A method for enhancing the synthesis of 1,5-pentanediamine by lysine decarboxylase using ionic liquid

Abstract

The present invention relates to a method for strengthening the synthesis of 1,5-pentanediamine by lysine decarboxylase using an ionic liquid, and belongs to the field of biochemical engineering. In the method for strengthening the synthesis of 1,5-pentanediamine by lysine decarboxylase using an ionic liquid, an ionic liquid, a substrate lysine, a coenzyme pyridoxal phosphate, a purified lysine decarboxylase or a genetically engineered bacterial cell suspension containing lysine decarboxylase and a buffer are mixed, and the target product 1,5-pentanediamine can be obtained by a one-step catalytic reaction, and the ionic liquid includes a choline ionic liquid and an imidazole ionic liquid, which can significantly improve the activity of lysine decarboxylase and improve the catalytic efficiency of lysine decarboxylase. The process steps of the method are very simple, and high-yield 1,5-pentanediamine can be obtained quickly and efficiently, which is conducive to the engineering amplification of the synthesis of pentamethylenediamine.

Description

Technical Field

[0001] The invention relates to a method for synthesizing 1,5-pentanediamine by lysine decarboxylase enhanced by ionic liquid, and belongs to the field of biochemical engineering. Background Art

[0002] Nylon, also known as polyamide (nylon), has significant application value in many fields such as fibers and engineering plastics. The market size has reached 7 million tons / year. In recent years, China's nylon production, production capacity and demand have all shown an upward trend. Currently, nylon 66 is the main variety, but foreign companies have completely monopolized the synthesis technology of its core monomer hexamethylenediamine precursor adiponitrile. 14 N2 (molecular weight 102.18, also known as cadaverine) and dibasic acids can be used to synthesize a variety of nylon 5X products, such as nylon 52, 5T, 54, 56, 510, 516, and 518. Nylon 56, among others, has similar properties to nylon 66, offering improved lightweighting, moisture wicking, excellent temperature resistance, high strength and wear resistance, low-temperature dyeability, and inherent flame retardancy. It has broad application prospects in defense and aerospace, and is recognized as a next-generation material to replace nylon 66. Pentamethylenediamine is also used in important chemical products such as polyurethanes, chelating agents, and additives. In agriculture, pentamethylenediamine participates in physiological processes such as plant cell division and growth, improving fruit set and development, and stress response. In medicine, pentamethylenediamine is used in drugs for treating arrhythmias and alleviating hypoglycemia, and is also effective in treating dysentery. Therefore, pentamethylenediamine has significant national defense, economic, and social benefits in the chemical, agricultural, and pharmaceutical sectors.

[0003] Chemical synthesis of pentamethylenediamine suffers from challenges such as demanding catalyst reaction conditions, low selectivity, and poor economic efficiency. For many years, achieving continuous and stable production remained elusive, resulting in high technical barriers to pentamethylenediamine synthesis. Biosynthesis of pentamethylenediamine reduces the use of fossil resources and instead utilizes renewable bioresources, offering low-carbon, environmentally friendly, and sustainable advantages. Currently, two pathways are primarily focused on: fermentation and enzymatic methods, with lysine decarboxylase playing a key role in both. Fermentation-based synthesis of pentamethylenediamine involves a complex metabolic pathway, resulting in unreacted components and microbial metabolic byproducts in the fermentation broth, leading to low glucose conversion and difficulties in subsequent purification. Enzymatic synthesis of pentamethylenediamine offers significant advantages, as it eliminates the need for complex fermentation control and eliminates the accumulation of byproducts. my country currently has high L-lysine production and significant overcapacity, making biosynthesis of pentamethylenediamine economical using L-lysine as a raw material. Kyoto University in Japan and Grenoble University in France have conducted in-depth crystal structure analysis of lysine decarboxylase and found that lysine decarboxylase is a decameric structure composed of 5 dimers. This complex structure leads to two problems in the practical application of biological synthesis of pentamethylenediamine: low lysine decarboxylase activity and instability.

[0004] Currently, patents CN105316270B, CN105368766A, CN104498519A, CN106148373A, EP3118312B1, and US7189543 generally study lysine decarboxylase, which is the only source of Escherichia coli. This enzyme is unstable under alkaline conditions, has low activity and catalytic strength during the high-concentration catalytic conversion of L-lysine hydrochloride, and has poor cell recycling efficiency, which increases operation time and production costs, reduces the yield of pentamethylenediamine, and seriously restricts its industrial application. Summary of the Invention

[0005] In view of the problems existing in the prior art, the object of the present invention is to provide a method for enhancing the synthesis of 1,5-pentanediamine by lysine decarboxylase using ionic liquids.

[0006] To achieve this object, the present invention adopts the following technical solutions:

[0007] The present invention provides a method for synthesizing 1,5-pentanediamine by using lysine decarboxylase enhanced by using ionic liquid. The method comprises: mixing an ionic liquid, a substrate L-lysine hydrochloride, pyridoxal phosphate, purified lysine decarboxylase or a genetically engineered bacterial cell suspension containing lysine decarboxylase, and a buffer solution; and subjecting the obtained mixed solution to a catalytic reaction to generate the 1,5-pentanediamine.

[0008] In the method for enhancing the synthesis of 1,5-pentanediamine by lysine decarboxylase using ionic liquids provided by the present invention, an ionic liquid, a substrate L-lysine hydrochloride, pyridoxal phosphate, a purified lysine decarboxylase or a genetically engineered bacterial cell suspension containing lysine decarboxylase and a buffer solution are mixed, and the target product 1,5-pentanediamine can be obtained by a one-step catalytic reaction; wherein the ionic liquid includes a choline ionic liquid and an imidazole ionic liquid, which can significantly improve the activity of lysine decarboxylase and enhance the catalytic efficiency of lysine decarboxylase. The process steps of the method are very simple, and 1,5-pentanediamine can be obtained quickly and efficiently with a high yield, which is conducive to the engineering amplification of pentamethylenediamine synthesis.

[0009] In the present invention, the choline ionic liquid includes choline glycine, choline alanine, choline valine, choline leucine, choline isoleucine, choline phenylalanine, choline proline, choline lysine, choline arginine, choline histidine, choline tryptophan, choline serine, choline oleic acid, choline dihydrogen phosphate, choline tyrosine, choline cysteine, choline glutamate, choline chloride, any one or a combination of at least two.

[0010] In the present invention, the imidazolium ionic liquid includes 1-carboxymethyl-3-methylimidazolium chloride, 1-dodecyl-3-methylimidazolium chloride, 1-allyl-3-methylimidazolium chloride, 1-ethyl-3-methylimidazolium bromide, 1-butyl-3-methylimidazolium bromide, 1-hexyl-3-methylimidazolium bromide, 1-dodecyl-3-methylimidazolium bromide, 1-ethyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide, 1-butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide, 1-butyl-3-methylimidazolium trifluoromethanesulfonate, 1-ethyl-3-methylimidazolium acetate, 1-butyl-3-methylimidazolium acetate, 1-ethyl-3-methylimidazolium dicyanamide, any one or a combination of at least two.

[0011] Preferably, the ionic liquid is a choline ionic liquid.

[0012] In the present invention, the mass volume percentage concentration of the ionic liquid in the mixed solution is 0.0001-50%, for example, 0.0003%, 0.0005%, 0.0008%, 0.001%, 0.003%, 0.005%, 0.008%, 0.01%, 0.03%, 0.05%, 0.08%, 0.1%, 0.3%, 0.5%, 0.8%, 1%, 3%, 5%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 48%, and specific values ​​between the above points. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific values ​​included in the range, and preferably 0.001-10%.

[0013] Preferably, the ionic liquid is a choline cysteine ​​ionic liquid or a choline glutamate ionic liquid.

[0014] Preferably, the substrate lysine comprises L-lysine hydrochloride.

[0015] Preferably, the concentration of the substrate L-lysine hydrochloride in the mixed solution is 1 to 3 mol / L, for example 1.2 mol / L, 1.4 mol / L, 1.5 mol / L, 1.6 mol / L, 1.8 mol / L, 2 mol / L, 2.2 mol / L, 2.4 mol / L, 2.5 mol / L, 2.6 mol / L, 2.8 mol / L or 2.9 mol / L, as well as specific values ​​between the above points. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively enumerates the specific values ​​included in the range.

[0016] Preferably, the concentration of pyridoxal phosphate in the mixed solution is 0.001-0.5 mM, for example, 0.002 mM, 0.004 mM, 0.006 mM, 0.008 mM, 0.01 mM, 0.012 mM, 0.015 mM, 0.018 mM, 0.02 mM, 0.04 mM, 0.05 mM, 0.07 mM, 0.09 mM, 0.1 mM, 0.15 mM, 0.2 mM, 0.25 mM, 0.3 mM, 0.35 mM, 0.4 mM or 0.45 mM, as well as specific values ​​between the above points. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific points included in the range.

[0017] Preferably, the concentration of pure lysine decarboxylase in the mixed solution is 0 to 3000 μg / mL, for example 0.1 μg / mL, 0.5 μg / mL, 0.8 μg / mL, 1 μg / mL, 3 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 35 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL, 500 μg / mL, 1000 μg / mL, 1500 μg / mL, 2000 μg / mL, 2500 μg / mL, 3000 μg / mL and specific values ​​between the above points. Due to space limitations and for the sake of brevity, the present invention no longer exhaustively lists the specific points included in the range.

[0018] Preferably, the OD of the mixed solution is 600 The range of 0.5 to 50 is as follows, for example, 0.6, 0.8, 1, 1.2, 1.5, 1.8, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 11, 13, 15, 17, 19, 20, 22, 24, 25, 27, 29, 30, 40 or 50, and specific point values ​​between the above point values. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values ​​included in the range.

[0019] In the present invention, the lysine decarboxylase is an enzyme that converts L-lysine hydrochloride into 1,5-pentanediamine.

[0020] Preferably, the lysine decarboxylase includes lysine decarboxylase derived from microorganisms and mutants thereof.

[0021] Preferably, the microorganisms include Escherichia coli, Bacillus subtilis, Hafnia alvei, Bacillus halodurans, Bacillus cereus, Bacterium cadaveris, Burkholderia vietnamensia, Chromobacterium violaceum, Vibrio cholerae, Streptomyces polosus, Selenomonas ruminantium, Salmonella typhimurium, Salmonella bongori, Serratia, Edwardsiella tarda, and Salmonella typhimurium. tarda), Bordetella, Aeromonas or Klebsiella, or a combination of at least two thereof.

[0022] In the present invention, the catalytic method of genetically engineered bacteria containing lysine decarboxylase specifically comprises: mixing substrate L-lysine hydrochloride, pyridoxal phosphate, a cell suspension of genetically engineered bacteria containing lysine decarboxylase and an ionic liquid to obtain OD600 The mixed solution has a concentration of 0.5 to 30 mol / L of substrate L-lysine hydrochloride, a concentration of 0.001 to 0.5 mM of pyridoxal phosphate, and a volume percentage concentration of 0.0001 to 50% of the ionic liquid.

[0023] The mixed solution is subjected to catalytic reaction at 35-65° C. under shaking conditions for 0.5-24 hours, and the reaction is terminated by centrifugation to obtain the 1,5-pentanediamine.

[0024] The method for constructing the genetically engineered strain comprises: transferring an expression vector carrying a lysine decarboxylase LDC gene and a lysine-pentanediamine reverse transporter CadB gene into an Escherichia coli strain to obtain the genetically engineered strain.

[0025] The genetically engineered strain is capable of expressing lysine decarboxylase and the lysine-pentanediamine antiporter CadB, thereby effectively increasing mass transfer during whole-cell catalytic synthesis. The present invention has no particular requirements for the type of expression vector, including various commonly used expression vectors capable of expressing the target gene in E. coli, such as plasmids. Expression vectors can be constructed using various methods commonly used in the art, such as enzymatic digestion of the target gene followed by ligation into the vector.

[0026] Preferably, the Escherichia coli strain is BL21 (DE3) strain.

[0027] Preferably, the preparation method of the expression vector is: inserting the lysine decarboxylase LDC gene and the lysine-pentanediamine reverse transporter CadB gene between the NcoI / SacI and BglII / PacI restriction enzyme sites of the pETDuet plasmid to obtain the plasmid pETDuet-ldc-cadB; then introducing pelB before the cadB sequence, and connecting through the NdeI / BglII restriction enzyme sites to obtain the expression vector pETDuet-ldc-pelB-cadB.

[0028] The preparation method of the genetically engineered bacterial cell suspension is as follows: the genetically engineered strain is placed in an LB culture medium containing ampicillin antibiotics to obtain a seed solution; the seed solution is inoculated into an LB culture medium containing ampicillin antibiotics and fermented to an OD of 0. 600 When the pH value is 0.6 to 1 (e.g., 0.7, 0.8, 0.9 or 1), an inducer is added to perform induction culture, and then the bacterial cells are collected by centrifugation and resuspended to obtain the genetically engineered bacterial cell suspension;

[0029] Preferably, the inoculation amount of the seed solution is 0.5-5%, for example 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.2%, 2.4%, 2.6%, 2.8%, 3%, 3.2%, 3.5%, 3.8%, 4%, 4.2%, 4.5%, 4.7% or 4.9%, as well as specific point values ​​between the above point values. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values ​​included in the range.

[0030] Preferably, the inducing agent comprises isopropyl-β-D-thiogalactopyranoside.

[0031] Preferably, the induction culture time is 5 to 30 hours, for example, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 15 hours, 17 hours, 19 hours, 20 hours, 22 hours, 24 hours, 25 hours, 27 hours or 29 hours, as well as specific point values ​​between the above point values. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values ​​included in the range.

[0032] Preferably, the induction culture temperature is 15-25°C, for example 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C or 24°C, as well as specific point values ​​between the above point values. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values ​​included in the range.

[0033] In the present invention, the solvent of the mixed solution is a buffer solution.

[0034] In the present invention, the buffer comprises any one of sodium acetate buffer, phosphate buffer, Tris-HCl buffer or sodium carbonate buffer, or a combination of at least two thereof.

[0035] Preferably, the pH value of the buffer solution is 5 to 11, for example, 5.5, 6, 6.2, 6.5, 6.8, 7, 7.2, 7.5, 7.8, 8, 8.5, 9, 9.5, 10, 10.5, and specific values ​​between the above points. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific points included in the range, and is further preferably 6 to 8.

[0036] In the present invention, the temperature of the catalytic reaction is 35 to 65°C, for example, 38°C, 40°C, 42°C, 45°C, 47°C, 49°C, 50°C, 51°C, 53°C, 55°C, 57°C, 59°C, 60°C, 62°C or 64°C, and specific values ​​between the above points. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific values ​​included in the range, and preferably 45 to 55°C;

[0037] Preferably, the catalytic reaction time is 0.5 to 24 h, for example, 0.8 h, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 3.5 h, 4 h, 5 h, 6 h, 8 h, 10 h, 12 h, 14 h, 15 h, 17 h, 19 h, 20 h, 21 h or 23 h, as well as specific point values ​​between the above point values. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values ​​included in the range.

[0038] Preferably, the catalytic reaction is carried out under shaking conditions.

[0039] Preferably, the oscillation speed is 100-800 rpm, for example, 150 rpm, 200 rpm, 250 rpm, 300 rpm, 350 rpm, 400 rpm, 450 rpm, 500 rpm, 550 rpm, 600 rpm, 650 rpm, 700 rpm or 750 rpm, as well as specific point values ​​between the above point values. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values ​​included in the range.

[0040] Preferably, after the catalytic reaction is completed, the method further includes a step of terminating the reaction.

[0041] Preferably, the method for whole-cell catalytic termination reaction is centrifugation.

[0042] Preferably, the method for terminating the reaction of the purified enzyme is 70° C. for 10 min.

[0043] Preferably, the catalytic method for purifying lysine decarboxylase specifically comprises: mixing substrate L-lysine hydrochloride, pyridoxal phosphate, purified lysine decarboxylase and ionic liquid; the concentration of substrate L-lysine hydrochloride in the mixed solution is 1-3 mol / L, the concentration of pyridoxal phosphate is 0.001-0.5 mM, and the volume percentage concentration of the ionic liquid is 0.0001-50%.

[0044] The preparation method of the purified lysine decarboxylase is as follows: the seed liquid is transferred into 50 mL of LB medium with 100 mg / L ampicillin antibiotic added at 1% volume, cultured at 37° C., IPTG is added at a final concentration of 0.1 mM for induction when the OD600 is 0.6, cultured at 20° C. for 20 hours, centrifuged at 4000 rpm, the bacteria are collected, and stored at -80° C. The cells are disrupted with an ultrasonic disruptor, centrifuged at 8000 rpm at a power of 40-60%, the cell fragments are precipitated, filtered with a 0.22 μm filter membrane, and protein is purified using a 5 mL Histrap purification column on an AKTA protein purifier. The storage solution is then replaced with a 5 mL HiTrap Desalting column, and the concentration of the purified lysine decarboxylase is determined using a BCA protein quantification method.

[0045] The mixed solution is subjected to catalytic reaction at 35-65° C. under shaking conditions for 0.5-24 hours, and the reaction is terminated by centrifugation to obtain the 1,5-pentanediamine.

[0046] Compared with the prior art, the present invention has the following beneficial effects:

[0047] The ionic liquid provided by the present invention strengthens the method for synthesizing 1,5-pentanediamine by lysine decarboxylase, and the ionic liquid, substrate L-lysine hydrochloride, coenzyme pyridoxal phosphate, purified lysine decarboxylase or genetically engineered bacterial cell suspension containing lysine decarboxylase and buffer are mixed, and the target product can be obtained by a one-step catalytic reaction. The ionic liquid can improve the activity and catalytic efficiency of lysine decarboxylase. Under the condition of not adding ionic liquid, the concentration of pentamethylenediamine obtained after 17.5 μg of purified lysine decarboxylase catalyzes L-lysine hydrochloride (1.5M) for 1h is 729.69mM, and the yield is 48.6%. After adding ionic liquid, such as choline cysteine ​​(concentration is 0.5%), the concentration of pentamethylenediamine obtained after 17.5 μg of purified lysine decarboxylase catalyzes L-lysine hydrochloride (1.5M) for 1h reaches 1250.86mM, and the yield is 83.4%. A certain concentration of choline cysteine ​​significantly improves the catalytic efficiency and yield of the enzyme. After adding 0.05% choline cysteine, the K m The value is 7.602, V max The value is 1541, K cat The value is 2101, K cat / K m The K value of the purified enzyme catalyzed reaction under the same conditions was 276.37. m , K cat 、V max and K cat / K m The values ​​are: 8.41, 1500, 2046 and 243.28 respectively. A certain concentration of choline cysteine ​​improves the catalytic efficiency, maximum reaction rate and substrate affinity of the enzyme. After adding 0.5% choline cysteine, for high substrate concentration (2M) catalysis, the product concentration after 2 hours of reaction is 1602.90mM, and the yield is 80.15%. Under the same conditions, the product concentration of the purified enzyme catalytic reaction is 1514.09mM, and the yield is 75.7%. 0.5% choline cysteine ​​improves the high substrate concentration catalytic ability and yield of the enzyme. Similarly, adding 0.01% (v / v) 1-dodecyl-3-methylimidazolium bromide can significantly improve the whole-cell catalytic yield of lysine decarboxylase. DETAILED DESCRIPTION

[0048] The technical solution of the present invention is further described below by way of specific embodiments. It should be understood by those skilled in the art that the embodiments are merely to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0049] Preparation Example 1

[0050] A genetically engineered strain, the preparation method is as follows:

[0051] (1) Construction of expression vector: The lysine decarboxylase gene (ldc) and the lysine-pentanediamine antiporter CadB (GenBank: WP_000092909.1) gene were respectively inserted between the NcoI / SacI and BglII / PacI restriction enzyme sites of the pETDuet plasmid to construct the plasmid pETDuet-ldc-cadB. Then, pelB was introduced before the cadB sequence and ligated through the NdeI / BglII restriction enzyme sites to obtain the expression vector pETDuet-ldc-pelB-cadB.

[0052] (2) Construction of genetically engineered strain: The expression vector pETDuet-ldc-pelB-cadB obtained in step (1) was transferred into the BL21 (DE3) strain to obtain genetically engineered strain I.

[0053] Preparation Example 2

[0054] The preparation method of purified lysine decarboxylase enzyme is as follows:

[0055] (1) The engineered strain I obtained in Preparation Example 1 was cultured in 5 mL of LB medium with 100 mg / L ampicillin at 37°C overnight to obtain a seed solution. The seed solution was transferred to 50 mL of LB medium with 100 mg / L ampicillin at a volume of 1.5% by volume and cultured at 37°C. When the OD 600 When the cell density was about 0.6-1, IPTG was added at a final concentration of 0.1 mM for induction. After further culturing at 15-25°C for 20 h, the cells were collected by centrifugation at 4000 rpm and stored at -80°C.

[0056] (2) The cells were disrupted using an ultrasonic disruptor at a power of 40-60%, sonicated for 2 seconds, paused for 3 seconds, and centrifuged at 8000 rpm to precipitate cell debris. The cell debris was filtered using a 0.22 μm filter membrane and protein was purified using a 5 ml Histrap purification column on an AKTA protein purifier. The preservation solution was then replaced with a 5 ml HiTrap Desalting column, and the concentration of the purified lysine decarboxylase was determined using the BCA protein quantification method.

[0057] Example 1

[0058] (1) preparing 16 choline ionic liquids, including choline glycine, choline alanine, choline valine, choline leucine, choline isoleucine, choline phenylalanine, choline proline, choline lysine, choline arginine, choline histidine, choline tryptophan, choline serine, choline oleic acid, choline dihydrogen phosphate, choline tyrosine, choline cysteine, choline glutamate, and choline chloride, and preparing a mother solution with a concentration of 10% (using water as the solvent);

[0059] (2) Add 200 μL of 2.5 M L-lysine hydrochloride (solvent is 50 mM, pH 8 phosphate buffer), 10 μL of 5 mM pyridoxal phosphate solution (PLP, solvent is pure water), 3.5 μL of 5 mg / mL lysine decarboxylase solution, 186.5 μL and 184 μL of 50 mM phosphate buffer, and 0 and 2.5 μL of the choline ionic liquid solution obtained in step (1) to a 2 mL centrifuge tube; catalyze the reaction of the mixed solution at 55° C. and 500 rpm in a constant temperature mixer for 1 h, centrifuge at 12000 rpm for 2 min, terminate the reaction, and obtain 1,5-pentanediamine.

[0060] The concentration and yield of 1,5-pentanediamine obtained in this example were tested by reverse phase high performance liquid chromatography.

[0061] Here’s how:

[0062] The reaction solution obtained in this example was diluted according to the injection requirements of high performance liquid chromatography to obtain a diluted reaction solution; 600 μL of boric acid buffer (50 mM, pH 9), 200 μL of methanol, 60 μL of the diluted reaction solution, 130 μL of double distilled water (ddH2O), and 10 μL of 1 M diethyl ethoxymethylene malonate (DEEMM, solvent: methanol) were reacted at room temperature for 10 min, then transferred to a 70°C water bath, allowed to stand for 2 h to terminate the reaction, and detected by high performance liquid chromatography (HPLC).

[0063] The chromatographic conditions are as follows:

[0064] HPLC detector: SPD-20A diode array detector; detection column: C18 column (2.1×100 mm); detection temperature: 35°C; injection volume: 5 μL; wavelength: 284 nm. Mobile phase A: acetonitrile; mobile phase B: sodium acetate buffer (25 mM, pH 4.8); flow rate: 0.5 mL / min; time program (mobile phase B proportion): 0 min 80%; 2 min 75%; 22 min 51.7%; 22.01 min 80%; 27 min 80%.

[0065] Yield of pentamethylenediamine = measured concentration × dilution factor / substrate concentration × 100%; wherein the measured concentration is the concentration obtained by liquid chromatography test.

[0066] Through the above detection and calculation, it can be known that the yield of 1,5-pentanediamine obtained in this example is as follows:

[0067] Serial number Types of ionic liquids Pentamethylenediamine yield Relative yield of pentamethylenediamine 1 No additives 48.65％ 100％ 2 Choline Glycine 55.68％ 114.45％ 3 Choline Alanine 53.76％ 110.50％ 4 Choline Valine 49.02％ 100.76％ 5 Choline Leucine 45.91％ 94.37％ 6 Choline Isoleucine 39.89％ 81.99％ 7 Choline Phenylalanine 34.10％ 70.09％ 8 Choline Proline 35.92％ 73.83％ 9 Choline Lysine 44.70％ 91.88％ 10 Choline Arginine 51.68％ 106.23％ 11 Choline Histidine 37.63％ 77.35％ 12 Choline Tryptophan 37.87％ 77.84％ 13 Choline Serine 46.80％ 96.20％ 14 Choline oleate 47.98％ 98.62％ 15 Choline dihydrogen phosphate 52.29％ 107.48％ 16 Choline Tyrosine 44.08％ 90.61％ 17 Choline Cysteine 83.39％ 171.41％ 18 Choline Glutamate 59.09％ 121.46％ 19 Choline chloride 50.73％ 104.28％

[0068] Example 2

[0069] (1) Prepare choline cysteine ​​and prepare a 10% (w / v) stock solution in H2O;

[0070] (2) In a 2 mL centrifuge tube, 300 μL of 2.5 M L-lysine hydrochloride (solvent: 50 mM, pH 8 phosphate buffer), 10 μL of 5 mM pyridoxal phosphate solution (PLP, solvent: pure water), and 3.5 μL of 5 mg / mL lysine decarboxylase solution were added. The added volumes of the ionic liquid obtained in step (1) were 0 μL, 0.5 μL, 2.5 μL, 25 μL, and 50 μL, respectively. The added volumes of 50 mM pH 8 phosphate buffer were 186.5 μL, 186 μL, 184 μL, 161.5 μL, and 136.5 μL, respectively. The mixed solution was catalyzed in a thermomixer at 55° C. and 500 rpm for 1 h, and then centrifuged at 12,000 rpm for 2 min to terminate the reaction to obtain 1,5-pentanediamine. The method for detecting the concentration of pentamethylenediamine was the same as in Example 1.

[0071] Through detection and calculation, it can be known that the yield of 1,5-pentanediamine obtained in this example is as follows:

[0072]

[0073] Example 3

[0074] The method for synthesizing 1,5-pentanediamine catalyzed by choline glutamate at different concentrations is different from Example 2 only in that choline cysteine ​​in steps (1) and (2) is replaced by choline glutamate.

[0075] Through detection and calculation, it can be known that the yield of 1,5-pentanediamine obtained in this example is as follows:

[0076]

[0077] Example 4

[0078] (1) preparing a choline cysteine ​​ionic liquid with a concentration of 0.2% (w / v);

[0079] (2) The substrate concentration range of the kinetic test was (0-40 mM). 0 μL, 10 μL, 20 μL, 30 μL, 40 μL, 50 μL, 80 μL, 100 μL, 150 μL, 200 μL, 250 μL, 300 μL and 400 μL of 50 mM L-lysine hydrochloride (solvent: 50 mM, pH 7 phosphate buffer), 10 μL of 5 mM pyridoxal phosphate solution (PLP, solvent: pure water), 3.5 μL of 0 1.13 mg / mL lysine decarboxylase solution, 2.5 μL of the choline ionic liquid solution obtained in step (1), 484 μL, 474 μL, 464 μL, 454 μL, 444 μL, 434 μL, 404 μL, 384 μL, 334 μL, 184 μL, and 84 μL of 50 mM phosphate buffer at pH 7. The mixed solution was catalyzed in a thermomixer at 55° C. and 500 rpm for 1 hour, and then centrifuged at 12,000 rpm for 2 minutes to terminate the reaction to obtain 1,5-pentanediamine. The method for detecting the concentration of pentamethylenediamine was the same as in Example 1.

[0080] The K of this reaction was calculated by nonlinear regression equation m , K cat and V max value, Km value is 7.602, V max The value is 1541, K cat The value is 2101, K cat / K m The K value of the purified enzyme catalyzed reaction under the same conditions was 276.37. m , K cat 、V max and K cat / K m The values ​​are: 8.41, 1500, 2046 and 243.28.

[0081] Example 5

[0082] (1) Prepare choline cysteine ​​by preparing a 10% (w / v) stock solution in 50 mM phosphate buffer (pH 8);

[0083] (2) In a 2 mL centrifuge tube, 800 μL of 2.5 M L-lysine hydrochloride (solvent: 50 mM, pH 8 phosphate buffer), 20 μL of 5 mM pyridoxal phosphate solution (PLP, solvent: pure water), and 40 μL of 5 mg / mL lysine decarboxylase solution were added. The added volumes of the ionic liquid obtained in step (1) were 0 μL and 50 μL, respectively, and the added volumes of 50 mM pH 8 phosphate buffer were 140 μL and 90 μL, respectively. The mixed solution was catalyzed in a constant temperature mixer at 55° C. and 250 rpm for 2 h to obtain 1,5-pentanediamine. The method for detecting the concentration of pentamethylenediamine was the same as in Example 1.

[0084] Through detection and calculation, it can be known that the yield of 1,5-pentanediamine obtained in this example is as follows:

[0085]

[0086] Example 6

[0087] The ionic liquid-enhanced whole-cell catalytic synthesis of 1,5-pentanediamine is as follows:

[0088] (1) The genetically engineered strain I provided in Preparation Example 1 was placed in 5 mL of LB medium containing 100 mg / L ampicillin and cultured overnight at 37° C. to obtain a seed solution; the seed solution was transferred into 50 mL of LB medium containing 100 mg / L ampicillin at a volume inoculation rate of 1% and fermented at 37° C. When the OD600 was approximately 0.6, isopropyl-β-D-thiogalactopyranoside (IPTG) was added at a final concentration of 0.1 mM for induction. The culture was induced at 20° C. for 20 h, centrifuged at 4000 rpm, and the cells were collected and resuspended in phosphate buffer (50 mM, pH 8) to obtain a genetically engineered bacterial cell suspension containing lysine decarboxylase;

[0089] (2) In a 2 mL centrifuge tube, 100 μL of 2.5 M L-lysine hydrochloride (solvent: 50 mM, pH 8 phosphate buffer), 10 μL of 5 mM pyridoxal phosphate solution (PLP, solvent: pure water), 25 μL of 10% ionic liquid, and 365 μL of the genetically engineered bacterial cell suspension obtained in step (1) were added and mixed by oscillation. The resulting mixture had an OD600 of approximately 1.5. The mixture was catalyzed in a thermomixer at 50° C. and 500 rpm for 1 hour. The reaction was terminated by centrifugation at 12,000 rpm for 2 minutes to obtain 1,5-pentanediamine. The method for detecting the concentration of pentamethylenediamine was the same as in Example 1.

[0090] Through detection and calculation, it can be known that the relative yield of 1,5-pentanediamine obtained in this example is as follows:

[0091]

[0092] The applicant states that while the above-described embodiments illustrate the present invention's method for whole-cell catalytic synthesis of 1,5-pentanediamine, the present invention is not limited to these embodiments. This does not necessarily mean that the present invention must rely on these embodiments for implementation. Those skilled in the art will understand that any improvements to the present invention, equivalent substitutions for raw materials in the present invention's products, additions of auxiliary ingredients, and selection of specific methods, etc., fall within the scope of protection and disclosure of the present invention.

Claims

1. A method for enhancing the synthesis of 1,5-pentanediamine by lysine decarboxylase using ionic liquids, the method comprising: An imidazole ionic liquid or a choline ionic liquid, a cell suspension of a specific genetically engineered strain I containing lysine decarboxylase or a purified lysine decarboxylase solution from the specific genetically engineered strain I, a substrate lysine, pyridoxal phosphate and a buffer solution are mixed, and the obtained mixed solution is subjected to a catalytic reaction to synthesize 1,5-pentanediamine; The mass volume percentage concentration of the ionic liquid in the mixed solution is 0.01% to 1%; The choline ionic liquid is choline glycine, choline alanine, choline valine, choline arginine, choline dihydrogen phosphate, choline cysteine, choline glutamate or choline chloride; the imidazole ionic liquid is 1-dodecyl-3-methylimidazolium chloride, 1-ethyl-3-methylimidazolium bromide, 1-butyl-3-methylimidazolium bromide, 1-hexyl-3-methylimidazolium bromide, 1-dodecyl-3-methylimidazolium bromide, 1-ethyl-3-methylimidazolium acetate or 1-butyl-3-methylimidazolium acetate; The specific genetic engineering strain I is constructed by transferring an expression vector carrying a lysine decarboxylase LDC gene and a lysine-pentanediamine reverse transporter CadB gene into an Escherichia coli BL21 (DE3) strain; The preparation method of the expression vector is specifically as follows: inserting the lysine decarboxylase LDC gene and the lysine-pentanediamine reverse transport CadB gene between the NcoI / SacI and BglII / PacI restriction enzyme cutting sites of the pETDuet plasmid to obtain the plasmid pETDuet-ldc-cadB; then introducing pelB in front of the cadB sequence, and connecting through the NdeI / BglII restriction enzyme cutting sites to obtain the expression vector pETDuet-ldc-pelB-cadB.

2. The method according to claim 1, characterized in that The method for preparing the cell suspension of the specific genetically engineered strain I containing lysine decarboxylase is as follows: the specific genetically engineered strain I is placed in an LB medium containing ampicillin antibiotics for cultivation to obtain a seed solution; Then the seed solution was inoculated into LB medium containing ampicillin antibiotics and fermented until OD 600 When the pH value is 0.6-1, an inducer is added to carry out induction culture, and then the bacterial cells are collected by centrifugation, and the bacterial cells are resuspended to obtain a cell suspension of the specific genetic engineering strain I containing lysine decarboxylase; The inoculation amount of the seed solution is 0.5-5% volume; The inducing agent is isopropyl-β-D-thiogalactopyranoside; The induction culture time is 5 to 30 h; The induction culture temperature is 15-25°C.

3. The method according to claim 1, characterized in that The preparation method of the purified lysine decarboxylase solution of the specific genetically engineered strain I is as follows: using an ultrasonic disruptor to disrupt a cell suspension containing the specific genetically engineered strain I, centrifuging at 8000 rpm at a power of 40-60%, precipitating cell fragments, filtering with a 0.22 μm filter membrane, using a 5 mL Histrap purification column to purify the protein on an AKTA protein purifier, then replacing the preservation solution with a 5 mL HiTrap Desalting desalting column, and using a BCA protein quantitative method to determine the concentration of the purified lysine decarboxylase.

4. The method according to claim 1, wherein the substrate lysine is L-lysine hydrochloride; The concentration of the substrate lysine in the mixed solution is 1-3 mol / L; The concentration of pyridoxal phosphate in the mixed solution is 0.001-0.5 mM; The OD of the mixed solution 600 0.5~30; The concentration of lysine decarboxylase in the mixed solution is 0-3000 μg / mL; The buffer included 50 mM phosphate buffer.

5. The method according to claim 1, characterized in that The temperature of the catalytic reaction is 35-65°C; The catalytic reaction time is 0.5 to 24 h; The catalytic reaction is carried out under oscillation conditions, and the oscillation speed is 100-800 rpm; After the catalytic reaction is completed, the step and method of terminating the reaction is also included, specifically, centrifuging at 12000 rpm to terminate the catalytic reaction.

6. The method according to any one of claims 1 to 5, characterized in that The method specifically comprises: mixing an imidazole ionic liquid or a choline ionic liquid, a cell suspension of a specific genetically engineered strain I containing lysine decarboxylase or a purified lysine decarboxylase solution from the specific genetically engineered strain I, a substrate lysine, pyridoxal phosphate and a buffer, and performing a catalytic reaction on the obtained mixed solution, wherein the concentration of the substrate lysine in the mixed solution is 1-3 mol / L, the concentration of the pyridoxal phosphate is 0.001-0.5 mM, and the mass volume percentage concentration of the ionic liquid is 0.01-1%, and the mixed solution is subjected to a catalytic reaction at 35-65° C. and under shaking conditions for 0.5-24 h, and the reaction is terminated by centrifugation to obtain 1,5-pentanediamine.

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