KR19980050070A - Thermophilic Strain Bacillus stiadermophilus KL-01 having D-amino acid aminotransferase activity - Google Patents

Abstract

The present invention can be usefully used for the synthesis of a new D-amino acid by having a D-amino acid aminotransferase (EC 2.6.1.21) having the activity of transferring the amino group of the D-amino acid to keto acid. Thermophilic microbial strains, more specifically Bacillus stearothermophilus KL-01 (KCTC 0285BP) and methods of making new D-amino acids using this novel thermophilic strain will be.

Description

Thermophilic Strain Bacillus stiadermophilus KL-01 having D-amino acid aminotransferase activity

Figure 2 shows the results of agarose gel electrophoresis of the gene fragments generated by PCR amplification for the D-AAT gene search of thermophilic microorganisms.

3 shows the reaction of D-alanine with α-ketoglutaric acid using D-AAT produced by the thermophilic strain Bacillus stearothermophilus KL-01 (KCTC 0285BP). It is a result of the liquid analyzing by HPLC.

The present invention provides a D-amino acid aminotransferase (EC 2.6.1.21) (hereinafter referred to as D-AAT) having the activity of transferring the amino group of D-amino acid to keto acid. It relates to a thermophilic microbial strain, more specifically Bacillus stearothermophilus KL-01 (KCTC 0285BP), which may be useful for the synthesis of new D-amino acids. The invention also relates to a process for the preparation of new D-amino acids using the novel pyrogenic strains according to the invention.

D-amino acids are generally not included in natural proteins, but are widely distributed in nature as components of microbial cell walls or peptide antibiotics.Industrially, synthetic sweeteners, β-lactam antibiotics, peptide hormones, insecticides, etc. It is widely used in food and medicine as an intermediate for producing

D-AAT is a novel method of transferring amino groups from various D-amino acids such as D-alanine, D-glutamic acid, and D-aminobutyric acid to keto acids such as pyruvic acid, α-ketoglutaric acid, and α-ketoisovaleric acid. It is an enzyme having the function of synthesizing D-amino acids. Soda et al. (1974) FEBS Lett. 46. 359-363. D-AAT also has a relatively broad substrate specificity and is capable of synthesizing various D-amino acids by a single process. This enzyme has been found mainly in microorganisms of the genus Bacillus. Tanizawa et al. In Japan have isolated two thermophilic Bacillus producing enzymes from thermophilic microorganisms [Tanizawa et al. (1989) J. Biol. Chem. 264. 2445-2449], recent studies on the reaction mechanism and protein structure of this enzyme have been reported (Sugio et al. (1995) Biochemistry, 34, 9661-9669).

In addition, the inventors of the present invention, microorganisms producing new substrate-specific D-AAT that is stable to heat and capable of enzymatic reaction at high temperature from high temperature bacterial habitats in Korea as well as exhibits high reactivity to branched amino acids such as D-valine. As a result, over 110 high-temperature microbial strains showing D-AAT activity were selected from a thousand high-temperature microorganisms isolated from Korean soil, and D-alanine and α-ketoglutaric acid were selected for these high-temperature microorganisms. By comparing the rate of catalyzing the reaction for producing D-glutamic acid from the two species of microorganisms showing the maximum activity was isolated. These two types of microorganisms showed very similar physiological characteristics to Bacillus sphaericus, a known strain, but they were thermophilic Bacillus sp. Named LK-1 and LK-2, the patent application has been completed as a novel strain (see Korean Patent Application Nos. 95-53557 and 96-709).

In bioprocesses using enzymes as biocatalysts, the stability of the biocatalyst is the most important factor in determining the productivity of the enzyme conversion reaction. In general, thermophilic microorganisms that live in a high temperature growth environment have heat-stable heat-resistant enzymes due to the high temperature environment of the habitat. In addition to the heat-stable enzymes, these heat-resistant enzymes have been reported to have stability to organic solvents, stability to pH and stability to chemical modifiers.

Bacillus sp., Which has been isolated by the inventors. Although LK-1 and LK-2 by themselves have a considerable degree of heat resistance, as mentioned above, the stability of the biocatalyst is the most important factor determining the productivity of the enzymatic conversion reaction. Further studies have been carried out to isolate new D-AAT producing strains which have higher thermal stability than 2 and can be used more advantageously in the enzymatic conversion process for synthesizing D-amino acids. As a result, Bacillus sp. We have successfully isolated 20 new thermophilic microorganisms that are capable of growing even at high temperatures of 70 ° C., which are not even LK-1 and LK-2, and exhibit excellent thermal stability.

The present inventors used Western blotting to select a strain that produces a new D-AAT different from the D-AAT derived from Bacillus sp LK-1 among the 20 D-AAT producing thermophilic microorganisms thus selected. Comparison of antigen-antibody reactivity with Western blotting) and homology of D-AAT gene sequences with PCR were performed. As a result, Bacillus sp. The present invention has been successful in separating one thermophilic microorganism with a new D-AAT having different characteristics from that of LK-1.

Accordingly, an object of the present invention is to provide a thermophilic strain that can be usefully used for synthesizing a new D-amino acid by having the ability to transfer the amino group of D-amino acid to keto acid. The thermophilic strain according to the present invention has a characteristic that can be grown even at a high temperature of 70 ℃.

The present invention also relates to a method for producing a new D-amino acid by transferring the amino group of D-amino acid to keto acid using the thermophilic strain as a biocatalyst.

As a result of an identification study on the thermophilic microorganisms isolated by the present inventors, the strain is grown on Bacillus stearothermophilus, which grows aerobicly at a temperature of 65 ° C. or higher and produces gram-positive and endogenous spores. It was identified as belonging microorganism.

Therefore, the obtained new heat-resistant D-AAT producing thermophile was named Bacillus stiadermophilus KL-01. To date, no studies have been made on the production of D-amino acids using a heat-resistant D-AAT derived from a thermophilic Bacillus stearothermophilus capable of growing at a high temperature of 65 ° C or higher.

Hereinafter, the isolation, screening process and characteristics of the novel strain according to the present invention will be described in detail.

Quantification of D-AAT Activity:

The reaction solution for measuring D-AAT activity included 0.2 ml of 0.1M Tris buffer (Pris-HCl, pH 8.5) and 10 mM D-alanine, α-ketoglutaric acid, and coenzyme solution corresponding to 0.1 ml of culture. What was prepared to use was used, and allowed to stand for 1 hour at 50 ℃. In addition, the amount of pyruvate produced by the catalytic reaction of D-AAT was developed by adding 0.2 ml of an aqueous solution of 60% potassium hydroxide and 0.1 ml of 2% salicyladehyde to the reaction solution and then standing still at 37 ° C. for 30 minutes. After dilution with water, quantification was performed by diluting with water twice and measuring the absorbance at 480 nm (Berntsson S. (1995) Anal. Chem. 27, 1659-1660.

Screening of D-AAT Active Strains from Soil-Separated Thermophiles:

First, samples from haystacks, humus, rivers, wastewater, and hot springs around the country were collected from 1.5% polypeptone, 0.2% yeast extract, 0.2% meat extract, and 0.2% glycerol. glycerol), 0.2% K ₂ HPO ₄ , 0.2% KH ₂ PO ₄ , 0.026% NH ₄ Cl was inoculated in MY medium and enriched at 55 ℃ (enrichment culture). This culture was inoculated in MY agar plate medium containing 2% agar (agar) and incubated at 55 ℃ to isolate the pure colonies. After the isolated strains were incubated for 8 to 10 hours in 5 ml MY liquid medium, the cells were harvested, and the cells were crushed with a sonicator to make coenzyme solution to measure D-AAT activity.

Over 110 species of microorganisms showing D-AAT activity were isolated during the first strain selection.

Selection of heat resistant D-AAT producing strains capable of growing at 70 ° C:

Among the 110 strains containing D-AAT activity, Bacillus sp. The thermal stability was higher than that of LK-1 and LK-2, and the following experiment was performed to select strains producing new D-AAT which have not been reported so far. The screening process of D-AAT producing strains with high thermal stability was performed by culturing the microorganisms previously isolated at 70 ° C. and separating 20 species in which microbial growth was observed. 20 thermophilic microorganisms were inoculated in 5 ml of MY medium and incubated at 60 ° C. for 8 to 10 hours. The cells were crushed into coenzyme solution and the D-AAT activity was measured. Among these high temperature microorganisms, the microorganisms having high D-AAT activity were as shown in Table 1 below.

TABLE 1

Comparison of selected D-AAT producing strains and isolation of new D-AAT producing thermophiles:

Among 20 D-AAT-producing thermophilic strains selected in the previous step, Bacillus sp. In order to select a strain producing a new D-AAT different from the D-AAT derived from LK-1 and LK-2, the following experiment was performed. The choice of new D-AAT producing strains was aimed at improving thermal stability and changing substrate specificity.

First, the crude extract of each thermophilic strain was developed by SDS-PAGE, and then the antigen-antibody reaction of rabbit serum prepared using D-AAT of LK-1 as an antigen. Western blotting was performed to compare D-AAT produced from each thermophilic microorganism and D-AAT derived from LK-1. At this time, the polyclonal antibody used is Bacillus sp. D-AAT derived from LK-1 was injected into rabbits to induce an immune response. As a result of Western blotting, as shown in FIG. 1, all 20 heat-resistant D-AAT producing strains capable of growing at 70 ° C. were all Bacillus sp. It was found to have the same molecular weight as D-AAT derived from LK-1 and to have a structurally similar D-AAT. Therefore, it was determined that it is difficult to select a strain having a new D-AAT different from the D-AAT derived from LK-1 using the antigen-antibody reactivity difference.

Next, the inventors used a polymerase chain reaction (PCR) method as another method for searching for and verifying a new D-AAT. That is, Bacillus sp. N-primers complementary to the 5'-terminal sequence of the LK-1-derived D-AAT gene and C-primers complementary to the 3'-terminal sequence, and PCR of the D-AAT gene in the total chromosome of each thermophile To amplify. In this case, when there is homology on the D-AAT gene sequence, the D-AAT gene is amplified to produce a 0.85 kb DNA fragment, but when the homology of the gene sequence is small, the amplification of the gene is not observed. D-AAT possessed by 20 selected D-AAT producing thermophils was Bacillus sp. It can be determined whether there is similarity with the D-AAT gene of LK-1. The two primers used in this method are shown in Table 2 below.

TABLE 2

After performing PCR for the search for the D-AAT gene for the total chromosome of each thermophile using the primers, the result of analyzing the produced gene fragments by agarose gel electrophoresis is shown in FIG. As can be seen from FIG. 2, thermophilic microorganism No. 257, in which the 0.85 kb gene fragment encoding D-AAT was not found, was found among the thermophilic strains. D-AAT produced by the pyrophilic microorganism 257 was found to be the same size protein as D-AAT produced by LK-1 in Western blotting.However, PCR analysis showed that the nucleotide sequence was different from LK-1. It was determined to be a microorganism producing D-AAT. Therefore, from the results so far, most of the D-AAT derived from the thermophilic microorganisms have similar molecular weights and gene sequences, but the thermophilic microorganisms No. 257 is believed to produce a new property of D-AAT that is completely different from the D-AAT derived from conventional pyrogenic microorganisms.

On the other hand, heat-resistant D-AAT production thermophile No. isolated and screened by the method described above. A taxonomic study to identify 257 was performed as follows.

Identification of Mycobacterium 257:

Isolated microorganism no. 257 formed creamy opaque colonies when incubated in 2% agar plates. It was also classified into Bacillus because it is a bacterium that grows well under aerobic conditions with a temperature of 30 ° C. or higher and 70 ° C. or lower, and is gram-positive and forms endogenous spores.

The results of physiological and biochemical identification of thermophilic Bacillus strains are as follows.

From the above results, the isolate isolated from the present invention was identified as belonging to Bacillus stearothermophilus (Bacillus stearothermophilus), and thus named Bacillus stearothermophilus KL-01. The inventors of the present invention deposited this new high temperature bacterium at the Gene Center in the Biotechnology Research Institute of the Korea Institute of Science and Technology on November 21, 1996, and received the accession number of KCTC 0285BP.

Hereinafter, the present invention will be described in more detail based on the following examples. However, these examples are only for the understanding of the present invention, and the scope of the present invention in any sense is not limited to these examples.

Example 1

The D-glutamic acid from D-alanine and α-ketoglutaric acid was produced using D-AAT produced by Bacillus stiadermophilus KL-01 (KCTC 0285BP) isolated and identified in the present invention as a biocatalyst. Prepared. As the reaction solution, D-alanine and α-ketoglutaric acid were added at concentrations of 0.9 g / l and 2.3 g / l, respectively, and the coenzyme solution of Bacillus stearothermophilus KL-01 (KCTC 0285BP) was used as a protein. 0.1M Tris buffer solution (pH8.5) added to the concentration of 1.0g / L was used. After the reaction solution was reacted at 55 ° C. for 1 hour, the reaction product was analyzed using an Enantio L1 optically active HPLC column (Tosoh, Japan). As shown in FIG. 3, not only D-glutamic acid but also L-alanine and L Glutamic acid was also confirmed to be produced. L-alanine is produced from D-alanine by an alanine racemase that acts specifically on alanine, and L-glutamic acid is reacted with L-alanine by the action of L-amino acid aminotransferase (L-AAT). It is known to be produced from α-ketoglutaric acid. The resulting D-glutamic acid can be produced solely by the action of D-amino acid aminotransferase (D-AAT), and HPLC analysis shows that Bacillus stiadermophilus KL-01 (KCTC 0285BP) isolated according to the present invention. ) Acts specifically on D-amino acids, clearly indicating that they are microorganisms that produce D-AAT enzymes that can be used for the production of various D-amino acids.

On the other hand, as an amino acid which donates an amino group, 1 type selected from D-alanine, D-aminobutyric acid, D-aspartic acid, D-glutamic acid, and Dn- valeric acid is used. Corresponding D-amino acids could be obtained by performing experiments under the same conditions as in Example 1 using one selected from glutaric acid, α-ketoisovaleric acid and α-keto-n-valeric acid. .

Therefore, from these results, it can be seen that the Bacillus stiadermophilus KL-01 (KCTC 0285BP) strain according to the present invention is suitable for enzymatic synthesis of D-amino acid and can be used industrially.

Claims (4)

Bacillus stearothermophilus KL-01 strain (KCTC 0285BP) having the ability to transfer the amino group of D-amino acid to keto acid. A method for preparing a new D-amino acid by transferring an amino group of D-amino acid as a substrate to keto acid using the strain according to claim 1. The method according to claim 2, wherein the substrate D-amino acid is one selected from D-alanine, D-aminobutyric acid, D-aspartic acid, D-glutamic acid, and D-n-valeric acid. The method according to claim 2, wherein the keto acid is one selected from pyruvic acid, α-ketoglutaric acid, α-ketoisovaleric acid and α-keto-n-valeric acid.