MXPA97002246A - Process for producing aspartase and l-aspartic acid - Google Patents

Abstract

Disclosed are a process for producing aspartase which comprises culturing a microorganism belonging to the genus Escherichia being capable of producing aspartase in a medium until aspartase is produced and accumulated in the culture, and recovering aspartase therefrom, wherein dissolved oxygen concentration of the medium is in the range of 0 to 1 ppm on the stage that microbial growth is middle and/or late logarithmic growth phase;and a process for producing L-aspartic acid which comprises converting fumaric acid and ammonia into L-aspartic acid in an aqueous medium in the presence of an enzyme source, wherein said enzyme source is a culture produced in accordance with said aspartase-producing process, cells isolated from the culture, or processed cells thereof.

Description

PROCESS FOR PRODUCING ASPARTASE AND PROCESS FOR PRODUCING ACID L-ASPART CO DESCRIPTION OF THE INVENTION The present invention relates to a process for producing aspartase using a microorganism belonging to the genus Escherichia that has the ability to produce and accumulate aspartase and with a process to produce L-aspartic acid using aspartase. Hitherto many reports have been made that relate to processes for producing L-aspartic acid from fumaric acid and ammonia using aspartase as produced by microorganisms (Japanese Patent Application, Examined, Published Nos. 54-12553 and 57). -18867). As such microorganisms, those belonging to the genera Escherichia and Corynebacterium are known and some attempts so far have been made to obtain mutants with high aspartase productivity by means of genetic recombination technology (Japanese Patent Application Unexamined, Published Nos. 60-133883 and 5-30977). In the process to produce aspartase in a large amount using such microorganisms of the genera Escherichia and Corynebacterium, fumaric acid, which is expensive, is used as the nutrient source. In the process to produce aspartase using a transformant with high aspartase productivity as obtained by means of genetic recombination, the antibiotics are added to the seed media to ensure the stability of the transformer's aspartase productivity. It is known that when a microorganism of the type capable of producing and accumulating aspartase together with fumarase is used to produce aspartase and aspartase is used to produce L-aspartic acid, some by-products other than L-aspartic acid are produced because the fumarase decreases the yield of the intended L-aspartic acid.

To avoid the reduction in the yield of L-aspartic acid, the system is treated with heat or treated with acids after the production of aspartase, thereby eliminating the fumarase activity of the system (Kagaku Kogyo, 58., 878 , 1994; Examined Japanese Patent Publication, Published No. 3-55103). The prior art techniques are problematic in that the processes, using microorganisms that are capable of producing aspartase to produce large amounts of aspartase, require fumaric acid, which is expensive, as the nutrient source; The processes that use transformants with high aspartase productivity as obtained by means of genetic recombination, require antibiotics to be added to the planting media to ensure the stability of aspartase productivity. the transformants; and in the processes for producing L-aspartic acid using the microorganisms that can produce and accumulate fumarases in addition to the intended aspartase, the production of by-products other than the intended L-aspartic acid is inevitable. 5 According to the present. invention, a process for producing aspartase is provided, which comprises cultivating a microorganism belonging to the genus Escherichia that is capable of producing aspartase in a medium until the aspartase is produced and accumulated in the culture. and recover from them the aspartase, in which the dissolved oxygen concentration of the medium is in the range of 0 to 1 ppm in the stage in which the microbial growth is in the middle and / or late logarithmic growth phase; and a process for producing L-aspartic acid, which comprises Converting fumaric acid and ammonia to L-aspartic acid in an aqueous medium in the presence of an enzyme source, in which the enzyme source is a culture produced according to the process for producing aspartase, the cells isolated from the culture or your processed cells. In the present invention, any microorganism can be used as long as it belongs to the genus Escherichia, preferably Escherichia coli, and is capable of producing aspartase, preferably producing a large amount of aspartase, it can be used.

In general, Escherichia coli may have the ability to produce an accumulated aspartase. As an example, Escherichia coli ATCC-11303 is mentioned. The suitable microorganism used in the present invention can be obtained as a mutant strain that produces a large amount of aspartase by subjecting the microorganisms that produce aspartase belonging to the genus Escherichia to conventional mutagenesis, screening the resulting mutants to select those capable of growing plus rapidly in minimal media containing L-aspartic acid as the sole nitrogen source (Appl. Env. Microbiol., 48., 1072, 1984). Such mutant strains include Escherichia coli EAPc7, Escherichia coli EAPc244, Escherichia coli EAPc28, Escherichia coli EAPcllO, 'Escherichia coli EAPcl30 (Japanese Application Without Ex: aminar, Published No. 60-133883) and Escherichia coli # H-9183 Suitable microorganisms used in the present invention can be obtained as a transformant that produces a large amount of aspartase by the use of genetic recombination technology. Such transformants include Escherichia coli TA5003, Escherichia coli TA5004 and Escherichia coli TA5005 (Japanese Unexamined Patent Application, Published No. 60-133883).

It is possible to produce aspartase by growing a microorganism of the present invention according to the process mentioned in the following. As the medium, any of a natural medium and a synthetic medium can be used as long as it contains carbon source, nitrogen sources, inorganic substances and other nutrients required by the microorganism used, etc., which can be assimilated by the microorganism used. and the microorganism can be cultivated efficiently in it. As the carbon source, the carbohydrate such as glucose, fructose, sucrose, melases containing these and still starch and starch hydrolysates; organic acid such as acetic acid, fumaric acid, lactic acid and acid propionic; and alcohol such as ethanol, glycerin and propanol, etc., can be used. As the source of nitrogen, ammonia, various inorganic or organic ammonium salts such as ammonium chloride, ammonium sulfate, ammonium acetate and phosphate ammonium; amines; other nitrogen-containing compounds; Peptone, meat extract, yeast extract, distilled corn liquor, casein hydrolyzate, bean turtle, hydrolyzed bean turtle, various cultured cells and digested product of cells, etc., can be used.

As the inorganic substance, the potassium diacid phosphate, dipotassium acid phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate and calcium carbonate, etc., can be used. The conditions of aeration or conditions of agitation during cultivation have great influences on the level of accumulation of aspartase. The microorganism of the present invention is grown under aerobic conditions until the early logarithmic growth phase, then it is grown in a controlled aiation condition in which the concentration of oxygen dissolved in the medium is in the range of 0 to 1 ppm, therefore, the production and accumulation of fumarase is reduced and the production and accumulation of the destined aspartase is increased. The cultivation is carried out at a temperature of 20 to 40 ° C, preferably 25 to 37 ° C. The pH of the medium is in the range of 5 to 9, preferably 6 to 8. The pH is adjusted with calcium carbonate, inorganic or organic acid, alkaline solution, ammonia, a pH buffering agent or the like. Generally, after cultivating for 3 to 24 hours, a large amount of aspartase accumulates in the cells of the microorganism.

After the end of the culture, the aspartase is isolated and purified from the culture by a conventional method. For example, the culture is centrifuged to collect the cells and the cells are washed and disintegrated with an ultrasonic disintegrator, a French press, a Manton-Gaulin homogenizer, a Dyno mill or the like to obtain an extract without cells. The extract without cells is centrifuged and the resulting supernatant is subjected to desalination with ammonium sulfate or the like, for the exchange chromatography anionic with diethylaminoethyl (DEAE) -Sepharose or the like, to hydrophobic chromatography with butyl-Sepharose, phenyl-Sepharose or the like, to gel permeation with a molecular sieve or the like, or to electrophoresis such as isoelectric electrophoresis, whereby a pure product of aspartase. The activity of the aspartase enzyme can be determined in the manner mentioned in the following. A sample is incubated in an aqueous solution comprising ammonium fumarate (160 g / liter, pH 8.7) and gCl ^ (0.2 mM) at 37 ° C for 60 minutes. The resulting L-aspartic acid was determined using high pressure liquid chromatography. The activity of the enzyme of aspartase is represented in units, with a unit (U) that is defined as the activity capable of producing 1 / mol of acid L-aspartic in one minute, is mentioned as a unit (U).

The fumarase activity can be determined in the manner mentioned in the following. A sample is incubated in an aqueous solution comprising sodium fumarate (1 M, pH 8.0) at 37 ° C for 30 minutes. The resulting malic acid is determined using high pressure liquid chromatography. The activity of the sample that produces 1 μmol of malic acid in one minute is mentioned as a unit (U). L-aspartic acid can be produced by using, as an enzyme source, a culture containing aspartase in a large amount obtained in the above process to produce aspartase, the cells isolated from the culture, or their processed cells, by the addition of fumaric acid and ammonia to an aqueous medium suitable for the reaction of the enzyme to produce and accumulate therein L-aspartic acid and by recovering them from the accumulated L-aspartic acid. The processed cells include dry cells, lyophilized cells, surfactant treated cells, enzymatically treated cells, ultrasonically treated cells, mechanically ground cells, solvent treated cells, protein-containing fractions of the cells and immobilized products of the processed cells or cells.

The enzyme which is obtained by extraction of the cells and has aspartase enzyme activity, its purified preparation and its immobilized products are also used as the processed cells. The aqueous medium includes water, buffer such as phosphate buffer, carbonate buffer, acetate buffer, borate buffer, citrate buffer and tris buffer, alcohol such as methanol and ethanol, ester such as ethyl acetate, ketone such as acetone and amide such as acetamide. The culture obtained in the above process to produce aspartase is also employed as the aqueous medium, either directly or after having been diluted 1 to 10 times, preferably 2 to 6 times. The activity of the enzyme source in the reaction system can be determined, for example, depending on the amount of the substrate used. In general, the activity of the enzyme in the aqueous medium can be in the range of 10 to 1000 U / liter, preferably 50 to 300 U / liter. The amount of fumaric acid in the aqueous medium can usually be in the range of 50 to 200 g / liter. The reaction of fumaric acid with ammonia in an aqueous medium in the presence of an enzyme source is usually carried out at 10 to 50 ° C and at a pH of 7.5 to 9.5 during 0.5 to 20 hours.

# After the end of the reaction, an acid such as sulfuric acid and hydrochloric acid is added to the reaction mixture to adjust the pH to 2.7, so that the L-aspartic acid formed crystallizes and the crystallized 5 L-aspartic acid it is collected from the reaction mixture. The present invention is described in more detail with reference to the following examples, which, however, are not intended to restrict the scope of the invention. invention. EXAMPLE Example 1: Production of Aspartase Escherichia coli ATCC-11303 (a wild-type strain) that is capable of producing aspartase and Escherichia coli H-9183 derived from E. coli ATCC-11303 and which is capable of producing a large amount of aspartase were used. Strain H-9183 was derived from strain ATCC-11303 according to the process mentioned in the following. Strain ATCC-11303 was subjected to mutagenesis in a Aqueous solution containing N-methyl-N1-nitro-N-nitrosoguanidine (0.25 mg / ml) at 30 ° C for 30 minutes. Approximately 80,000 strains were obtained by mutagenesis and were screened based on their growth rates in a minimal medium containing L-aspartic acid as the only source of nitrogen. Among the strains selected in this way, strain H-9183, which has a high growth rate, is obtained as a strain capable of producing a large amount of aspartase. Each of the strains ATCC-11303 and H-9183 were cultured with agitation aerobically at 30 ° C for 12 hours in a seed medium containing 2% glucose, 2% peptone, 0.3% meat lime and 0.5 % calcium carbonate. The resulting seed culture (10 ml) was inoculated into 1 liter of a medium containing 2% glucose, 0.5% distilled corn liquor, 0.03% ammonium sulfate and 0.3% potassium dihydrogen phosphate in a 2 1 fermenting vessel and the culture is carried out at 30 ° C with stirring at 800 rpm. During cultivation, the pH of the medium is maintained at approximately 6.5 + 0.2 with aqueous ammonia and the culture is carried out until the glucose in the medium was completely consumed. The cultivation was carried out in two ways; one that is to grow the microorganism aerobically by aeration of the medium at a rate of aeration of 1 liter / minute during the entire period of cultivation and the other that is to cultivate the microorganism with conversion of the aerobic condition at a rate of aeration of 1 liter / minute in a condition of controlled aeration during the course of the culture period. The conversion time of the aerobic condition into a controlled aeration condition was at the stage in the that microbial growth is in the growth phase? logarithmic medium. The amount of aeration in the medium was decreased to make the dissolved oxygen concentration of the medium in the range of 0 to 1 ppm as for the controlled aeration condition. After the end of the culture, the aspartase activity and the fumarase activity of the microorganism cells were determined. The results are shown in Table 1.

Table 1 Both of strain ATCC-11303 and strain H-9183 that have been cultured under controlled aeration conditions have increased aspartase activities and decreased fumarase activities, respectively. Example 2: Production of L-Aspartic Acid 15 The cultures of strain H-9183 as obtained in Example 1 were suitably diluted, for which fumaric acid was added at the concentration of 100 g / liter. Then, the ammonia is added to adjust the pH to 8.7. The reaction is carried out at 30 ° C with gentle agitation. Results are shown in table 2.

Table 2 F The results show that the L-aspartic acid productivity and the conversion in the product were increased in the crop as obtained in the condition through aeration followed by controlled aeration. Example 3: Production of L-aspartic acid The culture of strain H-9183 is obtained in the controlled aeration condition in Example 1, centrifuged to isolate the cells. The cells are suspended in a phosphate buffer (0.01 M potassium phosphate; of 7.4) and homogenized together with glass beads to disintegrate them into an extract. The extract is passed through a column filled with a resin, Duolite A-7 so it makes the existing protein in the extract adsorbed by the resin.

Then, an aqueous solution of fumaric acid (150 g / liter, adjusted to pH 8.5 with aqueous ammonia) is passed through the column at 37 ° C whereby a solution of L-aspartic acid (171 g) is obtained / liter). The pH of the solution (1 liter) is adjusted to 2.7 with sulfuric acid and 161 g of crystals of L-aspartic acid are obtained. INDUSTRIAL APPLICABILITY According to the process of the present invention to produce aspartase, it is possible to produce a large amount of aspartase with decrease of a fumarase / aspartase ratio. Furthermore, by using the aspartase produced in this form according to the invention, it is possible to produce pure L-aspartic acid containing less amount of by-products. fifteen i

Claims (2)

1. # CLAIMS 1. A process to produce aspartase, characterized in that it comprises cultivating a microorganism belonging to the genus Escherichia that is able to produce aepartase in a medium until the aspartase is produced and accumulated in the culture and recover the aspartase therefrom, in that the dissolved oxygen concentration of the medium is in the range of 0 to 1 ppm in the microbial growth stage is in the middle and / or late logarithmic growth phase. * 10
2. 2. A process for producing L-aspartic acid, which comprises converting fumaric acid and ammonia to L-aspartic acid in an aqueous medium in the presence of an enzyme source, in which the enzyme source is a culture produced according to the process in accordance with the 15 claim 1, the cells isolated from the culture or their processed cells.