阅读说明：本技术 一种体外催化ω-氨基酸生产二元羧酸的方法 (Method for producing dicarboxylic acid by in vitro catalysis of omega-amino acid ) 是由 高超 严金鑫 马翠卿 许平 于 2019-09-23 设计创作，主要内容包括：本发明公开了一种体外催化ω-氨基酸生产二元羧酸的方法,是以ω-氨基酸为底物,加入ω-氨基酸氧化酶Am-AOX,黄嘌呤氧化酶和过氧化氢酶,构建多酶反应体系,通过体外多酶高效催化ω-氨基酸生产一系列二元羧酸,如琥珀酸、戊二酸和己二酸。本发明构建的体外催化体系中只底物和3种酶,不需外源添加任何辅因子和辅助底物,并且所用酶的数量也少于天然合成途径,生产成本低、操作简便、污染低,产物得率高、分离方便,具有良好的应用价值。(the invention discloses a method for producing dicarboxylic acid by catalyzing omega-amino acid in vitro, which takes the omega-amino acid as a substrate, adds omega-amino acid oxidase Am-AOX, xanthine oxidase and catalase to construct a multi-enzyme reaction system, and efficiently catalyzes the omega-amino acid to produce a series of dicarboxylic acid such as succinic acid, glutaric acid and adipic acid by the multi-enzyme in vitro. The in vitro catalytic system constructed by the invention only contains the substrate and 3 enzymes, does not need to add any cofactor and auxiliary substrate from external sources, has the advantages of less enzyme quantity than that of a natural synthetic approach, low production cost, simple and convenient operation, low pollution, high product yield, convenient separation and good application value.)

1. A method for producing dicarboxylic acid by catalyzing omega-amino acid in vitro comprises the following steps:

(1) Using omega-amino acid as substrate, adding omega-amino acid oxidase Am-AOX (E.C.1.4.3.6), xanthine oxidase XOD (E.C.1.1.3.22) and catalase CAT (E.C.1.11.1.6) to establish multi-enzyme reaction system, and carrying out pure enzyme catalytic reaction to obtain conversion solution containing dicarboxylic acid;

(2) Heating the obtained conversion solution to denature and precipitate protein, centrifuging to remove protein, sucking supernatant and diluting to a multiple suitable for testing, respectively measuring the concentrations of a substrate and a product in a reaction system by using a high performance liquid chromatograph, and then separating and purifying the product to obtain dicarboxylic acid;

the method is characterized in that:

The substrate omega-amino acid in the step (1) is selected from 4-aminobutyric acid, 5-aminopentanoic acid or 6-aminocaproic acid; the omega-amino acid oxidase Am-AOX (E.C.1.4.3.6) is derived from Kluyveromyces marxianus DMKU3-1042 with the number of AP012218.1, the optimized nucleotide length of the omega-amino acid oxidase gene is 2034 basic groups, and the nucleotide sequence is shown in SEQ ID NO. 1; the multi-enzyme reaction system also contains buffer solution and divalent copper ions besides the substrate and the enzyme; the enzyme catalysis reaction condition is that under the conditions of 37 +/-1 ℃ and pH 7.4 +/-0.2, the shaking reaction is carried out for 3-30 hours in a water bath shaking table at 150 +/-10 revolutions per minute;

the heating condition of the step (2) is boiling at 102 +/-1 ℃ for 15 +/-2 minutes, and the protein-removing centrifugation condition is as follows: 14,680 +/-500 rpm/min, and the dicarboxylic acid is succinic acid, glutaric acid or adipic acid.

2. The method for in vitro catalyzing the production of dicarboxylic acids from omega-amino acids according to claim 1, wherein: the concentration of a substrate in the multi-enzyme reaction system is 10 +/-2 g/L, the dosage of the omega-amino acid oxidase is 0.7-3U/mL, the dosage of the xanthine oxidase is 0.7 +/-0.1U/mL, and the dosage of the catalase is 120 +/-10U/mL; the concentration of the buffer solution is 50mM, and the concentration of the divalent copper ions is 0.04 mM.

3. The method for the in vitro catalysis of omega-amino acids for the production of dicarboxylic acids according to claim 1 or 2, wherein: the buffer solution is PBS buffer solution with pH value of 7.4; the divalent copper ions are selected from CuSO₄Or CuCl₂。

4. the method for in vitro catalyzing the production of dicarboxylic acids from omega-amino acids according to claim 1, wherein: in the step (1), when the substrate is 4-aminobutyric acid and the concentration of the substrate is 10 g/L; the dosage of the omega-amino acid oxidase is 3U/mL, the dosage of the xanthine oxidase is 0.7U/mL, and the dosage of the catalase is 120U/mL; the condition of the enzyme catalytic reaction is that under the conditions of 37 ℃ and pH 7.4, the shaking table with water bath rotates at 150 r/min for oscillation reaction for 30 hours to obtain the conversion solution containing succinic acid.

5. The method for in vitro catalyzing the production of dicarboxylic acids from omega-amino acids according to claim 1, wherein: when the substrate is 5-aminopentanoic acid and the concentration of the substrate is 10g/L in the step (1); the dosage of the omega-amino acid oxidase is 1U/mL, the dosage of the xanthine oxidase is 0.7U/mL, and the dosage of the catalase is 120U/mL; the conditions of the enzyme catalytic reaction are that under the conditions of 37 ℃ and pH 7.4, the shaking table with water bath is used for oscillating reaction for 30 hours at 150 revolutions per minute to obtain conversion solution containing glutaric acid.

6. The method for in vitro catalyzing the production of dicarboxylic acids from omega-amino acids according to claim 1, wherein: when the substrate is 6-aminocaproic acid and the concentration of the substrate is 10g/L in the step (1); the dosage of the omega-amino acid oxidase is 0.7U/mL, the dosage of the xanthine oxidase is 0.7U/mL, and the dosage of the catalase is 120U/mL; the condition of the enzyme catalytic reaction is that under the conditions of 37 ℃ and pH 7.4, the water bath shaking table oscillates at 150 rpm for 30 hours to obtain conversion solution containing adipic acid.

Technical Field

The invention relates to a method for producing dicarboxylic acid, in particular to a method for producing dicarboxylic acid by catalyzing omega-amino acid with in vitro pure enzyme, belonging to the technical field of enzyme catalysis preparation of dicarboxylic acid.

Background

The dicarboxylic acid is an important component for synthesizing plastics, polyester and nylon, and has huge market and wide application^[1]. Dicarboxylic acids are currently predominantly synthesized from chemical petroleum. Such as general chemical process for the production of glutaric acidThe method is that butyrolactone and potassium cyanide are subjected to ring-opening hydrolysis to generate^[2]. It can also be prepared by the reaction of 1, 3-dibromopropane with sodium or potassium cyanide, or by oxidative cleavage of cyclopentene with ionic liquids^[3]. The adipic acid is prepared by oxidizing the mixture of cyclohexanone and cyclohexanol with nitric acid as catalyst^[4]. However, the chemical method has complex process, high toxicity and serious pollution. Therefore, researchers have been working on developing safe and non-polluting processes for producing dicarboxylic acids, and constructing biotechnological pathways to produce dicarboxylic acids from renewable feedstocks is currently the most attractive and sustainable alternative to chemical production^[5,6]。

Many strains have been engineered to synthesize dicarboxylic acids such as succinic, glutaric and adipic acids^[7-9]. However, since a large number of metabolic pathways competing with the target metabolic pathway exist in microbial cells, the microbes also need to perform a metabolic process to support their own growth, which may result in a decrease in the yield of the target product, thereby hindering the industrial production of dicarboxylic acids using engineered strains. Therefore, it is highly desirable to develop a new method for producing dicarboxylic acids with low pollution and high yield.

Omega-amino acids are widely distributed in nature and can be obtained from renewable resources^[10,11]. The search shows that the method for producing dicarboxylic acid by catalyzing omega-amino acid with pure enzyme in vitro is not reported.

Reference documents:

[1]Haushalter,R.W.,Phelan,R.M.,Hoh,K.M.,et al.(2017)Production of odd-carbon dicarboxylic acids in Escherichia coliusing an engineered biotin-fatty acid biosynthetic pathway.J.Am.Chem.Soc.139:4615-4618.

[2]Paris,G.,Berlinguet,L.,Gaudry,R.,et al.(2003)Glutaric acid and glutarimide.Org.Synth.47.

[3]Vafaeezadeh,M.,and Hashemi,M.M.(2016)A non-cyanide route for glutaric acid synthesis from oxidation of cyclopentene in the ionic liquidmedia.Process Saf.Environ.Prot.100:203-207.

[4]Kruyer,N.S.,and Peralta-Yahya,P.(2017)Metabolic engineering strategies to bio-adipic acid production.Curr.Opin.Biotechnol.45:136-143.

[5]Chae,T.U.,Ahn,J.H.,et al.(2019)Metabolic engineering for the production of dicarboxylic acids and diamines.Metab.Eng.https://doi.org/10.1016/j.ymben.2019.03.005.

[6]Chung,H.,Yang,J.E.,Ha,J.Y.,et al.(2015)Bio-based production of monomers and polymers by metabolically engineered microorganisms.Curr.Opin.Biotechnol.36:73-84.

[7]Zhu,L.W.,and Tang,Y.J.(2017)Current advances of succinate biosynthesis in metabolically engineered Escherichia coli.Biotechnol.Adv.35:1040-1048.

[8]Kim,H.T.,Khang,T.U.,Baritugo,K.A.,et al.(2019)Metabolic engineering of Corynebacterium glutamicum for the production of glutaricacid,a C5 dicarboxylic acid platform chemical.Metab.Eng.51:99-109.

[9]Skoog,E.,Shin,J.H.,Saez-Jimenez,V.,et al.(2018)Biobased adipic acid－The challenge of developing the production host.Biotechnol.Adv.36:2248-2263.

[10]Sattler,J.H.,Fuchs,M.,Mutti,F.G.,et al.(2014)Introducing an in situ capping strategy in systems biocatalysis to access 6-aminohexanoicacid.Angew.Chem.Int.Ed.Engl.53:14153-14157.

[11]Jorge,J.M.P.,Perez-Garcia,F.,and Wendisch,V.F.(2017)Anew metabolic route for the fermentative production of 5-aminovalerate fromglucose and alternative carbon sources.Bioresour.Technol.245:1701-1709.

Disclosure of Invention

aiming at the defects of serious pollution, low yield, high cost and difficult enlargement of industrial application of dicarboxylic acid in the prior art, the invention aims to provide a method for producing dicarboxylic acid by in vitro catalysis of omega-amino acid.

The invention relates to a method for producing dicarboxylic acid by catalyzing omega-amino acid in vitro, which comprises the following steps:

(1) Using omega-amino acid as substrate, adding omega-amino acid oxidase Am-AOX (E.C.1.4.3.6), xanthine oxidase XOD (E.C.1.1.3.22) and catalase CAT (E.C.1.11.1.6) to establish multi-enzyme reaction system, and carrying out pure enzyme catalytic reaction to obtain conversion solution containing dicarboxylic acid;

(2) Heating the obtained conversion solution to denature and precipitate protein, centrifuging to remove protein, sucking supernatant and diluting to a multiple suitable for testing, respectively measuring the concentrations of a substrate and a product in a reaction system by using a high performance liquid chromatograph, and then separating and purifying the product to obtain dicarboxylic acid;

The method is characterized in that:

The substrate omega-amino acid in the step (1) is selected from 4-aminobutyric acid, 5-aminopentanoic acid or 6-aminocaproic acid; the omega-amino acid oxidase Am-AOX (E.C.1.4.3.6) is derived from Kluyveromyces marxianus DMKU3-1042 with the number of AP012218.1, the optimized nucleotide length of the omega-amino acid oxidase gene is 2034 basic groups, and the nucleotide sequence is shown in SEQ ID NO. 1; the multi-enzyme reaction system also contains buffer solution and divalent copper ions besides the substrate and the enzyme; the enzyme catalysis reaction condition is that under the conditions of 37 +/-1 ℃ and pH 7.4 +/-0.2, the shaking reaction is carried out for 3-30 hours in a water bath shaking table at 150 +/-10 revolutions per minute;

the heating condition of the step (2) is boiling at 102 +/-1 ℃ for 15 +/-2 minutes, and the protein-removing centrifugation condition is as follows: 14,680 +/-500 rpm/min, and the dicarboxylic acid is succinic acid, glutaric acid or adipic acid.

In the method for producing dicarboxylic acid by catalyzing omega-amino acid in vitro: the concentration of a substrate in the multi-enzyme reaction system is 10 +/-2 g/L, the dosage of the omega-amino acid oxidase is 0.7-3U/mL, the dosage of the xanthine oxidase is 0.7 +/-0.1U/mL, and the dosage of the catalase is 120 +/-10U/mL; the concentration of the buffer solution is 50mM, and the concentration of the divalent copper ions is 0.04 mM.

Wherein: the buffer solution is preferably PBS buffer solution with the pH value of 7.4; the divalent copper ion is preferably CuSO₄Or CuCl₂。

A further preferred embodiment of the above process for the in vitro catalysis of omega-amino acids for the production of dicarboxylic acids is:

In the step (1), when the substrate is 4-aminobutyric acid and the concentration of the substrate is 10 g/L; the dosage of the omega-amino acid oxidase is 3U/mL, the dosage of the xanthine oxidase is 0.7U/mL, and the dosage of the catalase is 120U/mL; the condition of the enzyme catalytic reaction is that under the conditions of 37 ℃ and pH 7.4, the shaking table with water bath rotates at 150 r/min for oscillation reaction for 30 hours to obtain the conversion solution containing succinic acid.

When the substrate is 5-aminopentanoic acid and the concentration of the substrate is 10g/L in the step (1); the dosage of the omega-amino acid oxidase is 1U/mL, the dosage of the xanthine oxidase is 0.7U/mL, and the dosage of the catalase is 120U/mL; the conditions of the enzyme catalytic reaction are that under the conditions of 37 ℃ and pH 7.4, the shaking table with water bath is used for oscillating reaction for 30 hours at 150 revolutions per minute to obtain conversion solution containing glutaric acid.

When the substrate is 6-aminocaproic acid and the concentration of the substrate is 10g/L in the step (1); the dosage of the omega-amino acid oxidase is 0.7U/mL, the dosage of the xanthine oxidase is 0.7U/mL, and the dosage of the catalase is 120U/mL; the condition of the enzyme catalytic reaction is that under the conditions of 37 ℃ and pH 7.4, the water bath shaking table oscillates at 150 rpm for 30 hours to obtain conversion solution containing adipic acid.

In the above method for producing dicarboxylic acid by in vitro catalysis of omega-amino acid, the method for determining catalytic substrate omega-amino acid is as follows: respectively carrying out PITC pre-column derivatization on 4-aminobutyric acid, 5-aminopentanoic acid or 6-aminocaproic acid, and then detecting by adopting an HPLC method;

(1) PITC pre-column derivatization: boiling a sample to be tested at 102 +/-1 ℃ for 15 minutes, and centrifuging at 14,680 +/-500 revolutions per minute for 10-15 minutes; shaking and mixing 400 mu L of sample supernatant, 200 mu L of PITC-acetonitrile solution (100mM) and 200 mu L of triethylamine-acetonitrile solution (1M), standing for 5 minutes, shaking and mixing again, and standing at room temperature for derivatization for 1 hour; adding 800 mu L of n-hexane, performing vortex oscillation for 1 minute, and performing extraction at 12000 +/-500 revolutions per minute for 2 minutes after centrifugation; the lower layer solution was aspirated using a 1mL syringe, filtered through a 0.22 μm filter and analyzed by HPLC detection, during which aspiration of the upper layer solution was avoided.

(2) HPLC detection method: the chromatographic column adopted is ZORBAX SB-C18(5 μm, 4.6X 150mm), the column temperature is 38 ℃, the flow rate of the mobile phase is 0.6mL/min, the sample injection amount is 10 μ L, the detector is a photodiode array detector, the detection wavelength is set to 254nm, and the detection time is 40 minutes; mobile phase a was a 100mM ammonium acetate-acetonitrile solution (v/v, 97:3) at pH 6.5 and mobile phase B was an acetonitrile solution. Linear gradient elution: the proportion of the mobile phase B is increased from 18% to 30% in 0-20 minutes, and is maintained at 30% in the subsequent 20 minutes.

In the above method for producing dicarboxylic acid by in vitro catalysis of omega-amino acid, the method for determining the dicarboxylic acid as the catalytic product is as follows:

The HPLC is of the type Agilent 1100Hewlett-Packard equipped with a differential refractometer and a Bio-Rad Aminex HPX-87H analytical column (300X 7.8mM) at 55 ℃ with a mobile phase of 10mM H₂SO₄The flow rate is 0.4mL/min, the sample injection volume is 5 muL, and the detection time is 40 minutes.

The invention discloses a method for producing dicarboxylic acid by catalyzing omega-amino acid with in vitro pure enzyme, which takes the omega-amino acid as a substrate, and adds omega-amino acid oxidase Am-AOX, xanthine oxidase and catalase to construct a multi-enzyme reaction system, wherein the multi-enzyme catalysis approach comprises the following steps: oxidation of omega-amino acids to dicarboxylic acid semialdehydes by the omega-amino acid oxidase Am-AOX; dicarboxylic semialdehydes are oxidized by xanthine oxidase to dicarboxylic acids. The two reactions both use oxygen as direct electron acceptor, and produce H in the oxidation process of omega-amino acid oxidase Am-AOX and xanthine oxidase₂O₂Is cleaved by catalase to form H₂O and O₂(see FIG. 1). The results of the reactions for producing dicarboxylic acids from omega-amino acids catalyzed by the in vitro pure enzyme of the present invention are shown in FIGS. 3-5. The experiment shows that: when the catalytic reaction is carried out by taking 4-aminobutyric acid as a substrate, 8.31g/L of 4-aminobutyric acid is consumed in 30 hours, the accumulation of succinic acid is 8.05g/L, and the overall yield of the 4-aminobutyric acid to succinic acid is 0.97 g/g; when the catalytic reaction is carried out by taking 5-aminopentanoic acid as a substrate, 6.85g/L of 5-aminopentanoic acid is consumed in 30 hours of catalytic reaction, 6.33g/L of glutaric acid is accumulated, and the overall yield of 5-aminopentanoic acid to glutaric acid is 0.92g g^-1(ii) a When 6-aminocaproic acid is used as a substrate for catalytic reaction, the catalytic reaction is carried out for 30 hours6.40g/L of 6-aminocaproic acid was consumed, 7.07g/L of adipic acid was accumulated, and the overall yield of 6-aminocaproic acid to adipic acid was 1.10 g/g.

The invention has the outstanding characteristics and beneficial effects that:

(1) The in vitro catalytic route designed by the invention can produce three important dicarboxylic acids: succinic, glutaric and adipic acids, and the route can also be applied to the production of other dicarboxylic acids.

(2) The in vitro catalytic reaction for producing dicarboxylic acid from omega-amino acid does not need to add coenzyme factors and auxiliary substrates, and the quantity of the used enzyme is less than that of the enzyme produced by a natural synthetic route, so that the production cost is low.

(3) the in vitro catalytic reaction for producing the dicarboxylic acid has simple operation process and convenient product separation.

(4) the method for producing dicarboxylic acid has the advantages of high utilization rate of raw materials, high product yield and low pollution.

Drawings

FIG. 1 is a schematic diagram of an artificially designed in vitro enzymatic cascade catalytic reaction for the conversion of omega-amino acids to dicarboxylic acids;

Wherein Am-AOX is omega-amino acid oxidase; XOD is xanthine oxidase; CAT is catalase; n is 2,3,4.

FIG. 2 Am-AOX pure enzyme SDS-PAGE validation

FIG. 3 is a graph showing the in vitro catalytic process of producing succinic acid from 4-aminopentanoic acid.

FIG. 4 is a graph showing the in vitro catalytic process of 5-aminopentanoic acid to glutaric acid.

FIG. 5 is a graph of the in vitro catalysis of 6-aminocaproic acid to adipic acid.

Detailed Description

In order that the above objects, features and advantages of the present invention may be more clearly understood, the present invention will be further described with reference to specific embodiments. It should be noted that the embodiments and features of the embodiments of the present application may be combined with each other without conflict.

In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, however, the present invention may be practiced in other ways than those specifically described herein, and thus the present invention is not limited to the specific embodiments of the present disclosure.

in the following examples, the experimental methods used, which are not specifically described, are all conventional methods. The strains, materials, reagents and the like used were all obtained commercially unless otherwise specified.