KR100229284B1 - Novel thermophillic symbiobacterium sp and thermostable tyrosine phenol lyase and tryptonase - Google Patents

Abstract

The present invention relates to novel thermophilic symbiotic microorganisms and heat-resistant tyrosine phenolases and tryptopanases produced therefrom, and the genus Symbiobacterium sp. SC-1 (Symbiobacterium sp. ) Is an absolute symbiotic microbial strain that can only be grown by mixing with Bacillus sp. SK-1 (Bacillus sp. SK-1), and is used for the enzymatic synthesis of L-DOPA, a high-value medicinal substance. It produces a novel heat resistant tryptopanase for use in the enzymatic synthesis of heat resistant tyrosine phenolases and L-tryptophan, a valuable value added amino acid.

Description

Novel thermophilic symbiotic Symbiobacterium microorganisms and heat-resistant tyrosine phenolases and tryptopanases produced therefrom

The present invention relates to a novel high temperature symbiotic microorganism isolated from soil of Korea and heat resistant tyrosine phenolase and tryptopanase produced therefrom, and more particularly, a heat resistant tyrosine phenolic agent which decomposes tyrosine into phenol, pyruvic acid and ammonia. (thermostable L-tyrosine phenol-lyase, EC 4.1.99.2) and heat-resistant tryptophanase, L-tryptophan indole-lyase (EC 4.1.99.1), which breaks down tryptophan into indole, pyruvic acid and ammonia, Symbiobacterium sp. SC-, a thermophilic absolute symbiotic microorganism that can be grown only by mixing with Bacillus sp. SK-1 (Bacillus sp. SK-1; KCTC 0306BP) 1), and the enzymes produced thereby, and the mixed culture method of the symbiotic strain and the strain of the genus Bacillus.

r Mikrobiologie, 50, 20-31 (1967)), 그리고 폴리비닐알콜의 산화적 분해를 위해 협력하는 두 개의 슈도모나스 속 미생물(Shimao. et al., Argic. Biol. Chem., 48, 2873-2876 (1984)) 등을 들 수 있다. 한편, 기생의 관계에서는 기생하는 미생물의 생장을 위해 성장인자의 공급 등 파트너로서의 다른 미생물의 절대적 협력이 요구된다. 예를 들어, 브데로비브리오스(Bdellovibrios)의 경우, 이들의 성장에 필수적인 그램 음성 박테리아를 용혈(lysis)시켜 생육한다.In the natural world, many microorganisms are known to live in a symbiotic or parasitic relationship with each other in order to grow and express physiological functions. Examples include microorganisms in the genus Metanobacterium that cooperate with other microorganisms to produce methane from ethanol (Bryant. Et al., Archiv. F.

Mikrobiologie, 50, 20-31 (1967), and two Pseudomonas genus microorganisms (Shimao. et al., Argic. Biol. Chem., 48, 2873-2876 (C), which cooperate to oxidatively decompose polyvinyl alcohol 1984)). On the other hand, parasitic relationships require the absolute cooperation of other microorganisms as partners, such as supply of growth factors, for the growth of parasitic microorganisms. For example, Bdellovibrios grows by lysis of gram negative bacteria essential for their growth.

Tyrosine phenolase is an enzyme that is widely distributed in gram-negative enterobacteria such as Erwinia and Escherichia, which are cultured in media containing tyrosine as an inducer, and hydrolyzes tyrosine to phenol, pyruvic acid, and ammonia. catalyzes a variety of enzyme reactions, such as β-elimination and β-exchange, which produces phenol and serine from tyrosine (Enei et al., Argic. Biol. Chem., 37, 725-735 (1973)). Tryptopase is a multifunctional enzyme that catalyzes the α, β-removal reaction that hydrolyzes tryptophan to indole, pyruvic acid and ammonia, and the β-exchange reaction that produces indole and serine from tryptophan (Newton et al., Proc Natt.Acad. Sci. USA, 51, 382-389 (1964)), can be used as biocatalysts to synthesize L-tryptophan in indole, pyruvic acid and ammonia. Such tyrosine phenolases and tryptopanases induce enzymatic activity by L-tyrosine and L-tryptophan, respectively, in the medium.

Enzymatic synthesis of L-DOPA (L-dihydroxy phenylalanine), a high value-added pharmaceutical amino acid using tyrosine phenolase, uses an enzyme derived from mesophilic microorganisms. In the bioconversion process using enzymes, the stability of enzymes is a very important factor for productivity. Generally, it is known that thermophilic microorganisms that grow and adapt to a high temperature environment have heat-resistant enzymes that are stable to heat, and these heat-resistant enzymes are not only stable to heat but also stable to organic solvents and extreme hydrogen ion concentrations. It is also known to have stability against and chemically modified agents.

Therefore, the present inventors have attempted to search for a novel thermophilic microorganism that produces a heat-resistant tyrosine phenolase that can be usefully used in the enzymatic synthesis process of L-DOPA, a high-value pharmaceutical amino acid. From the samples collected in the environment, a mixed culture medium showing strong heat-resistant tyrosine phenolase activity was obtained, and the mixed culture medium was diluted with a physiological diluent and reinoculated, followed by two types of thermophilic microorganisms in bacilli form at the minimum dilution rate at which phenol was observed in the medium. After confirming that these two microorganisms are absolute symbiosis with gram negative thermophilic microorganisms having heat-resistant tyrosine phenolase activity and thermophilic Bacillus bacteria producing gram-positive spores, It came.

An object of the present invention is Symbiobacterium sp. SC-1 (Symbiobacterium sp. SC-1), a high-temperature absolute symbiotic microorganism that produces heat-resistant tyrosine phenolases and heat-resistant tryptopanases, and high value-added amino acids produced from the microorganisms. The present invention provides a heat-resistant tyrosine phenolase and tryptopanase, which can be usefully used for the enzymatic synthesis of phosphorus L-DOPA and L-tryptophan, and a mixed culture method of the symbiotic strain with the strain of the genus Bacillus.

Figure 1 is an electron micrograph (magnification: 35,000 times) of the absolute symbiotic microbial strain of the present invention Symbiobacterium SC-1 and Bacillus genus SK-1 of the symbiotic strain

FIG. 2 shows the growth according to time of each strain when cultivation of the mixed strain SC-1 and Bacillus SK-1 in the symbiotic bacterium of the present invention and Bacillus SK-1 in a symbiotic relationship. It is a graph showing the degree.

In order to achieve the above object, the present invention provides a novel high temperature symbiotic Symbiobacterium genus SC-1 having an absolute symbiotic relationship with the high temperature Bacillus genus SK-1 (KCTC 0306BP), isolated from the Korean soil.

The present invention also provides novel heat-resistant tyrosine phenolases and heat-resistant tryptopanases produced from the SC-1 genus SC-1.

According to the present invention there is also provided a method for culturing a novel high temperature Symbiobacterium genus SC-1, characterized by culturing by mixing the high temperature Bacillus genus SK-1 (KCTC 0306BP).

Hereinafter, the present invention will be described in detail.

Isolation and identification of novel high temperature symbiotic microorganisms of the present invention having heat resistant tyrosine phenolase and tryptopanase activity are as follows.

Samples collected from soils and high-temperature environments across Korea were inoculated in a liquid medium containing tyrosine and incubated at high temperature, and then the red color reaction of the phenol produced in the medium with 4-aminoantipyrine reagent (Lacoste, RJ, Anal. Chem., 31, 1246-1249 (1959)) can easily measure the activity of novel heat-resistant tyrosine phenolases produced by high-temperature microorganisms. As a result of the color development test, strong phenol was developed in the mixed culture grown at 60 ° C., and the mixed culture was spread on agar plate medium, and colonies having heat-resistant tyrosine phenolase activity could be separated. There was no. Colonies showing similar enzymatic activity could not be separated by control of the composition of the medium and agar concentration, so that the mixed culture was diluted with physiological saline and reinoculated, and then the bacilli form at the minimum dilution rate where phenol is observed in the medium. Two thermophilic microorganisms of were isolated. Investigation of two isolates of the bacterium type of thermophilic microorganisms revealed that one species was a gram negative thermophilic microorganism with heat-resistant tyrosine phenolase activity, and the other species was a thermophilic Bacillus bacterium that produces gram positive spores. The two thermophilic microorganisms were found to be in absolute symbiosis.

In order to identify the thermophilic symbiotic microorganisms having the heat-resistant tyrosine phenolase activity, the microbiological characteristics were examined, and they grow aerobicly at a temperature of 60 ° C. or higher, and are gram-negative and not yet registered in the Bergy's manual. It was identified as a microorganism belonging to Symbiobacterium (Suzuki et al., J. Gen. Microbiol., 134, 2353-2362 (1988)). The new high temperature symbiotic microorganism producing the heat resistant tyrosine phenolase and heat resistant tryptopanase is named Symbiobacterium sp. SC-1 in Symbiobacterium, and is located in the Biotechnology Research Institute of Korea Institute of Science and Technology. It was deposited in the Genetic Resource Center with accession number KCTC 0307BP dated January 24, 1997.

SC-1 of the high temperature symbiotic microbial Symbiobacterium of the present invention can not be obtained in the form of a single culture medium, and is obtained only as a mixed culture medium with the high temperature Bacillus microorganism, so that the strains of the Bacillus genus must be mixed and cultured. Due to the size difference of the bacteria, it can be separated by centrifugation. As a result of the identification of Bacillus sp. Strains having an absolute symbiotic relationship with the symbiotic microorganisms of the present invention, the thermophilic Bacillus sp. KCTC 0306BP). This thermophilic Bacillus strain is cultivable alone and does not produce tyrosine phenolases when cultured alone.

Heat-resistant tyrosine phenolase and tryptopanase produced from the high temperature symbiotic Symbiobacterium genus SC-1 of the present invention is mixed with the strain in Bacillus SK-1 and MTP liquid medium for 48 hours, followed by centrifugation By thermophilic Bacillus precipitated and the supernatant obtained by centrifugation again to harvest the SC-1 bacteria in the Symbiobacterium, the cells can be crushed to obtain a crude enzyme solution.

Using the crude enzyme solution obtained as described above, it was produced by enzymatic reaction by reacting with L-tyrosine and L-tryptophan as substrates in 0.1 M Tris buffer (Tris-HCl, pH 8.5) or 0.1 M phosphate buffer (pH 8.0), respectively. By measuring the amount of pyruvate which is the reaction product thus obtained, the activity of tyrosine phenolase and tryptopanase produced from the thermosymbiotic microorganism of the present invention can be measured.

L-DOPA is indole from catechol, pyruvic acid and ammonia by the reverse reaction of the enzymatic reaction catalyzed by using a heat resistant tyrosine phenolase and a heat resistant tryptopanase produced from the novel high temperature symbiotic microorganisms as described above. L-tryptophan can be enzymatically synthesized from pyruvic acid and ammonia, respectively.

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, Identification and Culture of Microorganisms

(Step 1) Screening of heat resistant tyrosine phenolase active strains from high temperature bacteria

First, samples collected from haystacks, humus, rivers, wastewater, and hot springs around the country were collected from 0.4% L-tyrosine, 0.3% K ₂ HPO ₄ , 0.1% KH ₂ PO ₄ , and 0.01% HN _4. Inoculated in a minimal liquid medium having a composition of Cl and enriched in culture for 48 hours at 60 ℃. The supernatant of this culture solution was taken, and the produced phenol was distinguished by red color reaction with 4-aminoantipyrine in alkaline aqueous solution to search for active strains of heat-resistant tyrosine phenolase. The sample thus selected was again purified for 0.05% L-tyrosine, 0.1% L-phenylalanine, 0.5% polypeptone, 0.1% yeast extract, 0.3% K ₂ HPO ₄ , After inoculation and incubation in MTP medium, a growth medium of high temperature microorganisms having a composition of 0.05% MgSO ₄ , plated on MTP agar plate medium containing 2% agar and incubated at 60 ° C. to obtain pure colony. Although separation was attempted, no colonies exhibiting heat resistant tyrosine phenolase activity could be obtained. Colonies showing tyrosine phenolase enzyme activity could not be isolated even by adjusting the components of the medium and agar concentration. Therefore, the mixed culture medium showing the first heat-resistant tyrosine phenolase activity was diluted 10 times with dilution with physiological saline and re-inoculated in MTP liquid medium, followed by two types of thermophilic in the form of bacilli at the minimum dilution rate at which phenol was observed in the medium. Microorganisms were identified.

As a result of investigating two types of bacterium type of microorganisms searched and separated as described above, one species is a gram-negative thermophilic microorganism having heat-resistant tyrosine phenolase activity, and the other is a high temperature that produces gram-positive spores. It was St. Bacillus. Selected two kinds of bacilli were mixed cultured in MTP liquid medium and observed by electron microscopy. According to the measurement of cells, one species was 0.2-0.3 μm in width and 1.5-6 μm in length, and the other species was 0.5-0.9 in width. It was 2.5 m in length. 1 is an electron micrograph (magnification: 35,000 times) of a thermophilic microorganism having heat-resistant tyrosine phenolase activity of the present invention and a Bacillus strain symbiotic thereto, wherein the thermophile of the present invention exhibits heat-resistant tyrosine phenolase activity. The microorganisms were identified as small Gram-negative bacilli.

(Step 2) Growth and isolation of absolute symbiotic microbial Symbiobacterium

Two microorganisms obtained by inoculating MTP liquid medium after diluting the sample in which phenol production was detected as in the condition of step 1 in accordance with the appropriate dilution in physiological saline were distinguished by size as shown in FIG. Therefore, paying attention to the difference precipitated by centrifugal force after mixed culture in MTP liquid medium, first, centrifugation was repeated twice at 500xg (centrifugal force) for 10 minutes to precipitate high temperature Bacillus. The obtained supernatant was further centrifuged at 8000xg for 10 minutes to obtain thermophilic microbial cells exhibiting heat resistant tyrosine phenolase activity.

The larger large bacilli were identified as SK-1 strain of Bacillus sp. (Bacillus sp. SK-1; KCTC 0306BP) that produces gram-positive and spores. Did not produce.

Small pyrogenic microorganisms grow aerobicly at temperatures above 60 ° C, gram negative, do not produce spores, and are not yet registered in the Bergy's manual Symbiobacterium. (Suzuki et al., J. Gen. Microbiol., 134, 2353-2362 (1988)). The microorganisms of this thermophilic Symbiobacterium genus include LB (1% tryptone, 0.5% yeast extract, 0.5% NaCl), MY (1% polypeptone, 0.2% yeast extract, 0.2% meat extract, 0.2 Various media such as% glycerol, 0.2% K ₂ HPO ₄ , 0.2% KH ₂ PO ₄ , 0.026% NH ₄ Cl), SLB (1.5% tryptone, 0.75% yeast extract, 1% NaCl, 0.1% casamino acid) and Even if nutrients such as vitamins and sugars were supplied, they could not grow alone. The Gram-negative bacillus was grown with a growth curve as shown in FIG. 2 only when mixed with the Bacillus SK-1 in MTP liquid medium, and mixed culture with another thermophilic Bacillus, such as Bacillus stearothermophilus. In one case, the high temperature symbiotic Symbiobacterium producing heat resistant tyrosine phenolases failed to grow. Figure 2 is the time of each strain when the mixed culture of the thermophilic symbiotic microorganism having a heat-resistant tyrosine phenolase activity of the present invention and SK-1 genus SK-1, which is a symbiotic relationship and SK-1 genus SK-1 alone culture time It is a graph showing the degree of growth according to.

From the above results, it was confirmed that the small Gram-negative bacilli having heat-resistant tyrosine phenolase activity had an absolute symbiotic relationship with the thermophilic Bacillus bacterium that produces gram-positive spores, and SC-1 (Symbiobacterium sp. SC-1).

Symbiobacterium sp. SC-1, a novel thermophilic symbiotic microorganism having heat-resistant tyrosine phenolase and tryptopanase activity according to the present invention, is a gene source in the biotechnology research institute affiliated to the Korea Institute of Science and Technology. It was deposited with the Center on January 24, 1997 and was granted Accession No. KCTC 0307BP.

Example 2 Activity Determination of Heat-Resistant Tyrosine Phenolyase and Tryptopanase

The novel high temperature symbiotic Symbiobacterium genus SC-1 of the present invention was mixed and cultured for 48 hours in Bacillus SK-1 genus MTP liquid medium, and then centrifuged to separate and harvest the Symbibacterium genus SC-1. After the cells were crushed to prepare a crude enzyme solution, heat-resistant tyrosine phenolase and tryptopanase activity were measured.

(1) Determination of heat resistant tyrosine phenolase activity

The reaction solution for measuring heat-resistant tyrosine phenolase activity was prepared to contain 10 mM L-tyrosine and coenzyme solution corresponding to 0.1 ml of culture medium in 0.2 ml of 0.1 M Tris buffer (Tris-HCl, pH 8.5), and 55 ° C. The reaction was allowed to stand for 1 hour at. The amount of pyruvate, a reaction product produced by the heat resistant tyrosine phenolase reaction, was added to the reaction solution by adding 0.2 ml of 60% potassium hydroxide solution and 0.1 ml of 2% salicylaldehyde (salicylaldehyde) for 30 minutes at 37 ° C. After color development, the absorbance was measured at 480 nm by diluting twice with water (Berntsson S., Anal. Chem., 27, 1659-1660 (1995)). The results are shown in Table 1.

(2) Quantification of heat resistant tryptopanase activity

The reaction solution for measuring heat-resistant tryptopanase activity was prepared in 0.2 ml of 0.1 M phosphate buffer (KPB, pH 8.0), containing 10 mM L-tryptophan and coenzyme solution corresponding to 0.1 ml of the culture, and at 60 ° C. It was left to react for 1 hour. The amount of the reaction product pyruvate produced by the heat-resistant tryptopanase reaction was added to the reaction solution by adding 0.2 ml of 60% potassium hydroxide solution and 0.1 ml of 2% salicylate, and then left to react at 37 ° C. for 30 minutes to develop color. After diluting twice with water, the absorbance was measured at 480 nm. The results are shown in Table 1.

One unit of enzyme activity was defined as the amount of enzyme that produced 1 μmol of the reaction product for 1 minute, and the protein content was determined by the Bradford method (Bradford, M., Anal. Biochem., 72, 248-254 (1976)).

Heat-resistant tyrosine phenolase dehydrogenase (Units / mg protein) 0.01 Heat-Resistant Tryptopanase Enzyme Inactivation (Units / mg Protein) 0.34

As described above, Symbiobacterium sp. SC-1 (Symbiobacterium sp. SC-1), a thermophilic symbiotic microorganism strain of the present invention, isolated from the soil of Korea according to the present invention, tyrosine, phenol, pyruvic acid and ammonia L-DOPA and high value-added medicinal substances with high value added by reverse reaction of enzymatic reaction catalyzed by producing heat-resistant tyrosine phenolase and tryptophan, which are decomposed into indole, pyruvic acid and ammonia. It can be used to enzymatically synthesize the amino acid L-tryptophan.

Claims (4)

Novel thermophilic symbiotic Symbiobacterium genus SC-1 (KCTC 0307BP) with absolute symbiosis with thermophilic Bacillus sp. SK-1 (KCTC 0306BP) isolated from Korean soil. A method of enzymatically producing L-DOPA (L-dihydroxy phenylalanine) using the SC-1 strain of the genus Symbiobacterium of claim 1. A method for producing L-tryptophan from indole, pyruvic acid and ammonia using the SC-1 strain of the genus Symbiobacterium of claim 1. A method of culturing the high temperature symbiotic Symbiobacterium genus SC-1 according to claim 1, wherein the high temperature Bacillus sp. SK-1 (KCTC 0306BP) strain is mixed and cultured.

Images