KR100674092B1 - Thermostable tyrosine phenol-lyase variant and method for fabricating the same - Google Patents

Abstract

A tyrosine phenol-lyase variant is provided to have increased activity and thermostability compared to a wild-type tyrosine phenol-lyase, thereby being useful as a biological catalyst and useful for industrially producing tyrosine. The thermostable tyrosine phenol-lyase variant has an amino acid sequence described as SEQ ID : NO. 6. The method for preparing the variant comprises the steps of: (a) culturing a transformed microorganism obtained by introducing an expression vector including a gene encoding the thermostable tyrosine phenol-lyase variant into a host cell such as bacteria, fungus and yeast so as to express the thermostable tyrosine phenol-lyase variant; and (b) isolating the thermostable tyrosine phenol-lyase variant from the thermostable tyrosine phenol-lyase variant expressed microorganism.

Description

Thermostable Tyrosine Phenylase Variants and Methods for Making the Same

FIG. 1 is a chemical reaction diagram showing the synthesis of L-tyrosine and L-DOPA by tyrosine phenol-lyase (TPL).

FIG. 2 is a schematic diagram illustrating an improvement process of TPL for preparing and screening a combinatorial mutagenesis TPL library through error-prone PCR and StEP shuffling. Here, wild-type TPL is shown as TTPL, variant TPL as mTTPL.

FIG. 3 is a gene map of pHCEIIB-mTTPL (2-9A5), a TPL variant expression plasmid prepared by introducing a gene encoding TPL variant into a plasmid vector pHCEIIB (Bioleaders, Korea). Here, variant TPL is expressed as mTTPL.

FIG. 4 is a mutant which searches for a first library prepared by mutagenesis PCR and first selects improved enzymes, and then further selects enzymes whose characteristics are further improved in a second library prepared by StEP shuffling them. It is a schematic diagram showing the amino acid mutation position of the variants shown in purple. The blue ones are the mutants that have been found to improve activity in the first library, and the red ones are the mutants that have been found to improve heat stability.

Figure 5 shows the nucleotide sequence and amino acid sequence of mutant 2-9A5 consisting of 1,377bp base (lower row) and 458 amino acids (upper row). The mutated regions of the base sequence and amino acid sequence compared to the wild type TPL are shown in red.

Figure 6 is a graph comparing the thermal stability of the purified mutant TPL (○) with wild-type TPL (●).

The present invention relates to a heat resistant tyrosine phenolase variant and a method for producing the same, and more particularly, tyrosine phenolase , tyrosine phenolase , which is a tyrosine degrading and synthesizing enzyme derived from Symbiobacterium toebii . .2: hereinafter, abbreviated as TPL), a gene encoding the same, an expression vector containing the gene, a microorganism transformed with the expression vector and a method for producing a high activity and heat resistant TPL variant using the transformed microorganism It is about.

TPL is an α, β-elimination that hydrolyzes tyrosine to phenol and pyruvate, and a β-exchange reaction that produces phenol and serine from the same substrate. enzymes catalyzing a variety of reactions (Enei, et al., Agri. Biol. Chem., 36: 1869, 1972). In addition, an enzyme having a function of reversibly decomposing or synthesizing aromatic amino acids such as tyrosine and L-DOPA and derivatives thereof is also called tyrosinase (FIG. 1). Intestinal microorganisms, such as Citrobacter freundii and Erwinia herbicola , cultured in medium containing tyrosine as an inducer, and thermophilic microorganisms such as Symbiobacterium toebii Commonly found TPL is used as an industrial biocatalyst for the biological synthesis of tyrosine, a therapeutic agent for the high-value medicinal amino acids L-dopa and Vasedou's disease, which are particularly noteworthy as representative Parkinson's disease therapies.

On the other hand, with the recent development of directed molecular evolution technology, not only the development of useful strains or vector improvement, but also researches on antibody engineering, vaccine development, gene therapy, new drug development, biocatalyst improvement, etc. In addition, research on the improvement of TPL, which is useful as an industrial biocatalyst, has emerged.

Directional molecular evolution refers to the evolution of evolution over time in the natural world and to its evolution in a short time in the artificial space of the laboratory. For directional molecular evolution, the enzyme variant library must be created, and the search for the enzyme variant with improved function and properties must be repeated to select variants with improved physical and biochemical properties. The purpose of generating the enzyme variant library is to form a collection group consisting of various nucleotide sequence variants by inducing mutations in the target enzyme, to secure a group of enzymes of various properties with different base sequences.

Directional molecular evolution of enzymes can proceed in many ways, including substrate specificity and activity to substrates and coenzymes and binding capacity, stability in heat or organic solvents, and selectivity to optical isomers. These library generation techniques continue to evolve and include random mutagenesis and DNA shuffling, such as error-prone PCR, chemical mutagenesis, UV irradiation, and the use of mutagenic host cells. Combinatorial mutagenesis and other methods of inducing mutations through the same recombination technique (Kuchner and Arnold, Trends in Biotechnol., 15: 523, 1997). In addition, kits for easily producing mutant libraries are commercially available and can be used for a purpose.

After obtaining gene diversity through the mutant library of enzymes, a technique is needed to search for variants with desired properties. In general, the search technology of the library can be automated by the resistance to antibiotics, the search using specific nutritionally demanding strains, the formation of transparent rings on agar medium, the expression of fluorescent substances, the solid phase selection by colored expression, and the robotic system. There is a wellplate method that can sensitively recognize the difference in activity (Olsen, et al., Curr. Opin. Biotechnol., 11 (4): 331, 2000).

Accordingly, the present inventors have carried out mutation-induced PCR and StEP to synthesize TPL, which synthesizes L-DOPA (L-DOPA), a high value-added medicinal amino acid, and tyrosine (Trosine), a treatment agent for Vasdou's disease. As a result of directional molecular evolution through shuffling, 2-9A5, a variant TPL substituted with valine, lysine, and alanine, respectively, was substituted for the 13th, 83rd, and 451th amino acids of the wild type TPL, and the 2-9A5 was selected from the wild type TPL. The present invention was completed by confirming that not only has high thermal stability but also significantly increased activity.

In conclusion, the main object of the present invention is to provide a variant TPL having significantly improved heat resistance and activity compared to wild type TPL.

Another object of the present invention is to provide a transformed microorganism obtained by introducing the gene encoding the variant TPL, the expression vector containing the gene and the expression vector into a host cell selected from the group consisting of bacteria, fungi and yeast. have.

Another object of the present invention to provide a method for producing a variant TPL using the transformed microorganism.

In order to achieve the above object, the present invention provides a variant TPL having the amino acid sequence of SEQ ID NO.

The present invention also provides a gene encoding a variant TPL having the amino acid sequence of SEQ ID NO: 6 and an expression vector containing the gene.

In the present invention, the gene may be characterized by having a nucleotide sequence of SEQ ID NO: 5, the expression vector may be characterized in that the pHCEIIB-mTTPL (2-9A5), the sequence according to the gist of the present invention If it is an expression vector capable of expressing the variant TPL having the amino acid sequence of No. 6, it is not limited thereto.

The present invention also provides a transformed microorganism obtained by introducing the expression vector into a host cell selected from the group consisting of bacteria, fungi and yeasts. The host cell may be characterized in that the E. coli, but is not limited thereto.

The present invention also comprises the steps of (a) culturing the transformed microorganism to express variant TPL; And (b) provides a method for producing a variant TPL comprising the step of separating the variant TPL from the microorganism expressing the variant TPL.

Hereinafter, the present invention will be described in more detail.

In the present invention, as shown in Figure 2, by inducing mutations in the wild-type TPL derived from Symbiobacterium toebii by a mutagenesis PCR method and a directional molecular evolution method of gene shuffling by StEP (Staggered Extension Process) A variety of high-quality mutant TPL gene libraries were prepared, and variant TPL was selected that greatly improved both activity and thermal stability as a biocatalyst for the synthesis of useful aromatic amino acids.

As a result of analyzing the base sequence of the selected variant TPL from Genotech (Korea), it was confirmed that the gene encoding the variant TPL was SEQ ID NO: 5 having a base sequence of size 1,377bp, and from the base sequence, SEQ ID NO: The amino acid sequence of 6 was estimated.

Comparing the amino acid sequence of the novel heat resistant and highly active variant TPL having SEQ ID NO: 6 with that of wild type TPL, alanine (A), the thirteenth amino acid of wild type TPL, was identified as valine (V), 83. The first amino acid glutamate (E) was confirmed that lysine (K) and the 451 th amino acid threonine (T) were each substituted with alanine, and the variant TPL was substituted with variant TPL 2-9A5 (or ' 2-9A5 '.

In order to prepare an expression vector comprising the gene encoding 2-9A5, the purified PCR product was sequentially processed with restriction enzymes Nde I and Hind III to make introduction DNA, and then the pHCE IIB always contained the gene using an expression vector. The expression vector pHCEIIB-mTTPL (2-9A5) was prepared, and the nucleotide sequence of the plasmid DNA isolated from the recombinant expression vector pHCEIIB-mTTPL (2-9A5) was confirmed. As a result, the vector contains a gene encoding a variant TPL. It was confirmed that there is.

In the present invention, a transformed microorganism obtained by introducing the expression vector into Escherichia coli JM83, which is a host microorganism commonly used for transformation, was prepared by using the electroporation method. Purification of 2-9A5 confirmed enzyme activity and thermal stability. As a result, the enzyme activity was increased about 2.2 times compared to wild-type TPL, the enzyme activity was maintained at 90% or more at 65 ℃, it was confirmed that the heat resistance of about 8.5 ℃ was increased by showing at least 60% enzyme activity at 70 ℃. .

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

In particular, the following examples describe only a method for preparing transformed microorganisms and variant TPLs by introducing a recombinant vector encoding variant TPLs into Escherichia coli JM83, which is a host microorganism commonly used for transformation. It will be apparent to those skilled in the art that transformed microorganisms and variant TPLs can be prepared using any cell selected from the group consisting of fungi as host cells.

Example 1 Preparation of Mutant TPL Gene Library

Mutation-induced mutations in wild-type TPL from Symbiobacterium toebii were induced by mutational PCR and StEP gene shuffling (Zhao, H., Methods in Enzymol ., 388: 42, 2004) TPL gene library was prepared (FIG. 2). Figure 2 is a schematic of the manufacturing process of the expression vector containing the mutant TPL gene by the mutagenesis method used in the present invention.

1) Preparation of random mutation library using mutation-induced PCR method

Genemorph II Random Mutagenesis Kit (Stratazine, USA) was used to prepare mutation libraries by error-prone PCR. As a template DNA, a TPL gene derived from Symbiobacterium compost was used, and a forward primer (SEQ ID NO: 1: 5'-CTCAAGACCCGTTTAGAGGCCC-3 ') and a reverse primer (SEQ ID NO: 2: 5'-ATGCGTCCGGCGTAGAGGAT-3') were used. . Mutagenesis PCR was inactivated for 2 minutes at 95 ° C, followed by 30 cycles of inactivation at 95 ° C for 30 seconds, primer binding at 55 ° C for 30 seconds, and DNA synthesis reaction at 72 ° C for 2 minutes. Finally, DNA synthesis was further performed at 72 ° C. for 10 minutes. The purified PCR product was used as introduction DNA by sequentially processing restriction enzymes Nde I and Hind III. As an expression vector, a pHCEIIB constitutive expression vector (Bioreaders, Korea) was used. The vector was treated with the same restriction enzyme as the introduced DNA, and then dephosphorylated with shrimp alkaline phosphatase (Rosh, USA). The prepared introduction DNA and the expression vector were mixed to a molar ratio of 4: 1, and then reacted with T4 DNA ligase (Takara, Japan) for 24 hours at 4 ° C. The reaction solution was purified to express the mutant TPL gene. Vector pHCEIIB-mTTPL was prepared (FIG. 2). The prepared expression vector was transformed into E. coli JM83, a host microorganism commonly used for transformation by electroporation, to prepare a mutant TPL library.

Preparation of cells for the introduction of genes (competent cells) is a very important process in order to maintain a stable library of high quality was always used to prepare new cells, was carried out in the following process. First, a single colony of E. coli JM83 was inoculated in 4 ml of LB medium and incubated overnight at 37 ° C., and the culture was inoculated at 0.5 ° C. (v / v) in 200 ml of LB medium and shaken at 37 ° C. After about 3 hours, when the OD ₆₀₀ of the culture medium reached 0.5 to 0.6, the mixture was put on ice and left to stand for 20 minutes. The culture solution was placed in a pre-cooled centrifuge container and centrifuged for 20 minutes at 5,000 × g at 4 ° C. After centrifugation, the supernatant was completely removed and suspended by adding cold sterile distilled water to the cell precipitate, and the cells were collected by centrifugation under the same conditions as above, and suspended by adding cold 10% (v / v) glycerol. Centrifugation for 20 minutes at 5,000 x g at 4 ° C. 10% (v / v) glycerol was added to the centrifuged cell precipitate so that the cell concentration (OD ₆₀₀ ) was 100, and then 50 μl aliquots were used.

Electroporation was performed by mixing DNA to the cells prepared above, adding an electric shock (18 kV / cm, 25 μs), and adding 1 ml of SOC medium (terrific broth) to incubate at 37 ° C. at 130 rpm for 1 hour.

The gene library manufacturing process is optimized based on repeated research, and has been used as a base technology for library manufacturing in molecular evolution research. Quality assessment of the prepared mutagenic PCR library was carried out by diluting the transformant recovery solution at various magnifications, plating it on LB (Luria Bertani) solid medium containing 50 µg / ml ampicillin and incubating for 16 hours at 37 ° C. After that, it was performed by a method of measuring the number of colonies. The total library size determined by the calculation was about 1.2 × 10 ⁵ , and 10 colonies were randomly selected and colony PCR was performed. As a result, it was confirmed that the library was a high-quality library containing most TPL genes.

2 ) recovery of the entire random mutation library

To recover the entire random mutant library, spread the cell concentration to about 50,000 cells in LB plate medium containing ampicillin (24 cm × 24 cm, Espiel, Korea) and incubate for 6 hours at 37 ° C. After transferring to the incubator at 12 ° C. for another 12 hours, the resulting colonies were dissolved in 2 × TY (16 g of tryptone, 10 g of yeast extract, and 5 g of NaCl) in distilled water to calibrate the pH to 7 with 5N NaOH, and the final volume was 1 liter. And then sterilized.) (15 ml), 50% glycerol (9 ml), 20% glucose (3 ml) and sterile water (3 ml) are suspended in 10 ml of storage buffer. Recovered. The sample was further centrifuged at 5,000 × g for 20 minutes at 4 ° C., and then added with a preservation solution to suspend the final OD ₆₀₀ to 100-200. DNA was separated and stored at -20 ° C.

3 ) Investigation of the rate of genetic variation by mutagenesis PCR

In order to investigate the ratio of genetic variation by mutation-producing PCR, 10 colonies were randomly taken from the library and sequenced. As a result, the mutation rate was 2-6 bases per 1.377 kb, and the change of 1-2 amino acids per 1 kb. Was expected to happen. It was confirmed that the position where the gene mutation occurred was evenly distributed in all genes without bias in some sections.

4) Screening of Random Mutant TPL Libraries

To screen for random mutant TPL libraries, the libraries were plated in LB plates containing ampicillin and then incubated at 37 ° C. for 16 hours to generate colonies. Into a 96-deep well plate (Bionia, Korea) was added LB liquid medium with ampicillin, and the resulting colonies were inoculated and shaken for 20 hours at 37 ℃ (Megagrow, Bioneer, Korea). After centrifugation of the culture solution at 3,000 rpm for 15 minutes at 4 ℃, the cells were suspended in 50mM Tris buffer solution (pH8) and washed once, followed by centrifugation at 3,000 rpm for 15 minutes at 4 ℃. After recovery, the suspension was suspended in 40 µl of 50mM Tris buffer solution (pH8), and then the same amount of Cellytic B (Sigma, USA) was added to the sample, mixed, and the cells were lysed at 37 ° C for 30 minutes to prepare an enzyme solution.

After taking a certain amount of the enzyme solution and heat-treated at 65 ° C. for 10 minutes, an equal amount of substrate solution (2 mM tyrosine, 20 μM pyridoxal-5-phosphate, 50 mM Tris buffer solution (pH8)) was added thereto, followed by 15 minutes at 37 ° C. After the enzyme reaction was conducted, the enzyme reaction was stopped by heat treatment at 94 ° C. for 3 minutes. Analysis of the amount of phenol produced by enzymatic activity was performed by color development with 4-aminoantipyrine (Arnold, FH and Georgiou, G. Method in Molecular Biology, vol . 230: Directed Enzyme Evolution: Screening and Selection Methods Human Press Inc., Totowa, NJ). The process is centrifuged to remove the cell fragments, the same amount of 0.1 N sodium hydroxide with the sample to the supernatant, 0.6% 4-aminoantipyrine, 0.6% potassium persulfonate (potassium persulfonate) is added After standing for 10 minutes, the absorbance at 490 nm was measured using a Microplate reader (Biorad, USA).

As a result, a total of 12,000 libraries were screened and seven mutants of 1-1C1, 1-2E12, 1-7A5, 1-20G12, 1-33D3, 1-39G3, and 1-39A9 were found to have increased activity and thermal stability. Screened. Among the mutants, four of 1-1C1, 1-2E12, 1-20G12, and 1-39A9 were mutants having increased activity compared to wild-type TPL, and were finally selected by investigating their activity in the range of 37-70 ° C. Tyrosine degrading activity at 37 ° C. was increased by up to 104% (Table 1). The remaining three of 1-7A5, 1-33D3 and 1-39G3 are mutants with improved thermal stability compared to wild-type TPL. It was confirmed that the thermal stability increased up to 5.6 ° C (Table 1).

Enzyme Activity and Thermal Stability of Mutant TPL Mutant Amino Acids Increase of enzyme activity ¹ Increase in thermal stability ² Control Wild type - -  Increased mutation 1-1C1 E42D, T129I + 104% -1.3 ℃ 1-20G12 A196T, T451A + 99% -1.3 ℃ 1-39A9 T129I, V262A  + 47% + 1.5 ℃ 1-41A11 T129I  + 69% -2.8 ℃  Thermostable mutations 1-7A5 T407A  -12% + 5.6 ℃ 1-33D3 E83K   + 5% + 3.1 ℃ 1-39G3 A13V, I457F  + 27% + 5.5 ℃

¹ Tyrosine degrading activity of coenzyme solution of variant TPL was measured at 37 ° C., and compared with that of S. toelii wild type TPL under the same conditions.

^The coenzyme solution containing ² variant TPL was heat-treated for 30 minutes to measure the temperature condition at which the tyrosine deactivation activity was halved, and the difference from the temperature at which the activity of S. toelii nocturnal TPL was halved.

5) Preparation of combinatorial mutation libraries via StEP shuffling

The random mutation library selected four mutants with increased activity and three mutants with increased thermal stability, but those with increased activity showed little improvement in thermostability, and mutants with improved thermostability showed changes in enzyme activity. Appeared to be absent. Therefore, in the present invention, a method for shuffling the mutants of Table 1 with StEP for the purpose of preparing a mutant TPL that is highly active and exhibits high thermal stability (Zhao, H., Methods in Enzymol., 388: 42, 2004 Directional molecular evolution studies were conducted.

The plasmid DNA was isolated from the seven mutants, and StEP PCR reaction was performed using DNA mixed as a template for molar ratios. In the StEP PCR reaction, a forward primer (SEQ ID NO: 3'5'-AACGGCGGCATGTCTTTCTATA-3 '), a reverse primer (SEQ ID NO: 4'5'-ATGCCTGGCAGTTCCCTACTCT-3') and a Vent DNA polymerase (Envy, USA) were used. . The PCR reaction was inactivated at 95 ° C. for 5 minutes, followed by 40 cycles of inactivation at 94 ° C. for 30 seconds, primer binding at 55 ° C. for 5 seconds, and DNA synthesis. Further PCR was performed using the purified StEP PCR product as a template to ensure a large amount of DNA for library preparation. Primers used for PCR were the same as those used for the StEP PCR, inactivated for 5 minutes at 94 ℃, then inactivated for 30 seconds at 95 ℃, primer binding for 30 seconds at 53 ℃, DNA synthesis for 90 seconds at 72 ℃ The procedure of the reaction was repeated 15 times, and finally, a DNA synthesis reaction was further performed at 72 ° C for 10 minutes. The following library preparation steps and recovery were performed in the same manner as described in the random mutation library preparation method of Example 1-1. The total size of the StEP library prepared by the above method was about 1.4 × 10 ⁶ , and 10 colonies were randomly selected and colony PCR was performed.

In order to investigate the degree of recombination of StEP genes, 10 colonies were randomly taken from the library and analyzed for sequencing. No additional mutations were found in addition to the mutations in the mutagenesis mutations. It was confirmed that the combinatorial mutation library prepared by StEP shuffling was of good quality.

6 ) Screening of Combinatorial Mutant Libraries by StEP Shuffling

To screen combinatorial mutant libraries, the libraries were plated in LB plates containing ampicillin and incubated at 37 ° C. for 16 hours to produce colonies. Into a 96-deep well plate (Bionia, Korea) was added LB liquid medium with ampicillin, and the resulting colonies were inoculated and shaken for 20 hours at 37 ℃ (Megagrow, Bioneer, Korea). The following method was carried out in the same manner as the screening of the random mutant TPL library of Example 1-4.

As a result, 2-9A5, a mutant strain having up to 2.8-fold improvement in tyrosine degrading activity at 37 ° C, was obtained from a total of 2,000 mutation libraries, and mutant strains 2-1F3 and 2-9H3 having improved thermal stability up to 11.2 ° C were obtained. (Table 2). However, as a biocatalyst for the synthesis of useful aromatic amino acids, the selection of mutant strains with improved enzymatic activity and thermal stability is desired. Mutation with excellent activity improvement effect (about 2.8 times) and thermal stability improvement effect (6.7 ℃) Host 2-9A5 was selected as the final stone variant in the present invention (Table 2 and FIG. 4).

Enzyme Activity and Thermal Stability of Mutant TPL Obtained by StEP Shuffling Mutant Amino Acids Increase of enzyme activity ¹ Increase in thermal stability ² Wild type - - 2-1F3 A13V, E83K, T407A, T451A  + 87% + 11.2 ℃ 2-6B1 A13V, E83K, I457F  + 42%  + 5.6 ℃ 2-7E2 A13V, E83K  + 42%  + 6.7 ℃ 2-8G3 A13V, T407A  + 12% + 10.1 ℃ 2-9A5 A13V, E83K, T451A + 184%  + 6.7 ℃ 2-9H3 A13V, E83K, T129I, T407A  + 99% + 11.2 ℃

¹ Tyrosine degrading activity of coenzyme solution of variant TPL was measured at 37 ° C., and compared with that of S. toelii wild type TPL under the same conditions.

^The coenzyme solution containing ² variant TPL was heat-treated for 30 minutes, and the temperature condition at which tyrosine degradation activity was halved was measured, and the temperature was different from that at which the activity of S. toelii wild type TPL was halved.

As a result of analyzing the amino acid sequence of the variants of Table 2 in Example 2, it was confirmed that mutants having improved activity and thermostability were recombined in a modular form during StEP shuffling. That is, 2-9A5 contained mutants of A13V, E83K, and T451A. Among them, A13V and E83K were identified from the thermostable mutants shown in Table 1, and T451A was derived from the active mutants. This proves that the StEP shuffling method is effectively applied to the genetic combination of useful mutant traits. On the other hand, heat resistance was improved by more than 10 ℃, the activity improvement was confirmed that all of the mutants (A13V, T407A) of 2-8G3 mutant strains of about 12% are all derived from the thermostable mutant strains (Fig. 4).

Example 2: Determination of the nucleotide sequence of a gene encoding variant TPL (2-9A5) and the amino acid sequence encoded from the gene

The base sequence of the variant TPL 2-9A5 was analyzed by Genotech (Korea). As a result, it was confirmed that the nucleotide sequence of the 2-9A5 gene contained in the recombinant expression vector pHCEIIB-mTTPL (2-9A5) according to the present invention was the sequence described in SEQ ID NO: 5 of FIG. Here, the amino acid sequence shown in FIG. 5 is named SEQ ID NO: 6, and the nucleotide sequence is SEQ ID NO: 5.

The plasmid DNA isolated from the recombinant vector pHCEIIB-mTTPL (2-9A5) having the nucleotide sequence was confirmed to contain a 1,377bp gene encoding TPL. In addition, the amino acid sequence of variant 2-9A5 of SEQ ID NO: 6 was estimated from the nucleotide sequence of the gene (FIG. 5).

Figure 5 shows the nucleotide sequence and amino acid sequence of 2-9A5, the lower part is the nucleotide sequence of SEQ ID NO: 5, the upper part is the amino acid sequence of SEQ ID NO: 6 encoded from the base sequence, compared with wild-type TPL The changed part is shown in red. Compared with the amino acid sequence of wild-type TPL, alanine (A), the 13th amino acid of wild-type TPL, valine (V), glutamate (E), the 83rd amino acid, lysine (K) and 451 The th amino acid threonine (T) was confirmed that each substituted with alanine.

Example 3: Production and Purification of Variant TPL (2-9A5)

Single colonies of wild type TPL and variant TPL 2-9A5 were inoculated in 5 ml of LB liquid medium and incubated overnight at 37 ° C., and then inoculated in 200 ml of LB liquid medium containing ampicillin for 24 hours at 37 ° C. Shake culture was performed. The cells were collected by centrifugation at 5,000 rpm for 10 minutes at 4 ° C., and then washed once with 50 mM Tris buffer solution (pH8) and suspended in 5 ml of the same buffer. Cellytic B (Sigma, USA) was added to the suspension in the same amount as the sample, and 20 μL and 100 μL of DNase (Roche, Germany: 10 U / ml) and lysozyme (Sigma, USA: 100 mg / mL), respectively. After addition and reaction at 37 ° C. for 30 minutes, the cells were completely ground by an ultrasonic cleaner. Cell debris was removed by centrifugation at 15,000 rpm for 30 minutes at 4 ° C., and the supernatant was reacted for 30 minutes at 48 ° C. to denature the protein of host E. coli, which is weak to heat. The mixture was centrifuged at 15,000 rpm for 30 minutes at 4 ° C., and the supernatant was filtered with a 0.2 μm filter, which was then used as a crude enzyme solution. The prepared coenzyme solution is passed through a Resouce Q column (Amersham Bioscience, Sweden), and then the active part is passed through a Phenyl superose column (Amersham Bioscience, Sweden) to purify wild type TPL and variant TPL. It was.

Example 4: Determination of Activity and Thermal Stability of Variant TPL (2-9A5)

In the present invention, the coenzyme solution of E. coli, which is used as the expression host of the variant TPL, contains various kinds of amino acids that may affect the activity of TPL, and unstable proteins or protease in the coenzyme solution affect the thermal stability observation of TPL. Can be crazy Therefore, in the present invention, in order to accurately verify the activity and thermal stability improvement effect of variant 2-9A5, the enzyme activity was measured from each enzyme purified in Example 2, the activity of TPL remaining after heat treatment at each temperature Was measured.

Specifically, to measure the activity of the enzyme, 5 μg of purified enzyme was added to 70 μl 50 mM Tris buffer solution (pH8) and the same amount of substrate solution (2 mM tyrosine, 20 μM pyridoxal-5-phosphate, 100 mM Tris buffer solution). (pH8)) was added and reacted at 55 ° C for 20 minutes. Deactivation of the enzyme was carried out by heat treatment at 94 ℃ for 5 minutes. The analysis of the amount of phenol produced by the enzymatic activity utilized the color development by 4-aminoantipyrine described above. The inactivation of wild type and variant TPL was 1.46U / mg and 3.28U / mg, respectively, and the mutant TPL showed an increase of about 2.2 times higher than that of wild type.

The results are different from those in which the activity improving effect of Example 1-6 was 2.8-fold, because it was determined that the degree of purification of enzymes was different. The difference in activity before and after purification resulted in the deactivation condition of enzyme activity during the purification process, or it was determined that the enzyme activity before purification was higher than after purification because the variant TPL had higher expression level of enzyme than wild type TPL.

To investigate the thermal stability of the variant TPL, 5 µg of purified enzyme was added to 70 µl 50 mM Tris buffer solution (pH8), followed by heat treatment at 25-75 ° C. for 30 minutes, followed by the same amount of substrate solution (2 mM tyrosine, 20 uM pyridoxal- 5-phosphate, 100 mM Tris buffer solution (pH8)) was added and the reaction was carried out at 60 ° C. for 20 minutes, and enzymatic activation was performed by heat treatment at 94 ° C. for 5 minutes. The analysis of the amount of phenol produced by the enzymatic activity utilized the color development by 4-aminoantipyrine described above. As a result, the enzymatic activity of wild-type TPL remained stable up to 60 ° C., but rapidly decreased at higher temperatures, whereas the enzyme activity of variant TPL was maintained at 90% or more at 65 ° C. and 60% or more at 70 ° C., about 8.5 ° C. It was confirmed that the heat resistance was increased (Fig. 6).

Similarly to the present invention, mutant TPL with increased organic solvent and heat resistance has been reported in Citrobacter Prundi (Korea Patent No. 10-0209785, 10-0184755), but no effect has been reported above 40 ° C. . In the case of mutant TPL reported in Symbiobacterium compost (Korean Patent Publication No. 10-2005-0044199), heat resistance and organic solvent stability are increased, and heat resistance is based on a temperature condition in which activity is halved by heat treatment at high temperature. While the heat stability of about 4 ~ 4.5 ℃ is increased, in the case of 2-9A5 of the present invention, the heat resistance is increased by about 8.5 ℃, the variant of the present invention is better than the above-described variants improved thermal stability In addition, 2-9A5 of the present invention was found to have a more excellent heat resistance and high activity because it simultaneously exhibits about 2.2-2.8-fold improvement in activity through gene shuffling with the mutant strain with improved enzyme activity.

The specific parts of the present invention have been described in detail above, and it should be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. will be. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

The present invention provides a novel heat resistant and highly active variant TPL using a directed molecular evolution technology, a gene encoding the same, an expression vector containing the gene, a microorganism transformed with the expression vector and a variant TPL using the transformed microorganism. It is effective to provide a method for producing. Variant TPL according to the present invention is the 13th amino acid alanine (alanine, A) of the wild type TPL (valine, V), the 83rd amino acid glutamate (glutamate, E) is the lysine (K) and 451st amino acid Threonine (T) is replaced by alanine, and the enzyme activity and heat resistance are significantly increased compared to wild type TPL, and it is a biocatalyst that produces L-dopa and Parkinson's disease, tyrosine, which is a treatment for Parkinson's disease. Would be very useful.

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Claims (8)

A tyrosine phenolase variant having the amino acid sequence of SEQ ID NO: 6. The gene encoding the tyrosine phenolase variant of claim 1. The gene according to claim 2, wherein the gene has a nucleotide sequence of SEQ ID NO. An expression vector containing the gene of claim 2. The expression vector according to claim 4, wherein the vector is pHCEIIB-mTTPL (2-9A5). A transformed microorganism obtained by introducing the expression vector of claim 4 or 5 into a host cell selected from the group consisting of bacteria, fungi and yeasts. The transforming microorganism according to claim 6, wherein the host cell is Escherichia coli. Process for preparing tyrosine phenolase variant comprising the following steps: (a) culturing the transformed microorganism of claim 6 to express tyrosine phenolase variant; And (b) separating the tyrosine phenolase variant from the microorganism in which the tyrosine phenolase variant is expressed.

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