KR19980078034A - Novel thermophilic Bacillus genus KLS-01 and heat-resistant D-alanine aminotransferase, L-aspartate aminotransferase and alanine racease produced therefrom - Google Patents

Abstract

The present invention relates to a novel thermophilic Bacillus sp . Microorganism and heat-resistant D-alanine aminotransferase, L-aspartate aminotransferase and alanine racease produced therefrom, and according to the present invention Bacillus sp . KLS-01 ( Bacillus sp.KLS-01) is a D-alanine aminotransferase which transfers an amino group of D-amino acid to keto acid, an alanine racease which catalyzes the isomer formation of alanine, and an amino group of L-aspartate to keto acid. L-aspartate aminotransferase can be used as a biocatalyst for enzymatic synthesis of new D-amino acid and L-amino acid.

Description

New Thermophilic Bacillus KLS-01 and Heat-Resistant D-Alanine Aminotransferases, L-Aspartate Aminotransferases and Alanine Raceases Produced therefrom

The present invention is to form isomers of D-amino acid aminotransferase (EC 2.6.1.21) (hereinafter, abbreviated as D-AAT) and alanine, an enzyme that transfers the amino group of D-amino acid to keto acid. Alanine racemase (hereinafter abbreviated as AlaRA), an enzyme that catalyzes the enzyme, and L-aspartate aminotransferase (L) which transfers the amino group of L-aspartate to keto acid. -Aspartate aminotransferase: KLS- genus of a new high temperature microbial strain Bacillus , which can produce EC 2.6.11) (hereinafter abbreviated as AspAT) and be useful as a biocatalyst for enzymatic synthesis of new D-amino and L-amino acids. 01 ( Bacillus sp.KLS-01).

D-amino acids are not included in proteins but are widely distributed in nature as a component of microbial cell walls or peptide antibiotics.Industrially, D-amino acids are used for the production of synthetic sweeteners, β-lactam antibiotics, peptide hormones, and insecticides. As an intermediate, it is widely used in food and medicine.

Methods for producing D-amino acids are known chemically synthesized and methods using microbial enzymes. The production of amino acids by chemical synthesis has a difficulty of undergoing a complicated optical pure process since the product is obtained in a racemic mixture of D, L-amino acids. On the other hand, D-amino acid synthesis using a biocatalyst has attracted attention as a low pollution clean technology because it can directly produce only optically pure D-amino acid and can reduce the production of by-products that are environmental pollution.

AlaRA and D-AAT are enzymes involved in the biosynthesis of D-amino acids, such as D-alanine and D-glutamic acid, which are components of microbial cell walls. AlaRA has a function of selectively transducing the amino group of alanine to selectively produce only D or L alanine, which is a stereoisomer of alanine, and D-AAT is a compound such as D-alanine, D-aspartic acid, D-glutamic acid, and D-aminobutyric acid. It is an enzyme having the function of synthesizing new D-amino acids by transferring amino groups from various D-amino acids to keto acids such as pyruvic acid, α-ketoglutaric acid, and α-ketoisovaleric acid (Soda et al., FEBS Lett 46 , 359-363 (1974). Thus, a combination of D-AAT and AlaRA can produce a variety of industrially useful D-amino acids from low-cost D, L-alanine mixtures.

In the bioconversion process using an enzyme as a biocatalyst, the stability of the biocatalyst is the most important factor in determining the productivity of the enzyme conversion synthesis reaction. In general, thermophilic microorganisms that live in hot growing environments produce heat-resistant enzymes that are thermally stable under the influence of high temperature extremes in the habitat. In addition to heat-stable enzymes, these heat-resistant enzymes have been reported to have stability to organic solvents, stability to pH and chemical modifiers.

Therefore, the present inventors grow at high temperature from the high temperature environment where the domestic high temperature bacteria inhabit in Korea, and are stable to heat to produce enzymes capable of biosynthesizing D, L-amino acid at high temperature (D-AAT, AlaRA, AspAT). Thermophilic microorganisms were searched. As a result, the present invention has been completed by separating new high temperature microorganisms that produce enzymes capable of growing at 70 ° C. and having high thermal stability and catalyzing amino acid biosynthesis from about a thousand high temperature microorganisms isolated from Korean soil. Was done.

The present invention provides novel thermophilic microorganisms producing heat-resistant D-AAT, AlaRA and AspAT, and novel such heat-resistant enzymes produced therefrom, which can be usefully used as biocatalysts for enzymatic synthesis of new D, L-amino acids. Its purpose is to.

Figure 1 shows the base sequence of the 16S rRNA of the Bacillus genus KLS-01 of the present invention compared to the 16S rRNA nucleotide sequence of several microorganisms previously reported,

Figure 2 shows the product L-alanine and D-glutamic acid produced by the heat-resistant D-alanine aminotransferase and alanine racease produced by KLS-01 of the high temperature Bacillus genus of the present invention to the enantio L1 optically active HPLC column The result of the analysis.

In order to achieve the above object, useful for enzymatic synthesis of an amino acid in the present invention (Bacillus sp. KLS-01), the Bacillus genus KLS-01 thermophilic microorganism strain of the novel isolated from Korea soil and which is produced from the microorganism Novel heat resistant D-AAT, AlaRA and AspAT can be used.

Hereinafter, the present invention will be described in detail.

AlaRA, which forms an isomer of amino acid, D-AAT which transfers amino group of D-amino acid to keto acid, and AspAT activity which transfers amino group of L-amino acid to keto acid, is useful for synthesis of new D, L-amino acid Isolation and identification of novel pyrogenic microorganisms of the present invention that can be used are as follows.

First, soil 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%. Inoculate into MY medium having a composition of glycerol, 0.2% K ₂ HPO ₄ , 0.2% KH ₂ PO ₄ , 0.026% NH ₄ Cl and enriched culture at 55 ° C. The culture was inoculated in MY plate agar plate medium containing 2% agar (agar) and incubated at 55 ℃ to isolate the pure colonies. The isolated strain is incubated at 70 ° C. to isolate colonies in which microbial growth is observed within 24 hours.

As a result of examining the microbiological characteristics of the isolated strains, when cultivated in agar plate medium, it forms a creamy opaque colony and grows well under aerobic conditions of 70 ° C or more. Spores were not produced upon culturing or solid culture. Also showed a thin film or the like to form a (pellicle), sseomeoseu microorganism culture unique characteristics of the genus (Thermus) when a long period of time static culture liquid culture when the filament (filament) type cells was observed, on the fluid medium. However, after determining the 16S rRNA base sequence of high temperature bacteria were compared to the nucleotide sequence homology with other microbes reported previously, Bacillus Thermo Denny tree 16S rRNA base sequence with high homology pecan switch (Bacillus thermodenitrificans) Indicated. Therefore, we named the microorganism strain of the invention in a Bacillus (Bacillus) classified into, and Bacillus KLS-01 (Bacillus sp. KLS -01)

As such, KLS-01, a genus of high-temperature microorganism Bacillus , with heat-resistant D-AAT, AlaRA and AspAT activity isolated from soil according to the present invention, was dated January 24, 1997 at the Genetic Resources Center of the Institute of Biotechnology, affiliated with the Korea Institute of Science and Technology. Deposited under accession no. KCTC 0302BP.

Heat-resistant D-AAT, AlaRA and AspAT produced from the thermophilic Bacillus genus KLS-01 were inoculated into 5 ml of MY liquid medium and incubated at 60 ° C. for 8-10 hours. The cells can be crushed to obtain a crude enzyme solution.

Using the coenzyme solution obtained as described above, in the case of D-AAT, D-alanine and α-ketoglutaric acid were reacted with the coenzyme solution in 0.1 M Tris buffer (Tris-HCl, pH 8.5) to generate a reaction by enzymatic reaction. The activity of AlaRA can be quantified by measuring the amount of pyruvic acid as a product, and the activity of AlaRA is reacted with D-alanine, α-ketoglutaric acid and PLP in 0.1 M Tris buffer and It can be quantified by analysis on an Enantio L1 optically active HPLC column. In addition, the activity of AspAT is β-sulfinopyruvate, a reaction product produced by enzymatic reaction by reacting coenzyme solution with L-cysteine sulfinate, α-ketoglutaric acid and PLP in 0.1 M phosphate buffer (pH 8.0). It can be quantified by measuring the amount of.

Amino acids such as D-alanine, D-glutamic acid, D-aminobutyric acid, D-aspartic acid, and pyruvic acid and α-ketoglue using heat-resistant D-AAT and AlaRA generated from the novel high-temperature microorganism of the present invention as described above. New amino acids can be enzymatically synthesized using keto acids such as taric acid and α-ketoisovaleric acid as substrates.

Hereinafter, the present invention will be described in more detail based on the following examples. However, these Examples are only for illustrating the present invention, the present invention is not limited to these.

Example 1 Isolation and Identification of Microorganisms

(Step 1) Separation of Microorganisms

Soil samples taken from haystacks, humus, rivers, wastewater and hot springs around the country were taken from 1.5% polypeptone, 0.2% yeast extract, 0.2% meat extract, 0.2% glycerol, 0.2% K ₂ HPO ₄ , 0.2% KH ₂ PO ₄ , 0.026% HN ₄ Cl was inoculated in MY medium and suspension cultured at 55 ℃. This culture was inoculated in MY plate medium containing 2% agar and incubated at 55 ° C. to isolate pure colonies. After cultivating the isolated strain at 70 ℃ colony was observed to select the growth of microorganism within 24 hours. The thermophilic microorganisms were inoculated in 5 ml MY liquid medium, incubated at 60 ° C. for 8-10 hours, and the cells were harvested by centrifugation.

(Step 2) Investigation and Identification of Microbial Characteristics

The thermophilic microorganism isolated in step 1 formed a creamy opaque colony when cultured in agar plate medium, and it was a gram-positive microorganism that grows well in aerobic conditions of 70 ° C. or higher, but liquid culture or solid culture for 57 hours or more. Did not produce spores. In addition, the cells were observed in filament form when liquid culture, were incubated with characteristics such as to form a thin film (pellicle) during long stationary culture, it can be easily found in a microorganism belonging to the genus sseomeoseu on the liquid medium.

Detailed physiological and biochemical properties of the microorganisms selected according to the present invention are shown in Table 1.

Growth temperature 40-70 ℃ Cell shape Rod shape motility none Endogenous spores none Gram dyeing positivity Catalase positivity Oxidase positivity Voges- Proskauer reaction voice PH of V-P solution 6 or less Nitrate reducing power voice Acid production from glucose voice Acid production from xylose voice Acid production from mannitol voice Gas generation from glucose voice Hydrolysis of casein weakness Gelatin hydrolysis positivity Starch hydrolysis weakness growth at pH 5.7 weakness Growth in the presence of sodium azide voice Growth in the presence of 7% NaCl voice Growth in anaerobic conditions voice

In order to more accurately identify the high temperature bacteria of the present invention having the microbiological characteristics as described above, by determining the nucleotide sequence of 16S rRNA known to be very effective in identifying microorganisms in the genome DNA of the high temperature bacteria Homology of 16S rRNA nucleotide sequences was compared with existing microorganisms.

First, the 16S rRNA gene of the microbial strain of the present invention uses a genomic DNA as a template, and a primer prepared based on the nucleotide sequence of the rRNA gene of Thermus thermophilus , whose base sequence is known. ) Was obtained by performing PCR (polymerase chain reaction). DNA base sequences of the used primers are shown in Table 2. The DNA fragment encoding the amplified 16S rRNA was subcloned into the plasmid pBluescript II and by dideoxy chain termination method (Sanger F., Proc. Natl. Acad. Sci. USA, 74 , 5463-5467 (1977)). Base sequence was determined. BLASTN searching (Stephen F. Altschul, J. Mol. Biol., 215 , 403-410 (1990)) of the 16S rRNA nucleotide sequence of the microorganism of the present invention to homology with the 16S rRNA nucleotide sequence of several microorganisms After a comparison, it was the most similar to Bacillus Thermo Denny pecan tree's (Bacillus thermodenitrificans). Figure 1 shows the base sequence of 16S rRNA of the thermophilic microorganisms screened according to the present invention compared to the 16S rRNA nucleotide sequence of several microorganisms previously reported.

Primers for PCR DNA sequencing N-terminal primer 5'-AGA GTT TGA TCC TGG CTC-3 ': 20mer C-terminal primer 5'-AGA AAG GAG GTG ATC CAG CC-3 ': 20mer

Results isolated strain of the present invention in more were identified as Bacillus (Bacillus species) and named as Bacillus KLS-01. The pyrobacterial was deposited on January 24, 1997 to the Genetic Resource Center in the Institute of Biotechnology, affiliated with the Korea Institute of Science and Technology and was granted accession number KCTC 0302BP.

Example 2 Activity Determination of Heat Resistant D-AAT, AlaRA and AspAT

KLS-01 of the thermophilic Bacillus of the present invention was inoculated in a 5 ml MY liquid medium, incubated at 60 ° C. for 8-10 hours, harvested cells by centrifugation, and the cells were crushed by an ultrasonic crusher to obtain a crude enzyme solution. The activity of each enzyme produced by the microorganism was measured.

(1) Quantification of D-AAT Activity

The reaction solution for measuring D-AAT activity included 10 mM D-alanine and α-ketoglutaric acid in 0.2 ml of 0.1 M Tris buffer (Tris-HCl, pH 8.5) and a coenzyme solution corresponding to 0.1 ml of the culture. It was prepared so as to, and allowed to stand at 55 ° C. for 1 hour. The amount of pyruvic acid produced by the D-AAT reaction was added to the reaction solution by adding 0.2 ml of 60% potassium hydroxide solution and 0.1 ml of 2% salicylaldehyde, and left to react at 37 ° C. for 30 minutes to develop color. Diluted fold and quantified by measuring absorbance at 480 nm (Berntsson S, Anal. Chem., 27 , 1659-1660 (1955)).

(2) Quantification of AlaRA Activity

The reaction solution for measuring AlaRA activity was prepared in 0.2 ml of 0.1 M Tris buffer (Tris-HCl, pH 8.5), corresponding to 10 mM D-alanine and α-ketoglutaric acid, 0.1 mM PLP, and 0.1 ml of culture. The reaction product was prepared to include an enzyme solution, and allowed to stand at 55 ° C. for 2 hours, and then the reaction product was analyzed using an Enantio L1 optically active HPLC column (Tosoh, Japan).

(3) Quantification of AspAT Activity

The reaction solution for measuring AspAT activity was prepared in 0.1 M phosphate buffer (KPB, pH 8.0) using 5 mM L-cysteine sulfinate, 10 mM α-ketoglutaric acid, 0.5 mM PLP, and 0.1 mL of the culture solution. After preparing to contain, it was made to react at 55 degreeC for 15 minutes. The amount of β-sulfinopyruvate produced by AspAT was terminated by adding 20 ml of the enzyme reaction solution and adding 0.1 ml of acidic para-rosaniline (ρ-rosaniline), followed by 0.98 ml of distilled water and 0.1 ml of 0.2% form. It was quantified by adding aldehyde (HCHO) and reacting at 37 ° C. for 30 minutes to measure absorbance at 560 nm (West, PW and Gaeke, GC, Anal. Chem., 28 , 1816 (1956)).

Activity measurement results of enzymes D-AAT, AlaRA and AspAT produced by Bacillus genus KLS-01 (KCTC 0302BP) selected in the present invention are shown in Table 3 below.

enzyme Enzyme deactivation (Units / mg protein) D-AAT 0.02 AspAT 0.11 AlaRA 0.17

Example 3 Synthesis of New Amino Acid Using Enzyme Produced from Microorganism of the Present Invention

Used for the enzyme production is selected separation Bacillus KLS-01 (KCTC 0302BP) was reacted with D- alanine (0.9 g / ℓ) and α- keto glutaric acid (2.3 g / ℓ) in accordance with the present invention, generation The reaction product was analyzed using an Enantio L1 optically active HPLC column (Tosoh, Japan).

Figure 2 is a result of analyzing the product obtained by reacting the coenzyme solution obtained from KLS-01 of the thermophilic Bacillus of the present invention with D-alanine and α-ketoglutaric acid as a substrate by an Enantio L1 optically active HPLC column. As shown in FIG. 2, not only D-glutamic acid but also L-alanine and L-glutamic acid were produced. The resulting D-glutamic acid can be produced solely by the reaction of D-AAT, and L-alanine is produced from D-alanine by AlaRA, an isomerase that specifically acts on alanine. L-glutamic acid is known to be produced from L-alanine and α-ketoglutaric acid by the action of L-alanine aminotransferase (L-AAT). From these results, the thermophilic Bacillus genus KLS-01 of the present invention specifically acts on D-amino acids, which is an isomerization enzyme AlaRA that specifically acts on the enzyme D-AAT and alanine, which can be used to produce various D-amino acids. It can be confirmed that it is a microorganism that produces.

As it described above, according to the invention in Bacillus novel thermophilic microorganism isolated from soil-KLS 01 is AlaRA which catalyzes the formation of D-isomer of alanine and ATT transition acid cake in the amino group of the D- amino acid, and L Produces AspAT, which transfers the amino group of aspartate to keto acid, and can be usefully used as a biocatalyst for enzymatic synthesis of new D- and L-amino acids.

Claims (6)

Bacillus sp. KLS-01 (KCTC 0302BP), a novel thermophilic Bacillus , isolated from Korean soil. A novel heat resistant D-alanine aminotransferase produced from Bacillus genus KLS-01. A novel heat resistant alanine racease produced from Bacillus genus KLS-01. A novel heat resistant L-aspartate aminotransferase produced from Bacillus genus KLS-01. The microorganism of claim 1 or the D-alanine aminotransferase of claim 2 produced by the microorganism and the alanine racease of claim 3 using D-amino acid and keto acid to react new D- and L-amino acids enzymatically. How to synthesize. 6. The method of claim 5, wherein the D-amino acid is at least one selected from D-alanine, D-glutamic acid, D-aminobutyric acid and D-aspartic acid, and the keto acid is pyruvic acid, α-ketoglutaric acid and α. -At least one selected from ketoisovaleric acid.