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WO2019091366A1 - Procédé de préparation de leucine l-tertiaire optiquement pure à l'aide d'un corps d'inclusion actif - Google Patents

Procédé de préparation de leucine l-tertiaire optiquement pure à l'aide d'un corps d'inclusion actif Download PDF

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WO2019091366A1
WO2019091366A1 PCT/CN2018/114105 CN2018114105W WO2019091366A1 WO 2019091366 A1 WO2019091366 A1 WO 2019091366A1 CN 2018114105 W CN2018114105 W CN 2018114105W WO 2019091366 A1 WO2019091366 A1 WO 2019091366A1
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moiety
fdh
leudh
inclusion body
linker peptide
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PCT/CN2018/114105
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Chinese (zh)
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方柏山
张永辉
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厦门大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C12P13/04—Alpha- or beta- amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
    • C12N9/0016—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with NAD or NADP as acceptor (1.4.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y102/00—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2)
    • C12Y102/01—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2) with NAD+ or NADP+ as acceptor (1.2.1)
    • C12Y102/01002—Formate dehydrogenase (1.2.1.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y104/00—Oxidoreductases acting on the CH-NH2 group of donors (1.4)
    • C12Y104/01—Oxidoreductases acting on the CH-NH2 group of donors (1.4) with NAD+ or NADP+ as acceptor (1.4.1)
    • C12Y104/01009—Leucine dehydrogenase (1.4.1.9)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present application belongs to the field of bioengineering technology, and particularly relates to a method for preparing optically pure L-tert-leucine by using active inclusion bodies.
  • L-Tle L-tertiary bright amino acid
  • the tert-butyl group in the structure of L-tertiary bright amino acid (L-Tle) facilitates the reaction from the back side due to its large steric hindrance, so L-Tle and its derivatives are often used as catalysts for inducing asymmetric reactions.
  • the products are highly selective, and are commonly used as templates for inducing asymmetric synthesis reactions, and are widely used in asymmetric synthesis.
  • the tert-butyl structure of L-Tle is highly hydrophobic and can effectively control the molecular configuration; in the polypeptide component, L-Tle is gradually replacing Val, Leu and Ile, because it can enhance the hydrophobicity of the polypeptide and Stability to prevent degradation by enzymes.
  • L-Tle has a wide range of applications in feed additives and nutritional supplements.
  • L-Tle and its derivatives are often used as metal chiral ligands or ligands for chemical enzyme catalysts to provide a more efficient catalytic mode for asymmetric amination reduction reactions.
  • Another important use of L-Tle is as a pharmaceutical intermediate, which is widely used in the synthesis of anti-AIDS drugs and biological inhibitors.
  • the methods for producing L-tert-leucine have been reported mainly as chemical reagent resolution, chiral source synthesis, chemical synthesis and biological enzymatic methods. Among them, the splitting method is limited by the yield, the chiral source method is limited by the natural product productivity, and the chemical synthesis method is costly. Therefore, these synthetic methods have not been successfully industrialized.
  • the bioenzymatic method is currently implementing L-tert-leucine. The main method of industrial production.
  • Inclusion bodies are often formed during the expression of heterologous proteins in prokaryotic expression systems and are generally considered to be adverse by-products, severely reducing the expression of soluble recombinant proteins.
  • studies on inclusion bodies in recent years have shown that they are mainly composed of recombinant proteins of interest, and that aggregation of recombinant proteins to form inclusion bodies does not mean loss of biological activity. According to reports in the literature and patents, it can be confirmed that inclusion bodies are still quite Biological activity of soluble recombinant proteins (Trends in Biotechnology 2012, 30: 65-7; Trends in Biochemical Sciences, 2017, 42(9): 726-737).
  • the target enzyme can be self-immobilized by fusion with an appropriate label to form a stable, reusable biocatalyst.
  • Nahlka et al. fused maltodextrin phosphorylase to the cellulose binding site of Clostridium cellulovorans to construct active inclusion bodies. 83% of maltodextrin phosphorylase was found in inclusion bodies and could be used for D-glucose-1.
  • - Repeated batch catalysis of phosphoric acid Journal of Industrial Microbiology and Biotechnology 2008, 35: 219-223); Diener et al.
  • the currently reported labels for inducing inclusion body formation include a cellulose binding site, a tetramerization site of the cell surface protein Tetrabrachion, a foot-and-mouth disease virus VP1 capsid protein, a green fluorescent protein, an elastin polypeptide, and the like.
  • L-Tle or other products by preparing bifunctional enzymes for the preparation of active inclusion bodies. Therefore, genetic engineering methods are used to construct bifunctional enzyme active inclusion bodies, and they are used as high-efficiency and economical biocatalysts to prepare optically pure L- Tle has an important meaning.
  • the purpose of the present application is to overcome the deficiencies of the prior art and to provide a process for the preparation of optically pure L-tert-leucine using active inclusion bodies.
  • a method for preparing optically pure L-tert-leucine using active inclusion bodies comprising the following steps:
  • the active component of the bifunctional enzyme activity inclusion body is a fusion bifunctional enzyme comprising a LeuDH moiety linked by a linker peptide and a regeneration enzyme for NAD +
  • the polymerase portion, wherein the LeuDH portion comprises a sequence as shown in SEQ ID NO: 01, wherein the linker peptide is a rigid linker peptide or a flexible linker peptide, and the rigid linker peptide is capable of forming an alpha helix to effectively isolate the LeuDH portion and the polymerase portion.
  • the flexible linker peptide described above does not have the ability to form a particular secondary structure, and is generally present in the form of a random coil to provide the flexibility required for the protein in the catalytic process;
  • the bifunctional enzyme activity inclusion body is resuspended in a reaction mixture having a pH of 6.0 to 10.0, and then reacted at 20 to 40 ° C, and the pH is controlled to be 6.0 to 10.0 during the reaction; the reaction mixture contains 50 to 1000 mM. Methylpyruvate, 50-1000 mM ammonium formate and 0.05-5 mM coenzyme NAD + .
  • the rigid linker peptide comprises a plurality of amino acid sequences as shown in SEQ ID NO 02, which are contiguously linked.
  • the flexible linker peptide comprises a plurality of amino acid sequences as shown in SEQ ID NO: 03.
  • the polymerase moiety is an FDH moiety, a glucose dehydrogenase moiety, a glycerol dehydrogenase moiety, an alcohol dehydrogenase moiety, a glucose-6-phosphate dehydrogenase moiety, and a lactate dehydrogenase. Department or hydrogenase part.
  • the polymerase moiety is an FDH moiety, and the FDH moiety comprises the amino acid sequence set forth in SEQ ID NO 04.
  • the pH of the reaction mixture in the step (2) is 8.5 to 9, the temperature is 30 ° C, and the pH is controlled to be 8.5 to 9 during the reaction.
  • the reaction mixture in the step (2) comprises 50 to 710 mM of trimethylpyruvate, 50 to 780 mM of ammonium formate, and 0.05 to 0.5 mM of the coenzyme NAD + .
  • the present application can greatly reduce the cost of catalyst preparation in a double enzyme system by constructing a fusion enzyme.
  • the method for constructing the bifunctional enzyme activity inclusion body of the present application is low in cost and easy for industrial application.
  • the bifunctional enzyme activity inclusion body in the present application belongs to the self-assembly immobilization without carrier, avoids the cost of enzyme immobilization, and facilitates separation and purification of downstream products.
  • the bifunctional enzyme activity inclusion body typically, the FDH-LeuDH bifunctional enzyme activity inclusion body has high optical selectivity, thermal stability is improved compared with the soluble bifunctional enzyme, and can be reused as an immobilized enzyme.
  • the cost-effective preparation of optically pure tert-leucine has a good industrial application prospect.
  • the present invention prepares a bifunctional enzyme active inclusion body by a genetic engineering bacteria containing a bifunctional enzyme expression vector, and can optimize the overall structure of the bifunctional enzyme activity inclusion body by adjusting the ligation peptide configuration, thereby improving the coupling efficiency of the double enzyme.
  • the application process is simple, no special requirements for equipment, suitable for industrial production.
  • Figure 1 is a diagram showing the agarose gel electrophoresis of the PCR product of the FDH-LeuDH bifunctional enzyme gene in Example 1 of the present application.
  • Example 2 is a whole cell SDS-PAGE diagram of FDH-LeuDH bifunctional enzyme genetically engineered bacteria mediated by different linker peptides in Example 2 of the present application.
  • Figure 3 is a SEM image of the bifunctional enzyme active inclusion body in Example 3 of the present application, wherein A is FDH-R1-LeuDH, B is FDH-R2-LeuDH, C is FDH-S1-LeuDH, and D is FDH-S2- LeuDH.
  • Example 4 is a recombinant protein distribution (A), an FDH enzyme activity distribution (B), and a LeuDH enzyme activity distribution (C) of the bifunctional enzyme activity inclusion body in Example 3 of the present application.
  • Example 5 is a comparison of the enzyme activities of the bifunctional enzyme activity inclusion body and the free enzyme in Example 3 of the present application, wherein A is a LeuDH moiety and B is an FDH moiety, and the relative enzyme activity is calculated by using the enzyme activity of the free single enzyme as 100%.
  • Figure 6 is a comparison of the catalytic ability of the FDH-R3-LeuDH soluble fraction and the active inclusion body in Example 4 of the present application.
  • Figure 7 is a graph showing the results of liquid chromatography in Example 4 of the present application, wherein A is a liquid chromatogram of standard L-Tle and standard D-Tle, and B is a liquid phase of a catalytic product of FDH-R3-LeuDH active inclusion body. Chromatogram, asterisk indicates D-Tle peak time.
  • Figure 8 is a SDS-PAGE diagram of FDH-R3-LeuDH active inclusion bodies of different IPTG concentrations in Example 5 of the present application.
  • Figure 9 is a comparison of the catalytic capabilities of FDH-R3-LeuDH active inclusion bodies of different IPTG concentrations in Example 5 of the present application.
  • Figure 10 is a comparison of the thermal stability of FDH-R3-LeuDH active inclusion bodies and soluble fractions in Example 6 of the present application, wherein A is FDH enzyme activity and B is LeuDH enzyme activity.
  • Figure 11 is a continuous recovery catalysis of FDH-R3-LeuDH active inclusion bodies in Example 7 of the present application.
  • the relative yield of the recovered catalysis was calculated as the first catalyzed yield of 100%.
  • the active ingredient of the bifunctional enzyme activity inclusion body of the present application is a fusion bifunctional enzyme comprising a LeuDH moiety linked by a linker peptide and a polymerase moiety for coenzyme NAD + regeneration.
  • the above-mentioned linker peptide is a rigid linker peptide or a flexible linker peptide capable of forming an ⁇ -helix to effectively isolate the above-mentioned LeuDH moiety and the polymerase moiety, and the flexible linker peptide has no ability to form a specific secondary structure, generally The form of the coil is present to provide the flexibility required for the protein in the catalytic process.
  • the polymerase part is the FDH part, the glucose dehydrogenase part, the glycerol dehydrogenase part, the alcohol dehydrogenase part, the glucose-6-phosphate dehydrogenase part,
  • the lactate dehydrogenase moiety or the hydrogenase moiety, the polymerized form of the above polymerase moiety, and the referenced PDB structure ID are shown in the following table.
  • the enzyme capable of forming a multimer and capable of being used for coenzyme regeneration is preferably FDH
  • the polymerase for coenzyme regeneration is preferably a polymer of FDH
  • the bifunctional enzyme activity inclusion body is preferably FDH-LeuDH bifunctional enzyme activity.
  • Inclusion body, that is, FDH is responsible for the regeneration of coenzyme.
  • OE-PCR Overlap extension polymerase chain reaction
  • P1 5'-GGAATTC CATATG AAAATTGTCCTGGTCCTGT-3' (SEQ ID NO 05), underlined for the NdeI restriction site sequence.
  • Ligation peptide primer 5'-GCCTATGGCAAACACGATAAAAAG XXX ATGACATTGG AAATCTTCGA-3', XXX refers to the linker peptide sequence, as shown in Table 1.
  • P3 5'-ATGACATTGGAAATCTTCGAATAT-3' (SEQ ID NO 06).
  • P4 5'-CCG CTCGAG TTACCGGCGACTAATGATGT-3' (SEQ ID NO 07), underlined for the XhoI restriction site sequence.
  • the FDH gene was amplified by P1 and ligation peptide primers, and the LeuDH gene was amplified by P3 and P4.
  • the PCR amplification system template 2uL, primers 1.5uL, PCR Mix 25uL, ddH2O 20uL.
  • PCR conditions 94 ° C pre-denaturation, 5 min; 94 ° C denaturation, 1 min, 56 ° C annealing, 1 min, 72 ° C extension, 15 s, 30 cycles; 72 ° C extension, 10 min.
  • the FDH and LeuDH genes were recovered using a gel recovery kit, and then PCR amplification was carried out using primers 1 and primers 4 using equimolar two enzyme genes as templates, and the fusion enzyme gene inserted into the linker peptide was obtained under the same conditions as above.
  • M represents a DNA marker
  • bands 1-7 are FDH-DL-LeuDH, FDH-S1-LeuDH, FDH-S2-LeuDH, FDH-S3-LeuDH, FDH-R1-LeuDH
  • the FDH-R2-LeuDH and FDH-R3-LeuDH genes were recovered from the fusion enzyme gene using a gel recovery kit.
  • the obtained fusion enzyme gene and pET-28a plasmid were digested with NdeI/XhoI, and the fusion enzyme gene and plasmid backbone were recovered by gel recovery kit, and the ligated plasmid was transformed into E. coli BL21 (DE3). Positive clones were screened using kanamycin resistant plates. The obtained positive clones were cultured at 37 ° C overnight, and the plasmid was extracted, and after double enzyme digestion verification, the strains were stored in a -80 ° C refrigerator.
  • amino acid sequence of the above FDH is shown in SEQ ID NO 04
  • nucleotide sequence is shown in SEQ ID NO: 08
  • amino acid sequence of the above LeuDH is shown in SEQ ID NO 01
  • nucleotide sequence is shown in SEQ ID NO 09.
  • Table 1 shows the amino acid sequences of different fusion enzyme linker peptides and the primer sequences used for insertion of the linker peptide.
  • a DL represents a direct linkage
  • R1-R3 represents an EAAAK linking peptide of 1 to 3 repeating units
  • S1-S3 represents a GGGGS linking peptide of 1 to 3 repeating units.
  • the underlined portion of b is a primer for the corresponding fusion enzyme construction.
  • 100 mg of the cells were suspended in 5 mL of ddH 2 O, and the bacterial cells were disrupted by an ultrasonic cell disrupter, centrifuged at 12,000 ⁇ g for 20 min, and the supernatant was temporarily stored at 4 ° C.
  • the precipitate obtained by centrifugation was first dissolved in PBS buffer supplemented with 1% by volume of ethylphenyl polyethylene glycol (NP-40), placed at 4 ° C for 45 min, and then 25 ⁇ L of DNAse and MgSO 4 (final concentration 10 mM) were added.
  • NP-40 ethylphenyl polyethylene glycol
  • the morphology of the inclusion bodies was observed directly by scanning electron microscopy.
  • the sample preparation method was as follows: 5 ⁇ L of the inclusion body sample was dropped on a single crystal silicon wafer, air-dried overnight, and then plated with about 2 nm thick platinum in a JFC-1600 (JEOL, Tokyo, Japan) sputtering apparatus (sputtering conditions) : 10 mA, 30 s), and the coated samples were placed in a field emission Sigma-type scanning electron microscope (Carl-Zeiss AG, Germany) for observation.
  • Figure 3 is a SEM structural diagram of a partial inclusion body, wherein A is FDH-R1-LeuDH, B is FDH-R2-LeuDH, C is FDH-S1-LeuDH, and D is FDH-S2-LeuDH.
  • the mediated bifunctional enzyme activity inclusion bodies exhibit a lamellar structure, while the flexible linker-mediated bifunctional enzyme activity inclusion bodies exhibit an irregular globular aggregate structure.
  • FIG. 4A shows the distribution of recombinant protein. It can be seen that more than 80% of the recombinant protein is present in the active inclusion body, and the recombinant protein distribution in the supernatant is higher. Less, Figure 4B and Figure 4C show the distribution of enzyme activity. It can be seen that more than 90% of FDH activity and LeuDH activity are distributed in the inclusion body part, and the FDH and LeuDH activity distribution in some bifunctional enzyme inclusion bodies exceeds 95. %, the experimental results indicate that most of the FDH-LeuDH bifunctional enzyme is expressed as an active inclusion body.
  • FIG. 5A shows the comparison of the LeuDH activity of the bifunctional enzyme activity inclusion body and the free enzyme
  • Fig. 5B shows the comparison of the FDH activity of the bifunctional enzyme activity inclusion body and the free enzyme.
  • the relative enzymatic activity was calculated by using the enzyme activity of LeuDH and FDH single enzymes as 100%. It can be seen that the activity of the FDH in the active inclusion body was significantly higher than that of the single enzyme (24.7%-146.6%), while the free enzyme fraction The enzyme activity of FDH showed a significant decrease.
  • the enzymatic activity of LeuDH is lower than that of free enzyme, but since the FDH is the rate-limiting enzyme in the L-Tle double enzyme catalytic system, the enzyme activity of LeuDH is much higher than that of FDH, so the decrease of LeuDH activity does not decrease. Overall catalytic efficiency.
  • the precipitate was added to the reaction mixture after the purification treatment, and 10 mL of the reaction mixture was suspended to start the reaction. The supernatant was added with 5 mL of the 2 ⁇ reaction mixture to keep the concentrations of the two experiments equal, and the two groups were placed at 30 ° C, 200 rpm. Reaction for 48 h.
  • the reaction mixture contained 50 mM trimethylpyruvate, 50 mM ammonium formate, 0.04 mM NAD + , adjusted to pH 8.5 with aqueous ammonia, and the solvent was H 2 O.
  • the soluble fraction of FDH-R3-LeuDH and the active inclusion body partially catalyze the catalytic ability of TMA to form L-Tle.
  • the results are shown in Fig. 6. It can be seen that the conversion of the soluble fraction under the same conditions is only 14.6%, and the activity includes The conversion rate of the bulk fraction was 93.5%, which was about 6.4 times that of the soluble fraction, and the L-Tle ee value obtained by the active inclusion body catalysis was more than 99%.
  • the results are shown in Fig. 4, wherein Fig. 7A is the standard L-Tle and D.
  • the HPLC spectrum of -Tle, Figure 7B is the spectrum of the catalytic product of FDH-R3-LeuDH active inclusion body.
  • FDH-R3-LeuDH was induced to culture using different IPTG concentrations, and then the distribution of recombinant protein was analyzed by SDS-PAGE, as shown in Figure 8, where M represents the protein standard, 1 is the supernatant (0 mM); (0 mM); 3 is the supernatant (0.01 mM); 4 is the inclusion body (0.01 mM); 5 is the supernatant (0.2 mM); 6 is the inclusion body (0.2 mM); 7 is the supernatant (1 mM); It is an inclusion body (1 mM). It can be seen that the content of recombinant protein in the inclusion body increases with the increase of IPTG concentration, while the soluble part of the recombinant protein shows an opposite trend.
  • the FDH-R3-LeuDH activity induced by 50 mg of different IPTG concentrations was suspended in 10 mL of the reaction mixture, and placed at 30 ° C, and reacted at 200 rpm for 16 h.
  • the reaction mixture contained 50 mM trimethylpyruvate, 50 mM ammonium formate, 0.01 mM NAD + , adjusted to pH 8.5 with aqueous ammonia, and the solvent was H 2 O.
  • Catalytic activity of the inclusion bodies As shown in Figure 9, it can be seen that the activity of inclusion bodies increased with the increase of IPTG concentration at the beginning, but when the concentration of IPTG increased to 1 mM, the catalytic efficiency decreased, at 0.2 mM. At the concentration of IPTG, the recombinant protein was expressed in the inclusion body at a high level and maintained a good active conformation.
  • the FDH-R3-LeuDH soluble fraction and the active inclusion body were placed in a water bath at 20-50 ° C for 1 h, respectively, and the residual enzyme activity was measured. The results are shown in Figure 10.
  • the FDH partial enzyme activity was maintained at 70-112% after the water bath, LeuDH Part of the enzyme activity was maintained at 87-108%, while the FDH activity of the soluble fraction was maintained at 47-94%, and the LeuDH enzyme activity was maintained at 74-94%.
  • the thermal stability of the active inclusion bodies FDH and LeuDH were better than The fusion enzyme, the increase in thermal stability may be due to the aggregation of the fusion enzyme in the cells to form aggregates to prevent the subunits of the enzyme from dissociating at high temperatures.
  • the enzyme activity of the active fusion enzyme inclusions increased slightly after incubation for 1 h at low temperature. The reason for this phenomenon may be that the misfolded recombinant protein in the inclusion body refolds under heat shock to form an active conformation.
  • the active inclusion bodies were centrifuged at 5000 x g for 10 min at 4 ° C, and the precipitate was washed twice with ddH 2 O to remove the reaction residue, followed by the addition of 10 mL of the reaction mixture, suspended, and the next reaction was started, and the batch catalysis was repeated for six rounds.
  • the yield of the first batch of catalysis is recorded as 100%, and the relative yield of the latter batch is calculated.
  • the results are shown in Fig. 11. It can be seen that the yield of L-Tle decreases with the increase of the number of times of recovery, without additional immobilization. In the case of medium and other modifications, after 2, 4, and 6 consecutive recovery catalysis, the yields were maintained at 86.0%, 72.0%, and 54.3%, respectively, of the first catalytic yield.
  • the experimental results show that the FDH-R3-LeuDH active inclusion body has good reusability, and the multi-enzyme active inclusion body can simultaneously realize the construction of the multi-enzyme catalytic system and the immobilization of the enzyme.

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Abstract

L'invention concerne un procédé de préparation d'une leucine L-tertiaire optiquement pure à l'aide d'un corps d'inclusion actif, le procédé comprenant les étapes suivantes, consistant à : (1) préparer un corps d'inclusion actif d'enzyme bifonctionnelle, l'ingrédient actif du corps d'inclusion actif d'enzyme bifonctionnelle étant une enzyme bifonctionnelle de fusion qui comprend une partie de LeuDH et une partie de polymérase, liées par un peptide de liaison, pour la régénération de la coenzyme NAD+; et (2) ajouter le corps d'inclusion actif d'enzyme bifonctionnelle mentionné ci-dessus à un liquide de réaction mixte de pH 6,0 à 10,0 pour la remise en suspension, puis faire réagir le mélange à 20°C jusqu'à 40°C, le pH étant régulé pour être de 6,0 à 10,0 pendant la réaction, le liquide de réaction mixte contenant 50 à 1000 mM en pyruvate de triméthyle, 50 à 1000 mM en formiate d'ammonium et 0,05 à 5 mM en coenzyme NAD+.
PCT/CN2018/114105 2017-11-08 2018-11-06 Procédé de préparation de leucine l-tertiaire optiquement pure à l'aide d'un corps d'inclusion actif WO2019091366A1 (fr)

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CN109628419A (zh) * 2018-12-29 2019-04-16 重庆大学 一种双功能融合蛋白及其生产d-苯乳酸的方法
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CN111518851B (zh) * 2019-11-06 2023-05-23 上海健康医学院 一种固定化酶连续制备[14/15n]-l-瓜氨酸的方法

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