Levodopa aminotransferase gene ldaa, its encoded enzyme and use

Abstract

The application discloses levodopa aminotransferase gene ldaA , its encoding enzyme and application. The application clones a functional gene related to levodopa degradation from a strain Z-3 ldaA , and proves that levodopa aminotransferase LdaA encoded by the gene can catalyze levodopa to generate an intermediate 3,4-dihydroxyphenylpyruvic acid (DHPPA) through an aminotransfer reaction, and the DHPPA is further spontaneously oxidized into 3,4-dihydroxyphenylacetic acid, thereby providing a key molecular basis for levodopa degradation mechanism research. The application determines suitable conditions of levodopa aminotransferase LdaA catalyzing levodopa conversion reaction, the optimal reaction temperature is 45 DEG C, the optimal reaction pH is 7.5, under the conditions, the enzyme activity of levodopa aminotransferase LdaA catalyzing levodopa conversion reaches 5.81 U / mg, thereby providing a technical basis for application of the enzyme in levodopa bioremediation.

Description

Technical Field

[0001] This invention relates to the field of environmental microbiology, specifically to the L-DOPA aminotransferase gene. ldaA Its encoded enzymes and applications. Background Technology

[0002] Levodopa (L-dopa) is an important drug widely used clinically to treat diseases such as Parkinson's disease. However, during its production, use, and discharge, it can enter environmental media such as water and soil, leaving residues. Studies have shown that levodopa is easily oxidized in the environment, generating biologically active intermediates that trigger oxidative stress, damaging cellular structure and metabolic function, and posing potential ecological risks. Therefore, developing biodegradation technologies for levodopa is of significant practical importance.

[0003] Microbial degradation has become an important technical route for the bioremediation of pharmaceuticals and organic pollutants due to its advantages such as low cost, wide applicability, and environmental friendliness. Existing studies have reported that some microorganisms have the ability to degrade levodopa (such as strain Z-3 disclosed in CN 118147010 B). However, current understanding of the functional genes and key enzymes in the levodopa degradation process is insufficient, and systematic studies on the relevant gene sequences, encoded proteins, and their catalytic effects are still lacking.

[0004] Aminotransferases are a class of important enzymes involved in the metabolism of amino acids and aromatic compounds, playing a crucial role in the transformation of various natural products and pollutants. However, there is currently a lack of clear reports on the functions of aminotransferase genes in the degradation of levodopa, especially a lack of direct experimental evidence on the involvement of specific aminotransferase genes and their encoded enzymes in levodopa degradation. These deficiencies limit the elucidation of the levodopa degradation mechanism and also restrict the development and application of related bioremediation agents, functional enzyme preparations, and genetically engineered strains. Therefore, there is an urgent need to provide a functional gene and its encoded enzyme related to levodopa degradation for use in the biodegradation of levodopa and related engineering applications.

[0005] The identification and functional analysis of levodopa degradation genes are crucial for the remediation of levodopa accumulation in the environment. These genes and their encoded enzymes can be used for the bioremoval of levodopa from soil and water. Furthermore, modern bioengineering techniques can be used to construct recombinant strains to further elucidate their enzymatic characteristics and degradation mechanisms, providing important support for the bioremediation of levodopa pollution. In conclusion, research on the functional genes and enzymes involved in the levodopa degradation process has significant theoretical and practical value. Summary of the Invention

[0006] The purpose of this invention is to provide a L-DOPA aminotransferase gene. ldaAThe study aims to investigate the encoded enzyme and its applications to address the shortcomings of existing technologies.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] The first aspect of this invention provides a L-DOPA aminotransferase gene. ldaA Its nucleotide sequence is shown in SEQ ID NO.3.

[0009] A second aspect of the present invention provides a levodopa aminotransferase LdaA, the amino acid sequence of which is shown in SEQ ID NO.4.

[0010] A third aspect of the present invention provides a method for obtaining the above-mentioned L-DOPA aminotransferase gene. ldaA The constructed recombinant carrier.

[0011] A fourth aspect of the present invention provides a recombinant microorganism obtained by transformation of the above-described recombinant vector.

[0012] The fifth aspect of this invention provides the above-mentioned L-DOPA aminotransferase gene. ldaA Application in L-DOPA biotransformation.

[0013] The sixth aspect of this invention provides the application of the above-mentioned L-DOPA aminotransferase LdaA in L-DOPA biotransformation.

[0014] The seventh aspect of this invention provides the application of the above-mentioned recombinant vector in levodopa biotransformation.

[0015] The eighth aspect of the present invention provides the application of the above-mentioned recombinant microorganisms in levodopa biotransformation.

[0016] The ninth aspect of the present invention provides a method for the biotransformation of levodopa, which uses the above-mentioned levodopa aminotransferase LdaA to catalyze the aminotransfer reaction of levodopa in the presence of pyridoxal phosphate and α-ketoglutarate to generate the intermediate 3,4-dihydroxyphenylpyruvic acid; the 3,4-dihydroxyphenylpyruvic acid is further spontaneously oxidized to 3,4-dihydroxyphenylacetic acid.

[0017] The beneficial effects of this invention are:

[0018] 1. Based on whole-genome sequencing results, this invention performs functional annotation and clones functional genes related to levodopa degradation from strain Z-3. ldaA Furthermore, it was confirmed that the L-DOPA aminotransferase LdaA encoded by it can catalyze the aminotransfer reaction of L-DOPA to generate the intermediate 3,4-dihydroxyphenylpyruvic acid (DHPPA), which is further spontaneously oxidized to 3,4-dihydroxyphenylacetic acid, providing a key molecular basis for the study of the degradation mechanism of L-DOPA.

[0019] 2. This invention clarifies the suitable conditions for the L-DOPA conversion reaction catalyzed by LdaA aminotransferase. The optimal reaction temperature is 45℃ and the optimal reaction pH is 7.5. Under these conditions, the enzyme activity of LdaA in the conversion of L-DOPA reaches 5.81 U / mg, providing a technical basis for the application of this enzyme in L-DOPA bioremediation. Attached Figure Description

[0020] Picture 1 This is an SDS-PAGE image of L-dopa aminotransferase LdaA. Lane M: Protein Marker; Lane 1: Empty vector control strain. E. coli (pET29a) Supernatant after disruption; Lane 2, recombinant strain E. coli (pET-ldaA) precipitate after crushing; Lane 3: recombinant strain E. coli (pET-ldaA) supernatant after disruption (i.e. crude L-DOPA aminotransferase LdaA enzyme solution); lane 4, the breakthrough solution of crude L-DOPA aminotransferase LdaA enzyme solution after affinity chromatography; lanes 5, 6, 7, 8, and 9 are elution fractions of 10, 30, 50, 75, and 100 mM imidazole, respectively.

[0021] Picture 2 The effect of temperature on the enzyme activity of L-DOPA aminotransferase (LdaA).

[0022] Picture 3 The effect of pH on the enzyme activity of L-DOPA aminotransferase (LdaA).

[0023] Picture 4 This is a mass spectrum of the L-DOPA conversion catalyzed by L-DOPA aminotransferase LdaA.

[0024] Picture 5 The HPLC chromatogram is for 3,4-dihydroxyphenylpyruvic acid (DHPPA).

[0025] Picture 6 This describes the pathway catalyzed by L-DOPA aminotransferase LdaA for the conversion of L-DOPA. Detailed Implementation

[0026] The present invention will be further explained below with reference to embodiments and accompanying drawings. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0027] The culture medium formulations involved in the following examples are as follows:

[0028] LB liquid medium, 1L formulation: NaCl 5.0 g, yeast extract 5.0 g, tryptone 10.0 g, ultrapure water to 1 L, sterilized at 121℃ for 20 min. pH 7.0. LB solid medium is prepared by adding 18 g agar powder to this formulation.

[0029] The PBS buffer (pH 7.2) 1L system formulation is as follows: NaCl 8.0 g, KCl 0.2 g, Na2HPO4 1.42 g, KH2PO4 0.24 g, and ultrapure water to a final volume of 1 L, with a pH of 7.2.

[0030] Example 1: L-DOPA aminotransferase gene ldaA Prediction, cloning and heterologous expression

[0031] 1.1 L-DOPA aminotransferase gene ldaA Prediction, cloning

[0032] Functional annotation based on whole-genome sequencing results suggests that strain Z-3 (strain Z-3 disclosed in CN 118147010 B) contains... orf5957 A gene encoding an aminotransferase was named L-DOPA aminotransferase gene ldaA, and the protein it encodes was named L-DOPA aminotransferase LdaA. Based on whole-genome sequencing results and KEGG functional annotation analysis, orf5957 The encoded protein belongs to the TyrB type of aminotransferase and participates in the biosynthesis and degradation pathways of tyrosine. TyrB family proteins are pyridoxal phosphate (PLP)-dependent aminotransferases, primarily involved in the biosynthesis and degradation of aromatic amino acids such as tyrosine, phenylalanine, and tryptophan. They catalyze the aminotransfer reaction between α-keto acids and amino acids to generate the corresponding amino acids. The L-DOPA aminotransferase gene is described above. ldaA The nucleotide sequence is shown in SEQ ID NO.3, and the amino acid sequence of the L-DOPA aminotransferase LdaA encoded by it is shown in SEQ ID NO.4.

[0033] 1.2 L-DOPA aminotransferase gene ldaA Heterogeneous expression

[0034] Designed for amplifying the L-DOPA aminotransferase gene ldaA Primers for the recombinant expression vector carrying a 6×His tag.

[0035] forward primer ldaA -F:

[0036] aagaaggagatatacatatgtcgctcttctccgctgtcca (SEQ ID NO. 1);

[0037] reverse primer ldaA -R:

[0038] tggtggtggtggtgctcgagcttcagcacagctgcgacg (SEQ ID NO. 2).

[0039] PCR amplification program: Stage 1, pre-denaturation at 95℃ (3 min); Stage 2, denaturation at 95℃ (15 s), annealing at 57℃ (15 s), extension at 72℃ (1 min), 30 cycles; Stage 3, final extension at 72℃ (5 min).

[0040] The pET29a(+) plasmid was digested with restriction endonucleases Nde I and Xho I at 37°C for 2 h. Then, the PCR-amplified gene fragment was ligated into the linearized pET29a(+) plasmid using homologous recombination to obtain the recombinant plasmid pET- ldaA The recombinant plasmid pET- ldaA Transform to E. coli BL21(DE3) cells were used to obtain heterologous expression strains, i.e., recombinant strains. E. coli BL21(pET- ldaA ).

[0041] Example 2: Purification and Enzyme Activity Assay of L-DOPA A

[0042] 2.1 Purification of L-DOPA aminotransferase LdaA

[0043] Recombinant strains E. coli BL21(pET- ldaA Add 1% (vol / vol) of the inoculum to 100 mL containing 50 mg·L⁻¹ -1 Kanamycin (Km) was cultured in LB liquid medium at 37°C and 180 rpm until the cell density reached 0.6-0.8 (OD). 600After adding isopropyl-β-D-thiogalactoside (IPTG) to a final concentration of 0.2 mM, the cells were cultured at 16℃ and 180 rpm for 12 h to induce protein expression. Then, the cells were centrifuged at 12000 rpm for 8 min, the supernatant was discarded, and the cells were collected. The cells were washed twice with PBS buffer (pH 7.2), resuspended in 15 mL of PBS buffer (pH 7.2), and sonicated at 550 W for 10 min (1 s of disruption followed by a 2 s pause). The cells were then centrifuged at 4℃ and 12000 rpm for 30 min, and the supernatant (crude LdaA aminotransferase solution) and precipitate were collected. A portion of the supernatant and precipitate was analyzed by SDS-PAGE. A portion of the supernatant was purified using a nickel ion affinity chromatography column (purified LdaA aminotransferase solution) before further SDS-PAGE analysis. The results showed that the obtained band size was approximately 43 kDa, which is basically consistent with the theoretical molecular weight of L-DOPA aminotransferase (LdaA) and can be used for subsequent experiments. Picture 1 ).

[0044] 2.2 Determination of the enzyme activity of L-dopa aminotransferase (LdaA)

[0045] To determine the optimal reaction temperature for L-DOPA aminotransferase (LdaA), its catalytic activity was evaluated within the temperature range of 20–50 °C. The enzymatic reaction system for LdaA (500 µL) was prepared using PBS buffer (pH 7.2) and contained 0.6 mM α-ketoglutarate, 0.12 mM pyridoxal phosphate (PLP), 0.5 mM L-DOPA, and 0.2 μM L-DOPA aminotransferase (LdaA, prepared according to section 2.1, pure L-DOPA enzyme). Reactions were carried out at 20 °C, 25 °C, 30 °C, 35 °C, 40 °C, 45 °C, and 50 °C for 30 min each. After the reaction, the enzyme reaction sample was boiled for 5 min, an equal volume of methanol was added, and the mixture was centrifuged at 12000 rpm for 5 min. The supernatant was filtered through a 0.22 μm filter, and the conversion of L-DOPA was detected by HPLC. Enzyme activity is defined as the amount of enzyme required to catalyze the conversion of 1 μmol of substrate (L-DOPA) per minute under specific assay conditions (1 U). The enzyme activity measured under the assay condition corresponding to the highest enzyme activity is defined as 100%. The relative enzyme activity under other assay conditions is calculated using the following formula: Relative enzyme activity (%) = (Enzyme activity under a specific assay condition / Highest enzyme activity) × 100%. The results showed that the highest enzyme activity (5.35 U / mg) of L-DOPA aminotransferase (LdaA) catalyzing the conversion of L-DOPA was observed at a reaction temperature of 45℃. Therefore, the optimal reaction temperature for L-DOPA conversion catalyzed by LdaA is 45℃. Picture 2 ).

[0046] The optimal reaction temperature for L-DOPA aminotransferase (LdaA) was determined using buffer solutions with a pH range of 3.0–9.0: 50 mM citrate-sodium citrate (pH 3.0–6.0) and 50 mM disodium hydrogen phosphate-potassium dihydrogen phosphate (pH 5.5–9.0). The enzymatic reaction system for LdaA (500 µL) consisted of buffer solutions at corresponding pH values ​​(pHs 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, and 9.0), including final concentrations of 0.6 mM α-ketoglutarate, 0.12 mM PLP, 0.5 mM L-DOPA, and 0.2 μM LdaA (prepared according to section 2.1, pure LdaA enzyme). The reaction was carried out at 45 °C for 30 min. After the reaction, the enzyme reaction sample was boiled for 5 min, an equal volume of methanol was added, and the mixture was centrifuged at 12000 rpm for 5 min. The supernatant was filtered through a 0.22 μm filter membrane, and the conversion of levodopa was detected by HPLC. Enzyme activity was defined as: 1 U is the amount of enzyme required to catalyze the conversion of 1 μmol of substrate (levodopa) per minute under specific assay conditions. The enzyme activity measured under the assay conditions corresponding to the highest enzyme activity was defined as 100%, and the relative enzyme activity under other assay conditions was calculated using the following formula: Relative enzyme activity (%) = (Enzyme activity under a certain assay condition / Highest enzyme activity) × 100%. The results showed that the highest enzyme activity (5.81 U / mg) of levodopa conversion was catalyzed by levodopa aminotransferase LdaA at a reaction pH of 7.5. Therefore, the optimal reaction pH for levodopa conversion catalyzed by levodopa aminotransferase LdaA is 7.5. Picture 3 ).

[0047] HPLC detection conditions for levodopa: Agilent ZORBAX SB C18 column (250 mm × 4.6 mm × 5 μm), column temperature 40°C, injection volume 20 μL, mobile phase methanol:water:acetic acid (20:80:0.5, V:V:V), flow rate 1.0 mL / min, isocratic elution, detection wavelength 280 nm.

[0048] Example 3 Analysis of the L-DOPA conversion pathway catalyzed by L-DOPA aminotransferase LdaA

[0049] The enzymatic reaction system for L-DOPA aminotransferase (LdaA) was prepared in 500 µL using PBS buffer (pH 7.2) and contained 0.6 mM α-ketoglutarate, 0.12 mM PLP, 0.5 mM L-DOPA, and 0.2 μM L-DOPA aminotransferase (LdaA) (prepared according to section 2.1, crude L-DOPA aminotransferase). The reaction was carried out at 45 °C, and samples were taken at 0, 2, and 12 h. Intermediate metabolites during the L-DOPA enzymatic reaction were analyzed using liquid chromatography-mass spectrometry (LC-MS). Mass spectrometry conditions: electrospray ionization (ESI), negative ion scanning mode, mass-to-charge ratio scan range 50–1200 m / z, capillary voltage 2.5 kV, sampling cone voltage 40.0 V, ion source temperature 120 °C, and desolvation gas temperature 400 °C. Liquid chromatography conditions: An ACQUITY UPLC BEH Shield RP18 column (2.1 mm × 100 mm, 1.7 μm) was used; the column temperature was 35℃; the injection volume was 2 μL; mobile phase A was acetonitrile; mobile phase B was ultrapure water containing 0.1% (vol / vol) formic acid; and the flow rate was 0.2 mL / min. -1 Gradient elution was employed, and the gradient elution program was as follows: 0-0.5 min: 5% (vol / vol) mobile phase A; 0.5-11.5 min: 5% (vol / vol) mobile phase A increased to 95% (vol / vol) mobile phase A; 11.5-13.5 min: 95% (vol / vol) mobile phase A decreased to 5% (vol / vol) mobile phase A, with the remainder being made up with mobile phase B.

[0050] LC-MS results showed that L-DOPA aminotransferase LdaA catalyzes the transamination of L-DOPA, initially generating the unstable intermediate 3,4-dihydroxyphenylpyruvic acid (DHPPA). This intermediate is poorly stable and will further spontaneously oxidize to 3,4-dihydroxyphenylacetic acid (DHPPA). Picture 4 HPLC comparison with DHPPA standard samples confirmed that DHPPA has poor stability and is easily oxidized to 3,4-dihydroxyphenylacetic acid (DHPPA). Picture 5 In summary, the L-DOPA conversion pathway catalyzed by LdaA aminotransferase is as follows: first, the intermediate DHPPA is formed via aminotransfer, followed by spontaneous oxidation of DHPPA, and finally, the major product that can be stably detected in the reaction system is 3,4-dihydroxyphenylacetic acid (DHA). Picture 6 ).

Claims

1. L-DOPA aminotransferase gene ldaA Its application in levodopa biotransformation is characterized by... The L-DOPA aminotransferase gene ldaA The nucleotide sequence is shown in SEQ ID NO.3; the L-DOPA biotransformation is carried out via an aminotransfer reaction.

2. The application of L-DOPA aminotransferase LdaA in L-DOPA biotransformation, characterized in that, The amino acid sequence of the L-DOPA aminotransferase LdaA is shown in SEQ ID NO.4; the L-DOPA biotransformation involves an aminotransfer reaction.

3. L-DOPA aminotransferase gene ldaA The application of the constructed recombinant vector in levodopa biotransformation is characterized by, The L-DOPA aminotransferase gene ldaA The nucleotide sequence is shown in SEQ ID NO.3; the L-DOPA biotransformation is carried out via an aminotransfer reaction.

4. L-DOPA aminotransferase gene ldaA The application of recombinant microorganisms obtained by transforming the constructed recombinant vector in levodopa biotransformation is characterized by, The L-DOPA aminotransferase gene ldaA The nucleotide sequence is shown in SEQ ID NO.3; the L-DOPA biotransformation involves an aminotransfer reaction.

5. A method for the biotransformation of levodopa, characterized in that, The levodopa aminotransferase LdaA is used to catalyze the aminotransfer reaction of levodopa in the presence of pyridoxal phosphate and α-ketoglutarate to generate the intermediate 3,4-dihydroxyphenylpyruvic acid; the 3,4-dihydroxyphenylpyruvic acid is further spontaneously oxidized to 3,4-dihydroxyphenylacetic acid; wherein, the amino acid sequence of the levodopa aminotransferase LdaA is shown in SEQ ID NO.4.

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