoculating the fermenter, about 7
At that time, base control was started using an aqueous Na/PPA solution. Preprogrammed pl-1 control set point is 7.2
and 7°8.

168 mmol (34°3CI) in the total volume of 350cc
) of Na @ PPA .H2O was contained as an aqueous solution. The volume of aqueous Na 2 PPA used as base control during the approximately 7 hour start period and approximately 24 hour end period was 175 CC. Acetic acid was added to control the upper pH range as in Example 1, and a total of 41 cc was added to the fermenter. After 24 hours of R, pH for the rest of the fermentation.
The lower set point of the 1liItllll instrument was increased to 7.5. Starting at this point, the remaining Na-'PPA solution (
175 CC) was pumped into the reactor after 2 hours. Fermentation was continued for an additional 24 hours, for a total of 48 hours.

The final culture volume was 1.025CG. The final measured volumes were as follows: Initial medium 700 cc Accessory material 50 cc Na-PPA 350 cc Acetic acid 41 cc Volume Glucose 100 cc NH40H (concentrated, 29% NHa) 2 cc small Total -1
,243cc removed sample 11xiscc = 165 QC-1,
Evaporative loss over the 078 cc 48 hour fermentation period was approximately 4.92%.

Samples of meat juice were analyzed as in Example 1.

The HPLC binding was as follows: 2 24 hours-16,19/'IL-phenylalanine 26
N-17,4Ml 29 n -19,5a/ 1 32 11-20.1 (1/1 48 n -21,89/ 1 Total 168.00 mmol Na-PPA・H2O (3
4,39), a total of 145.77 mmol of phenylalanine (24,089) was produced during the fermentation. The molar yield was 86.8%.

fi The following seed medium was prepared: Ingredient amount Glucose 20a Bakuto Peptone (D Hco) 10.

41 - Yeast Extract 101J Nail 30 The ingredients were combined and the pHII was adjusted to 7.2 with 5N NaOH. This medium was disinfected at 110°C for 10 minutes.

Brevibacterium lactofermentum ATCC No. 21420 for 18 hours according to the method of Example 1.
Seed cultures were inoculated from llX0-8 VA Qar (Dirco) slants. O, D, (64001
) reached 25, the seed culture was grown for 7 hours. The rlHlm of the fermenter medium is 6.5 to 6.5 at N1408.
Adjusted to 7.0. Also according to the method of Example 1, a volume of 201 volumes of seed culture broth was used to inoculate the fermenter. The fermenter was operated at 30° C., 400 RPM and 1. OLPM
It was operated with. The lower limit pH setting for the first 24 hours is 6.8.
and the upper pH set point was 7.8.

Potassium phenylbilhinate (K-PPA) aqueous solution was used as an alpha-keto acid precursor. The C42- solution was the crude sodium hydroxide hydrolysis product of 5-benzylidenehydantoin. This 5-pensylidenehytan 1-yne (HamspireChelc
The preparation of the hydrolysis products of al Co,) was as follows: deionized water stop 1598.8511 and KO1-1
168.3a was added to a 31 capacity stainless steel beaker and heated to boiling point. While continuing to boil, add 5-benzylidenehydantoin 188.20,
and mixed until dissolved. Boiling was continued for 2 hours. Water was added as needed to maintain the initial volume.

After cooling, the nine compound was rapidly cooled in an ice water bath. 1 mole of potassium phenylpyruvate hydrolysis product contains ~1
．． 2M KO+-1, ~0,5M potassium carbonate and ~0
．． Contains 5M urea. The hydrolysis product was then sterilized using a milli-bore filter (<0.22μ pore size).

This hydrolysis product, which has a pH value of approximately 13, was used for base control from the beginning of the fermentation. A total of 2251 were added by 40 hours.

Concentrated glacial acetic acid was added to lower the pH value. 51
volume was added during the first 17 hours. The broth flH value did not rise above 7.8 in the later stages of fermentation, so there was no need to add further acid.

After 24 hours of fermentation, I) l-1 set points were adjusted to 7.8 and 8°0. At this point 501 parts of glucose was added to bring the concentration in the broth to 10%.

At 65 hours 201 volumes of glucose were added.

Samples of meat juice were analyzed as in Example 1. l-IP
The l-C results were as follows: 40 hours - 6,8 g/l L-phenylalanine 43
n −7,8 (J/1 45 = −8,49/1 48 n−9,4o/I 65 N −10,29/1 From the total amount of 111.23 mmol of monophenylpyruvic acid supplied, A total of 75 mmol of phenylalanine was produced during the fermentation.

The molar yield was 68.0%. Evaporation loss was not considered in this experiment.

Example 4 In gravy seeds and growth medium (NH4) 2 SO 3
0 (l) and MOPS (morpholinopropane-sulfonic acid) 20.0 (l) and instead of cac03 as buffer.
The seeds and growth medium of Example 1 were used except that 9 (1) was used. Prehybacterium flavan ATCC No. 13
The culture and inoculation method of Example 1 was performed using 626.

The fermenter in this experiment was 500-600 ml for the first 18 hours.
It operated at 00 RPM and then at 650 RPM.

The base used for the p)-1 control was the monophenylpyruvate hydrolysis product described in Example 3; the IIH value was adjusted to 10 using Col. The lower 1'1■ set point was 7.2 and the upper set point was 7.8. This hydrolysis product was used from the beginning of the fermentation as a base control agent. A total of 400 ml was fed into the fermenter according to l'lH demand. After about 2545 hours of fermentation, glucose 5 was added to the remaining hydrolysis product-base solution 601, which was continued to be added as a base control agent
01 was added. A further 25 ml of glucose was added at about 48 hours.

Retaining the upper day 0 value and additional hydrolysis products -
Concentrated glacial acetic acid was used to supply the base solution to the culture medium. A total of 421 were added to the fermenter.

Samples of meat juice were analyzed as in Example 1.

The HPLC results were as follows = 11 hours-3
,33o/I l-phenylalanine 19 II-3
,96(]/1 22 11-6.19M+ 24 n-7,609/1 42 ll-11,609/1 45 /J -14,10al 1 65 n-11,9011/1 Low yield at 65 hours The rate was due to product decomposition.

A total of 111.99 mmol of 1-1-phenylalanine was produced by 45 hours from a total of 176.33 mmol of 1-1-phenylbirhinate fed. The molar yield at 45 hours was 63.5%.

The principles, preferred embodiments, and manner of operation of the present invention are set forth in the foregoing specification. However, the invention protected herein should not be construed as limited to the particular forms disclosed, because they are illustrative rather than limiting. Because there is. Various methods and modifications can be made by those skilled in the art without departing from the spirit of the invention.

Patent applicant: Tableau R. Brace &amp;
Company 47-AI

Claims (1)

Claims: 1. (a) growing a microorganism in a nutrient medium that converts an alpha-keto acid into its corresponding monoamino acid and that produces and secretes the acid during the growth phase; (b) providing a desired alpha-keto acid to a growing culture of microorganisms at a rate substantially corresponding to the rate of growth; and (C) causing the microorganisms to convert the alpha-keto acids into the corresponding amino acids. Method for producing sushi monoamino acid. 2. The method according to claim 1, wherein L-amino acids are recovered from fermented meat juice. 3. 1 ton of alpha keto acid in step (b)? = 3
The method according to claim 1, wherein the J degree is determined by the l)H value of the culture broth. 4. The method according to claim 3, wherein the alpha-keto acid supply is started about 6 to 10 hours after inoculation of the microorganism into the nutrient medium. 5. The method of claim 4, wherein the alpha-keto acid supply is started when the optical density of the growing culture (64011111) reaches about 10. 6. pH of the culture before starting alpha-keto acid supply
6. A process according to claim 5, wherein the wA value is controlled by adding a base compatible with the fermentation, said base being free or substantially free of alpha-keto acids. 7. The method according to claim 6, wherein the pH value of the culture solution is maintained in the range of about 6.8 to 7.2 before starting the supply of flufurketo acid. 8. The method of claim 6, wherein the base is selected from the group consisting of ammonium hydroxide, potassium hydroxide, sodium hydroxide, urea, and ammonia scum. 9. The method according to claim 3, wherein the alpha-keto acid is placed in a solution containing a base compatible with the fermentation. 10. The method according to claim 9, wherein the salt M is selected from the group consisting of ammonium hydroxide, potassium hydroxide and urea. 11. The method according to claim 9, wherein the alpha-keto acid co-feeding is started when the pH of the cultured meat juice drops to a range of about 4 or about 7.2 to about 7.5. 12. Adding alpha-keto acids as necessary to maintain the H value of the culture broth at or above a predetermined value in the range of about 6.8 to about 7.5. The method according to scope item 11. 13. The method of claim 12, wherein the basic alpha-acid solution is pumped into the meat juices by a signal from an automated gravy device. 14. The pf-111iI of the meat juice is within the range 8
14. The method of claim 13, wherein the discharge controller h1 automatically signals the pump to begin adding the chlorinated solution when the volume drops below a predetermined value. 15. The method of claim 3, wherein the addition of acid compatible with fermentation is initiated when the I)H value of the culture broth rises within the range of about 7.5 to about 7.8. 16. The method of claim 15, wherein nucleic acid is optionally added to maintain the 11)-1 value of the culture broth at about 8.0 or less. 17. The method according to claim 16, wherein the I)H value of the culture broth is controlled by an automated r)H control device. 18. The DHtt+lIm unit automatically signals a pump to add nucleic acids to the culture broth when the l)H value of the broth rises above a preset value within the range. The method according to scope item 17. 19. For biological transformation, a first portion of the total amount of alpha-keto acid desired to be fed to a growing culture is fed to the culture based on pH requirements, and the first portion of the alpha-keto acid is 13. The method of claim 12, wherein portions of 2 are supplied to the culture for a period of up to 24 hours following cessation of rlH demand supply. 20. The method of claim 19, wherein said first portion comprises 1/3 to 1/2 of said total amount. 21. The method of claim 19, wherein said second portion of alpha-keto acid is provided to said culture medium for a period of up to 8 hours. 22. The method of claim 21, wherein said second portion is provided to said 18 il liquid in one or more blobs or continuously. 23. Biologically converting alpha-keto acid precursors into 1-amino acids by feeding them to logarithmically growing cultures of microorganisms that are capable of biological conversion and secrete acids during the active growth phase. (a) monitoring the DI-(value change) of the broth of the growing culture; and (1) adding the alpha-keto acid precursor in an amount to offset the acid secreted by the microorganism. into a basic solution 5-. 24. A claim for starting addition of the alpha-keto acid when l')l-1111i of the cultured meat juice decreases to a predetermined value in the range of about 7.2 to about 7.5. The method according to item 23. 25. Claim No. 2, wherein the alpha-keto acid is added as necessary to maintain the IIH value of the cultured broth at or above a predetermined value in the range of about 6.8 to about 7.5. The method described in Section 23.