KR0184755B1 - Recombinant heat resistant tyrosine phenolase and method for preparing L-dopa using the same - Google Patents

Abstract

The present invention relates to a thermophilic tyrosine phenolase (thermostable tyrosine phenol-lyase, EC4.1.99.2) derived from high temperature microorganisms isolated from Korean soil, and to a method for preparing L-DOPA using the same. Selecting heat resistant tyrosine phenolase producing strains; Determining a base sequence of a gene encoding a heat resistant tyrosine phenolase from the high temperature microorganism; Transforming Escherichia coli by preparing a recombinant plasmid containing the heat resistant tyrosine phenolase gene; And culturing the transformant to obtain a recombinant heat resistant tyrosine phenolase, and producing 3,4-dihydroxyphenylalanine (L-DOPA) by using the same in the production method of L-DOPA of the present invention. It is possible to produce high yield of L-DOPA.

Description

Recombinant heat resistant tyrosine phenolase and method for preparing L-DOPA using the same

1 shows the nucleotide sequence of the gene encoding the recombinant heat resistant tyrosine phenolase of the present invention,

2 shows the amino acid sequence of the recombinant heat resistant tyrosine phenolase of the present invention.

The present invention relates to a thermophilic tyrosine phenolase (thermostable tyrosine phenol-lyase, EC4.1.99.2) derived from high temperature microorganisms isolated from soil in Korea, specifically tyrosine (or L-DOPA) to phenol (or catechol) The present invention relates to a heat resistant tyrosine phenolic agent capable of decomposing into dermatic acid and ammonia, and to a method for preparing 3,4-dihydroxyxalanlanine (hereinafter referred to as L-DOPA), which is a therapeutic agent for Parkinson's disease, using the same.

Tyrosine phenol-lyase is a microorganism cultured in a medium to which tyrosine is added as an inducer, for example, Kusagai and Yamada, J. Biol. Chem., 245, 1767-1772 (1970)), the genus Erwinia (Enei, et al., Agri. Biol. Chem., 37, 725-735 (1973)), the genus Citroacter (Kupletskaya MB, et al., Prinkl.Biokhim.Microbiol., 17, 278-283 (1981)), Symbiobacterium genus (Suzuki, et al., Biosci. Biotech. Biochem. 56, 84-89 (1992)), etc. These enzymes catalyze various enzymatic reactions, such as α, β-elimination reactions that hydrolyze tyrosine to phenol and matric acid, and β-exchange reactions that produce phenol and serine from tyrosine (Enei, et al., Agar Biol. Chem. 36, 1869-1876 (1972)).

Synthesis of L-DOPA using the microbial enzyme tyrosine phenolase was performed under conditions in which the equilibrium of the reaction was physically controlled by adding a high concentration of ammonium ions and pyruvic acid to the reaction solution to which catechol was added instead of phenol in the α, β-removal reaction. This is accomplished by performing an L-DOPA synthesis reaction which is the reverse of the α, β-removal reaction as follows:

Pyruvic acid + ammonium ion + catechol → L-DOPA + water

In bioprocesses that use enzymes as biocatalysts, stability of enzymes, or biocatalysts, is the most important factor. In the enzymatic process of synthesizing L-DOPA, which is a high value-added pharmaceutical amino acid, by enzymatic reaction of heat-resistant tyrosine phenolylase, the stability of the biocatalyst is very important for increasing productivity. In general, high temperature microorganisms that adapt to a high temperature environment are known to have heat-resistant enzymes that are stable to heat. These heat-resistant enzymes are stable to organic solvents, to extreme hydrogen ion concentrations, and to heat-stable enzymes. It is also known to have stability against chemical modifiers. Therefore, since tyrosine phenolia derived from high temperature microorganisms has such enzymatic stability, it may be usefully used as a biocatalyst in the synthesis and production of L-DOPA.

It is an object of the present invention to provide a method for producing tyrosine phenolases in high yield from high temperature microorganisms isolated from soil, and using this to produce L-DOPA.

In accordance with the above object, the present invention provides a polypeptide having heat-resistant tyrosine phenolase activity having the following amino acid sequence:

A gene encoding the polypeptide, an expression vector containing the gene, and E. coli transformed with the expression vector are provided.

The present invention also provides a method for producing a heat resistant tyrosine phenolase comprising culturing the strain and recovering the heat resistant tyrosine phenolase from the cultured cells; And it provides a method for producing L-DOPA comprising the step of adding and reacting the crude enzyme solution obtained by culturing the strain to the reaction solution containing pyruvic acid and catechol.

Hereinafter, the present invention will be described in detail step by step.

(1) Selection of heat resistant tyrosine phenolase producing strains from high temperature microorganisms isolated from soil

Among samples collected from various environments where high temperature microorganisms can grow, about 1,000 species of high temperature microorganisms that can grow under aerobic conditions of 55 to 60 ℃ are separated, shaken and cultured at 55 ℃, and the cells are recovered by heat resistance. Microorganisms having the activity of tyrosine phenolases were obtained. As a result, the microorganisms were found to be Gram-negative thermophilic microorganisms and Gram-positive thermophilic Bacillus microorganisms, which are absolute symbiotic microorganisms.

(2) Determination of the base sequence of a gene encoding a heat resistant tyrosine phenolase

The total chromosome is separated from the isolated high temperature symbiotic microorganism, partially hydrolyzed with restriction enzyme Sau3AI, and then electrophoresed to separate DNA fragments of about 3 to 10 kb in size. Subsequently, the isolated DNA fragment is combined with the vector pBR322 to prepare a recombinant plasmid, and then transformed into Escherichia coli JM105 by electroporation. A recombinant Escherichia coli exhibiting heat resistant tyrosine phenolase activity was obtained through a screening process for examining the heat resistant tyrosine phenolase activity of the transformants. The recombinant plasmid isolated therefrom contained a chromosomal DNA fragment derived from a high temperature bacterium of about 7.5 kb in size, hereinafter referred to as the recombinant plasmid pHT1.

After extracting and purifying the recombinant plasmid pHT1, a restriction map is prepared to determine the portion containing the heat resistant tyrosine phenolase gene. Unnecessary portions of the inserted DNA of pHT1 are digested with appropriate restriction enzymes, and the entire nucleic acid sequence is identified according to Sanger et al. (Sanger, F. et al., Science, 214, 1205-1210 (1981)). As a result, it was confirmed that this DNA fragment (recombinant plasmid pHT2) contained a 1,385 bp gene encoding a heat resistant tyrosine phenolase.

(3) Preparation of expression vector pHTL1 containing heat resistant tyrosine phenolase gene and transformation into E. coli

The N-terminal primer and the C-terminal primer are prepared by referring to the nucleotide sequence of the gene encoding the heat-resistant tyrosine phenolase determined above, and amplify the DNA fragment by PCR. The amplified DNA was recombined into plasmid pTrc99A to produce expression vector pHTL1, which was then transformed with E. coli JM105.

(4) Expression of Recombinant Heat Resistant Tyrosine Phenolic Agents and Production of L-DOPA Using the Same

Recombinant E. coli strain transformed with the recombinant plasmid pHLT1 was cultured in a medium containing 0.6 mM isopropylthiogalactoside (IPTG) to express a large amount of heat-resistant tyrosine phenolase, and then cells were recovered and crushed to heat-resistant tyrosine phenol. Prepare a lyase coenzyme solution.

This crude enzyme solution is added to a reaction solution containing an excess of ammonium acetate, sodium pyruvate and catechol and subjected to an enzymatic conversion reaction using tyrosine phenolase at 37 ° C. As a result of performing the reaction for 12 hours, 36.1 g / L of L-DOPA could be prepared.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

EXAMPLE

(Step 1) Selection of heat resistant tyrosine phenolase producing strains from high temperature microorganisms isolated from soil

First, soil samples were taken from compost, humus, rivers, wastewater, and hot springs throughout the country, and tyrosine phenolase-induced media (TMY medium; 15% polypepton, 0.2% yeast extract, 0.2% Inoculation in broth (meat extact), 0.2% glycerol, 0.2% K ₂ HPO ₄ , 0.2% KH ₂ PO ₄ , 0026% NH ₄ Cl, 0.2% L-tyrosine) and entry culture at 55-60 ° C. It was. This culture was inoculated in a tyrosine phenolase-inducing and selective plate medium containing 2% L-tyrosine and 2% agar (TMY-agar) and incubated at 55-60 ° C., after which tyrosine was decomposed to give clear ring. Colonies that produce (clear zones) were separated. It was confirmed that the isolated strain was a thermophilic symbiotic bacterium (Symbiobacterium sp.) In which gram-negative thermophilic microorganisms and gram-positive thermophilic Bacillus bacteria coexist and grow.

After the isolated high temperature symbiotic strains were incubated in 5 ml of TMY liquid medium for 8 to 10 hours, the cells were harvested, and the cells were crushed with an ultrasonic crusher to prepare a coenzyme solution, and heat-resistant tyrosine phenolase activity was measured.

The reaction solution for measuring heat-resistant tyrosine phenolase activity was prepared to include 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 at 50 ° C. It was left to react for 1 hour. The amount of pyruvate produced by the reaction of the heat-resistant tyrosine phenolic acid was developed by adding 0.2 ml of a 60% potassium hydroxide solution and 0.1 ml of 2% salicylaldehyde to the reaction solution and allowing it to stand at 37 ° C for 30 minutes to develop color. Then, it was determined by measuring the absorbance at 480 nm by diluting twice with water (Berntsson S. Anal. Chem. 27, 1659-1660 (1955)).

(Step 2) Determination of the base sequence of the gene encoding the heat resistant tyrosine phenolase

The high temperature symbiotic microorganism isolated in step 1 was inoculated in 100 ml of TMY medium and incubated at 55 ° C. for 24 hours, followed by centrifugation to recover the cells. The recovered cells were washed with SE (0.15M NaCl, 0.1M EDTA, pH 8.0), suspended in 10 ml of ST (0.1M NaCl, 0.01M EDTA, 0.1M Tris-HCl, pH 9.0), and then suspended in suspension. 5-10 mg of lysozyme, 2.5 ml of TS (0.14 M NcCl, 0.001 M EDTA, 0.02 M Tris-HCl, pH 7.5) and 5% SDS) and 12 mg of protease were added and left at 37 ° C. for 3 hours. Subsequently, 12.5 ml of phenol was added thereto, and the mixture was left at room temperature for 15 minutes, followed by centrifugation. After carefully separating the supernatant, two volumes of ethanol were added to separate chromosomal DNA with a glass rod. The chromosomal DNA was washed with TE (0.01M Tris-HCl (pH 7.5), 0.1 mM EDTA): ethanol (v / v) in a solution of 3: 7, 2: 8 and 1: 9, respectively, followed by SSC (( 0.1M NaCl, 0.15M sodium citrate), RNase A was added to the solution and left at 37 ° C., followed by centrifugation with the same amount of phenol and careful separation of the supernatant. After dissolving it in TE, it was dialyzed again in 100 times of TE overnight to obtain 2 ml of crude chromosomal DNA.

The crude chromosomal DNA was partially hydrolyzed with restriction enzyme Sau3AI and isolated by 0.7% agarose gel electrophoresis. The gel was stained with ethidium bromide (EtBr) and DNA fragments were isolated and purified (GENE CLEAN II KIT) by cutting a band of about 3 to 10 kb in size with a sterile knife. The isolated DNA fragment was hydrolyzed with restriction enzyme BamHI, 5'-terminal phosphate was removed, mixed with vector pBR322 (available from Pharmacia, Sweden) and reacted with T4 DNA ligase at 16 ° C. for 16 hours for recombination. The plasmid was allowed to generate. The recombinant plasmid was used for transformation of Escherichia coli JM105 (available from Pharmacia, Sweden) according to the electroporation method. The transformed Escherichia coli were cultured in a tyrosine phenolase selective separation medium containing ampicillin and 2% L-tyrosine. The selection of E. coli strains producing tyrosine phenolase is the same as the method used to select tyrosine phenolase producing strains from the high temperature bacteria in step (step 1). Through this screening process, one heat-resistant tyrosine phenolase active strain was obtained. After extracting and purifying the chromosomal DNA fragment (pHT1) of about 7.5 kb in size, a restriction enzyme map was prepared to determine the portion containing the heat resistant tyrosine phenolase gene. Unnecessary portions of the inserted DNA of pHT1 were digested with appropriate restriction enzymes to obtain fragment pHT2, and DNA sequences were synthesized according to Sanger et al. (Sanger, F. et al., Science, 214, 1205-1210 (1981)). The amino acid sequence was determined from this analysis.

FIG. 1 shows the nucleotide sequence of the gene encoding the recombinant heat resistant tyrosine phenolase of the present invention, and FIG. 2 shows the amino acid sequence of the recombinant heat resistant tyrosine phenolase of the present invention.

(Step 3) Preparation of the expression vector pHTL1 included in the heat resistant tyrosine phenolase gene and transformation into E. coli

N-terminal primer (5'-CAGCGACCCTGGGCGGAACC-3 ') and C-terminal primer (5'-TGACTAAGTCAAGCTTATTAGCTGATCGGCTCGAAGCG-3') were prepared based on the nucleotide sequence determination of the gene encoding the recombinant tyrosine phenolase cloned from the thermophilic microorganism. It was. Using these primers, a gene fragment encoding a heat resistant tyrosine phenolase using a recombinant fragment pHT2 as a template was amplified by PCR. The amplified DNA was recombined into plasmid pTrc99A (available from Pharmacia, Sweden) to prepare a high expression vector pHLT1, and then the expression vector pHLT1 was transformed into E. coli JM105. The transformed E. coli JM105 / pHLT1 was deposited on April 11, 1995 to the gene bank attached to the Korea Institute of Science and Technology Biotechnology Research Institute as Accession No. KCTC 0158BP.

(Step 4) Expression of recombinant heat resistant tyrosine phenolase and production of L-DOPA using the same

LB medium (1% tryptone, 0.5% yeast extract, 0.5%) with E. coli strain JM105 (KCTC 0158BP) containing recombinant plasmid pHLT1 for producing recombinant heat resistant tyrosine phenolase, and 100 ml / L of ampicillin NaCl) was inoculated at 200 ml and incubated at 37 ° C. with 200 rpm. When the cell concentration OD ₆₀₀ reached about 0.8, Isopropylthiogalactoside (IPTG) was added at a concentration of 0.6 mM to induce the production of heat-resistant tyrosine phenolases and continued incubation for 8 hours, followed by centrifugation at 5,000 rpm for 20 minutes. The cells were separated and recovered. The recovered cells were crushed using an ultrasonic mill and centrifuged to prepare a heat-resistant tyrosine phenolic coenzyme solution. The cells produced in LB medium had an absorbance at ₆₀₀ nm (OD ₆₀₀ ) of about 5.2, and the protein produced by SDS-PAGE analyzed the heat-resistant tyrosine phenolase about 15% of the total protein of recombinant E. coli. It was produced in the state. Meanwhile, the protein concentration of the recombinant heat-resistant tyrosine phenolase coenzyme solution was 16.4 mg / ml, and the specific activity was about 0.45 unit / mg protein by L-DOPA synthesis activity quantified at 37 ° C.

To synthesize and produce L-DOPA by enzymatic conversion, 0.65 M ammonium acetate (pH 8.5), 50 mM sodium pyruvate, 25 mM catechol, 0.1 mM pyridoxal-5-phosphate and 0.1% sodium sulfite, In addition, a recombinant heat-resistant tyrosine phenolase coenzyme solution was added at a concentration of 1.5 units / ml to prepare an enzyme conversion reaction solution for synthesis and production of L-DOPA. The reaction solution was sealed with a rubber stopper, filled with nitrogen gas, and then placed in a 37 ° C. water bath, followed by L-DOPA synthesis and production reaction with gentle stirring. In order to produce L-DOPA at high concentration through the enzymatic conversion reaction, catechol and sodium pyruvate as substrates were intermittently added and supplied to the reaction solution.

After 1 to 2 hours from the start of the L-DOPA synthesis and production reaction, white precipitate of L-DOPA began to be observed in the reaction solution, and the reaction solution became turbid due to the increased production of L-DOPA. After 2 to 3 hours, the reaction solution turned into a completely opaque solution.

As a result of performing the reaction for 12 hours, it was confirmed that a total of 36.1 g / L of L-DOPA was produced.

Claims (9)

Polypeptides having heat resistant tyrosine phenolase activity having the following amino acid sequence: A gene encoding the polypeptide of claim 1. The gene according to claim 2, which has the following nucleotide sequence: A heat resistant tyrosine phenolase expression vector comprising the gene of claim 2. The expression vector pHLT1 according to claim 4. E. coli transformed with the expression vector of claim 4. E. coli JM105 / pHLT1 (KCTC 0158BP). A method for producing a heat resistant tyrosine phenolase comprising culturing the strain of claim 6 and recovering the heat resistant tyrosine phenolase from the culture. A method for producing L-DOPA comprising the step of adding a crude enzyme solution obtained by culturing the strain of claim 6 to the reaction solution containing pyruvic acid and catechol.