WO2022001121A1 - Lysine décarboxylase pour synthétiser la pentanediamine et son utilisation - Google Patents

Lysine décarboxylase pour synthétiser la pentanediamine et son utilisation Download PDF

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WO2022001121A1
WO2022001121A1 PCT/CN2021/076305 CN2021076305W WO2022001121A1 WO 2022001121 A1 WO2022001121 A1 WO 2022001121A1 CN 2021076305 W CN2021076305 W CN 2021076305W WO 2022001121 A1 WO2022001121 A1 WO 2022001121A1
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lysine
lysine decarboxylase
buffer
amino acid
expression vector
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黄玉红
薛雅鞠
张锁江
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中国科学院过程工程研究所
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    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
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    • C12P13/00—Preparation of nitrogen-containing organic compounds
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/001—Amines; Imines
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y401/00—Carbon-carbon lyases (4.1)
    • C12Y401/01—Carboxy-lyases (4.1.1)
    • C12Y401/01018—Lysine decarboxylase (4.1.1.18)

Definitions

  • the application belongs to the technical field of genetic engineering, in particular to a lysine decarboxylase for synthesizing pentamethylene diamine and its application, in particular to a lysine decarboxylase gene sequence, protein sequence and The constructed expression vector and recombinant engineering bacteria.
  • Nylon is widely used in many fields such as fibers and engineering plastics due to its excellent mechanical properties, heat resistance, corrosion resistance and other properties.
  • China's nylon production, production capacity and demand have all shown an increasing trend.
  • the production and consumption of nylon 66 is huge, but because the synthesis technology of adiponitrile, the precursor of its core monomer hexamethylenediamine, has been monopolized by foreign companies, resources are tight, and the cost fluctuates greatly, and it is mainly derived from petroleum.
  • Pentamethylenediamine is the product of lysine decarboxylation, and it is a homologue with hexamethylenediamine.
  • a variety of nylon 5X products can be synthesized from pentamethylenediamine and dibasic acid, such as nylon 52, 5T, 54, 56, 510, 516, 518, etc., have better characteristics such as light weight loss, moisture absorption and perspiration, temperature resistance, abrasion resistance, dyeability and intrinsic flame retardancy, and have broad development prospects.
  • the synthesis of bio-nylon 5X can not only reduce the dependence on petroleum resources, but also break the monopoly of the output and technology of hexamethylenediamine products by multinational enterprises, and has broad application prospects in the fields of national defense and aerospace.
  • lysine decarboxylase The key to bio-based nylon 5X is the efficient synthesis of its core monomer, pentamethylenediamine. Efficient and stable lysine decarboxylase is the core of bio-based pentamethylene diamine synthesis. Lysine decarboxylase has a wide range of sources. Currently, the microorganisms reported to have lysine decarboxylase are mainly Escherichia coli (E.coli), Hafnia alvei, Bacillus halodurans, and Bacillus cereus.
  • Bacillus cereus Bacterium cadaveris, Burkholderia vietnamensia, Chromobacterium violaceum, Vibrio cholerae, Streptomyces polosus, rumination Bacteria such as Selenomonas ruminantium, Salmonella typhimurium, etc., but only a few sources of lysine decarboxylase have been studied in depth, such as lysine from Escherichia coli and Hafnia albicans acid decarboxylase.
  • CN105316270B, CN105368766A and CN104498519A disclose that different genetic engineering strains are constructed by using the inducible lysine decarboxylase CadA of Escherichia coli to catalyze the synthesis of pentamethylenediamine in whole cells.
  • Tianjin University of Science and Technology used the temperature-regulated promoter pR-pL and the signal peptide pelBs to transform the overexpression vector, and the Institute of Microbiology, Chinese Academy of Sciences integrated T7CadB into the chassis cell genome to increase the production of pentamethylenediamine.
  • CN106148373A, EP3118312B1 and US7189543 carried out sequence modification of the inducible lysine decarboxylase CadA of Escherichia coli.
  • Ajinomoto Company of Japan screened mutants with higher thermostability through the directed evolution of CadA, and Mitsui Chemicals Co., Ltd. Mutants with 10-20% increased activity are disclosed in the patent.
  • mutant enzyme lines are limited to the transformation of Escherichia coli inducible lysine decarboxylase CadA, and the source is very single.
  • the mutant strains have low activity and catalytic intensity in the process of catalytic conversion of high concentration of lysine, and the cell cycle utilization rate is poor. , increasing the operating time, production cost, reducing the yield of pentamethylenediamine and restricting the development of industrialization.
  • the purpose of this application is to provide a lysine decarboxylase for synthesizing pentamethylene diamine and its application, including the amino acid sequence of the lysine decarboxylase, the nucleotide sequence encoding the same , gene expression vectors and recombinant engineering bacteria.
  • the present application provides a lysine decarboxylase for synthesizing pentamethylenediamine, which can catalyze L-lysine to generate pentamethylenediamine, the lysine decarboxylase having (I), (II) or Amino acid sequence shown in any one of (III):
  • the application provides a novel high-efficiency lysine decarboxylase, the lysine decarboxylase can catalyze L-lysine to generate pentamethylene diamine, and the lysine decarboxylase can be induced by constructing an expression vector and genetically engineered bacteria The expression is obtained, and the whole cell catalyzes the synthesis of pentamethylene diamine from lysine hydrochloride. Meanwhile, the lysine decarboxylase provided by the present application is still stable at higher temperature and pH, and has higher yield.
  • the lysine decarboxylase represented by SEQ ID NO.1 is denoted as LdcEdw, and the similarity between the amino acid sequence and Escherichia coli CadA is 86.7%;
  • the lysine decarboxylase represented by SEQ ID NO.2 is named It is LdcAer, and its similarity with Escherichia coli CadA is 75.77%;
  • the lysine decarboxylase represented by SEQ ID NO.3 is named LdcSal, and the similarity between the amino acid sequence and Escherichia coli CadA is 92.3%; SEQ ID NO.
  • the lysine decarboxylase represented by 4 was named LdcKle, and the amino acid sequence was 94.4% similar to Escherichia coli CadA.
  • the amino acid sequence has a great influence on the three-dimensional structure and enzymatic properties of the enzyme, and sometimes a small amount of amino acid difference may lead to a large difference in properties between the two enzymes, and the impact is unpredictable. Therefore, it is the differences between the two that endow LdcEdw with different enzymatic properties from Escherichia coli CadA, and it is these differences that enable LdcEdw to achieve efficient conversion of high concentrations of lysine hydrochloride.
  • amino acid sequence shown in any one of SEQ ID NO. 88%, 90%, 92%, 94%, 96%, 98% or 99%, etc. homology of amino acid sequences can also achieve the conversion of lysine hydrochloride.
  • the lysine decarboxylase described in this application is derived from Hafnia alvei, Bacillus halodurans, Bacillus cereus, Bacterium cadaveris, Burke Burkholderia vietnamensia, Chromobacterium violaceum, Edwardsiella tarda, Vibrio cholerae, Streptomyces polosus, Lunamonas ruminants (Selenomonas ruminantium), Salmonella typhimurium, Salmonella bongori, Serratia, Bordetella, Vibrio cholerae, Aeromonas Lysine decarboxylase genes and mutants of bacteria of the genus Aeromonas and Klebsiella.
  • the lysine decarboxylase is derived from Edwardsiella tarda, Klebsiella, Aeromonas or Salmonella bongori.
  • a second aspect a nucleotide encoding the lysine decarboxylase of the first aspect, the nucleotide having the nucleotide shown in any one of (i), (ii) or (iii) sequence:
  • LdcEdw its nucleic acid sequence is the sequence shown in SEQ ID NO.5.
  • the nucleic acid sequence is codon-optimized, the GC content is 43%, and the similarity with the nucleic acid sequence of Escherichia coli CadA is 74.5%.
  • the nucleic acid sequence of described LdcAer is the sequence shown in SEQ ID NO.6.
  • the nucleic acid sequence is codon-optimized, the GC content is 45%, and the similarity with the nucleic acid sequence of Escherichia coli CadA is 69.2%.
  • the nucleic acid sequence of described LdcSal is the sequence shown in SEQ ID NO.7.
  • the nucleic acid sequence is codon-optimized, the GC content is 43%, and the similarity with the nucleic acid sequence of Escherichia coli CadA is 78.1%.
  • the nucleic acid sequence of described LdcKle is the sequence shown in SEQ ID NO.8.
  • the nucleic acid sequence is codon-optimized, the GC content is 43%, and the similarity with the nucleic acid sequence of Escherichia coli CadA is 78.6%.
  • the present application also provides a gene expression vector.
  • the gene expression vector comprises: a nucleotide sequence encoding the amino acid sequence as described in the first aspect or the nucleotide as described in the second aspect.
  • the gene expression vector is a pET plasmid, preferably a pETDuet plasmid.
  • the expression vector may be pETDuet, or may be various expression vectors commonly used in the art for expressing target genes in Escherichia coli.
  • the gene expression vector further comprises a nucleotide sequence encoding a lysine pentamethylene diamine antiporter.
  • the application also provides a construction method of the gene expression vector as described in the third aspect, the construction method comprising the following steps:
  • the gene expression vector is obtained by inserting the nucleotide sequence encoding the amino acid sequence described in the first aspect or the nucleotide sequence described in the second aspect between the restriction enzyme cleavage sites of the plasmid.
  • the construction method also includes the manipulation of inserting the lysine pentamethylene diamine antiporter gene.
  • the lysine pentamethylene diamine antiporter gene further includes a signal peptide.
  • the signal peptide comprises an E. coli periplasmic secretory signal peptide.
  • the expression vector takes pelB signal peptide as an example, but it is not limited to this signal peptide, which can be common in E. coli, such as dsbA, hlyA, lamB, malE, ompA, ompF, ompT, phoA, etc.
  • the construction method specifically includes the following steps:
  • the lysine decarboxylase gene ldc and the lysine pentanediamine antiporter gene cadB were inserted between the NcoI/SacI and Bgl II/Pac I restriction sites of the pETDuet plasmid, respectively, to construct the plasmid pETDuet- ldc-cadB, and then introduce pelB before the cadB sequence, and connect through the NdeI/Bgl II restriction enzyme site. Finally, the constructed expression vector is pETDuet-ldc-pelB-cadB.
  • a fifth aspect a recombinant engineering bacterium for synthesizing pentamethylenediamine, comprising the gene expression vector described in the third aspect and/or the nucleotide encoding the lysine decarboxylase described in the first aspect.
  • the engineered bacteria can be E. coli BL21 (DE3).
  • the present application also provides a method for preparing pentamethylenediamine using the host cell as described in the fifth aspect, the method comprising the steps of:
  • the bacterial liquid obtained after culturing and inducing the recombinant engineered bacteria is centrifuged and resuspended to obtain a bacterial suspension, which is mixed and reacted with a buffer containing lysine hydrochloride and pyridoxal phosphate (PLP). and centrifugation to obtain the pentamethylenediamine.
  • a buffer containing lysine hydrochloride and pyridoxal phosphate (PLP). pyridoxal phosphate
  • the temperature of the reaction is 35-65°C, for example, it can be 35°C, 40°C, 45°C, 50°C, 55°C, 60°C or 65°C, etc.
  • the time is 0.5-24h, For example, it can be 0.5h, 1h, 1.5h, 2h, 2.5h, 3h, 5h, 6h, 8h, 10h, 12h, 15h, 16h, 18h, 20h or 24h, etc., preferably 1-4h.
  • the shaking rate during the reaction is 400-800 rpm, such as 400 rpm, 500 rpm, 550 rpm, 600 rpm, 650 rpm, 700 rpm or 800 rpm, and the like.
  • the rotation speed during the centrifugation is 8000-12000rpm, for example, it can be 8000rpm, 8500rpm, 9000rpm, 10000rpm, 10500rpm, 11000rpm or 12000rpm, etc.
  • the time is 1-3min, for example, it can be 1min, 1.5min, 2min, 2.5min or 3min, etc.
  • the molar concentration of lysine hydrochloride in the buffer is 0.1-3M, such as 0.1M, 0.5M, 0.8M, 1M, 1.2M, 1.5M, 1.8M, 2M, 2.5M or 3M et al.
  • the molar concentration of PLP in the buffer is 0.1-0.5 mM, such as 0.1 mM, 0.15 mM, 0.2 mM, 0.25 mM, 0.3 mM, 0.35 mM, 0.4 mM, 0.45 mM or 0.5 mM, etc.
  • the buffer includes any one of sodium acetate buffer, phosphate buffer, Tris-HCl buffer or sodium carbonate buffer, preferably phosphate buffer.
  • the pH of the buffer solution is 5-11, for example, it can be 5, 6, 6.4, 7, 7.2, 7.5, 8, 9, 10 or 11, etc., preferably 6-10; The same solution was used for suspension.
  • cryopreservation operation is also included, and the cryopreservation is: cryopreservation at -80°C for more than 1 hour.
  • the method for preparing pentamethylene diamine comprises the steps:
  • the method for detecting lysine and pentamethylenediamine is:
  • the application also provides a bio-based pentamethylene diamine synthesis using the lysine decarboxylase as described in the first aspect, the gene expression vector as described in the third aspect or the recombinant host cell as described in the fifth aspect. applications in .
  • the application provides a novel lysine decarboxylase capable of efficiently synthesizing pentamethylene diamine, and the novel and efficient lysine decarboxylase can be induced by constructing an expression vector and genetically engineered bacteria at the same time. Expression obtained, when whole cell catalyzes lysine hydrochloride to synthesize pentamethylenediamine, it remains stable at pH 5-9 and temperature 40-60 °C, and the catalytic efficiency is high, and the catalytic strength can reach 136-204g/L /h;
  • the novel lysine decarboxylase provided by this application can realize near-complete conversion of high-concentration lysine hydrochloride.
  • the pH is 6.5 and the temperature is 50°C
  • the conversion rate of pentamethylenediamine is the largest, which can reach 100%.
  • the catalytic strength can reach 204g/L/h, and its activity and catalytic strength are significantly higher than those of the existing reported Escherichia coli CadA and its mutants, which is conducive to the efficient synthesis of high-concentration pentamethylenediamine, and has extremely high industrial application prospects.
  • Figure 1 is a schematic diagram of the pETDuet-ldcEdw-pelB-cadB expression vector constructed in Example 1.
  • FIG. 2 is a graph showing the variation of LdcEdw whole-cell catalysis with reaction time in Example 5.
  • FIG. 3 is a graph showing the variation of LdcAer whole-cell catalysis with reaction time in Example 5.
  • the type of the expression vector there is no special requirement for the type of the expression vector, and it can be various expression vectors commonly used in the art that can express the target gene in Escherichia coli, such as plasmids and the like.
  • the construction method of the expression vector can adopt various methods commonly used in the art, such as ligating the target gene into the vector after enzyme digestion.
  • the HPLC detector used is: SPD-20A diode array detector; the detection column is: C18 column (Shim-pack GIST-HP-C18 column, 2.1 ⁇ 100 mm, 3 ⁇ m particle size).
  • HPLC detector SPD-20A diode array detector; detection column: C18 column; detection temperature: 35°C; injection volume: 5 ⁇ L, wavelength: 284 nm.
  • mobile phase A is acetonitrile
  • mobile phase B is 25mM pH 4.8 sodium acetate buffer solution
  • flow rate 0.5mL/min
  • time program proportion of mobile phase B: 0min 80%; 2min 75%; 22min 51.7%; 22.01min 80%; 27min 80%.
  • This example provides a gene expression vector containing lysine decarboxylase and an engineered strain expressing the same.
  • the lysine decarboxylase is named LdcEdw, its amino acid sequence is SEQ ID NO.1, and the similarity with Escherichia coli CadA is 86.7%; after codon optimization, the nucleotide sequence of synthesizing LdcEdw is SEQ ID NO.5 , the GC content of the nucleotide sequence is 43%, and the similarity with the nucleic acid sequence of Escherichia coli CadA is 74.5%.
  • the lysine decarboxylase is named LdcAer, its amino acid sequence is SEQ ID NO.2, and the similarity with Escherichia coli CadA is 75.77%; after codon optimization, the nucleotide sequence of synthetic LdcAer is SEQ ID NO.6 , the GC content of the nucleotide sequence is 45%, and the similarity with the nucleic acid sequence of Escherichia coli CadA is 69.2%.
  • the lysine decarboxylase is named LdcSal, its amino acid sequence is SEQ ID NO.3, and the similarity with Escherichia coli CadA is 92.3%; after codon optimization, the nucleotide sequence of synthetic LdcSal is SEQ ID NO.7 , the GC content of the nucleotide sequence is 43%, and the similarity with the nucleic acid sequence of Escherichia coli CadA is 78.1%.
  • the lysine decarboxylase is named LdcKle, its amino acid sequence is SEQ ID NO.4, and the similarity with Escherichia coli CadA is 94.4%; after codon optimization, the nucleotide sequence of synthesizing LdcKle is SEQ ID NO.8 , the GC content of the nucleotide sequence is 43%, and the similarity with the nucleic acid sequence of Escherichia coli CadA is 78.6%.
  • this application also uses EcCadA (GenBank: WP_001295383.1) derived from Escherichia coli as a comparison; and LdcEdw, LdcAer, LdcSal, LdcKle and CadA are respectively constructed in pETDuet between NcoI/SacI of the plasmid; at the same time, the CadB (GenBank: WP_000092909.1) gene was constructed between BglII/PacI of the pETDuet plasmid; His tag was inserted before the nucleotide sequence encoding the protein;
  • the signal peptide pelB was introduced before the cadB sequence to construct pETDuet-ldcEdw-pelB-cadB (as shown in Figure 1), pETDuet-ldcAer-pelB-cadB, pETDuet-ldcSal-pelB-cadB, pETDuet-ldcKle-pelB-cadB and pETDuet-EccadA-pelB-cadB plasmids were transferred into E.coli BL21 (DE3) chassis cells respectively to construct genetically engineered strains that catalyze the synthesis of pentanediamine in whole cells, named EDW, AER, SAL, KLE and WT. Store at -80°C.
  • This example is a whole-cell catalysis comparison of gene expression vectors containing lysine decarboxylase LdcEdw, LdcAer and EcCadA, respectively.
  • the engineering strains EDW, AER and WT obtained in Example 1 were cultured in 5 mL of LB medium supplemented with 100 mg/L ampicillin at 37° C. overnight to obtain seed liquid.
  • the seed solution was transferred into 50 mL of LB medium with 100 mg/L ampicillin at a transfer volume of 1%, and cultured at 37 °C.
  • IPTG iso-isopropylamine
  • propyl- ⁇ -D-thiogalactopyranoside was induced, and cultured at 20°C for 20h. Centrifuge at 4000 rpm, collect the cells, and store at -80°C.
  • the bacteria were resuspended with 50mM sodium acetate buffer at pH 6, and 500 ⁇ L of whole-cell catalysis was performed, wherein PLP 0.1mM, lysine hydrochloride 1M, bacterial suspension OD was 1.5, 45°C, 500rpm for 1h, and 12000rpm for 2min centrifugation , take the supernatant and dilute to detect the content of lysine hydrochloride and pentamethylene diamine.
  • the engineering strain EDW obtained in Example 1 was cultured in 5 mL of LB medium supplemented with 100 mg/L ampicillin at 37° C. overnight to obtain seed liquid.
  • the seed solution was transferred into 50 mL of LB medium with 100 mg/L ampicillin at 1% volume, and cultured at 37 °C.
  • OD 600 was 0.6
  • IPTG IPTG with a final concentration of 0.1 mM was added for induction, and the temperature was 20 °C.
  • centrifuge 4000 rpm to collect bacterial cells and store at -80°C.
  • EDW is the highest when the pH of the reaction system reaches 6.5. At this time, the conversion rate can reach 99.47%, and lysine hydrochloride is basically completely converted.
  • AER reached the highest at pH 6 of the reaction system, and the conversion rate reached 92.26% at this time.
  • the pH increased to 7.5 the yield of pentamethylenediamine decreased to 86.92%.
  • the engineering strain EDW obtained in Example 1 was cultured in 5 mL of LB medium supplemented with 100 mg/L ampicillin at 37° C. overnight to obtain seed liquid.
  • the seed solution was transferred into 50 mL of LB medium with 100 mg/L ampicillin at 1% volume, and cultured at 37 °C. When the OD 600 was 0.6, IPTG with a final concentration of 0.1 mM was added for induction. Continue to culture for 20h, centrifuge at 4000rpm, collect the cells, and store at -80°C.
  • the bacteria were resuspended with pH 8 buffer, and 500 ⁇ L of whole cell catalysis was performed, wherein PLP was 0.1 mM, lysine hydrochloride was 1 M, and the OD of the bacterial suspension was 1.5.
  • Catalysis was carried out at 35-65°C and 500 rpm for 1 h, centrifuged at 12,000 rpm for 2 min, and the content of pentamethylene diamine was detected after the supernatant was diluted.
  • the whole-cell catalysis results of EDW and AER at 35-65 °C are shown in Table 2. It is not difficult to find that the production of pentamethylenediamine first increases and then decreases with the increase of temperature. is 95.95%.
  • the engineering strains EDW and AER obtained in Example 1 were cultured in 5 mL of LB medium supplemented with 100 mg/L ampicillin at 37° C. overnight to obtain seed liquid.
  • the seed solution was transferred into 50 mL of LB medium with 100 mg/L ampicillin at 1% volume, and cultured at 37 °C. When the OD 600 was 0.6, IPTG with a final concentration of 0.1 mM was added for induction. Continue to culture for 20h, centrifuge at 4000rpm, collect the cells, and store at -80°C.
  • the 20mL shake flask whole-cell catalysis was carried out with phosphate buffer of pH 8 and temperature of 50°C.
  • the OD of the concentrated bacteria in the system was about 10, and the substrate concentration was 2M.
  • Samples were taken at 0h, 1h, and 2h, respectively. The content of pentamethylenediamine was detected after the supernatant was diluted.
  • the engineering strains EDW and AER obtained in Example 1 were cultured in 5 mL of LB medium supplemented with 100 mg/L ampicillin at 37° C. overnight to obtain seed liquid.
  • the seed solution was transferred into 50 mL of LB medium with 100 mg/L ampicillin at 1% volume, and cultured at 37 °C.
  • OD 600 was 0.6
  • IPTG IPTG with a final concentration of 0.1 mM was added for induction, and the temperature was 20 °C.
  • centrifuge 4000 rpm to collect bacterial cells and store at -80°C.
  • the cells were disrupted with a sonicator, the power was 40-60%, centrifuged at 8000 rpm, the cell debris was precipitated, filtered with a 0.22 ⁇ m filter, and the protein was purified using a 5 ml Histrap purification column on an AKTA protein purifier, followed by 5 mL HiTrap Desalting desalting column was used to replace the preservation solution, and the concentration of purified lysine decarboxylase LdcEdw and LdcAer was determined by BCA protein quantification method.
  • the in vitro catalytic reaction system of lysine decarboxylase LdcEdw is 500 ⁇ L, the concentration of lysine hydrochloride is 1.5M, and the concentration of PLP is 0.1 mM.
  • pH 6.5 catalyzed at 500rpm for 1h, centrifuged at 12,000rpm for 2min, took the supernatant and diluted it to detect the content of pentamethylenediamine.
  • the reaction system was 500 ⁇ L, the concentration of lysine hydrochloride was 1.5 M, and the concentration of PLP was 0.1 mM.
  • the pure LdcAer enzyme was diluted 50 times and added to 190 ⁇ L.
  • the lysine decarboxylase LdcEdw provided by the present application can efficiently catalyze the synthesis of pentamethylenediamine.
  • the optimal catalytic temperature for whole-cell catalysis is 50 °C
  • the optimal pH of the catalytic system is 6.5
  • the catalytic strength can reach 204 g. /L/h, can achieve complete conversion of high concentration of lysine hydrochloride
  • the optimal catalytic temperature of LdcAer whole cell catalysis is 50 °C
  • the optimal pH of the catalytic system is 6, and the conversion rate of pentamethylene diamine can reach 97.2%.
  • a recombinant engineering bacterium is constructed with the amino acid sequences shown in SEQ ID NO.3 and 4 at the same time, which can also express pentamethylenediamine efficiently. Due to space limitations and for the sake of simplicity, only the experimental results are indicated here: SEQ ID NO.
  • the amino acid sequences shown in ID NO.3 and SEQ ID NO.4 were constructed by the methods of Examples 1 and 2.
  • the pH of the system was 5-10, and the temperature was The reaction is stable at 40-55°C, the optimum catalytic temperature is 50°C, the optimum pH of the catalytic system is 6 and 7.5 respectively, and the conversion rate of pentamethylene diamine can reach 72.5%.

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  • General Chemical & Material Sciences (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention concerne une lysine décarboxylase destinée à la synthèse de la pentylènediamine et son utilisation, comprenant les séquences de gènes et de protéines de la lysine décarboxylase, des vecteurs d'expression construits et des souches bactériennes issues du génie génétique, et une utilisation dans la synthèse de pentylènediamine d'origine biologique. La construction d'un vecteur d'expression et d'une souche bactérienne issue du génie génétiquea pour effet d'induire l'expression de la lysine décarboxylase, et la production de pentylènediamine par une synthèse catalytique de cellules entières. La lysine décarboxylase décrite peut atteindre 100 % de conversion de chlorhydrate de lysine à haute concentration, et l'intensité de production de la pentanediamine peut atteindre 204 g/L/h.
PCT/CN2021/076305 2020-07-02 2021-02-09 Lysine décarboxylase pour synthétiser la pentanediamine et son utilisation WO2022001121A1 (fr)

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