A lactam hydrolase mutant, a decarboxylase mutant and application thereof in preparation of 5-methoxytryptamine

Abstract

The application belongs to the field of synthesis of pharmaceutical intermediates and discloses a lactam hydrolase mutant, a decarboxylase mutant and application of the lactam hydrolase mutant and the decarboxylase mutant in preparation of 5-methoxytryptamine. The lactam hydrolase mutant and the decarboxylase mutant with high activity are obtained by mutating specific positions of the lactam hydrolase and the decarboxylase. When the lactam hydrolase mutant and the decarboxylase mutant are used in preparation of 5-methoxytryptamine, the conversion rate and the yield are improved, the conditions are mild, and the use of acid and alkali reagents is reduced.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical intermediate synthesis, specifically relating to a lactam hydrolase mutant, a decarboxylase mutant, and their application in the preparation of 5-methoxytryptamine. Background Technology

[0002] Melatonin, or N-acetyl-5-methoxytryptamine, is a hormone primarily secreted by the pineal gland. It is commonly used to treat insomnia and jet lag, and is characterized by its endogenous nature, low toxicity, non-addictiveness, and lack of withdrawal symptoms. As a well-known raw material in the sleep field, it enjoys high levels of public education and its efficacy has been thoroughly studied. For sleep disorders caused by endogenous factors, such as shallow sleep and difficulty falling asleep, melatonin is more effective than GABA. 5-Methoxytryptamine is a key intermediate in the synthesis of melatonin and also an important starting material for the preparation of various indole alkaloids. Therefore, research on the synthesis methods of 5-methoxytryptamine is of great significance.

[0003] In the study by Hu Songlin et al. (Chemical Synthesis of N-acetyl-5-methoxytryptamine [J]. Chemical World, 2016, (11): 699-702), 1,2,3,4-tetrahydro-6-methoxy-1-oxo-β-carboline (compound I) was hydrolyzed with NaOH using 60% ethanol aqueous solution as solvent and refluxed at 80-85℃ for 5 h. After the reaction, the pH was adjusted to 6-7 with glacial acetic acid, and 2-carboxyl-5-methoxytryptamine (compound II) was obtained by filtration. Then, the product was decarboxylated with 5% hydrochloric acid at reflux temperature for 3 h, and the pH was adjusted with NaOH to precipitate the product, yielding 5-methoxytryptamine (compound III) with a yield of 84%.

[0004] The reaction formula is as follows:

[0005]

[0006] The process described in patent CN114195696A is as follows: Compound I reacts with NaOH at 85℃ for 10 hours, then cools to room temperature and adjusts the pH to 5 to obtain compound II; subsequently, it reacts with hydrochloric acid aqueous solution at 100℃ for 4 hours, cools to 50℃, first adjusts the pH to 5.5 and reacts for 10 minutes, then adjusts the pH to 10 and reacts for 10 minutes, and finally filters and collects the residue to obtain compound III.

[0007] In patent CN114478353A, compound I is reacted with NaOH in an ethanol-water solution at 110°C under reflux for 12 hours. After the reaction, the pH is adjusted to 5 with 20% hydrochloric acid, and the temperature is further increased to 110°C under reflux for 10 hours. After centrifugation, the mixture is dried in a vacuum drying oven at 50°C for 1 hour to obtain compound III, with a yield of 85%. Patent CN104496882A uses a 50% ethanol-water solution as the solvent. Compound I is reacted with NaOH at 83°C for 7 hours. After cooling to 40°C, the pH is adjusted to 6 with glacial acetic acid, and the mixture is centrifuged to obtain compound II. Compound II is then decarboxylated with hydrochloric acid at 102°C for 3.5 hours, neutralized with NaOH to pH 6, and centrifuged to obtain compound III, with a yield of approximately 80%.

[0008] In the preparation of the key melatonin intermediate 5-methoxytryptamine, the traditional method uses 1,2,3,4-tetrahydro-6-methoxy-1-oxo-β-carboline as raw material and obtains the target product through two-step reactions of amide hydrolysis and decarboxylation. However, it has obvious drawbacks: the reaction requires multiple acid-base treatments in the form of "base-acid-base", which requires a large amount of acid and base, resulting in a low yield; at the same time, it generates a large amount of wastewater and waste salt, and the subsequent treatment cost is high. Summary of the Invention

[0009] To address the aforementioned problems, this invention provides a lactam hydrolase mutant, a decarboxylase mutant, and their application in the preparation of 5-methoxytryptamine. Applying these lactam hydrolase mutants and decarboxylase mutants to the synthesis of 5-methoxytryptamine not only results in high catalytic yield but also simplifies the post-processing, aligns with green and environmentally friendly principles, and can significantly reduce the production cost of melatonin.

[0010] The technical solution of the present invention is as follows:

[0011] A lactam hydrolase mutant, obtained by mutating a lactam hydrolase with NCBI accession number WP_059035869.1; said mutation is one of the following:

[0012] G32F; A124G; L126A; L130Y; I233K; L234A; G32F / L126A / L130Y / I233K; G32F / L130Y / I233K / L234A; G32F / L126A / L130Y / I233K. Preferably, it is one of G32F / L126A / L130Y / I233K; G32F / L130Y / I233K / L234A; G32F / L126A / L130Y / I233K, with G32F / L130Y / I233K / L234A being the most preferred.

[0013] This invention also provides a decarboxylase mutant, obtained by mutating the decarboxylase with NCBI accession number AEY82397.1; the mutation is one of the following: W94F; W94Y; A105G; T264S; T264Y; T371A; T371G; W94Y / T264S; W94Y / A105G / T371G; T264Y / T371A; W94F / A105G / T264S / T371A; W94Y / A105G / T264S / T371A. Preferably, it is one of W94F / A105G / T264S / T371A; W94Y / A105G / T264S / T371A, and most preferably W94Y / A105G / T264S / T371A.

[0014] The present invention also provides a gene encoding the above-mentioned lactam hydrolase mutant or a gene encoding the above-mentioned decarboxylase mutant.

[0015] The present invention also provides a method for preparing 5-methoxytryptamine, a key intermediate of melatonin, comprising: under the catalysis of the decarboxylase mutant, 2-carboxyl-5-methoxytryptamine undergoes a decarboxylation reaction, and after the reaction is complete, the 5-methoxytryptamine is obtained.

[0016] Preferably, the decarboxylation reaction is carried out in a mixed solvent of water and DMSO;

[0017] The reaction is carried out at a pH of 5.0 to 6.0, a temperature of 35 to 39°C, and a time of 8 to 24 hours.

[0018] Preferably, the preparation method further includes a step of preparing 2-carboxy-5-methoxytryptamine:

[0019] Under the catalysis of the lactam hydrolase mutant, 1,2,3,4-tetrahydro-6-methoxy-1-oxo-β-carboline undergoes a hydrolysis reaction to obtain the 2-carboxy-5-methoxytryptamine.

[0020] Preferably, the hydrolysis reaction is carried out in a mixed solvent of water and DMSO;

[0021] The reaction is carried out at a pH of 7.0 to 8.0, a temperature of 35 to 39°C, and a time of 8 to 24 hours.

[0022] Furthermore, the decarboxylation reaction and hydrolysis reaction are carried out in a one-pot process; that is, after the hydrolysis is completed, the decarboxylase mutant is directly added and the pH is adjusted to carry out the decarboxylation reaction.

[0023] This invention also provides another method for preparing 5-methoxytryptamine, a key intermediate of melatonin. Under the combined action of the lactam hydrolase mutant and the decarboxylase mutant, 1,2,3,4-tetrahydro-6-methoxy-1-oxo-β-carboline undergoes a series of hydrolysis and decarboxylation reactions to obtain the 5-methoxytryptamine.

[0024] Preferably, the hydrolysis and decarboxylation reactions are carried out in a mixed solvent of water and DMSO;

[0025] The reaction is carried out at a pH of 5.0 to 8.0, a temperature of 35 to 39°C, and a time of 8 to 24 hours.

[0026] Preferably, the above-mentioned catalyst lactam hydrolase mutant and the decarboxylase mutant are used in the form of crude enzyme solution after cell disruption, or in the form of engineered bacteria resting cells expressing recombinant enzymes, or in the form of purified enzymes or immobilized enzymes, which are conventional methods in the art.

[0027] Compared with the prior art, the beneficial effects of the present invention are reflected in:

[0028] (1) The present invention modifies and improves wild-type enzymes through enzyme engineering, thereby greatly improving enzyme activity.

[0029] (2) By using a new lactam hydrolase mutant and the aforementioned decarboxylase mutant, the present invention achieves a higher yield, is more mild, and requires less acid and alkali when preparing 5-methoxytryptamine. Detailed Implementation

[0030] Example 1: Construction of recombinant strain expressing lactam hydrolase

[0031] Lactamases specifically catalyze the hydrolysis and cleavage of lactam bonds, possessing a broad spectrum of compounds and efficiently hydrolyzing β-, γ-, and δ-lactams. First, the amino acid sequence of a lactamase (NCBI accession number: WP_059035869.1, abbreviated as Gd-LAase) derived from *Gordonia desulfuricans* was obtained (see below). After codon optimization by *E. coli*, it was chemically synthesized and subsequently ligated into the plasmid vector pET-28a(+) via NcoⅠ and XhoⅠ restriction sites. Take E. coli BL21(DE3) Competent Cells (Sangon Biotech, catalog number: B528414) from the -80℃ freezer, quickly place them on ice, and let them thaw for 10 minutes. Under aseptic conditions in a clean bench, add 100 ng of plasmid DNA, incubate on ice for 30 minutes, then heat shock in a 42℃ water bath for 45 seconds. Quickly return them to ice and let them thaw for 2 minutes. Add 1 mL of antibiotic-free LB medium, and incubate at 37℃ with shaking for 1 hour. Take 100 μL and spread it on an LB solid medium plate containing kanamycin (50 μg / mL). Incubate upside down at 37℃ overnight. The single colony transformant obtained is the recombinant expression strain of lactam hydrolase. This strain will be used for the first step of enzyme catalyst production.

[0032] The amino acid sequence of Gd-LAase is as follows:

[0033] MGYITVGTENSTDIELYYEDHGTGQAVVLIHGYPLDGHSWELQSRALIDAGYRVITYDRRGFGQSSKAGIGYDYDTFAADLDTVLTTLDLRDVILVGFSMGTGELARYTRNHGHDRVARFAFLASLEPFLLKTDDNPTGV DKEVFDGIHTAAYTDRYAWFTQFYDNFYNLGENLGTRISHEVVDANRNTAVASAPAAAYKVVPPTWIEDFRPDVAAVRDSGKPTLILHGTADNILPIDATGRPFHEAVPAARYVEIEGAPHGLLWTHAEEVTAALLDFVRS

[0034] Example 2 Construction of recombinant strains expressing decarboxylase

[0035] Decarboxylases catalyze the removal of carboxyl groups from compounds, releasing carbon dioxide. First, the amino acid sequence of a decarboxylase derived from Vincaminor (NCBI accession number: AEY82397.1, abbreviated as Vm-DCase) was obtained (see below). After codon optimization, it was chemically synthesized and subsequently ligated into the plasmid vector pET-28a(+) via NcoⅠ and XhoⅠ restriction sites. Take E. coli BL21(DE3) Competent Cells (Sangon Biotech, catalog number: B528414) from the -80℃ freezer, quickly place them on ice, and let them thaw for 10 minutes. Under aseptic conditions in a clean bench, add 100 ng of plasmid DNA, incubate on ice for 30 minutes, then heat shock in a 42℃ water bath for 45 seconds. Quickly return them to ice and let them thaw for 2 minutes. Add 1 mL of antibiotic-free LB medium, and incubate at 37℃ with shaking for 1 hour. Take 100 μL and spread it on an LB solid medium plate containing kanamycin (50 μg / mL). Incubate upside down at 37℃ overnight. The single colony transformant obtained is the decarboxylase recombinant expression strain, which will be used for the second step of enzyme catalyst production.

[0036] The amino acid sequence of Vm-DCase is as follows:

[0037] MGSIDSTNDVALSNGSSVGEFKPLEAEEFRKQAHCMVDFIADYYKNVESYPVLSQVEPGYLRERLPETAPYLPESLDKIMSDIQKDIIPGMTHWMSPNFYAFFPATVSSAAFLGEMLSTALNSVG FTWVSSPAATELEMIVMDWLAKMLKLPECFMFSGTGGGVIQNTTSESILCTIIAARERVLENLGPNSIGKLVCYGSDQTHTMFPKTCKLAGIFPDNIRLIPTTLETDFSIDPHVLREMVKADVDA GLIPLFLCATLGTTSTTATDPVSSLSEITNEFNIWMHVDAAYAGSACICPEFRHYLDGIERVDSLSPHKWLLAYLDSTCLWVKNPNLLLRALTTNPEYLKNKQSDLDKVVDFKNWQIATGRKF RSLKLWLILRSYGVANLQTHIRSDVAMAKMFEGFVRSDPRFEVVVPRNFSLVCFRLKPLPGSDVEILNKKLNDMLNSTGRVYMTHTIVGGIYMLRLAVGSSLTEEHHVRAVWELIKKLADDLLKEA

[0038] Example 3: Fermentation production by strain

[0039] The recombinant strain was inoculated at a 1% inoculum into 5 mL of LB medium containing 50 mg / L kanamycin and cultured at 37°C and 200 rpm for 15 h to obtain primary seed culture. Then, the primary seed culture was transferred at a 4% inoculum into 50 mL of TB medium containing 50 mg / L kanamycin and cultured at 37°C and 220 rpm until the OD value of the bacterial culture reached 0.6–0.8. IPTG at a final concentration of 0.1 mM was then added to induce expression at 25°C and 220 rpm for 24 h. After fermentation, the supernatant was removed by centrifugation at 4000 rpm for 15 min, and the bacterial cells were collected to obtain the enzyme catalyst.

[0040] Example 4: Validation of lactam hydrolase catalysis

[0041] The activity verification system of lactamase is as follows: The reaction system is 25 mL of DMSO-water mixed solvent (DMSO volume ratio 50%), in which the concentration of compound I is 20 g / L, the cell concentration is 10 g / L, and the pH of the system is adjusted to 7.5. After reacting at 35℃ for 24 h, the hydrolysis conversion rate of compound I is detected by high performance liquid chromatography (HPLC). The results show that the catalytic conversion rate of Gd-LAase can reach 42.9%.

[0042] Example 5: Decarboxylase Catalysis Verification

[0043] The activity verification system of decarboxylase is as follows: The reaction system is 25 mL of DMSO-water mixed solvent (DMSO volume ratio 50%), in which the concentration of compound II is 20 g / L, the amount of pyridoxal phosphate (PLP) is 0.02%, the cell concentration is 10 g / L, and the pH of the system is adjusted to 5.5. After reacting at 35℃ for 24 h, the decarboxylation conversion rate of compound II is detected by HPLC. The results show that the catalytic conversion rate of Vm-DCase can reach 89.1%.

[0044] Example 6: Molecular Modification of Lactam Hydrolase

[0045] Considering the low activity of Gd-LAase, directed evolution was employed to enhance its activity, open up the catalytic pathway, and improve catalytic conversion rate. By analyzing the amino acid composition of the enzyme-substrate binding region, G32, A124, L126, L130, I233, and L234 were selected as site-directed mutagenesis targets to construct the modified strain. Wild-type pET-28a(+)-Gd-LAase was used as the PCR template, and PCR was performed using the primers listed in the table below. The reaction conditions were as follows: a 50 μL system containing 25 μL PrimeStar Max enzyme, 2 μL each of forward and reverse primers, 1 μL template DNA, and water to a final volume of 50 μL. PCR reaction conditions: 98℃ denaturation for 5 min followed by cycling, then 98℃ denaturation for 30 s, 58℃ annealing for 15 s, and 72℃ extension for 1 min, for a total of 30 cycles, followed by a final extension at 72℃ for 10 min. Agarose gel electrophoresis confirmed that the PCR product band size was approximately 6 kb. The PCR fragment was recovered using a PCR product recovery kit (Sangon Biotech, catalog number B518141), and then the PCR product was transformed into E. coli BL21(DE3) under the same transformation conditions as in Example 1. After successful mutation was confirmed by plasmid sequencing, enzyme production fermentation was performed under the same fermentation conditions as in Example 3.

[0046]

[0047] The enzyme activity assessment system was as follows: 5 mL DMSO-water mixture (DMSO volume percentage 50%), compound I concentration 20 g / L, cell concentration 10 g / L, pH 7.5, reaction at 35℃ for 2 h, and HPLC was used to detect the hydrolysis conversion rate of compound I. Enzyme activity definition: Under the above reaction conditions, the amount of enzyme required to catalyze the conversion of 1 μmol of compound I per minute is defined as 1 enzyme activity unit (U); unit enzyme activity definition: enzyme activity units (U / g) contained in one gram of wet cells; relative enzyme activity definition: the relative activity of each mutant was calculated based on the unit enzyme activity of wild-type Gd-LAase (100%), and the results are shown in Table 1.

[0048] By screening for the best mutant strains for each target site and performing random combination mutations, the mutant strain GdLA-8 (GdLA-G32F / L130Y / I233K / L234A) with the most significant increase in enzyme activity was finally obtained, with a unit enzyme activity of 39.8 U / g and a relative enzyme activity of 323.6% compared to the wild type.

[0049] Table 1

[0050]

[0051] Example 7 Molecular modification of decarboxylase Vm-DCase

[0052] Similarly, the catalytic pocket residue composition of the decarboxylase Vm-DCase was determined through compound molecular docking simulation: W94, F102, F103, A105, V124, F126, T264, H320, L327, T371, G372. A saturated mutant library was constructed based on these sites. Using wild-type pET-28a(+)-Vm-DCase as a template for PCR, PCR was performed using the primers listed in the table below. PCR conditions were the same as in Example 6. The PCR product was transformed into E. coli BL21(DE3) under the same transformation conditions as in Example 1. Transformants from the saturated mutant library were picked into 96-well plates, and 200 μL of LB liquid medium containing 50 mg / L kanamycin was added to each well. The plates were then incubated overnight at 37°C and 250 rpm to obtain the primary seed culture. 40 μL of the primary seed culture was transferred into TB liquid medium containing 50 mg / L kanamycin and cultured at 37°C until OD600 = 0.6-0.8. Induction was performed using IPTG at a final concentration of 0.1 mM, and the induction temperature was adjusted to 25°C. The culture was then incubated overnight to obtain the enzyme-producing bacterial culture.

[0053]

[0054] A high-throughput screening strategy was employed, using the PEPC enzymatic carbon dioxide assay kit. The detection principle is as follows: low-concentration CO2 produced by the decarboxylase-catalyzed reaction exists in the form of carbonate ions. In the system, HCO3⁻ reacts with phosphoenolpyruvate (PEP) in the presence of phosphoenolpyruvate carboxylase and Mg²⁺ to generate oxaloacetate and phosphate. Oxaloacetate further reacts with malate dehydrogenase to generate malate, while simultaneously oxidizing NADH to NAD⁺. Since the rate of NADH consumption is directly proportional to the carbon dioxide content in the sample, the amount of carbon dioxide generated in the sample can be quantitatively analyzed by measuring the rate of change in absorbance at 340 nm, thus reflecting the catalytic activity of the enzyme.

[0055]

[0056] The high-throughput screening system was as follows: A 0.8 mL DMSO-water mixture (DMSO 50% by volume) was constructed in a 96-well plate, with compound II at a concentration of 10 g / L and PLP at 0.02%. 0.2 mL of bacterial culture from the 96-well plate was added, and the pH was adjusted to 5.5. The reaction was blocked at 35°C for 2 h. After the reaction, 50 μL of the reaction solution was added to a new 96-well plate containing 500 μL of CO2 assay kit solution. The mixture was incubated at 37°C for 5 min, and the absorbance at 340 nm was measured. The screening criteria were: mutants with an absorbance reduction of more than 20% compared to the control strain were considered beneficial mutants, and the mutation type was determined by plasmid sequencing.

[0057] Beneficial mutants were rescreened, and mutations showing enhanced activity were randomly combined for further mutation. The rescreening evaluation system was as follows: 5 mL DMSO-water mixture (DMSO 50% by volume), compound II concentration 20 g / L, cell concentration 10 g / L, pH 5.5, reaction at 35℃ for 2 h, and HPLC was used to detect the decarboxylation conversion rate of compound II. Enzyme activity was defined as the amount of enzyme required to catalyze the conversion of 1 μmol of compound II per minute under the above reaction conditions; unit enzyme activity was defined as the enzyme activity units (U / g) contained in each gram of wet cells; relative enzyme activity was defined as the relative activity of each mutant calculated based on the unit enzyme activity of wild-type Vm-DCase (100%).

[0058] The optimal mutant strain VmDC-12 (VmDC-W94Y / A105G / T264S / T371A) was finally obtained through screening, with a unit enzyme activity of 89.6 U / g and a relative enzyme activity of 409.1% compared to the wild type.

[0059] Table 2

[0060]

[0061] Example 8 Catalytic Example:

[0062] Preparation of Compound II via Gd-LAase Enzyme Catalysis: 100g of Compound I was added to a 3L reactor and dissolved in a mixture of 400g pure water and 400g DMSO. 20g of GdLA mutant strain 8 cells were added, and the pH was adjusted to 7.5 before adding pure water to bring the volume to 1L. The reaction was carried out at 37℃ and 300rpm for 12h with stirring. Sampling and analysis showed that the residual amount of Compound I was 0.9g / L, and the amount of product Compound II generated was 101.2g / L, with a conversion rate of 99.1% and a yield of 93.4%.

[0063] Example 9 Catalytic Example:

[0064] Vm-DCase enzyme-catalyzed preparation of compound III: 100g of compound II was added to a 3L reactor and dissolved in a mixture of 400g pure water and 400g DMSO; 20g of Vm-DCase mutant cells 12 were added, the pH of the system was adjusted to 5.5, and then pure water was added to bring the volume to 1L. The pH was maintained stable during the reaction. The reaction was carried out at 37℃ and 300rpm for 12h with stirring. Sampling and analysis showed that the residual amount of compound II was 0g / L, the amount of product compound III generated was 80.5g / L, the conversion rate reached 100%, and the yield was 99.0%.

[0065] Example 10 Catalytic Example:

[0066] Two-enzyme tandem catalysis: 100g of compound I was added to a 3L reactor and dissolved in a mixture of 400g pure water and 400g DMSO. 20g of GdLA mutant strain 8 cells were added, and the pH was adjusted to 7.5 before adding pure water to bring the volume to 1L. The reaction was stirred at 37℃ and 300rpm for 12h. Then, 20g of VmDC mutant strain 12 cells and 0.2g of PLP were added, and the pH was adjusted to 5.5 with dilute sulfuric acid. The reaction was continued at 37℃ and 300rpm for another 12h. Sampling analysis showed that the residual amount of compound I was 0.2g / L, the amount of product compound III generated was 86.7g / L, the conversion rate reached 99.8%, and the yield was 98.5%.

[0067] Comparative Example 1: Double Wild-type Enzyme Control Experiment

[0068] 100g of compound I was added to a 3L reactor and dissolved in a mixture of 400g pure water and 400g DMSO. 20g of wild-type GdLA cells were added, and the pH was adjusted to 7.5. Pure water was then added to bring the volume to 1L. The reaction was stirred at 37℃ and 300rpm for 12 hours. Then, 20g of wild-type VmDC cells and 0.2g of PLP were added, and the pH was adjusted to 5.5 with dilute sulfuric acid. The reaction was continued at 37℃ and 300rpm for another 12 hours. Sampling analysis showed that the residual amount of compound I was 78.2g / L, the amount of product compound III formed was 15.6g / L, the conversion rate was 21.8%, and the yield was 17.7%.

[0069] In this invention, the amino acid sequence of the lactam hydrolase derived from *Gordonia desulfuricans* is as follows:

[0070] Gd-LAase

[0071] MGYITVGTENSTDIELYYEDHGTGQAVVLIHGYPLDGHSWELQSRALIDAGYRVITYDRRGFGQSSKAGIGYDYDTFAADLDTVLTTLDLRDVILVGFSMGTGELARYTRNHGHDRVARFAFLASLEPFLLKTDDNPTGV DKEVFDGIHTAAYTDRYAWFTQFYDNFYNLGENLGTRISHEVVDANRNTAVASAPAAAYKVVPPTWIEDFRPDVAAVRDSGKPTLILHGTADNILPIDATGRPFHEAVPAARYVEIEGAPHGLLWTHAEEVTAALLDFVRS

[0072] Nucleotide sequence of lactam hydrolase from Gordonia desulfuricans:

[0073] >Gd-LAase

[0074] ATGGGCTATATCACTGTTGGCACTGAAAACTCTACGGATATCGAACTGTATTACGAAGACCACGGTACCGGTCAAGCAGTAGTTCTGATTCACGGCTACCCGCTGGATGGTCACTCCTGGGAACTGCAGTCTCGTGCGCTGATTGACGCCGGTTACCGCGTCATCACCTACGACCGTCGTGGCTTTGGTCAGTCCTCTAAAGCGGGCATCGGCTATGATTACGATACCTTCGCGGCGGATCTGGATACCGTGCTGACCACCCTGGACCTGCGTGATGTTATCCTGGTGGGCTTCTCTATGGGTACTGGTGAACTGGCTCGTTATACCCGTAACCACGGTCACGACCGTGTAGCGCGTTTCGCGTTCCTGGCAAGCCTGGAACCGTTCCTGCTGAAAACCGACGACAACCCTACCGGCGTAGACAAGGAAGTTTTCGATGGCATCCACACCGCCGCTTACACTGACCGTTACGCGTGGTTCACTCAGTTTTACGACAACTTCTACAACCTGGGCGAGAACCTGGGCACCCGTATCAGCCACGAAGTGGTTGATGCGAACCGTAACACCGCAGTTGCATCCGCTCCGGCGGCGGCCTACAAAGTAGTTCCGACCTGGATCGAGGATTTCCGCCCGGATGTAGCGGCGGTTCGTGATAGCGGCAAACCAACGCTGATTCTGCACGGTACCGCAGATAACATTCTGCCGATCGACGCAACCGGCCGCCCGTTCCACGAAGCCGTTCCGGCAGCTCGTTACGTTGAGATCGAAGGTGCACCGCACGGTCTGCTGTGGACTCACGCCGAGGAAGTTACCGCAGCGCTGCTGGACTTCGTGCGCTCC

[0075] Amino acid sequence of decarboxylase from Vincaminor:

[0076] >Vm-DCase

[0077] MGSIDSTNDVALSNGSSVGEFKPLEAEEFRKQAHCMVDFIADYYKNVESYPVLSQVEPGYLRERLPETAPYLPESLDKIMSDIQKDIIPGMTHWMSPNFYAFFPATVSSAAFLGEMLSTALNSVGFTWVSSPAATELEMIVMDWLAKMLKLPECFMFSGTGGGVIQNTTSESILCTIIAARERVLENLGPNSIGKLVCYGSDQTHTMFPKTCKLAGIFPDNIRLIPTTLETDFSIDPHVLREMVKADVDAGLIPLFLCATLGTTSTTATDPVSSLSEITNEFNIWMHVDAAYAGSACICPEFRHYLDGIERVDSLSLSPHKWLLAYLDSTCLWVKNPNLLLRALTTNPEYLKNKQSDLDKVVDFKNWQIATGRKFRSLKLWLILRSYGVANLQTHIRSDVAMAKMFEGFVRSDPRFEVVVPRNFSLVCFRLKPLPGSDVEILNKKLNDMLNSTGRVYMTHTIVGGIYMLRLAVGSSLTEEHHVRAVWELIKKLADDLLKEA

[0078] Decarboxylase nucleotide sequence derived from Vincaminor:

[0079]

Claims

1. A lactam hydrolase mutant, characterized in that, It was obtained by a mutation of the lactam hydrolase with NCBI accession number WP_059035869.1; the mutation is one of the following mutations: G32F; A124G; L126A; L130Y; I233K; L234A; G32F / L126A / L130Y / I233K; G32F / L130Y / I233K / L234A; G32F / L126A / L130Y / I233K.

2. A gene encoding a lactam hydrolase mutant as described in claim 1.

3. A decarboxylase mutant, characterized in that, It was obtained by a decarboxylase mutation with NCBI accession number AEY82397.1; the mutation is one of the following: W94F; W94Y; A105G; T264S; T264Y; T371A; T371G; W94Y / T264S; W94Y / A105G / T371G; T264Y / T371A; W94F / A105G / T264S / T371A; W94Y / A105G / T264S / T371A.

4. A gene encoding the decarboxylase mutant as described in claim 3.

5. A method for preparing 5-methoxytryptamine, a key intermediate in melatonin production, characterized in that, include: Under the catalysis of the decarboxylase mutant described in claim 3, 2-carboxyl-5-methoxytryptamine undergoes a decarboxylation reaction, and after the reaction is complete, 5-methoxytryptamine is obtained.

6. The method for preparing 5-methoxytryptamine, a key intermediate in melatonin, according to claim 5, is characterized in that, The decarboxylation reaction is carried out in a mixed solvent of water and DMSO; The reaction is carried out at a pH of 5.0 to 6.0, a temperature of 35 to 39°C, and a time of 8 to 24 hours.

7. The method for preparing 5-methoxytryptamine, a key intermediate in melatonin, according to claim 5, is characterized in that, It also includes the preparation steps of 2-carboxy-5-methoxytryptamine: Under the catalysis of the lactam hydrolase mutant of claim 1, 1,2,3,4-tetrahydro-6-methoxy-1-oxo-β-carboline undergoes a hydrolysis reaction to obtain the 2-carboxy-5-methoxytryptamine after the reaction is completed.

8. The method for preparing 5-methoxytryptamine, a key intermediate in melatonin, according to claim 7, is characterized in that, The hydrolysis reaction is carried out in a mixed solvent of water and DMSO; The reaction is carried out at a pH of 7.0 to 8.0, a temperature of 35 to 39°C, and a time of 8 to 24 hours.

9. A method for preparing 5-methoxytryptamine, a key intermediate in melatonin production, characterized in that, Under the combined action of the lactam hydrolase mutant of claim 1 and the decarboxylase mutant of claim 3, 1,2,3,4-tetrahydro-6-methoxy-1-oxo-β-carboline undergoes a series of hydrolysis and decarboxylation reactions to obtain the 5-methoxytryptamine.

10. The method for preparing 5-methoxytryptamine, a key intermediate in melatonin, according to claim 9, is characterized in that, The hydrolysis and decarboxylation reactions are carried out in a mixed solvent of water and DMSO; The reaction is carried out at a pH of 5.0 to 8.0, a temperature of 35 to 39°C, and a time of 8 to 24 hours.

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