Escherichia coli putrescine aminotransferase mutant and application thereof

Abstract

The present invention discloses a mutant of Escherichia coli putrescine aminotransferase and its application, belonging to the field of bioengineering technology. The present invention overcomes the limitation of low catalytic stability of the wild-type enzyme by mutating the 91st phenylalanine to tyrosine in the putrescine aminotransferase PaTA derived from Escherichia coli, and the obtained mutant F91Y has an enzyme activity of 2.56U / mg. The recombinant bacteria expressing the mutant F91Y use 10g / L 1,4-cyclohexanedicarboxaldehyde as a substrate and the conversion rate reaches 50.48%. It has high production intensity and high catalytic stability in the preparation of 1,4-cyclohexanedimethylamine, reduces the amount of bacteria used in the conversion, and greatly saves the production cost of industrialization.

Description

Technical Field

[0001] The invention relates to an Escherichia coli putrescine aminotransferase mutant and application thereof, belonging to the technical field of bioengineering. Background Art

[0002] 1,4-cyclohexanedimethylamine (1,4-BAC) is mainly used in the production of pharmaceutical intermediates, epoxy resin curing agents and polyurethane intermediates, and has high industrial value.

[0003] Currently, it is primarily produced by chemical methods, which are subject to issues such as expensive metal catalysts, harsh reaction conditions, and high environmental pollution. Greener alternative synthesis methods are needed. The biological preparation of 1,4-cyclohexanedimethylamine offers stable and safe product quality, mild process conditions, high efficiency, and environmental friendliness. This can alleviate environmental and resource pressures and promote the development of a low-carbon and circular economy in my country. Therefore, an effective and efficient biological method for the preparation of 1,4-cyclohexanedimethylamine is urgently needed.

[0004] The microbial production of 1,4-cyclohexanedimethylamine involves a key enzyme, putrescine aminotransferase (PaTA), which has high substrate specificity and can catalyze the reductive amination of 1,4-cyclohexanedicarboxaldehyde to form 1,4-cyclohexanedimethylamine. Currently, the enzymatic conversion method has more industrial application value due to its advantages such as high yield, high conversion rate and short conversion cycle. However, the large-scale production of 1,4-cyclohexanedimethylamine by enzymatic conversion is subject to the following limitations: (1) the cost of the substrate 1,4-cyclohexanedicarboxaldehyde is high, which is not economical for industry; (2) high concentrations of 1,4-cyclohexanedicarboxaldehyde are toxic to cells; (3) as the catalytic reaction proceeds, the catalytic stability of PaTA decreases, which greatly limits the development of the industrialization of 1,4-cyclohexanedimethylamine and is a key issue in current research. Therefore, from an industrial perspective, there is an urgent need to solve the problem of reduced catalytic stability of PaTA so that the preparation of 1,4-cyclohexanedimethylamine by enzymatic conversion can be realized on a large scale.

[0005] In recent decades, protein engineering has become an effective strategy to improve the properties of enzymes at the molecular level, such as the most effective method to expand the substrate range, enhance enzyme activity and improve enzyme stability. Therefore, the problem of poor catalytic stability may be solved by designing PaTA through protein engineering. Protein engineering can be mainly divided into four categories: traditional directed evolution (i.e., irrational design), semi-rational design, rational design (based on structure and computer technology) and the combined application of multiple strategies. At present, certain research progress has been made on the use of protein engineering to modify transaminases. However, the effect of improving catalytic stability is still limited and far from meeting the actual industrial needs.

[0006] Furthermore, multienzyme cascade reactions are also an important method in enzymatic biotransformations, offering many advantages. For example, they can avoid the accumulation of reaction intermediates, utilize inexpensive and readily available raw materials as starting substrates, and the synergy between multienzyme reactions can be modulated by regulating the expression ratio of the enzymes. Summary of the Invention

[0007] The present invention provides a PaTA mutant that can be used to prepare 1,4-cyclohexanedimethylamine. This mutant protein catalyzes the conversion of 1,4-cyclohexanedicarboxaldehyde to 1,4-cyclohexanedimethylamine. The strain constructed in the present invention exhibits high production efficiency and catalytic stability in the preparation of 1,4-cyclohexanedimethylamine, reduces the amount of bacteria used in the conversion, and significantly reduces industrial production costs.

[0008] The present invention provides a putrescine aminotransferase PaTA mutant, which uses the putrescine aminotransferase PaTA derived from Escherichia coli shown in SEQ ID NO.1 as the parent sequence, and mutates the phenylalanine at position 91 to tyrosine.

[0009] In one embodiment, the amino acid sequence of the mutant is shown as SEQ ID NO.2.

[0010] The present invention also provides a gene encoding the mutant, and the nucleotide sequence of the gene is shown in SEQ ID NO.4.

[0011] The present invention also provides an expression vector carrying the gene.

[0012] In one embodiment, the vector includes but is not limited to pET28a(+).

[0013] The present invention provides a method for preparing the PaTA mutant, the method comprising:

[0014] (1) Designing site-directed mutagenesis primers, using a vector carrying the Escherichia coli putrescine aminotransferase PaTA gene as a template for site-directed mutagenesis; constructing a plasmid vector containing the mutant;

[0015] (2) transforming the mutant-containing plasmid constructed in step (1) into a host cell;

[0016] (3) Select the positive clone prepared in step (2) for fermentation culture, and collect the putrescine aminotransferase PaTA in the culture.

[0017] In one embodiment, the host cell is a bacterial or fungal cell.

[0018] In one embodiment, the host cell is Escherichia coli.

[0019] The present invention also provides a cell catalyst comprising microbial cells expressing the mutant.

[0020] In one embodiment, the cell catalyst further comprises recombinant microbial cells co-expressing glutamate dehydrogenase derived from Escherichia coli and formate dehydrogenase derived from Candida species.

[0021] The invention provides a recombinant Escherichia coli for co-expressing glutamate dehydrogenase gene and formate dehydrogenase gene. The recombinant Escherichia coli uses pACYCDuet-1 as an expression vector.

[0022] In one embodiment, the nucleotide sequence of the glutamate dehydrogenase gene is shown as SEQ ID NO.5; the nucleotide sequence of the formate dehydrogenase gene is shown as SEQ ID NO.6.

[0023] The present invention provides a method for preparing 1,4-cyclohexanedimethylamine. In the method, 1,4-cyclohexanedicarboxaldehyde is used as a reaction substrate, BL21-PaTA cells expressing the mutant and pACYCDuet-GLUDH-cbFDH cells expressing glutamate dehydrogenase and formate dehydrogenase are added to a reaction system, and the reaction is carried out at pH 6-8.5 and 16-45° C. for 24 hours.

[0024] The present invention also provides use of the mutant or the cell catalyst in converting 1,4-cyclohexanedicarboxaldehyde to produce 1,4-cyclohexanedimethylamine.

[0025] In one embodiment, the pH of the conversion reaction is 6.5 to 7.5.

[0026] In one embodiment, the temperature of the conversion reaction is 20-37°C.

[0027] In one embodiment, the concentration of the substrate 1,4-cyclohexanedicarboxaldehyde is 10-15 g / L.

[0028] The present invention also claims the use of the mutant in producing products containing 1,4-cyclohexanedimethylamine.

[0029] Beneficial Effects: The mutant of putrescine aminotransferase PaTA constructed in the present invention can be used to catalyze the production of 1,4-cyclohexanedimethylamine, increasing the production capacity per unit catalyst and effectively reducing production costs. The present invention also provides a method for catalyzing the production of 1,4-cyclohexanedimethylamine using the mutant. This conversion reaction uses only water as the catalytic medium, offering advantages such as mild reaction conditions, ease of operation, and high yield. The mutant obtained in the present invention can produce 1,4-cyclohexanedimethylamine at a yield of 5.12 g / L using 1,4-cyclohexanedicarboxaldehyde as a substrate, with a conversion rate of 50.48%. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the reaction pathway diagram of the present invention.

[0031] Figure 2 This is a high performance liquid chromatogram of the product 1,4-cyclohexanedimethylamine OPA involved in the present invention after derivatization.

[0032] Figure 3 The figure is an SDS-PAGE diagram of the induced expression of PaTA enzyme of the present invention; lanes 1 to 3 respectively show the band sizes of target proteins in whole cells, supernatant and precipitate after induced expression at 0.2 mM IPTG concentration at 25°C. DETAILED DESCRIPTION

[0033] Gene Sources: The enzyme PaTA gene used in this invention is derived from Escherichia coli; the pET28a(+) plasmid was purchased from Novagen (Madison, WI, USA); restriction endonucleases, T4 DNA ligase, primeSTAR, and others were purchased from TaKaRa (Dalian, China). O-diacetylbenzene and standards were purchased from SIGMA. BsPanD mutants were all molecularly engineered, and all other reagents were commercially available.

[0034] Prepare LB medium: peptone 10 g / L, yeast powder 5 g / L, sodium chloride 10 g / L, and sterilize at 121°C for 20 min.

[0035] Prepare fermentation medium: tryptone 12 g / L, yeast extract (Angel Yeast Powder 802) 24 g / L, glycerol 4 mL / L, KH2PO4 2.31 g / L and K2HPO4 12.31 g / L.

[0036] Determination of 1,4-cyclohexanedimethylamine by HPLC: o-phthalaldehyde (OPA) pre-column derivatization reversed-phase HPLC was used to determine the yield of the product 1,4-cyclohexanedimethylamine in the conversion solution. Specifically, a Dionex high-performance liquid chromatograph and an Agilent ZORBAX SB-aq column (250×4.6mm, 5μm) were used. Mobile phase A: 0.01M KH2PO4, KOH adjusted to pH 5.3, organic membrane filtration and ultrasonic degassing for 15 minutes; mobile phase B: phase A: acetonitrile: methanol = 1:5:3, adjusted to pH 5.3 with acetic acid. Elution was performed using the gradient elution program shown in Table 1; column temperature was 35°C, flow rate was 1 mL·min -1 The injection volume was 10 μL, the chromatographic retention time was 23 min, and the detection wavelength was 338 nm.

[0037] Table 1 Gradient elution of 1,4-cyclohexanedimethylamine

[0038]

[0039]

[0040] Prepare pH 7.5 HEPES buffer: 0.1 mol / L HEPES buffer. For the specific formula, see "Industrial Microbiology Experimental Technology Manual" (China Light Industry Press, edited by Zhuge Jian).

[0041] Specific enzyme activity assay: PaTA enzyme activity is measured using HPLC. One unit of PaTA activity is defined as the amount of enzyme (U) required to produce 1 μmol of 1,4-cyclohexanedimethylamine product. Enzyme activity is calculated by measuring the 1,4-cyclohexanedimethylamine content.

[0042] Specific enzyme activity is defined as the number of enzyme activity units per milligram of protein (U / mg protein) and is calculated according to the following formula:

[0043]

[0044] Determination of 1,4-cyclohexanedimethylamine content: The reaction product was determined by high performance liquid chromatography (HPLC). The liquid phase detection conditions were similar to those for the HPLC method for determining 1,4-cyclohexanedimethylamine.

[0045] Calculation method of molar yield:

[0046] Molar yield = mass of product / molecular weight of product ÷ mass of substrate / molecular weight of substrate.

[0047] Example 1: Construction and screening of single mutants and construction of pACYCDuet-GLUDH-cbFDH

[0048] 1. Construction of single mutants: Design of PaTA F91Y The primers for the mutation sites are shown in Table 2 , and mutants were constructed by whole-plasmid PCR.

[0049] Table 2 Sequences of primers for single mutants

[0050]

[0051] The PCR amplification system was constructed as follows: 0.5 μL of PrimSTAR enzyme, 10 μL of 5× PrimeSTAR Buffer, 4 μL of dNTP, 1 μL of each primer for each mutation site, and template (PaTA WT,SEQ ID NO.3)4μL, water 32.5μL; reaction conditions: ①94℃3min; ②98℃10s; ③55℃30s; ④72℃3min; ⑤recycle ②~④3 steps 29 times; ⑥72℃5min; ⑦12℃keeping.

[0052] The above reaction system was incubated at 37°C for 3 h to digest the plasmid template (digestion system: DpnI 0.5 μL, PCR product 45 μL, 10×T Buffer 5 μL). The digestion product was introduced into E. coli BL21 competent cells by chemical transformation. The specific steps of chemical transformation are as follows:

[0053] (1) Introduce 10 μl of homologous recombination product into 100 μl of BL21 competent cells;

[0054] (2) Ice bath for 15-30 minutes;

[0055] (3) Heat shock in a 42°C water bath for 90 seconds, then quickly place in ice and let stand for 3-5 minutes;

[0056] (4) Add 800 μl of resistance-free LB medium, mix well, and incubate at 37°C, 200 rpm for 1 h;

[0057] (5) Centrifuge at 5000 rpm for 2 min to collect the bacteria;

[0058] (6) Remove the supernatant and mix the remaining 100-200 μl by pipetting. Apply the mixture to a plate containing 0.05 mg / mL kanamycin resistance and incubate at 37°C for about 12 h.

[0059] (7) Single clones were picked and placed in LB containing 0.05 mg / mL kanamycin resistance. After incubation at 200 rpm and 37°C for 12 h, the clones were sent to the company for sequencing. The ones with correct sequencing were considered positive transformants.

[0060] 2. Construction of pACYCDuet-GLUDH-cbFDH

[0061] (1) Add about 2000-3000 ng of plasmid (pACYCDuet), 10 uL Buffer, 5 uL EcoRI, and 5 uL SalI to a 100 uL system, and make up the rest with water. Incubate at 37°C for 3 h to perform double enzyme digestion on the plasmid.

[0062] (2) The result in (1) was recovered by gel-cleavage to obtain the homologous recombination vector.

[0063] (3) Add 65uL water, 20uL 5×Ps Buffer, 8uL dNTP, 2uL upstream primer, 2uL

[0064] The downstream primers, 2 μL template (PET-28a plasmid with the Glu-DH sequence shown in SEQ ID NO. 5), and 1 μL Primestar enzyme were used to perform the PCR reaction according to the following procedure: ① 94°C for 5 min; ② 98°C for 10 s; ③ 55°C for 30 s; ④ 72°C for 2 min; ⑤ cycle steps ② to ④ 29 times; ⑥ 72°C for 5 min; ⑦ keep warm at 12°C.

[0065] (4) The results in (3) were subjected to gel recovery to obtain homologous recombinant fragments.

[0066] (5) Homologous recombination of the structures of (2) and (4)

[0067] (6) The plasmid obtained after homologous recombination in (5) was introduced into the competent cell, cultured on a shaking platform at 37°C for 50 min, plated on an LB plate, and a single colony was picked for sequencing.

[0068] (7) Referring to (1)-(6), cbFDH (shown in SEQ ID NO. 6) was ligated into pACYCDuet-GLUDH, and then pACYCDuet-GLUDH was transformed into the microbial cells expressing the single mutant constructed in Part 1 of this Example.

[0069] Example 2: Expression and purification method of mutant enzyme

[0070] The positive transformants of the recombinant strain prepared in Example 1 were inoculated into LB medium and cultured at 37°C until OD 600 When the pH value was 0.6 to 1.0, lactose was added at a final concentration of 5 g / L to induce enzyme expression. The induction temperature was 25°C for 12 hours to obtain a fermentation broth. The fermentation broth was centrifuged at 4°C and 6000 rpm for 10 minutes, and the cells were collected as whole-cell catalysts.

[0071] Prepare bacterial cells according to the above method and add 10 mL of Binding Buffer A (20 mM sodium phosphate, 0.5 mM NaCl, 20 mM imidazole, 1% glycerol, adjusted to pH 7.4 with HCl) to thoroughly resuspend the cells. Place the centrifuge tube in an ice bath and place it in an ultrasonic cell disruptor. Ultrasonic disruption is performed using a 4-second on-time and 4-second interval for a total of 10 minutes. The resulting disrupted liquid is subjected to low-temperature high-speed centrifugation at 8000 rpm at 4°C for 30 minutes to obtain a crude enzyme solution. Filter through a 0.22 μm microporous filter membrane and set aside.

[0072] Prepare the nickel ion affinity chromatography column. First, use a constant flow pump at 4°C to pump ultrapure water into the column to rinse the column (about 6 to 12 times the column volume), and then balance the column environment with 10mL of binding solution A. When the effluent at the lower end of the column is consistent with the pH value of the low salt concentration buffer pumped into the column (about 5 times the column volume of buffer is required), add the obtained membrane-permeable crude enzyme solution to the column. First, use binding solution A to rinse the impurities to baseline equilibrium, and then elute with eluent B (20mM sodium phosphate, 0.5mM NaCl, 500mM imidazole). Collect the eluate of the absorption peak, determine the enzyme activity, and obtain the target protein that is electrophoretically pure. It has been determined that 10g of wet bacteria can express 63mg of enzyme protein, with a specific enzyme activity of 2.56U / mg.

[0073] Example 3: Whole-cell conversion and production of 1,4-cyclohexanedimethylamine at different pH

[0074] In a 100 mL conical flask, 30 g / L whole cells (whole cells were prepared according to the method of Example 2), 10 g / L 1,4-cyclohexanedicarboxaldehyde, 100 mM 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), 200 mM ammonium formate, 20 mM glutamic acid, 1 mM NAD + 5 mL reactions were prepared with 0.5 mM pyridoxal phosphate (PLP) in HEPES buffer at pH 6.0, pH 6.5, pH 7.0, pH 7.5, pH 8.0, and pH 8.5. The reactions were incubated at 30°C, 200 rpm, and in a shaker for 24 hours. The yield of 1,4-cyclohexanedimethanol was determined using the above assay method, and the molar yield was calculated.

[0075] The results showed that PaTA's cyclization activity increased with increasing pH from pH 6.0 to pH 7.5, reaching a peak around pH 7.5 with a 1,4-cyclohexanedimethylamine production of 4.32 g / L and a molar yield of 42.40%. The cyclization activity then decreased with further increases in pH. This suggests that a slightly alkaline environment is more favorable for PaTA-catalyzed transamination reactions, and that whole cells exhibit enhanced transamination activity at pH 7.5.

[0076] Table 3 pH optimization results

[0077]

[0078] Example 4: Whole-cell conversion and production of 1,4-cyclohexanedimethylamine at different temperatures

[0079] For a specific embodiment, see Example 3, except that the yield of 1,4-cyclohexanedimethylamine converted by PaTA over 24 hours at different temperatures (16, 20, 25, 30, 37, and 45°C) was measured in a buffer solution at pH 7.5, and the molar yield was calculated. The results showed that PaTA's transamination activity increased with increasing temperature within the 16-30°C range, while its cyclization activity decreased within the 30-45°C range, reaching a peak at 30°C. The yield of 1,4-cyclohexanedimethylamine was 4.60 g / L, for a molar yield of 45.35%.

[0080] Table 4 Reaction temperature optimization results

[0081]

[0082] Example 5: Whole-cell conversion and production of 1,4-cyclohexanedimethylamine at different substrate concentrations

[0083] For a specific embodiment, see Example 3, except that the yield of 1,4-cyclohexanedimethylamine converted by PaTA at different substrate concentrations (5, 10, 15, 20, and 25 g / L) over 24 hours was measured at 30°C in a buffer solution at pH 7.5, and the molar yield was calculated. The results showed that the cyclization activity of PaTA increased with increasing temperature within the 5-10 g / L range, while it decreased within the 10-25 g / L range. At 10 g / L, the 1,4-cyclohexanedimethylamine yield was 4.82 g / L, for a molar yield of 47.52%.

[0084] Table 5 Substrate concentration optimization results

[0085]

[0086] Comparative Example 1:

[0087] The specific embodiment is the same as Examples 1-2, except that the mutation site is replaced at position 327 or 419, respectively, mutating phenylalanine at position 327 to tyrosine, or mutating leucine at position 419 to alanine. Using the wild-type enzyme as a control, the specific enzyme activities of the mutants were determined using the same method as Example 5. The specific enzyme activities of the mutants are shown in Table 6.

[0088] Table 6 Specific enzyme activities of different mutants

[0089] mutant Specific enzyme activity wild type 1.86 U / mg F327Y 1.72 U / mg L419A 1.43 U / mg

[0090] Comparative Example 2:

[0091] The specific implementation method is the same as that of Example 5, except that the wild enzyme PaTA is expressed WTThe recombinant bacteria containing the recombinant plasmid pACYCDuet-GLUDH-cbFDH were used as cell catalysts, and the reaction was carried out under the condition of substrate concentration of 10 g / L. The results showed that the 1,4-cyclohexanedimethylamine production of the wild enzyme was 3.12 g / L, and the molar yield was 30.76%.

[0092] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. Putrescine aminotransferase Pa A TA mutant, characterized in that Putrescine aminotransferase from Escherichia coli shown in SEQ ID NO.1 Pa TA is the parent sequence, and the phenylalanine at position 91 was mutated to tyrosine.

2. A gene encoding the mutant according to claim 1.

3. An expression vector carrying the gene according to claim 2.

4. A recombinant Escherichia coli, characterized in that The mutant according to claim 1 is expressed using Escherichia coli BL21 as a host.

5. A cell catalyst, characterized in that The cell catalyst contains microbial cells expressing the mutant according to claim 1; the microbial cells use Escherichia coli BL21 as a host.

6. The cell catalyst according to claim 5, characterized in that The invention also contains recombinant microbial cells expressing glutamate dehydrogenase derived from Escherichia coli and formate dehydrogenase derived from Candida species.

7. The cell catalyst according to claim 5, characterized in that The invention contains recombinant Escherichia coli expressing glutamate dehydrogenase shown in SEQ ID NO.5 and formate dehydrogenase shown in SEQ ID NO.

6.

8. A method for preparing 1,4-cyclohexanedimethylamine, characterized in that: Using 1,4-cyclohexanedicarboxaldehyde as a reaction substrate, the cell catalyst according to any one of claims 6 to 7 is used to react at pH 6-8.5 and 16-45° C. for 24 hours.

9. Use of the mutant according to claim 1 or the cell catalyst according to any one of claims 6 to 7 in converting 1,4-cyclohexanedicarboxaldehyde to produce 1,4-cyclohexanedimethylamine.

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