Patent Application
20110003346
1. Field of the Invention
The present invention relates to a synthesis method of aromatic amino acids, more particularly, to a stable and easy method for synthesizing and processing of homophenylalanine.
2. Description of the Related Art
Hypertension, also called high blood pressure, means patient's blood pressure higher than normal standard. Severely high pressure is defined as 50% or over 50% higher than standard. Persistent hypertension might lead to strokes, heart attacks, heart failure and arterial aneurysm. Even moderate hypertension would cause negative effects to people's health. In industrialized countries, hypertension is a main reason of cardiovascular morbidity and mortality.
Renin-Angiotensin system (RAS) is a crucial moderating factor for blood pressure. In the RAS reaction, Angiotensin-Converting Enzyme (ACE), which is a circulating enzyme, mediates extracellular volume, and arterial vasoconstriction. ACE has two primary functions: (1) ACE can catalyze the conversion of angiotensin I to angiotensin II. Angiotensin II is a vasoconstrictor in a substrate concentration dependent manner. (2) ACE can catalyze the degradation of a vasodilator-bradykinin. Therefore, ACE is a good target for medicines to therapy some multiple failures including high blood pressure, heart failure, diabetic nephropathy and type 2 diabetes mellitus. Angiotensin-Converting Enzyme Inhibitors (ACEIs) can inhibit the activity of ACE, decreasing the formation of angiotensin II and the degradation of bradykinin; therefore, ACEIs have been used to prevent RAS overactive and led to a systematic dilation of blood vessels and lower blood pressure. One of raw materials for synthesizing ACEIs is homophenylalanine (HPA). HPA is sorted into a D-form homophenylalanine (D-HPA) and a L-form homophenylalanine (L-HPA). Due to its slow metabolism and easily remain in organisms; the safety issue and medicines' kinetics of the D-HPA needs further evaluation. Consequently, most medicines of ACEIs use L-HPA as a raw material, such as Enalapril, Ramipril, Quinapril and Lisinopril. HPA is not a natural occurred amino acid and can be synthesized artificially. The conventional synthesis methods of amino acids includes an enantioseletive hydrogenation method, a full chemical synthesis method and a biochemical synthesis method.
The enantioseletive hydrogenation method uses organic solvents to extract D-HPA and L-HPA from a kinetic resolution. Sequentially, D-HPA can be isolated by different optical activity, but the D-HPA is not a good raw material for current ACEIs. Therefore, the synthetic HPA by the enantiotioseletive hydrogenation method is limited.
The full chemical synthesis method is widely used in pharmaceutical industries for massive synthesis of aromatic amino acids. The full chemical synthesis method uses an intermediate product beta-benzoylacrylic acid with 1-arylethylamine to synthesize HPA through a reduction. However, the product of HPA includes D-HPA and L-HPA so that an additional separation is required to separate D-HPA and L-HPA. The full chemical synthesis method for HPA is complicated and uses many kinds of chemicals and organic solvents that result in high cost and environmental pollution.
The biochemical synthesis method is a biotransformation method in which an aminotransferase is used as a biocatalyst to selectively synthesize stereospecific amino acids. The biochemical synthesis method has some advantages: simple to synthesize stereospecific amino acids and using few chemicals and low environmental pollution. A previous report showed that an E. coli aspartate aminotransferases can be used to synthesize L-HPA. It uses an aspartate as an amino donor and an 2-oxo-4-phenylbutyric acid (OPBA) as an amino acceptor in the amino-transferring reaction at 37° C. However, E. coli aspartate aminotransferase has following defects in synthesizing L-HPA,
(1) Low Efficiency in Synthesizing Amino Acid:
A higher temperature leads to a higher reaction rate; however, E. coli aspartate aminotransferase is less stable, easily get inactive over 37° C. or after hours of working time, so that the E. coli aspartate aminotransferase reaction can not be carried out in higher temperature. Moreover, even E. coli aspartate aminotransferase reaction is carried out at 37° C., E. coli aspartate aminotransferase is going to be inactive after few hours. Therefore, much more E. coli aspartate aminotransferase or a longer reaction time are expected to use in synthesizing amino acids. On the other hands, a lower reaction temperature also causes a lower solubility of aromatic amino acceptors, which results in lower efficiency in amino acids synthesizing reaction.
(2) High Cost in Keeping Enzyme Active:
E. coli aspartate aminotransferase is easily inactive over 37° C., so that coolers or freezers are required for its preparation, storage and also for its transportation. The enzyme activity of E. coli aspartate aminotransferase will be decreased if proper procedures are not taken in the preparation, storage and transportation. Thus, the cost to obtain active E. coli aspartate aminotransferase will be high.
An objective of the present invention is to provide a synthesis method of aromatic amino acids which can process in high temperature with shorter time and higher efficiency.
Another objective of present invention is to provide a synthesis method of aromatic amino acids by using more active aminotransferase with an easy-storage and easy-transportation character, so that the amount of aminotransferase needed in reaction and the cost in manufacturing are low.
A synthesis method of aromatic amino acids according to the aspect of present invention includes a process of preparing thermostable enzyme, a process of amino-transferring reaction and a process of product precipitation and enzyme recycling. The process of preparing thermostable enzyme comprises an isolation step of the gene of Thermus thermophilus aspartate aminotransferase (TtAspAT), a construction step of the TtAspAT gene in an expression vector, and a transformation step of host cells with the TtAspAT gene-harboring vector, for producing a plurality of TtAspAT. The process of amino-transferring reaction performs a TtAspAT-catalyzing reaction, in which an amino group from an amino donor is transferred to a carbonyl group of a keto acid (the amino acceptor) through an amino-transferring reaction at a temperature between 50° C. and 80° C., for a reaction time from 30 to 90 minutes to produce aromatic amino acids. The process of product precipitation and enzyme recycling is used to recover and purify the synthesized amino acids and recycle the active aminotransferase in a supernatant of a reaction mixture for using one more time in another cycle of synthesis reaction.
Further scope of the applicability of present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferable embodiments of the invention, are given by way of illustration only, since various will become apparent to those skilled in the art from this detailed description.
The illustrative embodiments may best be described by reference to the accompanying drawings where:
All figures are drawn for ease of explanation of the basic teachings of the present invention only; the extensions of the figures with respect to number, position, and relationship, of the parts to form the preferred embodiments will be explained or will be within the skill of the art after the following teachings of the present invention have been read and understood. Further, the exact dimensions and dimensional proportions to conform to specific force, weight, strength, and similar requirements will likewise be within the skill of the art after the following teachings of the present invention have been read and understood.
Where used in the various figures of the drawings, the same numerals designate the same or similar parts. Furthermore, when the terms “S1”, “S2”, “S3”, and similar terms are used herein, it should be understood that these terms have reference only to the structure shown in the drawings as it would appear to a person viewing the drawings and are utilized only to facilitate describing the invention.
Referring to
The process of preparing thermostable enzyme S1 provides a thermostable and easy-purifying aspartate aminotransferase (AspAT), which is a product by expressing the TtAspAT (Thermus thermophilus aspartate aminotransferase) gene in E. coli. The construction of TtAspAT gene in an E. coli expression vector with binding tag is obtained by recombinant gene cloning techniques, which is well known to persons skilled in the art. The TtAspAT gene cloning includes an isolation step of the TtAspAT gene in which a target DNA (TtAspAT gene) was amplified from desired organism's genomic DNA; a construction step of the TtAspAT gene in an expression vector in which the target DNA was constructed into an appropriate vector for protein expression in suitable host; and a transformation step of host cells with the TtAspAT gene-harboring vector, for producing a plurality of TtAspAT. Polymerase chain reaction (PCR) is the most common used techniques for amplifying specific sequences. In the isolation step of the TtAspAT gene, the PCR reaction is performed by the target DNA from a genomic DNA sample, unwinding the two strands thereof, hybridizing the unwound strands with primers, and amplifying the target DNA (TtAspAT) with DNA polymerase. The parameters and reagents are well known to persons skilled in the art. The oligonucleotide sequences used as primers can be optional modified in part, for example, to introduce endonuclease restriction site or eliminate stop codon for fusing to downstream sequences. The sequence information of TtAspAt gene can be obtained directly from GenBank and used to design primers which make the construction fused to a suitable tag in the C-terminal of the recombinant TtAspAT. To simplify the work, the tag chosen in this invention is 6× histidine tag, which can bind to Ni-chelating resin to facilitate purification of the expressed recombinant TtAspAT. In a preferred embodiment, genomic DNA is prepared from Thermus thermophilus and TtAspAT gene is amplified from the genomic DNA by the PCR technique. Normally, the expression vector can be chosen by those skilled in the art as desired. After proper manipulations by gene cloning techniques, which is well known to persons skilled in the art, a construction with the DNA sequence of 6× histidine tagged TtAspAT can be obtained. The construction (vector DNA with TtAspAT gene) can be transformed in to suitable host for protein expression. Protein expression and purification for TtAspAT preparation are performed by processes well known in the art.
In the process of amino-transferring reaction S2 in the present invention, the amino donor is L-aspartate and the amino acceptor can be chosen from α-ketoglutarate (α-KG), homophenylpyruvate, phenylpyruvate, indole-3-pyruvic acid or p-hydroxyphenylpyruvate individually. The TtAspAT can transfer an amino group from the L-aspartate to a carbonyl group of α-KG, homophenylpyruvate, phenylpyruvate, indole-3-pyruvic acid or p-hydroxyphenylpyruvate to generate L-HPA, L-phenylalanine, L-tryptophan and L-tyrosine respectively. A advantageous condition for the process of amino-transferring reaction is at a temperature between 50° C. and 80° C. and for a reaction time from 30 to 90 minutes. The most advantageous temperature is 70° C. The preferred embodiment of the present invention is that the TtAspAT transfers the amino group from L-aspartate to the carbonyl group of homophenylpyruvate to generate L-HPA. The L-HPA can be a raw material of ACEI.
In the most advantageous time of TtAspAT activity test, using a high performance liquid chromatography (HPLC) examine the concentration of synthesized L-homophenylalanine (L-HPA). According to
After amino-transferring reaction catalyzed by TtAspAT, product was precipitated by lowing the temperature of reaction mixture and the active aminotransferase was recycled from the supernatant of reaction mixture. The present invention provided a higher temperature environment for catalyzing reaction, not only enhanced the activity of TtAspAT, but also made the substrate more soluble and the synthesis of amino acids more complete. The synthesized L-HPA was easily precipitated from the reaction mixture in process S2 with a temperature of 0 degree centigrade in ice-water bath. Moreover, the supernatant of the reaction mixture still have high aminotransferase activity after amino-transferring reaction (see the hale life test in process S2). As the results, the supernatant can be recycled 2-3 times at least, which makes enzyme-catalyzing synthesis more economical. L-phenylalanine, L-tryptophan and L-tyrosine synthesized in process S2 are hydrophobic compounds, and consequently, are extracted through organic solvents, following by preparative C18 column chromatography for the HPLC isolation, which are well known methods to persons skilled in the art.
As has been discussed above, the TtAspAT can be used to synthesize aromatic amino acids at high temperature between 50 and 80° C., and increase the solubility of amino donors and amino acceptors. The TtAspAT decreases the reaction time for synthesizing aromatic amino acids and increases the efficiency in synthesizing aromatic amino acids. Moreover, The TtAspAT is a thermostable enzyme, which makes the TtAspAT easily storage and convenient transportation at room temperature. Furthermore, TtAspAT is more active in higher temperature, hence the less TtAspAT can be used to synthesize more amino acids in method of present invention and save the cost in manufactory.
Although the invention has been described in detail with reference to its presently preferable embodiment, it will be understood by one of ordinary skill in the art that various modifications can be made without departing from the spirit and the scope of the invention, as set forth in the appended claims.
Isolation of genomic DNA from Thermus thermophilus
A Thermus thermophilus used in the invention was bought from Bioresource collection and research center (BCRC), Hsinchu, Taiwan. The genomic DNA of Thermus thermophilus was isolated according to the method as described by Sambrook et al. 1989. The Thermus thermophilus was incubated in a 10 ml Thermus medium at 60° C. overnight. The T. thermophilus was harvested by centrifuging 5 ml of the overnight culture at 14,000 rpm for 30 seconds. Removing the supernatant and adding 377 μl of 50 mM TrisHCl (pH8.0) to suspend the pellet. Then 2 μl of proteinase K (20 mg/ml) and 20 μl of 10% SDS was added, mixed well and incubated at 37° C. for 1 hour. The treated mixture was then added with 95 μl of 4.1 M NaCl and 55 μl of 10% CTAB sequentially and incubated at 65° C. for 10 minutes. TrisHCl buffer-saturated phenol was used to extract the mixture once. Transfer the supernatant to a new microcentrifuge tube, and phenol-chloroform solution (1:1) was used to extract the mixture one more time. Transfer the supernatant to a new microcentrifuge tube, adding 330 μl isopropanol and inverts several times to precipitate a genomic DNA of T. thermophilus. Wash the precipitated genomic DNA of T. thermophilus with 70% ethanol for 3 times and dry it. Finally the precipitated genomic DNA of T. thermophilus was resuspended in 50 μwater.
Two primers, TtAspAT-F and TtAspAT-R, were designed based on the sequence of the TtAspAT gene of T. thermophilus HB8. The sequence of the forward primer TtAspAT-F is (5′-ACTTAGCATATGCGCGGCCTTTCCC-3′) and of the reverse primer TtAspAT-R (5′-CCCACGACCCCGCGCGGTTCGAACCCAA-3′) to amplify TtAspAT gene. A PCR reaction mixture (50 μl) contained 1 μl of genomic DNA of T. thermophilus as template, 5 μl of 10X buffer, 4 μl of dNTP (2.5 mM), 1 μl of primer TtAspAT-F (1 pmole/μl), 1 μl of primer TtAspAT-R (1 pmole/μ1), 1 μl of DNA polymerase and 40 μl water was prepared. The PCR reaction mixture was put into a thermocycler to perform PCR reaction in 40 cycles to amplify the TtAspAT gene.
Cloning of TtAspAT Gene into Expression Vector
A plasmid pET21b and the fragment of TtAspAT gene amplified by PCR were digested with two restriction endonucleases (NdeI and HindIII) and ligated by a T4 DNA ligase at 16° C. for 12 hours to obtain a ligated DNAs. The ligated DNAs were transformed into an E. coli DH5α to select clones with correct insert, and then a correct clone was transformed into E. coli BL21(DE3) for massive express TtAspAT.
E. coli BL21(DE3) harboring the TtAspAT gene was incubated in a LB broth containing 100 μl/ml ampicillin at 37° C. overnight and then subcultured until the OD600 of a culture reached 0.6. Then isopropyl-β-D-thiogalactopyranoside (IPTG) was added to final concentration of 1 mM and incubated for another 18 hours for induction. The culture was harvested by centrifugation at 7,500 rpm for 10 minutes at 4° C. to obtain a pellet. After washing the pellet with 0.9% NaCl, the pellet was resuspended in a PLP buffer (1 mM PLP in 0.1 M TrisHCl buffer, pH 8.0) and disrupted by ultra-sonication. The suspension was then centrifuged at 13,000 rpm for 20 minutes at 4° C. to remove the cell debris and clear supernatant was used as a crude extract. The crude extract was heated at 60° C. water bath for 20 minutes and centrifuged at 13,000 rpm for 20 minutes at 4° C. to remove the denatured proteins. The clear supernatant was subjected to a Ni-chelating resin column for further purification by one-step chromatography, since the recombinant TtAspAT contains six histidine tagged in C-terminal which can bind to Ni-chelating resin and be simple eluted by imidazole solution to obtain Thermus thermophilus aspartate aminotransferase (TtAspAT).
This activity of TtAspAT was determined by a modified malate dehydrogenase (MDH) coupling method (Ziehr, H. and M. R. Kula, 1985). A mixture contained 200 mM L-aspartate, 25 mM α-KG, 0.15 mM NADH, 0.1 mM PLP, 5 U of MDH, and 100 mM TrisHCl buffer (pH 8.0), was prepared in a total volume of 1 ml. The reaction was initiated by adding a diluted TtAspAT and proceeded at specific temperature. The decrease of absorbance at 340 nm due to consumption of NADH was monitored for 3-5 minutes and was then converted to enzyme activity. One unit of enzyme activity was defined as the amount of enzyme that catalyzed the production of oxaloacetate equal to 1 μmole NADH consumed for converting oxaloacetae to malate per minute. The preferred embodiment of the present invention examines the optimal catalysis temperature and stability of TtAspAT
The TtAspAT activity in different temperature was determined under standard reaction mixture in every 5° C. increased temperature by MDH-coupling method. The enzymatic activity was measured from 25° C. to 95° C. (Table1), the related results are illustrated in
aOne unit of enzyme activity was defined as the amount of enzyme that produced the oxaloacetate equal to 1 μmole NADH consumed for converting oxaloacetae to malate per minute.
The stability of TtAspAT was measured by the way that the enzyme was conducted a heat treatment at 37° C., 50° C., 60° C., 70° C. and 80° C. for a period of time. After the heat treatment, MDH-coupling method was used to determine the remaining activity of the heat-treated TtAspAT. By plotting treating time to the remaining activity of TtAspAT (illustrated in
The high performance liquid chromatography (HPLC) analysis of TtAspAT against homophenylpyruvate was performed by the method modified from Houng, J. Y. and C. L. Hsieh (1996, U.S. Pat. No. 5,552,317). A reaction mixture contained 200 mM aspartate, 25 mM homophenylpyruvate, 100 mM Tris-HCl buffer (pH8.0), 5 U of TtAspAT in a final reaction volume of 1 ml was prepared. The reaction mixture was aliquoted into 0.12 ml for each microtube and incubated at 60° C. for 1, 2, 4, 6, 8 and 12 hours individually. Reaction was stopped by adding 35 μl of 6N HCl. The amount of homophenylalanine (HPA) produced by TtAspAT catalyzed bioconversion was then measured by the HPLC using a reverse phase C18 column (Ascentis C18, 250 mm×4.6 mm, particle size 5 μm, SUPELCO, PA). The mobile phases were an A solvent contained 10% methanol/perchloric acid (pH 2.0) and a B solvent contained 80% methanol/perchloric acid (pH 2.0). The flow rate was set at 0.7 ml/min. The gradient was set from 0% to 100% of the B solvent. HPA of the concentration 0.5 mM, 1 mM, 1.5 mM and 2 mM was used to set standard curve. The yield of the bioconversion reaction was calculated by the total amount of produced HPA divided with the amount of inputted HPP. The HPLC analysis showed that the yield after 6 hours slowed down and reached 83.42% after 12 hours reaction. (Table 3) The relationship between reaction time and yield of the HPA (illustrated in
| Number | Date | Country | Kind |
|---|---|---|---|
| 098122547 | Jul 2009 | TW | national |
