CN114686537A

CN114686537A - Method for producing N-methyl-L-amino acid by catalyzing L-amino acid through multi-enzyme cascade and application thereof

Info

Publication number: CN114686537A
Application number: CN202210389939.6A
Authority: CN
Inventors: 娄文勇; 高胜寒; 徐培; 宗敏华
Original assignee: South China University of Technology SCUT
Current assignee: South China University of Technology SCUT
Priority date: 2022-04-14
Filing date: 2022-04-14
Publication date: 2022-07-01
Anticipated expiration: 2042-04-14
Also published as: CN114686537B

Abstract

The invention discloses a method for generating N-methyl-L-amino acid by catalyzing L-amino acid through multi-enzyme cascade and application thereof. The method takes natural L-amino acid as a substrate, and utilizes L-amino acid oxidase, N-methyl amino acid dehydrogenase and NADPH regenerating enzyme to synchronously cascade and catalyze the L-amino acid reaction to generate the N-methyl-L-amino acid. The method has high efficiency, low cost and wide substrate range, can be suitable for L-phenylalanine, L-glutamic acid, L-leucine, L-lysine, L-valine, L-isoleucine and the like, converts the L-phenylalanine, L-glutamic acid, L-leucine, L-lysine, L-valine, L-isoleucine and the like into corresponding N-methyl-L-amino acid, has high product conversion rate and has good industrial application prospect.

Description

Method for producing N-methyl-L-amino acid by catalyzing L-amino acid through multi-enzyme cascade and application thereof

Technical Field

The invention belongs to the technical field of enzyme engineering, and particularly relates to a method for generating N-methyl-L-amino acid by catalyzing L-amino acid through multi-enzyme cascade and application thereof.

Background

N- α -alkylation is a mechanism for modulating the activity of chiral α -amino acids. N-methyl-alpha-amino acid is widely applied in the fields of pharmacy, fine chemical engineering and the like, and is an important component for preparing various bioactive molecules, such as antibiotics vancomycin, immunosuppressive agent cyclosporine, cytostatic actinomycin, siderophore aerobic protein and the like. Therefore, the research on the synthetic method of the N-methyl-L-amino acid has important value.

Similar to other chiral compounds, the preparation method of N-methyl-L-amino acid is mainly divided into chemical synthesis and biological synthesis. Chemical synthesis relies on either an N-alkylation process or a reductive amination process. These synthetic routes generally require the use of toxic reagents and hazardous solvents, such as pyrophoric metal hydrides or genotoxic alkylating agents, the reaction process generates a large amount of environmentally polluting waste, some reactions also require special temperature and pressure environments, and some processes yield products of low purity and require complicated work-up procedures.

With the development of biotechnology, the biological method for preparing N-methyl-L-amino acid is receiving more and more attention. The biological method for synthesizing the N-methyl-L-amino acid has the advantages of green and clean property, mild reaction condition and high conversion rate. Current synthetic methods involve the use of N-methyltransferases or several dehydrogenases. N-methyltransferase (EC2.1.1) is responsible for N- α -methylation of many amino acids, peptides and proteins in nature, producing monomethylated, dimethylated or trimethylated products, with the disadvantage that this enzyme is generally highly selective for substrates, and has a narrow substrate range. Some dehydrogenases catalyze the reductive amination of α -keto acids or imines with amine donors to produce N-methyl-L-amino acids, including Opines dehydrogenases, N-methyl amino acid dehydrogenases, ketimine reductases, imine reductases, and the like. The reaction needs specific substrates, and some substrates have poor solubility and high price, so the method is not suitable for industrial production.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a method for producing N-methyl-L-amino acid by catalyzing L-amino acid through multi-enzyme cascade, wherein the method combines L-amino acid oxidase and N-methyl amino acid dehydrogenase to produce corresponding N-methyl-L-amino acid by taking natural L-amino acid as a substrate.

Another object of the present invention is to provide the use of said multiple enzyme cascade for catalyzing the production of N-methyl-L-amino acids from L-amino acids.

The purpose of the invention is realized by the following technical scheme:

a method for producing N-methyl-L-amino acid by catalyzing L-amino acid through multi-enzyme cascade is characterized in that L-amino acid is used as a substrate, and L-amino acid oxidase, N-methyl amino acid dehydrogenase and NADPH (nicotinamide adenine dinucleotide phosphate) regenerating enzyme are used for catalyzing L-amino acid reaction to produce N-methyl-L-amino acid through synchronous cascade catalysis.

The L-amino acid comprises at least one of L-phenylalanine, L-glutamic acid, L-leucine, L-lysine, L-valine and L-isoleucine; preferably at least one of L-phenylalanine, L-glutamic acid and L-leucine.

The N-methyl-L-amino acid is at least one of N-methyl-L-phenylalanine, N-methyl-L-glutamic acid, N-methyl-L-leucine, high proline, N-methyl-L-valine and N-methyl-L-isoleucine; preferably at least one of N-methyl-L-phenylalanine, N-methyl-L-glutamic acid and N-methyl-L-leucine.

The L-amino acid oxidase is preferably ancestral L-amino acid oxidase, and the amino acid sequence of the L-amino acid oxidase is shown in SEQ ID NO. 1.

The N-methyl amino acid dehydrogenase is preferably N-methyl amino acid dehydrogenase derived from pseudomonas putida, and the amino acid sequence of the N-methyl amino acid dehydrogenase is the accession number AB190215.1 at NCBI.

The NADPH regenerating enzyme is preferably formic acid dehydrogenase derived from Burkholderia, and the amino acid sequence of the NADPH regenerating enzyme is ACF35003.1 at the NCBI accession number.

The L-amino acid oxidase, the N-methyl amino acid dehydrogenase and the NADPH regenerating enzyme can be obtained by conventional methods, such as prokaryotic expression and protein purification.

The host bacterium for prokaryotic expression is preferably Escherichia coli; more preferably E.coli BL21(DE 3).

The protein can be purified by adopting Ni column affinity chromatography.

The reaction system is a Tris-HCl buffer solution system with the pH value of 7.5-9.0 or Na with the pH value of 9.5-10.0₂CO₃-NaHCO₃A buffer system; preferably Na with pH of 9.5-10.0₂CO₃-NaHCO₃A buffer system; more preferably Na at pH 9.5₂CO₃-NaHCO₃A buffer system.

The reaction system is as follows: 0.01-1 mg/mL L-amino acid deaminase, 1-5 mg/mL N-methyl amino acid dehydrogenase, 0.2-1 mg/mL NADPH-regenerating enzyme, 10-100 mM L-amino acid, 10-200 mM amine donor, 10-300 mM formate, 1-10 mM NADP ⁺20 to 200U/mL catalase, pH7.5 to 10.

The formate is preferably sodium formate.

The molar ratio of the formate to the L-amino acid is 1-5: 1; preferably 3: 1.

The amine donor is methylamine.

The molar ratio of the amine donor to the L-amino acid is 1-5: 1; preferably 2: 1.

The NADP⁺The molar ratio of the L-amino acid to the L-amino acid can be 1: 1-10; preferably 1: 5-10; more preferably 1: 10.

The NADP⁺Is oxidized coenzyme II.

The reaction system is preferably: 0.01mg/mL L-amino acid deaminase, 1mg/mL N-methyl amino acid dehydrogenase, 0.4mg/mL NADPH-regenerating enzyme, 10-100 mM L-amino acid, 20-200 mM amine donor, 30-300 mM formate, 5-10 mM NADP⁺50U/mL catalase, pH 9.5.

The reaction temperature is 20-50 ℃; preferably 37 deg.c.

The rotating speed of the reaction is 100-200 rpm; preferably 160 rpm.

The reaction time is 6-24 h; preferably 12-24 h; more preferably 12 h.

The method for producing N-methyl-L-amino acid by catalyzing L-amino acid through multienzyme cascade is applied to preparing N-methyl-L-amino acid.

The N-methyl-L-amino acid is at least one of N-methyl-L-phenylalanine, N-methyl-L-glutamic acid, N-methyl-L-leucine, L-high proline, N-methyl-L-valine and N-methyl-L-isoleucine; preferably at least one of N-methyl-L-phenylalanine, N-methyl-L-glutamic acid and N-methyl-L-leucine.

In the method for catalyzing L-amino acid to generate N-methyl-L-amino acid, a cofactor regeneration system exists, wherein the cofactor regeneration system comprises the following components: l-amino acid oxidase catalyzes the oxidation of L-amino acid to produce alpha-keto acid, and N-methyl amino acid dehydrogenase catalyzes the reduction of alpha-keto acid to N-methyl-L-amino acid and simultaneously oxidizes NADPH to NADP⁺NADPH-regenerating enzyme NADP⁺Reduced to NADPH, which is formed to take part in the reductive amination of the a-keto acid again (FIG. 1).

Compared with the prior art, the invention has the following advantages and effects:

(1) the invention develops a new method for preparing N-methyl-L-amino acid in vitro with high efficiency and low cost, which takes natural L-amino acid which is cheap and easy to obtain as a substrate and utilizes L-amino acid oxidase, N-methyl amino acid dehydrogenase and NADPH regenerating enzyme to catalyze and synthesize the N-methyl-L-amino acid; wherein, the substrate has wider range (converting substrate L-amino acid into corresponding N-methyl-L-amino acid), can be suitable for L-phenylalanine, L-glutamic acid, L-leucine, L-lysine, L-valine, L-isoleucine and the like, has the yield of 47 to 100 percent, and has better industrial application prospect.

(2) The method for synthesizing the N-methyl-L-amino acid by multi-enzyme cascade catalysis takes the L-amino acid as a substrate, optimizes various influencing factors of the reaction, including reaction temperature, pH value, sodium formate, methylamine concentration and the like, and improves the conversion rate of the substrate and the conversion rate of the product (N-methyl-L-amino acid).

Drawings

FIG. 1 is a schematic diagram showing the coupling of a cascade reaction pathway for producing a corresponding N-methyl-L-amino acid from an L-amino acid to an NADPH regeneration pathway (in the figure, the R group is: -CH)₂-C₆H₅、-(CH₂)₂-COOH、-C₄H₈、-CH₂-CH(CH₃)₂or-CH- (CH)₃)₂)。

FIG. 2 is a graph showing the results of SDS-PAGE verifying the expression of L-amino acid oxidase, N-methyl amino acid dehydrogenase and formate dehydrogenase (M: DNAmarker; lane 1: AncLAAO; lane 2: NMAADH; lane 3: BsFDH).

FIG. 3 is a graph showing the effect of reaction temperature and pH on the catalysis of L-phenylalanine to N-methyl-L-phenylalanine in a cascade reaction; wherein A is the influence of the reaction temperature on the cascade reaction catalysis of L-phenylalanine to generate N-methyl-L-phenylalanine; and B is the influence of pH on the cascade reaction for catalyzing L-phenylalanine to generate N-methyl-L-phenylalanine.

FIG. 4 is a graph of initial molar ratios of sodium formate and methylamine to substrate with the effect of catalyzing the cascade of L-phenylalanine to N-methyl-L-phenylalanine; wherein A is the influence of the initial molar ratio of sodium formate to a substrate on catalyzing L-phenylalanine to generate N-methyl-L-phenylalanine through a cascade reaction; b is the influence of the initial molar ratio of methylamine to substrate on the catalysis of L-phenylalanine to generate N-methyl-L-phenylalanine by cascade reaction.

FIG. 5 is a graph showing the results of HPLC detection of the cascade reaction catalyzing L-amino acid to produce N-methyl-L-amino acid; wherein (a) to (F) are a standard substance liquid phase detection spectrum and a sample detection spectrum (in the figure, A is a standard substance liquid phase detection spectrum of N-methyl-L-phenylalanine and L-phenylalanine, B is a sample L-phenylalanine detection spectrum, C is a standard substance liquid phase detection spectrum of N-methyl-L-glutamic acid and L-glutamic acid, D is a sample L-glutamic acid detection spectrum, E is a standard substance liquid phase detection spectrum of N-methyl-L-leucine and L-leucine, F is a sample L-leucine detection spectrum, G is a standard substance liquid phase detection spectrum of L-homoproline and L-lysine, H is a sample L-homoproline detection spectrum, and I is a standard substance liquid phase detection spectrum of N-methyl-L-valine and L-valine, j: a sample L-valine detection map; k: liquid phase detection spectrum of N-methyl-L-isoleucine and L-isoleucine standard substance, L: sample L-isoleucine detection profile).

Detailed Description

The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. Test methods without specifying specific experimental conditions in the following examples are generally performed according to conventional experimental conditions. Unless otherwise specified, reagents and starting materials for use in the invention are commercially available.

Example 1: recombinant expression of enzymes involved in the production of N-methyl-L-amino acids from L-amino acids

(1) Ancestral L-amino acid oxidase (AncLAAO, SEQ ID NO.1) (the sequence is from DOI:10.1038/s42004-020-00432-8), coding gene of Pseudomonas putida N-methyl amino acid dehydrogenase (NMAADH) (accession number of NCBI: AB190215.1) and coding gene of Burkholderia formamida dehydrogenase (BsFDH) (accession number of NCBI: ACF35003.1) are subjected to codon optimization and synthesis by Kinry, inserted between Xho I site and Nde I site of conventional commercially available pET-28a vector respectively, and subjected to enzyme digestion analysis and identification to obtain corresponding recombinant plasmids.

Ancestral L-amino acid oxidase (SEQ ID NO. 1):

MGTHYTFGKEITDKPLPTQVKVAIVGAGMSGLYSAWRLQQEANCQDLAIFERSNRTGGRLDSDLIEFKNLRSETPKTITVKEEQGGMRFLFDGMDDLMALFLKLNLQDDIVPFPMNSGGNNRLFFRGESFSVEDAQQDDYAIWSHLYNLDQSEQGVNPKDIVNVVFNRILEANPQFQQRPEVRGPEFWQAFRLECQWQGQTLNEWTLWDLYTDMGYSQECINMLYRVLGFNGTFLSQMNAGVAYQLLEDFPAGVQFKTFKDGFSTLPNKLVEEVGTDNIHLQTSIEEIDFAEESGLYSLHYSHTDEHGRVHKGQVKAEKVILGLPRLALEKLFVRSNAFNRLDKDRSEQLWNTLQSASNQPLLKINLYYDSAWWGRGTTGRPAVEFGPNFADLPTGSVYPFYAVNDELAAALMYQERSTNPSKAVQAKLDRIGNEKYERPAALTIYCDYLNINFWSNLQNIGETYHHPHQDDYVEDVPADIYPASTAVVEQATRFFKDIFNTHYVPEPILTSARIWEGSVRFDIPASRQFGFGVHQWAVGANDKEVMATLAEPLPNLFTCGEAFSDYQGWVEGALRSTDLALEKGFGLKPLSQVYFENTNISSSDAIKAVYEENSSKLINQYIETNFSANTAPIEKTADVDSVIGVNLSYFDTK。

(2) chemically introducing each recombinant plasmid into competent cells of escherichia coli BL21(DE3), coating the competent cells on LB solid medium containing 50ug/mL kanamycin, inverting the competent cells at 37 ℃ for overnight culture, selecting monoclonal strains, inoculating the monoclonal strains into 5-20 mL LB liquid medium, performing shaking culture at 37 ℃ and 180rpm, respectively inoculating the strains into 100mL LB liquid medium containing 50ug/mL kanamycin at 1% (v/v), culturing at 37 ℃ and 180rpm for 2-3 h until OD 600 is 0.6-0.8, adding isopropyl-beta-D-thiogalactoside (IPTG) with the final concentration of 0.1mM for induction, continuing induction at 16 ℃ for 20h, centrifugally collecting PB (8000rpm, 5min), resuspending the cells with buffer solution of pH 6.5-9.5 or Tris-HCl buffer solution, performing ultrasonic disruption (350W, working for 2s, pause for 3s, 20min), centrifuging (12000rpm, 10min) after the crushing is finished, and collecting soluble supernatant expression components (crude enzyme liquid).

Example 2: protein purification of N-methyl-L-amino acid related enzymes by catalyzing L-amino acid with multi-enzyme cascade

And (3) separating and purifying the crushed soluble supernatant expression component by using a Ni column affinity chromatography to obtain L-amino acid oxidase, N-methyl amino acid dehydrogenase and formate dehydrogenase. The specific operation is as follows: equilibrating the media with 5-10 column volumes of equilibration buffer (pH 7.550 mM Tris-HCl, 50mM imidazole, 500mM NaCl); the crude enzyme solution obtained by disruption in example 1 was filtered through a 0.45 μm filter; loading the sample at the flow rate of 1 mL/min; after the loading is finished, washing the hybrid protein by using 10-20 times column volume buffer solution (pH 7.550 mM Tris-HCl, 50mM imidazole and 500mM NaCl); then eluting with an elution buffer (pH 7.550 mM Tris-HCl, 200mM imidazole, 500mM NaCl) with 5-10 times column volume, and collecting the eluent; the eluate was washed with 50mM Na pH 9.5₂CO₃-NaHCO₃Diluting with a buffer solution, performing ultrafiltration concentration (10KDa), repeating for 5-10 times, and removing imidazole; the concentration of each target protein obtained after ultrafiltration was measured by the Bradford method, and the target protein was stored at-80 ℃ after being dispensed. Each target protein was verified by SDS-PAGE, and the results are shown in FIG. 2.

Example 3: influence of reaction temperature and buffer pH value on catalytic synthesis of N-methyl-L-phenylalanine

The method researches the influence of reaction temperature and pH on the synthesis of N-methyl-L-phenylalanine by catalyzing L-phenylalanine through multienzyme cascade, and comprises the following specific steps:

(1) the reaction system contained 50mM L-phenylalanine (L-Phe), 100mM methylamine, 100mM sodium formate, 5mM NADP⁺(oxidized coenzyme II, purchased from Mecanum Biotechnology Co., Ltd., Shanghai) and 50U/mL catalase (purchased from Shanghai (Co., Ltd.)) were added to the mixture to react with 0.1mg/mL L-amino acid deaminase, 1mg/mL N-methyl amino acid dehydrogenase, and 0.4mg/mL formate dehydrogenase at 20-45 deg.C (specifically, 20 deg.C, 30 deg.C, 37 deg.C, and 45 deg.C), and Tris-HCl buffer solution (pH 7.5, 8.0, 8.5, and 9.0) and Na (pH 9.5-10.0) at pH 7.5-9.0₂CO₃-NaHCO₃Buffer (pH 9.5, 10.0).

(2) The L-amino acid oxidase catalyzes L-phenylalanine to be oxidized to generate phenylpyruvic acid (PPA), and the PPA is detected by adopting an iron ion color development method. Preparation of 10mL of iron ion color developing agent: FeCl₃Dissolved in 6mL of dimethyl sulfoxide (DMSO), diluted with 3.8mL of deionized water, and acidified with 200. mu.L of glacial acetic acid₃The final concentration was 100 mM. Adding a proper amount of sample into the iron ion color developing solution, uniformly mixing, standing at room temperature, and detecting the light absorption value of the sample at the wavelength of 640nm by using an ultraviolet spectrophotometer after the color is stable. Calibration curve for phenylpyruvic acid concentration: y is 0.1619x +0.0003361 and x is phenylpyruvic acid concentration, mM.

Detecting N-methyl-L-phenylalanine (N-Me-L-Phe) by using High Performance Liquid Chromatography (HPLC), wherein the detection conditions are as follows: the chromatographic column is xylonite

CR (+); the mobile phase is perchloric acid aqueous solution with the pH value of 1.1-1.25; the detection wavelength is 210 nm; the column temperature is 30 ℃; the flow rate is 0.5 mL/min; the amount of the sample was 20. mu.L. The liquid phase detection method is utilized to measure the concentration of N-methyl-L-phenylalanine generated by catalyzing L-phenylalanine through multienzyme cascade.

As shown in FIG. 3, the catalytic rate was high at 37 to 45 ℃ and 37 was preferable in view of the influence of temperature on the enzyme stabilityThe temperature is the temperature of the cascade reaction; the yield is highest when the pH is 9.5-10.0, and Na with the pH of 9.5 is preferred₂CO₃-NaHCO₃The buffer solution was used as a reaction solution.

Example 4: effect of initial molar ratio of sodium formate and methylamine to substrate on catalytic Synthesis of N-methyl-L-phenylalanine

Ammonium formate is a substrate for coenzyme cycling and methylamine is an amine donor for reductive amination reactions, both concentrations having an effect on the reaction rate. The influence of the initial molar ratio of the reaction components to the substrate on the synthesis of N-methyl-L-phenylalanine by catalyzing L-phenylalanine with a multiple enzyme cascade was investigated with the reaction system of example 3. The molar concentration ratio of methylamine or sodium formate to substrate (L-Phe) is selected to be 1:1, 2:1, 3:1, 4:1, 5:1, i.e. the concentration of L-Phe in the system is 50mM, and 50mM, 100mM, 150mM, 200mM, 250mM of sodium formate or methylamine are added, respectively. Then, phenylpyruvic acid (PPA) is detected by using an iron ion chromogenic method, and N-methyl-L-phenylalanine (N-Me-L-Phe) is detected by using a high performance liquid chromatography, and the specific steps are the same as in example 3.

As shown in fig. 4, when the molar concentration ratio of sodium formate to the substrate is 3:1, the yield of the product is significantly increased, and therefore, the molar concentration ratio of sodium formate to the substrate is preferably 3: 1; the yield of N-methyl-L-phenylalanine increases with increasing methylamine concentration, and a methylamine to substrate molar concentration ratio of 2:1 is chosen for economy.

Example 5: tri-enzyme coupling catalysis of L-phenylalanine to generate N-methyl-L-phenylalanine

The final concentration of the amino acid is 0.1mg/mL L-amino acid deaminase, 1mg/mL N-methyl amino acid dehydrogenase, 0.4mg/mL formate dehydrogenase, 100mM L-phenylalanine, 200mM methylamine, 300mM sodium formate, 10mM NADP⁺And Na at pH 9.5 of 50U/mL Catalase₂CO₃-NaHCO₃Carrying out multi-enzyme cascade catalytic reaction in buffer solution, and reacting at 37 ℃ and 160rpm for 12 h.

Example 6: tri-enzyme coupling catalysis of L-glutamic acid to generate N-methyl-L-glutamic acid

The final concentration of the amino acid solution was 0.1mg/mL L-amino acid deaminase, 1mg/mL N-methyl amino acid dehydrogenase, 0.4mg/mL formate dehydrogenase, and 10mM L-glutamic acidAlanine, 20mM methylamine, 30mM sodium formate, 1mM NADP⁺And Na at pH 9.5 of 50U/mL Catalase₂CO₃-NaHCO₃Carrying out multi-enzyme cascade catalytic reaction in buffer solution, and reacting at 37 ℃ and 160rpm for 12 h.

Example 7: tri-enzyme coupling catalysis of L-leucine to generate N-methyl-L-leucine

The final concentration of the amino acid is 0.1mg/mL L-amino acid deaminase, 1mg/mL N-methyl amino acid dehydrogenase, 0.4mg/mL formate dehydrogenase, 10mM L-leucine, 20mM methylamine, 30mM sodium formate, 1mM NADP⁺And Na at pH 9.5 of 50U/mL Catalase₂CO₃-NaHCO₃Carrying out multi-enzyme cascade catalytic reaction in buffer solution, and reacting at 37 ℃ and 160rpm for 12 h.

Example 8: tri-enzyme coupling catalysis of L-lysine to generate L-high proline

The final concentration of the amino acid is 0.1mg/mL L-amino acid deaminase, 1mg/mL N-methyl amino acid dehydrogenase, 0.4mg/mL formate dehydrogenase, 10mM L-lysine, 20mM methylamine, 30mM sodium formate, 1mM NADP⁺And Na at pH 9.5 of 50U/mL Catalase₂CO₃-NaHCO₃Carrying out multi-enzyme cascade catalytic reaction in buffer solution, and reacting at 37 ℃ and 160rpm for 12 h.

Example 9: tri-enzyme coupling catalysis of L-valine to generate N-methyl-L-valine

The final concentration of the amino acid is 0.1mg/mL L-amino acid deaminase, 1mg/mL N-methyl amino acid dehydrogenase, 0.4mg/mL formate dehydrogenase, 10mM L-valine, 20mM methylamine, 30mM sodium formate, 1mM NADP⁺And Na at pH 9.5 of 50U/mL Catalase₂CO₃-NaHCO₃Carrying out multi-enzyme cascade catalytic reaction in buffer solution, and reacting at 37 ℃ and 160rpm for 12 h.

Example 10: tri-enzyme coupling catalysis of L-isoleucine to generate N-methyl-L-isoleucine

The final concentration of 10. mu.g/mL L-amino acid deaminase, 1mg/mL N-methyl amino acid dehydrogenase, 0.4mg/mL formate dehydrogenase, 10mM L-isoleucine, 20mM methylamine, 30mM sodium formate, 1mM NADP⁺And Na at pH 9.5 of 50U/mL Catalase₂CO₃-NaHCO₃Buffer solutionThe multi-enzyme cascade catalytic reaction is carried out in the process, and the reaction is carried out for 12 hours at 37 ℃ and 160 rpm.

Example 11: method for detecting product

Detecting the L-amino acid and the N-methyl-L-amino acid in the examples 5 to 10 by using a High Performance Liquid Chromatography (HPLC), wherein the detection conditions are as follows: the chromatographic column is xylonite

CR (+); the mobile phase is perchloric acid aqueous solution with the pH value of 1.1-1.25; the detection wavelength is 210 nm; the column temperature is 30 ℃; the flow rate is 0.5 mL/min; the amount of the sample was 20. mu.L. By using the liquid phase detection method, the conversion rate of the multi-enzyme cascade catalysis L-amino acid to generate the corresponding N-methyl-L-amino acid is measured, and the detection results are shown in figure 5 and table 1.

TABLE 1 results of conversion rates of multiple L-amino acids to the corresponding N-methyl-L-amino acids by multiple enzyme cascades

Note:

"*": substrate conversion ═ C₀-C_Substrate)/C₀×100％；

In the formula, C₀Represents the molar concentration, mM, of the starting substrate of the reaction; c_SubstrateRepresents the molar concentration of the substrate, mM, after t time of reaction;

"**": conversion of product ═ C_{Product of}/C_{Theory of the invention}×100％；

In the formula, C_{Product of}Represents the molar concentration, mM, of the target product after time t of the reaction; c_{Theory of the invention}Represents the theoretical molar concentration of the target product, mM.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

<110> south China university of science and technology

<120> method for catalyzing L-amino acid to generate N-methyl-L-amino acid by multi-enzyme cascade and application thereof

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<170> SIPOSequenceListing 1.0

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Met Gly Thr His Tyr Thr Phe Gly Lys Glu Ile Thr Asp Lys Pro Leu

1 5 10 15

Pro Thr Gln Val Lys Val Ala Ile Val Gly Ala Gly Met Ser Gly Leu

20 25 30

Tyr Ser Ala Trp Arg Leu Gln Gln Glu Ala Asn Cys Gln Asp Leu Ala

35 40 45

Ile Phe Glu Arg Ser Asn Arg Thr Gly Gly Arg Leu Asp Ser Asp Leu

50 55 60

Ile Glu Phe Lys Asn Leu Arg Ser Glu Thr Pro Lys Thr Ile Thr Val

65 70 75 80

Lys Glu Glu Gln Gly Gly Met Arg Phe Leu Phe Asp Gly Met Asp Asp

85 90 95

Leu Met Ala Leu Phe Leu Lys Leu Asn Leu Gln Asp Asp Ile Val Pro

100 105 110

Phe Pro Met Asn Ser Gly Gly Asn Asn Arg Leu Phe Phe Arg Gly Glu

115 120 125

Ser Phe Ser Val Glu Asp Ala Gln Gln Asp Asp Tyr Ala Ile Trp Ser

130 135 140

His Leu Tyr Asn Leu Asp Gln Ser Glu Gln Gly Val Asn Pro Lys Asp

145 150 155 160

Ile Val Asn Val Val Phe Asn Arg Ile Leu Glu Ala Asn Pro Gln Phe

165 170 175

Gln Gln Arg Pro Glu Val Arg Gly Pro Glu Phe Trp Gln Ala Phe Arg

180 185 190

Leu Glu Cys Gln Trp Gln Gly Gln Thr Leu Asn Glu Trp Thr Leu Trp

195 200 205

Asp Leu Tyr Thr Asp Met Gly Tyr Ser Gln Glu Cys Ile Asn Met Leu

210 215 220

Tyr Arg Val Leu Gly Phe Asn Gly Thr Phe Leu Ser Gln Met Asn Ala

225 230 235 240

Gly Val Ala Tyr Gln Leu Leu Glu Asp Phe Pro Ala Gly Val Gln Phe

245 250 255

Lys Thr Phe Lys Asp Gly Phe Ser Thr Leu Pro Asn Lys Leu Val Glu

260 265 270

Glu Val Gly Thr Asp Asn Ile His Leu Gln Thr Ser Ile Glu Glu Ile

275 280 285

Asp Phe Ala Glu Glu Ser Gly Leu Tyr Ser Leu His Tyr Ser His Thr

290 295 300

Asp Glu His Gly Arg Val His Lys Gly Gln Val Lys Ala Glu Lys Val

305 310 315 320

Ile Leu Gly Leu Pro Arg Leu Ala Leu Glu Lys Leu Phe Val Arg Ser

325 330 335

Asn Ala Phe Asn Arg Leu Asp Lys Asp Arg Ser Glu Gln Leu Trp Asn

340 345 350

Thr Leu Gln Ser Ala Ser Asn Gln Pro Leu Leu Lys Ile Asn Leu Tyr

355 360 365

Tyr Asp Ser Ala Trp Trp Gly Arg Gly Thr Thr Gly Arg Pro Ala Val

370 375 380

Glu Phe Gly Pro Asn Phe Ala Asp Leu Pro Thr Gly Ser Val Tyr Pro

385 390 395 400

Phe Tyr Ala Val Asn Asp Glu Leu Ala Ala Ala Leu Met Tyr Gln Glu

405 410 415

Arg Ser Thr Asn Pro Ser Lys Ala Val Gln Ala Lys Leu Asp Arg Ile

420 425 430

Gly Asn Glu Lys Tyr Glu Arg Pro Ala Ala Leu Thr Ile Tyr Cys Asp

435 440 445

Tyr Leu Asn Ile Asn Phe Trp Ser Asn Leu Gln Asn Ile Gly Glu Thr

450 455 460

Tyr His His Pro His Gln Asp Asp Tyr Val Glu Asp Val Pro Ala Asp

465 470 475 480

Ile Tyr Pro Ala Ser Thr Ala Val Val Glu Gln Ala Thr Arg Phe Phe

485 490 495

Lys Asp Ile Phe Asn Thr His Tyr Val Pro Glu Pro Ile Leu Thr Ser

500 505 510

Ala Arg Ile Trp Glu Gly Ser Val Arg Phe Asp Ile Pro Ala Ser Arg

515 520 525

Gln Phe Gly Phe Gly Val His Gln Trp Ala Val Gly Ala Asn Asp Lys

530 535 540

Glu Val Met Ala Thr Leu Ala Glu Pro Leu Pro Asn Leu Phe Thr Cys

545 550 555 560

Gly Glu Ala Phe Ser Asp Tyr Gln Gly Trp Val Glu Gly Ala Leu Arg

565 570 575

Ser Thr Asp Leu Ala Leu Glu Lys Gly Phe Gly Leu Lys Pro Leu Ser

580 585 590

Gln Val Tyr Phe Glu Asn Thr Asn Ile Ser Ser Ser Asp Ala Ile Lys

595 600 605

Ala Val Tyr Glu Glu Asn Ser Ser Lys Leu Ile Asn Gln Tyr Ile Glu

610 615 620

Thr Asn Phe Ser Ala Asn Thr Ala Pro Ile Glu Lys Thr Ala Asp Val

625 630 635 640

Asp Ser Val Ile Gly Val Asn Leu Ser Tyr Phe Asp Thr Lys

645 650

Claims

1. A method for catalyzing L-amino acid to generate N-methyl-L-amino acid by multi-enzyme cascade, which is characterized in that: the L-amino acid is taken as a substrate, and L-amino acid oxidase, N-methyl amino acid dehydrogenase and NADPH regenerating enzyme are synchronously cascaded to catalyze the reaction of the L-amino acid to generate the N-methyl-L-amino acid.

2. The method of claim 1, wherein:

the L-amino acid oxidase is ancestor L-amino acid oxidase, and the amino acid sequence of the L-amino acid oxidase is shown in SEQ ID NO 1;

the N-methyl amino acid dehydrogenase is derived from pseudomonas putida, and the amino acid sequence of the N-methyl amino acid dehydrogenase is represented by the accession number AB190215.1 at NCBI;

the NADPH regenerating enzyme is formate dehydrogenase derived from Burkholderia, and the amino acid sequence of the NADPH regenerating enzyme is ACF35003.1 at the NCBI login number.

3. The method of claim 1, wherein:

the L-amino acid is at least one of L-phenylalanine, L-glutamic acid, L-leucine, L-lysine, L-valine and L-isoleucine;

the N-methyl-L-amino acid is at least one of N-methyl-L-phenylalanine, N-methyl-L-glutamic acid, N-methyl-L-leucine, high proline, N-methyl-L-valine and N-methyl-L-isoleucine;

the reaction system is a Tris-HCl buffer solution system with the pH value of 7.5-9.0 or Na with the pH value of 9.5-10.0₂CO₃-NaHCO₃A buffer system.

4. The method of claim 3, wherein:

the L-amino acid is at least one of L-phenylalanine, L-glutamic acid and L-leucine;

the N-methyl-L-amino acid is at least one of N-methyl-L-phenylalanine, N-methyl-L-glutamic acid and N-methyl-L-leucine;

the reaction system is Na with pH of 9.5-10.0₂CO₃-NaHCO₃A buffer system.

5. The method of claim 1, wherein:

the reaction system is as follows: 0.01-1 mg/mL L-amino acid deaminase, 1-5 mg/mL N-methyl amino acid dehydrogenase, 0.2-1 mg/mL NADPH-regenerating enzyme, 10-100 mM L-amino acid, 10-200 mM amine donor, 10-300 mM formate, 1-10 mM NADP⁺20-200U/mL catalase, pH 7.5-10;

the formate is sodium formate;

the molar ratio of the formate to the L-amino acid is 1-5: 1;

the amine donor is methylamine;

the molar ratio of the amine donor to the L-amino acid is 1-5: 1;

the NADP⁺The molar ratio of the L-amino acid to the L-amino acid is 1: 1-10;

the NADP⁺Is oxidized coenzyme II.

6. The method of claim 5, wherein:

the reaction system is as follows: 0.01mg/mL L-amino acid deaminase, 1mg/mL N-methyl amino acid dehydrogenase, 0.4mg/mL NADPH-regenerating enzyme, 10-100 mM L-amino acid, 20-200 mM amine donor, 30-300 mM formate, 5-10 mM NADP⁺Catalase 50U/mL, pH 9.5;

the molar ratio of the formate to the L-amino acid is 3: 1;

the molar ratio of the amine donor to the L-amino acid is 2: 1;

the NADP⁺The molar ratio of the L-amino acid to the L-amino acid is 1: 5-10.

7. The method of claim 1, wherein:

the rotating speed of the reaction is 100-200 rpm;

the reaction temperature is 20-50 ℃;

the reaction time is 6-24 h.

8. Use of the method of claims 1 to 7 for the preparation of N-methyl-L-amino acids by the multi-enzyme cascade catalysis of L-amino acids to N-methyl-L-amino acids.

9. Use according to claim 8, characterized in that:

the N-methyl-L-amino acid is at least one of N-methyl-L-phenylalanine, N-methyl-L-glutamic acid, N-methyl-L-leucine, L-high proline, N-methyl-L-valine and N-methyl-L-isoleucine.

10. Use according to claim 9, characterized in that:

the N-methyl-L-amino acid is at least one of N-methyl-L-phenylalanine, N-methyl-L-glutamic acid and N-methyl-L-leucine.