Amine dehydrogenase mutant and application thereof

Abstract

The invention relates to the technical field of gene engineering, in particular to an amine dehydrogenase mutant and application thereof. The amino acid sequence of the amine dehydrogenase mutant is as shown in SEQ ID NO. 1. Compared with wild type amine dehydrogenase or other catalytic enzymes, the amine dehydrogenase mutant has excellent catalytic activity and stability. The asymmetric amination reaction of high-concentration acetophenone can be efficiently catalyzed, and the conversion rate gt is realized; the method has the advantages that the purity of the R-alpha-phenylethylamine enantiomer is more than 99%, the excess value (e.e. Value) of the R-alpha-phenylethylamine enantiomer generated by catalysis is more than 99.5%, the optical purity is extremely high, and the harsh requirements of chiral drug intermediates on the optical purity are met. The mutant can realize soluble expression in host cells, and is easy to prepare on a large scale through microbial fermentation.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of genetic engineering, in particular to an amine dehydrogenase mutant and application thereof. BACKGROUND

[0002] Alpha-phenethylamine (CAS#: 98-84-0618-36-0) is an important chemical intermediate raw material. Its derivatives are widely used in the fields of dyes, medicines, perfumes, emulsifiers and the like, and have very good application prospects. The phenethylamine molecule contains a chiral carbon atom, and thus can be divided into R-alpha-phenethylamine (CAS#: 3886-69-9) and S-phenethylamine (CAS#: 2627-86-3) two enantiomers and racemic phenethylamine. Optically active phenethylamine is an important intermediate, and the preparation and resolution thereof have very important scientific significance and great market value. The optical enantiomers R-phenethylamine or S-phenethylamine can be used as a chiral resolution reagent to resolve some racemic organic acids, phenols, esters or alcohols, and can also be used as a chiral auxiliary and a chiral raw material for some asymmetric synthesis. Therefore, the optical enantiomers of phenethylamine are one of the very important chiral intermediates. Only by improving the yield and optical purity thereof, can the phenethylamine be better applied in fine chemical industry and play a more important role in actual life.

[0003] At present, the synthesis of phenethylamine mainly includes chemical synthesis and biological catalysis. The chemical synthesis has the problems of long time, high energy consumption and more by-products. The biological catalysis often uses transaminase and amine transferase, however, in actual production, the concentration of added phenylacetone in the catalytic system is generally low, resulting in low concentration of generated phenethylamine; increasing the concentration of phenylacetone will lead to low conversion rate and incomplete substrate reaction due to the reversibility of the enzyme; secondly, the chiral alpha-phenethylamine generated in most enzyme catalytic reactions is not good; thirdly, the catalyst in biological catalysis is often disposable, which needs single fermentation and single catalysis, thereby increasing the production cost.

[0004] Therefore, in view of the above difficulties, there is an urgent need for a biological catalyst with high catalytic activity and high specificity to catalyze the synthesis of R-alpha-phenethylamine from phenylacetone, so as to meet the needs of industrial production.

[0005] In view of this, the present application is proposed. SUMMARY

[0006] The present application aims to provide an amine dehydrogenase mutant and application thereof. The amine dehydrogenase mutant has excellent catalytic activity and stability.

[0007] In order to achieve the above-mentioned purpose of the present application, the following technical solutions are adopted: In one aspect of the present application, an amine dehydrogenase mutant is provided, which has an amino acid sequence as shown in SEQ ID NO. 1.

[0008] The amine dehydrogenase mutant has excellent catalytic activity and stability compared to wild-type amine dehydrogenase or other catalytic enzymes. It can efficiently catalyze the asymmetric amination reaction of high-concentration acetophenone, achieve a conversion rate of >99%, and completely solve the industry problem of "low substrate concentration and incomplete reaction" caused by substrate inhibition and thermodynamic equilibrium in the background art. The R-alpha-phenethylamine enantiomer produced by the mutant has an enantiomeric excess value (e.e. value) of greater than 99.5%, and has extremely high optical purity, which meets the stringent requirements of chiral drug intermediates for optical purity and overcomes the problem of "poor chirality of alpha-phenethylamine". The mutant can be expressed in a host cell, and is easy to be produced in large quantities by microbial fermentation, thereby solving the problem of high cost of enzyme catalyst preparation.

[0009] SEQ ID NO. 1 is: MTLEIFEYLEKYDYEQVVFCQDKESGLKAIIAIHDTTLGPALGGTRMWTYDSEEAAIEDALRLAKGMTYSNAAAGLNLGGAKTVIIGDPRKDKSEAMFRALGRYIQGLNGRYITAVDVGTTVDDMDIIHEETDFVSGISPSFGSSGNPSPVTAYGVYRGMKAAAKEAFGTDNLEGKVIAVQGVGNVAYHLCKHLHAEGAKLIVTDINKEAVQRAVEEFGASAVEPNEIYGVECDIYAPCALGATVNDETIPQLKAKVIAGSALNQLKEDRHGDIIHEMGIVYAPDYVINAGGAINCADELYGYNRERALKRVESIYDTIAKVIEISKRDGIATYVAADRLAEERIASLKNSRSTYLRNGHDIISRR.

[0010] Further, the nucleotide sequence encoding the amine dehydrogenase mutant is shown in SEQ ID NO. 2.

[0011] SEQ ID NO. 2 is:

[0012] Another aspect of the present application also relates to a recombinant expression vector comprising a nucleotide sequence encoding the amine dehydrogenase mutant.

[0013] The recombinant expression vector comprising the gene encoding the amine dehydrogenase mutant can efficiently express the amine dehydrogenase mutant with the above-mentioned excellent performance in a suitable host cell, and provides a reliable technical tool and genetic basis for efficient and large-scale production of the mutant enzyme.

[0014] Further, the recombinant vector comprises a plasmid.

[0015] Any conventional plasmid in the art can be used to implement the technical solution of the present application. Further, the plasmid includes but is not limited to at least one of pET-21a(+), pET-28a(+), pET-22b(+), pETDuet-1 or pACYCDuet-1.

[0016] Further, the plasmid is pET-28a(+).

[0017] Another aspect of the present application also relates to a recombinant bacterium comprising a nucleotide sequence encoding the amine dehydrogenase mutant or the recombinant expression vector.

[0018] The recombinant bacterium can efficiently express the amine dehydrogenase mutant of the present application. The engineered strain is simple to culture, fast to grow and genetically stable, and is suitable for large-scale fermentation production. Using the recombinant bacterium, a high-activity and high-selectivity biological catalyst can be obtained in the form of wet bacterium, crude enzyme liquid or pure enzyme liquid at low cost and in large quantities, which provides an adequate enzyme source for industrial application and effectively reduces the production cost.

[0019] Further, the recombinant bacterium can be obtained by transforming the nucleotide sequence encoding the amine dehydrogenase mutant or the recombinant expression vector into a host bacterium.

[0020] The present application does not make specific limitations on the host bacterium, and any conventional host bacterium in the art can be used to implement the technical solution of the present application. Further, the host bacterium includes but is not limited to at least one of Escherichia coli, Bacillus subtilis or Saccharomyces cerevisiae.

[0021] Another aspect of the present application also relates to a preparation method of amine dehydrogenase, comprising the following steps: Fermenting the recombinant bacterium.

[0022] The method described above for preparing amine dehydrogenase significantly improves the unit yield and soluble expression ratio of amine dehydrogenase mutants. This method is stable, simple to operate, and highly reproducible, easily scaled up from the laboratory to industrial production, and can economically and efficiently prepare enzyme preparations that meet the needs of industrial applications.

[0023] Furthermore, an inducing agent is added during the fermentation process.

[0024] Furthermore, the OD of the fermentation system... 600 The inducer is added when the concentration is 15-20 (e.g., but not limited to 15, 16, 17, 18, 19, or 20). By precisely controlling the induction timing, the expression conditions of the exogenous protein are optimized. This approach balances the relationship between cell growth and protein expression, achieving high cell density while maximizing the yield and soluble proportion of the target protein, and avoiding metabolic burden or inclusion body formation caused by premature product expression and accumulation.

[0025] Furthermore, the inducer includes, but is not limited to, IPTG.

[0026] Furthermore, the fermentation time is 15-24 hours, including but not limited to 15 hours, 18 hours, 20 hours, 22 hours or 24 hours.

[0027] Furthermore, wet cells containing intracellular expression of amine dehydrogenase mutants are extracted from the fermentation products, or crude enzyme solutions containing amine dehydrogenase mutants are obtained by breaking the cells.

[0028] Further, the crude enzyme solution is separated and purified. The separation and purification methods can be those conventional in the field.

[0029] Another aspect of the present invention relates to amine dehydrogenase prepared by the aforementioned method.

[0030] The amine dehydrogenase described above has high yield, good activity, and is easy to obtain. It can be directly used as a highly efficient catalyst for the synthesis of R-α-phenylethylamine, ensuring the efficiency and economy of subsequent biocatalytic processes.

[0031] Another aspect of the present invention relates to a method for preparing R-α-phenylethylamine, comprising the following steps: A buffer solution, glucose, and coenzyme were added to an acetophenone solution to obtain a substrate reaction solution; glucose dehydrogenase and a catalyst were then added to the substrate reaction solution to carry out a catalytic reaction. The catalyst comprises: the amine dehydrogenase mutant and / or the amine dehydrogenase.

[0032] The preparation method of the R-phenethylamine has high production efficiency, avoids high energy consumption, heavy metal catalysts and a large amount of waste generated in the traditional chemical method, and meets the principle of green chemistry. The whole cell or crude enzyme liquid is used as a catalyst, and a high-efficiency coenzyme regeneration system is used, so that the reaction can be driven by a small amount of coenzyme, thereby greatly reducing the production cost. After the reaction is completed, the product can be efficiently recovered through a simple alkalization and extraction step, has high yield and high product purity, and can meet the subsequent application requirements without a complex purification process.

[0033] Further, the concentration of the acetophenone solution is 10-100 g / L, including but not limited to 10 g / L, 20 g / L, 30 g / L, 35 g / L, 40 g / L, 45 g / L, 50 g / L, 55 g / L, 60 g / L, 65 g / L, 70 g / L, 75 g / L, 80 g / L, 85 g / L, 90 g / L, 95 g / L or 100 g / L. This concentration range marks a major breakthrough of the present application. The substrate concentration is greatly increased from usually less than 20 g / L in traditional biological catalysis to a maximum of 100 g / L, which greatly improves the production capacity of the unit volume reactor, reduces the solvent consumption and energy cost of subsequent separation and purification, and meets the requirement of high production intensity for industrial production. The mutant can maintain high activity at such a high substrate concentration, which significantly overcomes the substrate inhibition effect, which is difficult to achieve by wild type or ordinary enzymes, and embodies the superiority of the mutant of the present application.

[0034] Further, 880-920 mL of buffer, 8-12 g of glucose and coenzyme with a final concentration of 0.8-1.2 mM are added per liter of the acetophenone solution.

[0035] Further, the coenzyme includes NADP.

[0036] Further, the addition amount of glucose dehydrogenase is 0.2%-2% of the mass of the substrate reaction solution, and the addition amount of the catalyst is 0.5%-5% of the mass of the substrate reaction solution.

[0037] Further, the temperature of the catalytic reaction is 25-60℃, including but not limited to any one of 25℃, 30℃, 35℃, 40℃, 45℃, 50℃, 55℃ or 60℃, or a range value between any two of them. The mutated amine dehydrogenase has high catalytic activity at this temperature condition.

[0038] Further, the time of the catalytic reaction is 3-16 h, including but not limited to any one of 3 h, 4 h, 5 h, 6 h, 8 h, 10 h, 12 h, 14 h or 16 h, or a range value between any two of them.

[0039] Further, the catalytic reaction is carried out under stirring at a speed of 100-600 rpm / min, including but not limited to 100 rpm / min, 200 rpm / min, 300 rpm / min, 400 rpm / min, 500 rpm / min or 600 rpm / min.

[0040] Further, the buffer solution comprises NH4CL and / or NH4OH. The addition of the buffer solution not only regulates the pH of the catalytic reaction, but also provides an available ammonia source for the catalytic reaction.

[0041] Further, the pH of the buffer solution is 8-10, including but not limited to 8, 8.5, 9, 9.5 or 10, any one of which is a point value or a range value between any two of them.

[0042] Further, the catalytic reaction further comprises extracting and purifying the reaction solution after the catalytic reaction. The method of extraction and purification can be carried out by using conventional methods in the art.

[0043] Further, the substrate is added to the catalytic reaction system in a one-time feeding manner.

[0044] Further, when the catalyst is a wet bacterial body, the amount of the catalyst is 5-50 g / L of the wet bacterial body.

[0045] Another aspect of the present application also relates to the use of the recombinant expression vector or the recombinant bacteria in at least one of the following aspects: (1) preparing an amine dehydrogenase mutant; (2) improving the yield of the amine dehydrogenase mutant; (3) preparing R-α-phenethylamine.

[0046] Compared with the prior art, the present application has the following advantages: (1) The amine dehydrogenase mutant provided by the present application has far superior catalytic performance to natural enzymes. In actual application systems, it can efficiently catalyze the asymmetric amination reaction of high-concentration substrate phenylacetone, achieve a conversion rate of more than 99%, and the enantiomeric excess value (e.e. value) of the product R-α-phenethylamine is greater than 99.5%. This solves the two core technical problems of "low conversion rate under high substrate concentration" and "poor chiral selectivity" that have long existed in existing biological catalysis technology, and provides reliable guarantee for obtaining chiral amine products with high optical purity.

[0047] (2) The amine dehydrogenase mutant provided by the present application can be expressed in a soluble form in a host cell, avoiding the formation of inclusion bodies, so that it can be prepared on a large scale and at a low cost through conventional microbial fermentation technology. This not only simplifies the downstream enzyme preparation production process, but also greatly reduces the production cost of the catalyst, thereby removing the economic barriers for industrial application.

[0048] (3) The method for preparing R-α-phenylethylamine provided by the present invention is carried out under mild conditions of room temperature and pressure, without the need for precious metal catalysts or harsh reaction conditions. The process uses ammonia water as the amino donor and water as the main byproduct, which has high atom economy and conforms to the principles of green chemistry and sustainable development. Compared with traditional chemical synthesis methods, it generates less waste and is more environmentally friendly.

[0049] (4) This invention successfully integrates the advantages of high substrate concentration, high conversion rate, high chiral selectivity and low catalyst cost. The method is simple to operate, stable and easy to scale up, perfectly meeting the core requirements of industrial production for efficiency, cost and quality, and providing a practical and highly competitive technical path for the bio-based industrial production of R-α-phenylethylamine. Detailed Implementation

[0050] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. However, those skilled in the art will understand that the embodiments described below are some embodiments of the present invention, but not all embodiments, and are only used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0051] The specific formulation of the culture medium involved in the embodiments of the present invention is as follows: Seed culture medium: consisting of 20 g / L glucose, 1.5 g / L yeast extract, 1.2 g / L peptone, 1.5 g / L (NH4)2SO4, 1.2 g / L K2HPO4, 0.8 g / L MgSO4, 1 g / L citric acid, 10 mg / L MnSO4·H2O, and 8 mg / L FeSO4·7H2O.

[0052] Fermentation medium: comprising 1.5 g / L anhydrous disodium hydrogen phosphate, 1.8 g / L potassium dihydrogen phosphate, 0.7 g / L ammonium chloride, 0.4 g / L anhydrous magnesium sulfate, 120 g / L glucose, 4 g / L peptone, 2.4 g / L yeast extract, 0.8 g / L anhydrous sodium sulfate, 30 mg / L manganese chloride tetrahydrate, 20 mg / L ferrous sulfate, 1 g / L citric acid, 2 mg / L vitamin mixture, and biotin. The vitamin mixture includes vitamins B1, B3, B5, and B12.

[0053] Example 1: Construction of recombinant expression plasmid pET-28a(+)-AMDH In this embodiment, the expression plasmid pET-28a(+)-AMDH was constructed using pET-28a(+) and an amino acid dehydrogenase mutant as shown in SEQ ID NO.1. The plasmid was synthesized by Suzhou Genewiz Biotechnology Co., Ltd., and the expression host was BL21(DE3).

[0054] Example 2: Fermentation and preparation of amine dehydrogenase mutants (Preparation of amine dehydrogenase) Using pET-28a(+)-AMDH constructed in Example 1 and engineered E. coli BL21(DE3), fermentation expression was performed according to the following steps: Plate activation: Dip an inoculation loop into the bacterial culture from the glycerol tube and streak it in four zones on an LB solid plate containing 50 μg / mL kanamycin. Incubate overnight in an inverted incubator at 37°C.

[0055] Activation in shaker: After plate activation in the above steps, pick large single clones and inoculate them into LB liquid medium containing 50 μg / mL kanamycin for activation. Then, streak the plates again on LB solid plates containing 50 μg / mL kanamycin. The plate should be used within one week.

[0056] Seed culture: Large single colonies were picked from solid plates and inoculated into seed culture medium containing 50 μg / mL kanamycin at a volume of 30 mL / 250 mL. The culture was carried out at 37°C and 200 rpm for 12 h to obtain the seed culture.

[0057] Fermentation culture: The seed culture was inoculated into a 10L fermenter at a 1% inoculum rate. 6L of fermentation medium (containing 50μg / mL kanamycin) was added. The initial culture temperature was 37℃. The pH was maintained at 7.0±0.2 by automatic addition of ammonia solution. The dissolved oxygen level was maintained at 30%~50% by adjusting the stirring speed or aeration rate. When the OD... 600 When the temperature reaches 18°C, add IPTG inducer to a final concentration of 0.1 mM and lower the culture temperature to 30°C to start fermentation.

[0058] The glucose concentration in the tank was controlled to ≤0.5 g / L by adding 80% glucose solution. 600 Once the temperature reaches 100°C, the cells are collected in a container to obtain the AMDH catalyst. The catalyst system is then resuspended in water at a wet cell mass fraction of 1%.

[0059] It should be noted that, in practice, a crude enzyme solution can also be obtained by homogenization, disruption, and centrifugation. Considering the simplicity of the reaction, whole cells were used in subsequent examples.

[0060] Example 3: Amine dehydrogenase mutant catalyzes the production of R-α-phenylethylamine from acetophenone. In this embodiment, the catalytic system (AMDH whole cell) prepared in Example 2 was used as the catalyst to catalyze the production of R-α-phenylethylamine from acetophenone. The specific operation is as follows: Acetophenone was dissolved in 10% v / v DMSO to prepare acetophenone solutions with concentrations of 500 g / L and 1000 g / L. 900 mL of a 0.1 M buffer solution containing NH₄Cl and NH₄OH (pH 9.5) was added to 100 mL of the acetophenone solution. Then, 2 eq of glucose and 1 mL of 1 M NADP were added, mixed well, and 0.2 wt% GDH and 0.5 wt% AMDH (the catalytic system prepared in Example 2) were added. The catalytic reaction was carried out at 30 °C until the reactants were completely converted, at which point the reaction was stopped. Table 1 compares the effect of different concentrations of acetophenone on the product concentration.

[0061] Table 1. Effect of different raw material concentrations on product concentration

[0062] The results showed that when the concentration of acetophenone solution was 50 g / L, the reaction conversion rate could be >99%.

[0063] Example 4: Extraction of R-α-phenylethylamine after biocatalysis Accurately measure the volume of the fermentation broth in the fermenter, for example, 5L of reaction liquid. Adjust the pH of the reaction liquid to 10 using 5M sodium hydroxide, add 30% volume of methyl ether and mix thoroughly. Let stand for 7 hours, separate the upper organic phase and extract it three times with methyl ether. Combine the three liquids and use a rotary evaporator to evaporate the methyl ether in the organic phase at 40°C under vacuum conditions to obtain 198g of crude R-α-phenylethylamine powder.

[0064] Comparative Example 1 The only difference between this comparative example and Example 3 is that R-α-phenylethylamine was prepared using unmutated wild-type AMDH.

[0065] Comparative Example 2 The only difference between this comparative example and Example 3 is that the temperature of the catalytic reaction is 65°C.

[0066] Experimental Example The reactants from Examples 3, 1, and 2 after the catalytic reaction were completed were purified using the purification method provided in Example 5. The purity and mass concentration of the purified products were statistically analyzed by high performance liquid chromatography, and the results are shown in Table 2.

[0067] Table 2

[0068] As shown in Table 2, the mutant provided by this invention has significant advantages in terms of product purity, mass concentration, yield, and optical purity.

[0069] Although the present invention has been illustrated and described with specific embodiments, it should be understood that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; those skilled in the art should understand that modifications can be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein, without departing from the spirit and scope of the present invention; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention; therefore, this means that all such substitutions and modifications that fall within the scope of the present invention are included in the appended claims.

Claims

1. An amine dehydrogenase mutant, characterized in that, The amino acid sequence of the amine dehydrogenase mutant is shown in SEQ ID NO.

1.

2. A recombinant expression vector, characterized in that, The recombinant expression vector comprises a nucleotide sequence encoding the amine dehydrogenase mutant of claim 1.

3. A recombinant bacterium, characterized in that, Includes the nucleotide sequence encoding the amine dehydrogenase mutant of claim 1 or the recombinant expression vector of claim 2.

4. A method for preparing an amine dehydrogenase, characterized in that, Includes the following steps: The recombinant bacteria described in claim 3 are fermented.

5. The method for preparing amine dehydrogenase according to claim 4, characterized in that, The OD of the fermentation system 600 When the temperature is 15-20, add an inducing agent.

6. The amine dehydrogenase prepared by the method for preparing amine dehydrogenase as described in claim 4 or 5.

7. A method for preparing R-α-phenylethylamine, characterized in that, Includes the following steps: Add buffer solution, glucose and coenzyme to acetophenone solution to obtain substrate reaction solution; The reaction was carried out by adding glucose dehydrogenase and a catalyst to the substrate reaction solution; The catalyst comprises: the amine dehydrogenase mutant of claim 1 and / or the amine dehydrogenase of claim 6.

8. The method for preparing R-α-phenylethylamine according to claim 7, characterized in that, Includes at least one of the following technical features: (1) The concentration of the acetophenone solution is 10~100g / L; (2) The buffer solution includes: NH4Cl and / or NH4OH.

9. The method for preparing R-α-phenylethylamine according to claim 7, characterized in that, Includes at least one of the following technical features: (1) The temperature of the catalytic reaction is 25~60℃; (2) The time for the catalytic reaction is 3 to 16 hours.

10. The use of the recombinant expression vector of claim 2 or the recombinant bacteria of claim 3 in at least one of the following: (1) Preparation of amine dehydrogenase mutants; (2) Increase the yield of amine dehydrogenase mutants; (3) Preparation of R-α-phenylethylamine.

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