KR100330688B1 - Gene Coding for Heat-resistant Alanine Racemase of Aquifex pyrophilus, Heat-resistant Alanine Racemase Expressed therefrom, and Method for Preparing the Same - Google Patents

Abstract

The present invention is a gene of alanine racease enzyme having a strong heat resistance isolated from Aquifex pyrophilus, a sedative bacterium of ultra-high temperature microorganisms, alanine racease having an amino acid sequence expressed therefrom, containing the gene It relates to a method for producing the alanine racemes in E. coli transformed with an expression vector.

Alanine racease produced in E. coli transformed with an expression vector comprising the gene of alanine racease of Aquipex pyrophyllus isolated in the present invention is a higher temperature, pH and organic solvent than enzymes isolated from conventional mesophilic bacteria. Since it has high stability even under such conditions, it can be usefully used industrially, such as production of pharmaceuticals and production of food materials.

Description

Gene Coding for Heat-resistant Alanine Racemase of Aquifex pyrophilus, Heat-resistant Alanine Racemase Expressed therefrom, and Genes encoding the heat resistant alanine racease of Aquipex pyrophyllus Method for Preparing the Same}

The present invention relates to a gene of alanine racease enzyme having a strong heat resistance derived from aquipex pyrophyllus which is a very high temperature microorganism, an alanine racease having an amino acid sequence expressed therefrom and a host transformed with an expression vector containing the gene. It relates to a method of preparing said alanine racease in a cell. More specifically, the present invention relates to a heat resistant alanine racease having a gene having the nucleotide sequence shown in SEQ ID NO: 1 and an amino acid sequence expressed therefrom.

Alanine racease enzyme is an enzyme that converts D-alanine to L-alanine, or L-alanine to D-alanine. D-alanine produced in this process is used to form cell membranes of microorganisms.

D-amino acids form precursors for the production of antibiotics and are used as raw materials for antibiotic synthesis, and their use in the food industry such as sweeteners, seasonings and food additives is increasing. Such D-amino acids have been mainly produced by organic synthesis until now, but recently, a method for producing D-amino acids using enzymes such as amino acid racemes has been sought. In particular, alanine racease is one of the key enzymes in the production process of D-amino acid using amino acid racemease, and development of highly stable enzyme is required to develop a more efficient method for producing D-amino acid. Bacillus subtilis (Ferrari, E., Henner, DJ, and Yang, MY (1985) Isolation of an alanine recemase gene from Bacillus subtilis and its use for plasmid maintainance on B. subtilis.Biotechnology 3, pp 1003-1007) or Bacillus stearothermophilus (Inagaki, K., Tanizawa, K., Bade, B., Walsh, c. T., Tanaka, h., And Soda, K. (1986) Thermostable alanine recemase from Bacillus stearothermophilus: Molecular cloning of the gene, enzyme purification, and characterization.Biochemistry Vol 25, pp3268-3274) There is no research on alanine racemes.

Biological resources used in enzymatic engineering up to now are enzymes originating from living organisms growing at room temperature and high temperature. These enzymes have limited stability and activity in environments such as high temperature, acidic, basic or high salinity. However, recently, very high temperature microorganisms that can grow near or above the boiling point of water of 100 ° C. have been reported, and enzymes isolated from these microorganisms are known to remain active even at high temperatures. Therefore, there is a need to develop enzymes that can be used to produce D-amino acids in these ultra-high temperature microorganisms.

Accordingly, an object of the present invention is to isolate alanine racease genes from ultra-high temperature bacteria and express them in host cells to provide alanine raceases having high stability even under conditions such as high temperature, pH, and organic solvents.

1 shows an alanine racease gene and a DNA sequence around the gene and an amino acid sequence encoded thereby.

Figure 2 is a block diagram of pAR1 which is a plasmid expressing the alanine racease gene cloned from Aquipex pyrophyllus.

Figure 3 is a photograph showing the results of electrophoresis on the modified polyacrylamide gel of alanine racemate purified by heat treatment.

Figure 4 is a graph showing the results of measuring the reactivity to convert the D-alanine of the alanine racease enzyme of Aquipex pyrophyllus expressed and purified in E. coli to L-alanine at each pH.

5 shows the results of measuring the reactivity of converting D-alanine of alanine racease enzyme of Aquipex pyrophyllus expressed and purified in Escherichia coli into L-alanine at 37, 60, 85, 90, 95, and 100 ° C. Graph shown.

Figure 6 is a graph showing the results of measuring the heat resistance of the alanine racease enzyme isolated from the ultra-high temperature Aquipex pyrophyllus.

The present inventors have conducted in-depth studies to achieve the above object. As a result, the inventors have found that alanine racemes isolated from the very high temperature bacterium Aquipex pyrophyllus have activity while maintaining a stable state even at a very high temperature. It was completed.

That is, the present invention cloned the alanine racease gene that can be used for the production of D-amino acid in the high temperature microorganism Aquipex pyrophyllus and characterized the structure of the gene and then cloned the cloned gene by genetic recombination method It relates to a method for mass production of the enzyme by expression in E. coli.

The present invention provides a gene of heat resistant alanine racease isolated from Aquipex pyrophyllus which is an ultra high temperature bacterium having heat resistant alanine racease. More specifically, the present invention provides a gene encoding the heat resistant alanine racease that is stable and has high reactivity even at a high temperature of 80 ° C. or more, having the nucleotide sequence of SEQ ID NO: 1.

In another aspect, the present invention provides a heat resistant alanine racease having the amino acid sequence of SEQ ID NO: 2 expressed from the gene of the heat resistant alanine racease.

In addition, the present invention includes an expression vector comprising the gene and a host cell transformed with the expression vector. E. coli is particularly preferred as the host cell.

The present invention also provides a method for producing heat-resistant alanine racease, comprising culturing the transformed host cell, crushing the cultured E. coli, and recovering the heat-resistant alanine racease expressed from E. coli.

Alanine racemese derived from Aquipex pyrophyllus produced by the present invention has the advantage that it is more stable to heat and pH than the enzyme in the mesophilic bacteria existing in the production of pharmaceuticals and food materials.

The alanine racease gene of Aquipex pyrophyllus is expressed by Kim, S., Choi, I.-G., Kim, S.-H., and Yu, YG (1999) Molecular cloning, expression, and characterization of a thermostable glutamate recemase from a hyperthermophilic bacterium, Aquifex pyrophilus.Extremophiles Vol. 3 pp175-183). As reported by Choi, et al. (Choi, I.-G., Kim, SS, Ryu, J.-R., Han, YS, Bang, W.-G., Kim, for the isolation of alanine racease enzyme). S.-H., Yu, YG (1997) Random sequence analysis of genomic DNA of a hyperthermophile: Aquifex pyrophilus.Extremophiles Vol. 1, pp125-134), Alanine of Escherichia coli and Bacillus subtilis in Aquipex pyrophilus AQpU219, a clone containing a nucleotide sequence highly similar to racease in the pUC19 vector, is used as a probe. In this plasmid DNA, aquipex pyrophyllus DNA in the pUC19 vector is amplified by polymerase chain reaction using M13 forward primer and M13 reverse primer. Using the amplified DNA fragments as probes, hybridization is performed in the genome library of Aquipex pyrophyllus to search for and isolate clones showing one positive signal. The base sequence of the site corresponding to the alanine racease gene in the isolated clone can be determined by chain termination.

The base sequence of the alanine racease gene cloned according to the present invention and the sequence of amino acids encoded therefrom are as shown in FIG. 1. The determined base sequence consists of 1468 bases, of which 1023 bases have been identified that encode a protein consisting of 341 amino acids.

As a result of comparing and analyzing the determined nucleotide sequence with alanine racease of another known species, the amino acid sequence encoded by the Aquipex pyrophyllus alanine racease gene isolated in the present invention was 29.3% compared with E. coli, and Bacillus stearothermophilus), 38.4% and Lactobacillus (Lactobacillus) and 33.4% identity. In particular, the amino acid sequence of the lysine site, the 33rd amino acid contributing to the alanine racease enzyme activity was confirmed that the high similarity.

In order to mass express alanine racemes of Aquipex pyrophyllus isolated as described above, the gene of this protein is cloned into pET28a, an E. coli expression vector. First, primers are prepared by adding recognition sites of NdeI and XhoI restriction enzymes to the 5 'and 3' terminal regions of the alanine racease gene.

Using these two primers, the alanine racease gene is amplified from the genomic DNA of Aquipex pyrophyllus by polymerase chain reaction, and the amplified DNA is cleaved with restriction enzymes NdeI and XhoI and then applied to the NdeI and XhoI sites of the pET28a vector. In combination, pAR, an alanine racease expression vector, is prepared.

After confirming the exact nucleotide sequence of the alanine racease gene present in the prepared vector, the vector is used to transform host cells, such as Escherichia coli BL21 (DE3). Recombinant host cells transformed with pAR1 were cultured in LB containing 50 μg / ml of kanamycin at 37 ° C. under conditions suitable for growth, and 1 mM of isopropyl β- when OD ₆₀₀ reached 0.6. Cells are harvested by centrifugation after inducing the expression of proteins using D-thiogalactoside (IPTG).

Subsequently, the cells recovered by the centrifugation method are suspended in a dilution solution, and then the cell extracts generated by crushing the cells are centrifuged to remove the unbroken cells. The supernatant is heat treated, centrifuged to remove the denatured protein recovered as a precipitate, and the supernatant is chromatographed to purify the expression product.

The alanine racemes of the expressed Aquipex pyrophyllus were found to have a size of about 38 kDa as shown in FIG. 3 on a 15% modified polyacrylamide gel and had an expression level of 5% or less of the total protein. It was confirmed.

As a result of investigating the D-amino acid conversion activity of the purified protein to L-amino acid, the highest conversion activity of L-alanine to D-alanine, and showed a slight activity of converting L-serine to D-serine, It was found that the conversion activity for amino acids is very small. This result demonstrates that the cloned gene is an alanine racease gene of Aquipex pyrophyllus.

The optimal pH conditions of the expressed alanine racease enzyme activity can be investigated by measuring the enzyme activity at various pHs using different buffers, and it can be seen that the highest activity is at pH 9.5 as shown in FIG. .

Optimum enzyme reaction temperature can be determined by measuring the activity of the enzyme at 37, 60, 85, 90, 95, 100 ℃, as shown in Figure 5 showing the highest activity at 85 to 90 ℃ Aquipex Pyro Optimal conditions for the enzymatic activity of P. alanine racemese were found to be buffer pH 9.5, reaction temperature 85-90 ° C.

The high heat resistance seen in proteins isolated from very high temperature microorganisms can be investigated by measuring the expression of alanine racemes expressed at 95 ° C. for several hours to remove denatured proteins and measuring the activity of the remaining enzyme. As shown in FIG. 6, alanine racease enzyme of Aquipex pyrophyllus shows little change in activity when left at 95 ° C. for 60 minutes and maintains about 50% of activity when left for 4 hours. Can be.

These characteristics indicate that alanine racemes isolated from Aquipex pyrophyllus have high heat resistance and retain their enzymatic activity in the original ultra high temperature bacteria even when expressed in E. coli.

Hereinafter, the present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto.

<Example 1>

Cloning of Alanine Racease Gene

The alanine racease gene of Aquipex pyrophyllus was isolated from a genomic library of Aquipex pyrophyllus strains prepared by the method of Kim Sang-suk as described above. In order to isolate the alanine racease enzyme, a probe containing AQpU219, a clone containing a nucleotide sequence highly similar to alanine racease of Escherichia coli and Bacillus subtilis, was isolated from the gene fragment of Aquipex pyrophyllus as reported by Choi In-Geul et al. Used as.

In this plasmid DNA, Aquipex pyrophyllus DNA in the pUC19 vector was subjected to 30 ° C of 96 ° C for 1 minute, 55 ° C for 30 seconds, and 72 ° C for 1 minute using M13 forward primer and M13 reverse primer. It was amplified by the polymerase chain reaction repeated several times. Using the amplified DNA fragments as probes, plaque hybridization and southern hybridization were performed in the genome library of Aquipex pyrophyllus to search for and isolate clones showing one positive signal.

The base sequence of the site corresponding to the alanine racease gene in the isolated clone was determined by chain termination. The base sequence of the cloned alanine racease gene and the sequence of amino acids encoded therefrom are shown in FIG. 1. The determined base sequence consists of 1468 bases, of which 1023 bases have been identified that encode a protein consisting of 341 amino acids.

As a result of comparing the determined nucleotide sequence with alanine racease of another known species, the amino acid sequence was 29.3% with E. coli, 38.4% with Bacillus stearothermophilus, and 33.4% with Lactobacillus. In particular, the amino acid sequence of the lysine site, the 33rd amino acid contributing to the alanine racease enzyme activity was confirmed that the high similarity.

<Example 2>

Expression Vector Construction and Expression of Alanine Racease Protein

The gene of this protein was cloned into pET28a, an E. coli expression vector, to produce an active protein from the alanine racease gene of Aquipex pyrophyllus. First, primers in which the recognition sites of NdeI and XhoI restriction enzyme were added to the 5 'and 3' terminal regions of the alanine racease gene were prepared.

Using these two primers, alanine racease gene was amplified by polymerase chain reaction from genomic DNA of Aquipex pyrophyllus. The DNA thus obtained was digested with restriction enzymes NdeI and XhoI, and then bound to the NdeI and XhoI sites of the pET28a vector to prepare pAR, an alanine racease expression vector. After confirming the exact nucleotide sequence of the alanine racease gene present in the prepared vector, the vector was used to transform the host E. coli BL21 (DE3). Recombinant BL21 (DE3) transformed with pAR1 was cultured in LB containing 50 μg / ml kanamycin at 37 ° C., and when OD ₆₀₀ reached 0.6, 1 mM isopropyl β-D-thiogalactoside ( Cells were harvested by centrifugation 3 hours after inducing protein expression using IPTG).

Figure 3 shows the results of electrophoresis of alanine raceme purified by heat treatment on a modified polyacrylamide gel, column 1 is a marker indicating the size of the protein, column 2 before the expression of the alanine racease gene E. coli protein sample, column 3 is E. coli protein sample after the expression of the alanine racease gene, column 4 is a protein sample after heat treatment, column 5 is an alanine racease enzyme isolated by cation exchange resin. The position of the alanine racease protein is indicated by the arrow.

As shown in FIG. 3, the alanine racease of the expressed Aquipex pyrophyllus was found to have a size of about 38 kDa, as determined by a 15% modified polyacrylamide gel, and the expression level of 5% or less of the total protein. It was confirmed to have.

<Example 3>

Purification of Alanine Racease Enzyme Produced in Escherichia Coli

Alanine racemes derived from Aquipex pyrophyllus expressed in E. coli were isolated by the following method. As shown in Example 2, E. coli expressing alanine racemes was recovered by centrifugation, and the cells were suspended in dilute solution (50 mM Tris-HCl buffer, pH7.8 and 50 mM NaCl), followed by a French press. Pressure was applied to disrupt the cells. The cell extracts were centrifuged at 16000 rpm for 20 minutes to remove unbroken cells. The supernatant was heat-treated at 84 ° C. for 1 hour, centrifuged at 16000 rpm for 20 minutes to remove denatured E. coli protein recovered as a precipitate, and the supernatant was purified using Ni-NTA agarose (Quiagen, Germany).

The alanine racease enzyme expressed in E. coli retains its original properties showing high thermal stability, while most of the E. coli proteins are removed during the heat treatment, and the alanine racease expressed through the heat treatment is purified to about 55%. When purified by chromatography it was purified to about 80%. Purification levels of alanine racemes after heat treatment and Ni-NTA agarose are shown in FIG. 3.

<Example 4>

Determination of Enzyme Activity and Characterization of Alanine Racease of Aquipex Pyrophyllus

Purified alanine racemes were used to determine the racease enzyme activity of 19 L-amino acids including L-alanine and excluding glycine by measuring the circular dichroism (CD) signal of the amino acid at 204 nm. As a result, it was confirmed that the racease activity for L-alanine was the highest and that there was racemease activity for L-serine, and the activity was hardly observed in other amino acids. From this result, it was confirmed that the protein expressed in E. coli from the gene isolated from Aquipex pyrophyllus is an alanine racease enzyme.

PH 6-6.5 (50 mM, Na-PO ₄ ), pH 7-9.5 (50 mM, Tris-HCl), Ph 10-11 (50 mM, 3- to investigate the optimal pH conditions for alanine racease enzyme activity As a result of measuring enzyme activity at the pH of [cyclohexylamino] -1-propanesulfonic acid; CAPS), the highest activity was shown at pH 9.5 as shown in FIG.

In order to investigate the optimum enzyme reaction temperature, enzyme activity was measured at 37, 60, 85, 90, 95, and 100 ° C., as shown in FIG. 5, showing the highest activity at 85 to 90 ° C. FIG. Thus, the optimum conditions for Aquipex pyrophyllus alanine racease enzyme activity were found to be buffer pH 9.5, reaction temperature 85-90 ° C.

Subsequent determination of alanine raceme activity was performed by Badet et al. (Badet, B., Roise, D., and Walsh, CT (1984) Inactivation of the dadB Salmodella typhimurium alanine recemase by D and L isomer of α-substitute alanine: Kinetics, stoichiometry, active site peptide sequencing, and reaction mechanism.Biochemistry Vol 23, pp5188-5194), were performed according to the optimum conditions identified above.

Determination of the activity of alanine racease enzyme was carried out using 10 μg of purified protein in 100 μl of reaction buffer containing 10 mM D-alanine (50 mM Tris-HCl, pH 9.5, 5 μM pyridoxal 5′-phosphate, 2 mM dithiotretol). After the addition, the reaction was carried out at 85 ° C. for 1 hour. 0.3 times of 7% perchloric acid was added to stop the reaction, followed by neutralization by addition of 0.2 times of 1N NaOH. A 0.1-fold solution containing 25 units of L-alanine dehydrogenase, 0.5M Tris-HCl, pH 9.5, and 5 mM NAD ⁺ was added to the finished sample. The amount of L-alanine produced was calculated by measuring the amount of NADH produced as L-alanine is converted to pyruvate at 340 nm.

In order to examine the heat resistance of the isolated alanine racease enzyme, the enzyme was left at 95 ° C. for several hours to remove the denatured protein, and the activity of the remaining enzyme was measured. As shown in FIG. 6, alanine racease enzyme of Aquipex pyrophyllus showed almost no change in activity when left at 95 ° C. for 60 minutes, and maintained at about 50% even when left for 4 hours. These characteristics indicate that the alanine racease enzyme of the present invention has higher heat resistance than the protein isolated from E. coli and Bacillus subtilis having about 50% of activity when left at 80 ° C. for 60 minutes. Even if it was confirmed to maintain the stability expected in the original ultra high temperature bacteria.

As described above, alanine racease of Aquipex pyrophyllus produced in Escherichia coli transformed with an expression vector comprising the gene of alanine racease isolated in the present invention is higher than the enzyme isolated from the existing mesophilic bacteria. And because it has high stability even under conditions such as high pH, it can be usefully used industrially, such as the production of pharmaceuticals and the production of food materials.

Claims (5)

A gene of heat resistant alanine racease having the nucleotide sequence of SEQ ID NO: 1 isolated from Aquipex pyrophyllus. A heat resistant alanine racease protein having the amino acid sequence of SEQ ID NO: 2 encoded from the gene of claim 1. An expression vector pAR comprising the gene of claim 1. E. coli transformed with the expression vector of claim 3. A method for producing heat-resistant alanine racemes comprising culturing E. coli of claim 4 and crushing the cultured E. coli to recover heat-resistant alanine racemes expressed from E. coli.