CN116949024A - Phenylalanine ammonia lyase mutant and application thereof in synthesis of (S) -2-chloro-phenylalanine - Google Patents
Phenylalanine ammonia lyase mutant and application thereof in synthesis of (S) -2-chloro-phenylalanine Download PDFInfo
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- C12Y—ENZYMES
- C12Y403/00—Carbon-nitrogen lyases (4.3)
- C12Y403/01—Ammonia-lyases (4.3.1)
- C12Y403/01005—Phenylalanine ammonia-lyase (4.3.1.5)
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Abstract
The invention discloses a phenylalanine ammonia lyase mutant and application thereof in synthesizing (S) -2-chloro-phenylalanine, belonging to the technical fields of enzyme engineering and microbial engineering. The phenylalanine ammonia lyase mutant is obtained by mutating histidine 240 of an amino acid sequence shown as SEQ ID NO.1 into phenylalanine or mutating leucine 80 into valine, and catalyzing a substrate to generate (S) -2-chloro-phenylalanine in a system without adding any cosolvent, so that the inhibition effect of the substrate and the product can be greatly reduced, and the conversion rate is as high as 61.2% or 92%.
Description
Technical Field
The invention relates to a phenylalanine ammonia lyase mutant and application thereof in synthesizing (S) -2-chloro-phenylalanine, belonging to the technical fields of enzyme engineering and microbial engineering.
Background
L-phenylalanine is used as one of the essential amino acids of human body, is commonly used as a nutrition enhancer, an amino acid transfusion and a component of a compound amino acid preparation, can be used as a synthetic raw material of an additive aspartame in the food industry, and can be used for synthesizing medicines such as L-DOPA (L-DOPA), epinephrine and the like in the medicine industry. Enantiomerically enriched phenylalanine compounds are important intermediates in the above pharmaceutical preparations, wherein (S) -2-chloro-phenylalanine is the main raw material for the production of angiotensin I converting enzyme inhibitors (ACE) for the treatment of hypertension (hypertension).
WO2006/069799A1 discloses a process for the preparation of enantiomerically enriched phenylalanine compounds in one of which 2-Cl-cinnamic acid is reacted in aqueous ammonia using a phenylalanine ammonia lyase derived from Rhodotorula glutinis to produce (S) -2-chloro-phenylalanine. The disadvantage of this process is that strong substrate and product inhibition occurs and therefore needs to be carried out at low concentrations. If a large-scale production of the product is required, a larger volume or more complex feeding scheme is required, and the process is not competitive from an industrial production point of view.
CN101517089a discloses an improved process for the preparation of phenylalanine compounds using phenylalanine ammonia lyase derived from Idiomarina loihiensis for catalyzing the reaction of the corresponding cinnamic acid compounds with an amino donor, which process results in an effective reduction of substrate inhibition and a higher activity compared to phenylalanine ammonia lyase derived from r. However, in the actual production process, the catalytic activity of phenylalanine ammonia lyase derived from I.loihiensis is still low, and cannot meet the requirements of industrial production.
CN106755160a discloses an improved process for preparing phenylalanine compounds, the histidine ammonia lyase used is derived from Pseudoalteromonas sp.p1-26, and is used for producing L-phenylalanine compounds, and compared with the above process, the method can reduce production cost and improve production efficiency. The method is used for production, and although the yield is improved to a certain extent compared with the two methods, the histidine ammonia lyase obtained by screening has limited substrate selection, and the required enzyme amount in production is too high, so that the method is not beneficial to large-scale popularization.
Disclosure of Invention
In order to solve the problems, the invention provides a phenylalanine ammonia lyase mutant with raised catalytic activity, reduced substrate and product inhibition and wider substrate spectrum, which mutates the 240 th histidine of an original amino acid into phenylalanine or mutates the 80 th leucine into valine, and the conversion rate of the mutant to the substrate reaches 61.2 percent and 92 percent respectively compared with that of the wild phenylalanine ammonia lyase, and the substrate conversion rate of the wild phenylalanine ammonia lyase is only 33.4 percent, so that the phenylalanine ammonia lyase mutant of the invention has good performance in the catalytic process and industrial application value.
The first object of the present invention is to provide a phenylalanine ammonia lyase mutant obtained by mutating histidine 240 of the amino acid sequence of phenylalanine ammonia lyase shown in SEQ ID NO.1 to phenylalanine or leucine 80 to valine.
It is a second object of the present invention to provide a gene encoding the phenylalanine ammonia-lyase mutant.
A third object of the present invention is to provide a recombinant plasmid carrying the gene.
Further, the recombinant plasmid vector is pET-28a (+) plasmid, pET-28b (+) plasmid or pET-20b (+) plasmid.
It is a fourth object of the present invention to provide a host cell carrying said gene or said recombinant plasmid.
Further, the host cell is a bacterial, fungal, plant cell or animal cell.
It is a fifth object of the present invention to provide a method for producing the phenylalanine ammonia-lyase mutant, the method comprising the steps of: inoculating the recombinant cells into a fermentation medium for fermentation to obtain fermentation liquor; centrifuging the fermentation liquor, and collecting thalli; crushing thalli, and centrifuging to obtain cell crushing supernatant; and extracting cell disruption supernatant to obtain the phenylalanine ammonia lyase mutant.
A sixth object of the present invention is to provide a method for producing (S) -2-chloro-phenylalanine, comprising the step of performing catalytic conversion using the phenylalanine ammonia-lyase mutant or an expression system containing the mutant.
Further, the reaction system is carried out in ammonia water.
Further, the temperature of the reaction is 30-40 ℃, and the pH value is 9.0-10.5.
The invention has the beneficial effects that:
the invention modifies the molecular structure of phenylalanine ammonia lyase based on phenylalanine ammonia lyase from microorganism Idiomarina abyssalis by site-directed mutagenesis technique to finally obtain phenylalanine ammonia lyase mutant with increased catalytic activity and increased catalytic substrate range. Compared with other biocatalysts for preparing (S) -2-chloro-phenylalanine, the phenylalanine ammonia lyase mutant provided by the invention can catalyze high-concentration substrates, has higher catalytic activity and wider substrate range, and greatly reduces the inhibition effect of the substrates and products, so that the phenylalanine ammonia lyase mutant has extremely high application prospect in the production and preparation of (S) -2-chloro-phenylalanine.
Detailed Description
The present invention will be further described with reference to specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the present invention and practice it.
The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications. Wherein the room temperature is a room temperature conventional in the art, and the room temperature ranges from 20 ℃ to 40 ℃.
The expression plasmid pET28a was purchased from Novagen, shanghai. E.coli DH 5. Alpha. Competent cells, E.coli BL21 (DE 3) competent cells, 2X Taq PCR MasterMix, agarose gel DNA recovery kit and DNA Marker were all purchased from Beijing Tian Gen Biochemical technology Co. Restriction endonucleases NdeI and XhoI were purchased from DaLibao biosystems, inc.
Example 1: cloning of phenylalanine ammonia lyase wild-type Gene
Based on the predicted Idiomarina abyssalis gene sequence (Genebank accession number: WP_ 054488769.1) recorded in Genbank, PCR primers were designed as follows:
an upstream primer:
5'-GTGCCGCGCGGCAGCCATATGACAACTTCAATTATAGCTTTTGGG-3'
a downstream primer:
5'-GTGGTGGTGGTGGTGCTCGAGTGCCGGCTCTTGATAC-3'
wherein the template is genomic DNA of Idiomarina abyssalis.
The PCR system is as follows: 2X Taq PCR MasterMix. Mu.L, 1. Mu.L (0.3. Mu. Mol/L) each of the upstream primer and the downstream primer, 1. Mu.L (0.1. Mu.g) of the DNA template, and 7. Mu.L of deionized water.
The PCR amplification procedure was: (1) pre-denaturation at 95℃for 3min; (2) denaturation at 94℃for 30s; (3) annealing at 55 ℃ for 30s; (4) extending at 72 ℃ for 1min; repeating steps (2) - (4) for 30 cycles; (5) continuing to extend at 72 ℃ for 10min, and cooling to 4 ℃.
And purifying the PCR product by agarose gel electrophoresis, and recovering the PCR product by using an agarose gel DNA recovery kit to obtain a complete phenylalanine enzyme full-length gene sequence. The sequence is sequenced by DNA, the total length is 1542bp, and the sequence is named IaPAL. The nucleotide sequence of the gene is shown as SEQ ID NO.2 in the sequence table.
The obtained phenylalanine ammonia lyase wild type IaPAL full-length gene sequence is subjected to 3-base mutation, histidine at 240 th position of the phenylalanine ammonia lyase wild type gene coding sequence is mutated into phenylalanine, leucine at 80 th position is mutated into valine, and the nucleotide sequences of the obtained mutated genes are shown as SEQ ID NO.3 and SEQ ID NO. 4.
Example 2: preparation of phenylalanine ammonia lyase recombinant plasmid and recombinant expression transformant and mutant
The phenylalanine ammonia lyase gene DNA fragment obtained in example 1 and pET28a empty plasmid were treated with restriction endonucleases NdeI and XhoI for 2 hours at 37℃and purified by agarose gel electrophoresis, and the target fragment was recovered by using agarose gel DNA recovery kit. The target fragment is connected overnight at 4 ℃ under the action of T4 DNA ligase to obtain recombinant expression plasmid pET28a IaPAL.
The recombinant expression plasmid is transformed into E.coli DH5 alpha competent cells of Escherichia coli, positive recombinants are screened on a resistance plate containing kanamycin, monoclonal colonies are selected, and positive clones are verified by a PCR method. Culturing recombinant bacteria, extracting plasmids after the plasmids are amplified, re-transforming the plasmids into competent cells of E.coli BL21 (DE 3), coating a transformation solution on an LB plate containing kanamycin, performing inversion culture at 37 ℃ overnight to obtain positive recombinant transformant E.coli BL21 (DE 3)/pET 28a IaPAL, and performing colony PCR and gene sequencing to verify positive clones. The mutant plasmids were transformed as described above.
Example 3: expression of phenylalanine ammonia lyase mutants
The recombinant E.coli obtained in example 2 was inoculated into LB medium (peptone 10g/L, yeast extract 5g/L, naCl 10g/L, pH 7.0) containing kanamycin, shake-cultured overnight at 37℃and 1% (v/v) of the inoculum size was inoculated into a 500mL Erlenmeyer flask containing 100mL of LB medium, and shaking culture was carried out at 37℃and 180rpm, when the OD 600 of the culture solution reached 0.6, IPTG was added as an inducer at a final concentration of 0.2mmol/L, after induction at 25℃for 12 hours, the culture solution was centrifuged, the cells were collected, washed twice with normal saline to obtain resting cells, and freeze-dried for 24 hours to obtain freeze-dried cells, which were stored at 4 ℃. Suspending the obtained resting cells in buffer solution with pH value of 7.0, performing ultrasonic disruption in ice bath, and centrifuging to collect supernatant, namely the crude enzyme solution of phenylalanine ammonia lyase. The crude enzyme solution was analyzed by polyacrylamide gel electrophoresis, and phenylalanine ammonia lyase was present in a soluble form.
Example 4: determination of the Activity of phenylalanine ammonia lyase wild type and mutant
High Performance Liquid Chromatography (HPLC) is adopted to detect the activity and selectivity of phenylalanine ammonia lyase, and the mobile phase condition is methanol: water=95; 5, flow rate 1mL/min, column temperature 35 ℃, wavelength 222nm, the used column is amino acid chiral column, model is Astec CHIRBIOTIC 15cm ﹡ 4.6.6 mm,5 μm. The method for measuring the vitality comprises the following steps: 10mM 2-Cl-cinnamic acid, an appropriate amount of pure enzyme was added to 0.5mL of the reaction system (6M aqueous ammonia, pH=9.6), and the change in the peak area of the product was detected in the reverse phase. The concentration of pure enzyme protein is determined based on the fact that most proteins have a maximum absorption peak at 280nm, so that concentration data can be directly obtained by a Nanodrop instrument. After the purified protein is concentrated and desalted, 5 mu L of pure enzyme is dripped on an instrument by utilizing the molar extinction coefficient and the protein molecular weight of the protein obtained by website https:// web. Expasy. Org/protparam, and the protein concentration is read according to the molar extinction coefficient and the protein molecular weight. The proteins are diluted in sequence by different times, and the determination results under different dilution times are verified to have good linear relationship, so that the protein concentration of the pure enzyme can be obtained.
Under the above reaction conditions, the activity (U) per unit phenylalanine ammonia lyase was defined as the amount of enzyme required to produce 1. Mu. Mol of product (S) -2-chloro-phenylalanine per minute.
TABLE 1 phenylalanine ammonia lyase wild-type and mutant Activity
Example 5: determination of phenylalanine ammonia lyase wild type and mutant conversion
High Performance Liquid Chromatography (HPLC) was used to detect the conversion and selectivity of phenylalanine ammonia lyase. The mobile phase conditions were methanol: water = 95:5, flow rate 1mL/min, column temperature 35 ℃, wavelength 222nm, the used column is amino acid chiral column, model is Astec CHIRBIOTIC 15cm ﹡ 4.6.6 mm,5 μm. The conversion rate is determined as follows: 18.26 g.L was added to 10mL of the reaction system (6M aqueous ammonia, pH 9.6) -1 Substrate, 0.73 g.L -1 Freeze-drying the cells. Samples were taken after 24h and the change in peak area of the substrate was detected in reverse phase. The conversion rate of the phenylalanine ammonia lyase wild type and mutant is calculated according to the following method:
conversion (%) = [ (S substrate initiation-S substrate termination) ×100% ] S substrate initiation
TABLE 2 phenylalanine ammonia lyase wild-type and mutant conversion
Example 6: determination of specific activity of phenylalanine ammonia lyase wild type and mutant substrate
The specific activity of phenylalanine ammonia lyase catalyzed different substrates was determined as in example 4, as shown in Table 3, L80V was higher for ortho substituent substrates and H240F was higher for ortho, meta and para substituent substrates and for trans cinnamic acid, the natural substrate, than for WT.
TABLE 3 specific activities of phenylalanine ammonia lyase wild type and mutant for different substrates
Note that: n.a.: non available.
Example 7: determination of substrate spectrum conversion rate of phenylalanine ammonia lyase wild type and mutant
The conversion of phenylalanine ammonia lyase to various substrates was determined as in example 5, as shown in Table 4, with higher L80V conversion to ortho-substituent substrates and higher H240F conversion to ortho-, meta-and para-substituent substrates and trans-cinnamic acid as a natural substrate compared to WT.
TABLE 4 conversion of phenylalanine ammonia-lyase wild-type and mutant to different substrates
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations and modifications of the present invention will be apparent to those of ordinary skill in the art in light of the foregoing description. It is not necessary here nor is it exhaustive of all embodiments. And obvious variations or modifications thereof are contemplated as falling within the scope of the present invention.
Claims (10)
1. A phenylalanine ammonia-lyase mutant characterized in that: the phenylalanine ammonia lyase mutant is obtained by mutating histidine 240 of an amino acid sequence shown as SEQ ID NO.1 into phenylalanine or mutating leucine 80 into valine.
2. A gene encoding the phenylalanine ammonia-lyase mutant according to claim 1.
3. The gene according to claim 2, characterized in that: the nucleotide sequence is shown as SEQ ID NO.3 or SEQ ID NO. 4.
4. A recombinant expression vector comprising the gene of claim 2 or 3.
5. A host cell comprising the phenylalanine ammonia-lyase mutant according to claim 1.
6. The host cell of claim 5, wherein: the host cell is of the type bacterial, fungal, plant or animal.
7. Use of the phenylalanine ammonia-lyase mutant according to claim 1, the gene according to claim 2 or 3, the recombinant expression vector according to claim 4 or the host cell according to claim 5 for the synthesis of (S) -2-chloro-phenylalanine.
8. A method for synthesizing (S) -2-chloro-phenylalanine, comprising the steps of: the phenylalanine ammonia-lyase mutant or the expression system containing the mutant according to claim 1, which is added to a reaction system containing a substrate for reaction.
9. The method according to claim 8, wherein: the reaction temperature is 30-40 ℃, and the reaction pH value is 9.0-10.5.
10. The method according to claim 8, wherein: the substrate comprises trans-cinnamic acid, 2-Cl-cinnamic acid, 3-Cl-cinnamic acid, 4-Cl-cinnamic acid, and 2-NO 2 Cinnamic acid, 3-NO 2 Cinnamic acid, 4-NO 2 -cinnamic acid, 2-Br-cinnamic acid, 3-Br-cinnamic acidAcid, 4-Br-cinnamic acid, 2-CF 3 Cinnamic acid, 3-CF 3 Cinnamic acid, 4-CF 3 -one or more of cinnamic acid, 2, 4-2-Cl-cinnamic acid and 2, 5-2-Cl-cinnamic acid.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103409449A (en) * | 2012-09-18 | 2013-11-27 | 江南大学 | Method for gene cloning of rhodotorula glutinis phenylalanine deaminase and high-efficiency expression method of gene in coliform strain |
| CN105238825A (en) * | 2006-09-12 | 2016-01-13 | DPx控股有限公司 | Method for producing optically active phenylalanine compounds from cinnamic acid derivatives employing a phenylalanine ammonia lyase derived from idiomarina loihiensis |
| US20160362711A1 (en) * | 2014-02-06 | 2016-12-15 | Japan Science And Technology Agency | 4-amino cinnamic acid production method using enzyme |
| CN113817697A (en) * | 2021-10-20 | 2021-12-21 | 江南大学 | Phenylalanine dehydrogenase mutant and application thereof in synthesis of L-homophenylalanine |
| CN115873837A (en) * | 2022-11-18 | 2023-03-31 | 安徽盈科生物科技有限公司 | High-expression novel phenylalanine ammonia lyase |
| CN116103323A (en) * | 2023-02-16 | 2023-05-12 | 安徽中医药大学 | Polygonum multiflorum phenylalanine ammonia lyase gene FmPAL, and encoding product and application thereof |
-
2023
- 2023-07-24 CN CN202310908301.3A patent/CN116949024B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105238825A (en) * | 2006-09-12 | 2016-01-13 | DPx控股有限公司 | Method for producing optically active phenylalanine compounds from cinnamic acid derivatives employing a phenylalanine ammonia lyase derived from idiomarina loihiensis |
| CN103409449A (en) * | 2012-09-18 | 2013-11-27 | 江南大学 | Method for gene cloning of rhodotorula glutinis phenylalanine deaminase and high-efficiency expression method of gene in coliform strain |
| US20160362711A1 (en) * | 2014-02-06 | 2016-12-15 | Japan Science And Technology Agency | 4-amino cinnamic acid production method using enzyme |
| CN113817697A (en) * | 2021-10-20 | 2021-12-21 | 江南大学 | Phenylalanine dehydrogenase mutant and application thereof in synthesis of L-homophenylalanine |
| CN115873837A (en) * | 2022-11-18 | 2023-03-31 | 安徽盈科生物科技有限公司 | High-expression novel phenylalanine ammonia lyase |
| CN116103323A (en) * | 2023-02-16 | 2023-05-12 | 安徽中医药大学 | Polygonum multiflorum phenylalanine ammonia lyase gene FmPAL, and encoding product and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| 无: "aromatic amino acid ammonia-lyase [Idiomarina abyssalis] NCBI Reference Sequence: WP_054488769.1", GENBANK, 18 May 2021 (2021-05-18) * |
| 李楠 等: "L-苯丙氨酸解氨酶产生菌分离及酶促反应研究", 食品科学, vol. 26, no. 12, 31 December 2005 (2005-12-31), pages 74 - 77 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117965514A (en) * | 2024-02-01 | 2024-05-03 | 江南大学 | Aromatic amino acid ammonolysis enzyme mutant and method for preparing trans-cinnamic acid and p-coumaric acid by using same |
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