WO2020233451A1 - 一种l-谷氨酸脱氢酶突变体及其应用 - Google Patents

一种l-谷氨酸脱氢酶突变体及其应用 Download PDF

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WO2020233451A1
WO2020233451A1 PCT/CN2020/089775 CN2020089775W WO2020233451A1 WO 2020233451 A1 WO2020233451 A1 WO 2020233451A1 CN 2020089775 W CN2020089775 W CN 2020089775W WO 2020233451 A1 WO2020233451 A1 WO 2020233451A1
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glufosinate
ammonium
reaction
dehydrogenase
glutamate dehydrogenase
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PCT/CN2020/089775
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French (fr)
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田振华
程占冰
丁少南
焦琦
徐文选
黄瑶
江枫
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弈柯莱生物科技(上海)股份有限公司
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Priority to JP2021569424A priority Critical patent/JP2022533444A/ja
Priority to EP20809767.5A priority patent/EP3974522A4/en
Publication of WO2020233451A1 publication Critical patent/WO2020233451A1/zh
Priority to US17/531,081 priority patent/US11905534B2/en

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    • 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)
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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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    • 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)
    • C12N9/0026—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on CH-NH groups of donors (1.5)
    • C12N9/0028—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on CH-NH groups of donors (1.5) with NAD or NADP as acceptor (1.5.1)
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    • 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
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    • 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)
    • C12Y104/01002—Glutamate dehydrogenase (1.4.1.2)
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    • 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)
    • C12Y104/01004—Glutamate dehydrogenase (NADP+) (1.4.1.4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

Definitions

  • the invention belongs to the field of biotechnology, and specifically relates to an L-glutamate dehydrogenase mutant and its application.
  • Glufosinate-ammonium (2-amino-4-[hydroxy(methyl)phosphono]butyric acid) is a broad-spectrum contact herbicide developed by Hearst in the 1980s. At present, the three major herbicides in the world are glyphosate, glufosinate, and paraquat. Compared with glyphosate and paraquat, glufosinate has excellent herbicidal performance and less side effects. There are two optical isomers of glufosinate-ammonium, namely D- glufosinate-ammonium and L- glufosinate-ammonium, but only L- glufosinate-ammonium has herbicidal activity. Therefore, the method of developing L- glufosinate-ammonium is important for improving atomic economy , It is of great significance to reduce the cost of use and reduce environmental pressure.
  • the main methods for preparing L-Glufosinate-ammonium include chiral resolution, chemical synthesis and biocatalysis.
  • Chiral resolution methods such as CN1053669C discloses the use of quinine alkaloids as resolving agents to recrystallize L-glufosinate quinine salt, and then neutralize the salt with acid to obtain L-glufosinate ammonium.
  • quinine alkaloids as resolving agents to recrystallize L-glufosinate quinine salt, and then neutralize the salt with acid to obtain L-glufosinate ammonium.
  • 5-nitrosalicyaldehyde or 3,5-dinitrosalicylic aldehyde is used as racemization reagent to racemize unreacted D- glufosinate-ammonium to obtain DL- glufosinate-ammonium, which is then used in the resolution reaction .
  • this method requires expensive chiral resolution reagents and multi-step recrystallization, and the operation is cumbersome, and it is not an ideal method.
  • EP0382113A describes a method for catalytically cleaving the carboxylate of N-acetyl- glufosinate-ammonium by acyltransferase to obtain L- glufosinate-ammonium, but the enzyme in this method is not specific to free N-acetyl- glufosinate-ammonium Therefore, N-acetyl-glufosinate-ammonium must be esterified, which increases the reaction steps and correspondingly increases the production cost.
  • Another part is the use of 2-oxo-4-(hydroxymethyl phosphinyl) butyric acid (PPO) as a substrate to prepare L-glufosinate-ammonium catalyzed by transaminase.
  • PPO 2-oxo-4-(hydroxymethyl phosphinyl) butyric acid
  • US5221737A and EP0344683A describe the use of glutamine Acid as the amino donor, the method of obtaining L-glufosinate ammonium from the corresponding keto acid 4-(hydroxymethylphosphinyl)-2-oxobutyric acid through the action of aminotransaminase derived from Escherichia coli, the reaction system needs to wait The amount or excess of amino donor glutamic acid makes it difficult to purify the product.
  • CN1284858C improved the above method, using aspartic acid as the amino donor, from the corresponding keto acid 4-(hydroxymethylphosphinyl)-2-oxobutyric acid through the action of aspartate aminotransferase to obtain L-
  • the reaction requires only equimolar amino donor and amino acceptor.
  • most amino donors used in the method using transaminase are amino acids, and the cost is relatively high.
  • CN108588045A discloses the application of multiple mutants of glutamate dehydrogenase in the preparation of L-glufosinate-ammonium. It was found that the glutamate dehydrogenase (NCBI accession number: NP_742836.1) derived from pseudomonas putida (NCBI accession number: NP_742836.1) was placed at position 167. The mutation of alanine to glycine or the mutation of valine at position 378 to alanine both improved the catalytic ability of the enzyme on PPO. Then, the 167 and 378 positions of glutamate dehydrogenase from other sources were studied.
  • Source site mutants it was found that such mutants can also improve the catalytic ability of glutamate dehydrogenase for PPO; however, there are no mutants at homologous sites of glutamate dehydrogenase from other sources. Combine mutations for research. In addition, although the two sites of pseudomonas putida's glutamate dehydrogenase were mutated in the patent application at the same time, the enzyme activity of the resulting double mutant was only equivalent to that of the single mutant, and due to biological The field is unpredictable, and the effect of mutants with double mutation sites is not necessarily better than that of their respective single mutation mutants.
  • the technical problem to be solved by the present invention is that the existing L-glutamate dehydrogenase has the disadvantages of low catalytic efficiency when preparing L- glufosinate-ammonium or its salt. Therefore, the present invention provides an L-glutamate dehydrogenase Mutant and its application in preparing L- glufosinate-ammonium or its salt.
  • the enzyme activity is Higher, so that the efficiency of the enzyme is improved, the cost of the reaction is reduced, and it is conducive to industrial production.
  • the source of the wild-type L-glutamate dehydrogenase used in the present invention is the glutamate dehydrogenase of Lysinibacillus sphaericus, and the amino acid sequence is shown in SEQ ID NO. 1, Genbank accession number WP_012293812.1.
  • the glutamate dehydrogenase has not been subjected to double mutations at two sites at the same time, and due to the unpredictability in the biological field, the effect of the mutants at the double mutation site is not necessarily better than their respective ones. Single mutant mutants.
  • the present inventors made a combined mutation on the 175th and 386th positions of the above wild-type enzyme against the substrate 2-oxo-4-(hydroxymethylphosphinyl) butyric acid (PPO), and unexpectedly found that when the 175th position was After amino acid residue A is mutated to G, and amino acid residue V at position 386 is mutated to an amino acid residue with less steric hindrance, the resulting mutant has a significant increase in the specific enzyme activity of the substrate PPO.
  • PPO 2-oxo-4-(hydroxymethylphosphinyl) butyric acid
  • a mutant of L-glutamate dehydrogenase the sequence of the mutant of L-glutamate dehydrogenase is as described in SEQ ID NO.1
  • the 175th amino acid residue A of the sequence was mutated to G, and the 386th amino acid residue V was mutated to a sequence with less sterically hindered amino acid residues.
  • the L-glutamate dehydrogenase mutant has Catalyzes the activity of 2-oxo-4-(hydroxymethylphosphinyl) butanoic acid or its salt.
  • amino acid sequence of the L-glutamate dehydrogenase mutant is shown in SEQ ID NO. 7 or SEQ ID NO. 9 in the sequence list.
  • nucleotide sequence of the L-glutamate dehydrogenase mutant is shown in SEQ ID NO. 8 or SEQ ID NO. 10 in the sequence list.
  • the said less steric hindrance means that the amino acid residue after mutation is less sterically hindered than the amino acid residue in the wild sequence.
  • the amino acids can be modified or unmodified natural amino acids; the present invention takes natural amino acids as an example.
  • the second technical solution of the present invention to solve the above technical problem is: an isolated nucleic acid encoding the above-mentioned L-glutamate dehydrogenase mutant.
  • nucleotide sequence encoding the nucleic acid is shown in SEQ ID NO. 8 or SEQ ID NO. 10.
  • the third technical solution of the present invention to solve the above technical problems is: a recombinant expression vector containing the nucleic acid.
  • the fourth technical solution of the present invention to solve the above technical problems is: a transformant containing the nucleic acid or the recombinant expression vector.
  • the fifth technical scheme of the present invention to solve the above technical problems is: a preparation method of L- glufosinate ammonium salt, which includes the following steps: in a reaction solvent, L-glutamate dehydrogenase mutant, inorganic amino donor and In the presence of reduced coenzyme NADPH (nicotinamide adenine dinucleotide phosphate), 2-oxo-4-(hydroxymethyl phosphinyl) butyrate is subjected to ammoniating reaction to obtain L-glufosinate-ammonium salt.
  • NADPH nicotinamide adenine dinucleotide phosphate
  • the preparation method of the L- glufosinate ammonium salt may further include the following steps: in the presence of D-amino acid oxidase (DAAO), the D- glufosinate ammonium salt is subjected to an oxidation reaction to obtain the 2-oxygen Substitute 4-(hydroxymethyl phosphinyl) butyrate.
  • DAAO D-amino acid oxidase
  • the cation of the D-glufosinate ammonium salt may be a conventional cation in the art, such as ammonium ion, sodium ion and/or potassium ion. It can also be the cation of the buffer used.
  • the D- glufosinate ammonium salt can exist alone, or coexist with L- glufosinate ammonium salt (the L- glufosinate ammonium salt may not react at this time), for example: D type Enriched glufosinate ammonium salt (that is, the content of the D-type enantiomer> 50%, even pure D- glufosinate ammonium salt), L-type enriched glufosinate ammonium salt (that is, the content of L- glufosinate ammonium Content>50%, excluding the case of pure L- glufosinate-ammonium salt) or racemic glufosinate-ammonium salt, etc.
  • D type Enriched glufosinate ammonium salt that is, the content of the D-type enantiomer> 50%, even pure D- glufosinate ammonium salt
  • L-type enriched glufosinate ammonium salt that is, the content
  • the concentration of the D amino acid oxidase (DAAO) can be conventional in the art, preferably 0.6-6 U/mL, more preferably 1.8 U/mL.
  • the concentration of the D- glufosinate ammonium salt may be conventional in the art, and is preferably 100-600 mM, more preferably 200 mM.
  • the oxidation reaction can also be carried out in the presence of catalase.
  • the oxidation reaction can also be carried out under aeration conditions.
  • the ventilation is preferably air or oxygen.
  • the rate of said ventilation is preferably 0.5-1VVM.
  • the air may be conventional air in the field, and generally contains oxygen, and the content of oxygen contained is also conventional in the field. It is oxygen in the air that participates in the reaction.
  • the oxidation reaction can also be carried out under aeration conditions, the oxidation reaction can also be carried out in the presence of a defoamer.
  • the pH of the reaction system is preferably 7-9, more preferably 8.
  • the pH can be achieved by using a buffer.
  • the pH can also be adjusted by using alkali (or alkali solution).
  • the buffer is preferably phosphate buffer or Tris-HCl buffer, and the phosphate buffer is preferably disodium hydrogen phosphate-sodium dihydrogen phosphate buffer or dipotassium hydrogen phosphate-dibasic phosphate buffer. Potassium hydrogen buffer.
  • the alkali solution is preferably ammonia water.
  • the temperature of the reaction system can be conventional in the art, preferably 20-50°C, more preferably 20°C.
  • the oxidation reaction and the amination reaction can be carried out separately or simultaneously (in the same reaction system).
  • the simultaneous process is for example: in the presence of D-amino acid oxidase (DAAO), L-glutamate dehydrogenase mutant, inorganic amino donor and reduced coenzyme NADPH, the D- glufosinate ammonium salt is oxidized Reaction and amination reaction to obtain L-glufosinate ammonium salt.
  • DAAO D-amino acid oxidase
  • L-glutamate dehydrogenase mutant L-glutamate dehydrogenase mutant
  • inorganic amino donor and reduced coenzyme NADPH reduced coenzyme
  • the cation of the L-glufosinate ammonium salt may be a conventional cation in the art, such as ammonium ion, sodium ion and/or potassium ion. It can also be the cation of the buffer used.
  • the cation of the 2-oxo-4-(hydroxymethylphosphinyl) butyrate may be a conventional cation in the art, such as ammonium ion, sodium ion and/or potassium Ions etc. It can also be the cation of the buffer used.
  • the dosage of the L-glutamate dehydrogenase mutant can be conventional in the art, and the concentration of the L-glutamate dehydrogenase mutant is preferably 0.05- 2.3 U/ml, more preferably 0.1-1 U/ml, for example 0.23 U/ml.
  • the amount of the inorganic amino donor can be conventional in the art, and the concentration of the inorganic amino donor is preferably 100-2000 mM, more preferably 200 mM.
  • the concentration of the 2-oxo-4-(hydroxymethylphosphinyl) butyrate is preferably 100-600 mM, more preferably 200 mM.
  • the dosage of the 2-oxo-4-(hydroxymethylphosphinyl) butyrate can be conventional in the art, and the reduced coenzyme NADPH and the 2-
  • the quality of oxo-4-(hydroxymethylphosphinyl) butyrate is preferably 1:100-1:20000, more preferably 1:1000-1:15000, and still more preferably 1:5000 .
  • the inorganic amino donor is one or more of ammonia, ammonium sulfate, ammonium chloride, diammonium hydrogen phosphate, ammonium acetate, ammonium formate and ammonium bicarbonate.
  • the temperature of the reaction can be conventional in the art.
  • the temperature for performing the ammoniating reaction is preferably It is 20-50°C, more preferably 37°C.
  • the temperature of the ammoniating reaction is lower than 20°C, the ammoniating reaction is slow; when the temperature of the ammoniating reaction is higher than 50°C, the enzyme will be irreversible Degeneration and inactivation.
  • reaction solvent is water.
  • the pH for carrying out the amination reaction is preferably 7-9, more preferably 8.5.
  • the pH can be achieved by using a buffer.
  • the pH can also be adjusted by using alkali (or alkali solution).
  • the buffer is preferably phosphate buffer or Tris-HCl buffer, etc.
  • the phosphate buffer is preferably disodium hydrogen phosphate-sodium dihydrogen phosphate buffer or dipotassium hydrogen phosphate-phosphate Potassium dihydrogen buffer, etc.
  • the alkali solution is preferably ammonia water.
  • the preparation method of the L-glufosinate ammonium salt also includes the following steps: in the dehydrogenase (such as glucose dehydrogenase, alcohol dehydrogenase or formate dehydrogenase, etc.) and hydrogen donor (such as glucose, isopropanol) Or formate, etc.), the oxidized coenzyme NADP + is subjected to a reduction reaction to obtain the reduced coenzyme NADPH.
  • dehydrogenase such as glucose dehydrogenase, alcohol dehydrogenase or formate dehydrogenase, etc.
  • hydrogen donor such as glucose, isopropanol
  • the oxidized coenzyme NADP + is subjected to a reduction reaction to obtain the reduced coenzyme NADPH.
  • the dehydrogenase has a one-to-one correspondence with the hydrogen donor, for example:
  • the hydrogen donor is isopropanol
  • the hydrogen donor is glucose
  • the hydrogen donor is formate.
  • the concentration of the dehydrogenase can be conventional in the art, preferably 0.6-6 U/mL, more preferably 2 U/mL.
  • the concentration of the hydrogen donor can be conventional in the art, preferably 100-1000 mM, more preferably 240 mM.
  • the concentration of the oxidized coenzyme NADP + can be conventional in the art.
  • the pH for performing the reduction reaction is preferably 7-9, and more preferably 8.5.
  • the pH can be achieved by using a buffer.
  • the pH can also be adjusted by using alkali (or alkali solution).
  • the buffer is preferably phosphate buffer or Tris-HCl buffer, etc.
  • the phosphate buffer is preferably disodium hydrogen phosphate-sodium dihydrogen phosphate buffer or dipotassium hydrogen phosphate-phosphate Potassium dihydrogen buffer, etc.
  • the alkali solution is preferably ammonia water.
  • the temperature of the reaction system can be conventional in the art, preferably 20-50°C, more preferably 37°C.
  • the reduction reaction and the amination reaction can be carried out separately or simultaneously (in the same reaction system).
  • the said simultaneous progress is shown in the preferred embodiment of the present invention: in the presence of glucose dehydrogenase, glucose, oxidized coenzyme NADP + , L-glutamate dehydrogenase mutant, inorganic amino donor, The 2-oxo-4-(hydroxymethylphosphinyl) butyrate undergoes an amination reaction (the reduction reaction of NADP + exists at the same time) to obtain L- glufosinate ammonium salt.
  • the NADPH used in the amination reaction can be generated through the reduction reaction cycle.
  • concentration of the oxidized coenzyme NADP + can be conventional in the art. In order to ensure that the reaction can proceed normally, it is compared with the quality of the 2-oxo-4-(hydroxymethylphosphinyl) butyrate.
  • the ratio is 1:100-1:20000, preferably 1:1000-1:15000, and more preferably 1:5000.
  • the reduction reaction, the oxidation reaction and the amination reaction can be carried out separately or simultaneously (in the same reaction system).
  • the described simultaneous progress is shown in the preferred embodiment of the present invention: in D-amino acid oxidase (DAAO), dehydrogenase, hydrogen donor, oxidized coenzyme NADP + , L-glutamate dehydrogenase mutant ,
  • DAAO D-amino acid oxidase
  • dehydrogenase hydrogen donor
  • NADP + oxidized coenzyme
  • L-glutamate dehydrogenase mutant L-glutamate dehydrogenase mutant
  • the D- glufosinate ammonium salt is subjected to oxidation reaction and amination reaction (the reduction reaction of NADP + exists at the same time) to obtain L- glufosinate ammonium salt.
  • the NADPH used in the amination reaction can be generated through the reduction reaction cycle.
  • the concentration of the oxidized coenzyme NADP + can be conventional in the art.
  • the concentration of NADP + and the 2-oxo-4-(hydroxymethylphosphinyl) butyrate The mass ratio is 1:100-1:20000, preferably 1:1000-1:15000, and more preferably 1:5000.
  • the reaction time of the preparation method is detected by a conventional method
  • the final concentration of the raw material or the final concentration or the conversion rate of the product can be stopped when the desired goal is reached;
  • the conventional method includes pre-column derivatization of high-performance liquid phase Chromatography or ion pair chromatography, etc.
  • the sixth technical solution of the present invention to solve the above technical problems is: a preparation method of L-glufosinate-ammonium, which includes the following steps:
  • L-glufosinate-ammonium salt is prepared
  • step (1) The L- glufosinate-ammonium salt prepared in step (1) is acidified to obtain L- glufosinate-ammonium.
  • the seventh technical scheme of the present invention for solving the above technical problems is: an application of the L-glutamate dehydrogenase mutant prepared above in the preparation of L- glufosinate-ammonium or its salt.
  • the application may include the following steps: reacting 2-oxo-4-(hydroxymethylphosphinyl) butyrate in the presence of L-amino acid dehydrogenase, inorganic amino donor and reduced coenzyme , That is, L- glufosinate ammonium salt.
  • the application may include the following steps: in the presence of L-amino acid dehydrogenase, inorganic amino donor and reduced coenzyme, 2-oxo-4-(hydroxymethylphosphinyl) butyric acid The reaction yields L-Glufosinate-ammonium.
  • the application may include the following steps: in the presence of L-amino acid dehydrogenase, inorganic amino donor and reduced coenzyme, the 2-oxo-4-(hydroxymethylphosphinyl) butyrate The reaction is carried out to obtain L- glufosinate-ammonium salt, and then the acidification reaction is carried out to obtain L- glufosinate-ammonium.
  • the L- glufosinate ammonium salt may generally exist in the form of L- glufosinate ammonium salt.
  • the concentration of the above compound is the concentration of the compound in the entire reaction system before the reaction.
  • the reagents and raw materials used in the present invention are all commercially available.
  • the L-glutamate dehydrogenase mutant of the present invention prepares L-glufosinate-ammonium or its salt, compared with the glutamate dehydrogenase mutant at the single mutation site (the 175th or the 386th)
  • the enzyme activity is higher, so that the efficiency of the enzyme is improved (for example, the conversion rate when participating in the reaction is higher, and the stereoselectivity is stronger), which reduces the cost of the reaction and facilitates industrial production.
  • Figure 1 The Marfey reagent pre-column derivatization HPLC analysis result of the racemic glufosinate-ammonium standard, where the last two peaks are the peaks of the Marfey reagent itself.
  • Figure 2 HPLC analysis result of Marfey reagent pre-column derivatization of D- glufosinate-ammonium and L- glufosinate-ammonium in the prepared product.
  • FIG. 5 Ion pair HPLC analysis results of the reaction solution after the reaction.
  • the experimental methods in the present invention are conventional methods unless otherwise specified, and specific gene cloning operations can be found in the "Molecular Cloning Experimental Guide” edited by J. Sambrook et al.
  • codons corresponding to the amino acids are also conventional in the art, and the corresponding relationships between specific amino acids and codons are shown in Table 2.
  • pET28a was purchased from Novagen; NdeI enzyme and HindIII enzyme were purchased from Thermo Fisher Company, E.coli BL21(DE3) competent cells were purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.; Catalase was purchased from Shandong Fengtai Biotechnology Co., Ltd.; NADPH was purchased from Shenzhen Bangtai Biological Engineering Co., Ltd.; NH 4 Cl was purchased from Shanghai Titan Technology Co., Ltd.
  • the chiral analysis of the product is carried out by pre-column derivatization high performance liquid chromatography (High Performance Liquid Chromatography, HPLC), the specific analysis method is:
  • Chromatographic conditions Agilent ZORBAX Eclipse plus C18, 3.5 ⁇ m, 150*4.6mm.
  • Mobile phase A 0.1% TFA+H 2 O
  • mobile phase B 0.1% TFA+CAN.
  • Detection wavelength 340nm
  • flow rate 1.0mL/min
  • column temperature 30°C.
  • the descriptions are all “glufosinate-ammonium”, but since “glufosinate-ammonium” is in the reaction system, the technicians refer to “glufosinate-ammonium salt” as “glufosinate-ammonium” by default.
  • Glufosinate-ammonium refers to "glufosinate-ammonium ammonium salt”
  • the corresponding glufosinate-ammonium standards also refer to glufosinate ammonium salt standards
  • the corresponding PPO is also PPO ammonium salt.
  • LsGluDH glutamate dehydrogenase sequence SEQ ID NO.1 from Lysinibacillus sphaericus retrieved from NCBI, Genbank accession number WP_012293812.1, according to the nucleotide sequence SEQ ID NO of the mutant gene in Table 3 .4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10 synthetic gene, the gene synthesis company is Suzhou Jinweizhi Biotechnology Co., Ltd. (Suzhou Industrial Park Xinghu Street 218 Bio-Nano Science Park C3 Floor).
  • mutant genes were enzymatically linked to pET28a, and the restriction sites were NdeI&HindIII.
  • the vector linked to the enzyme is transformed into the competent host E. coli BL21 cells.
  • the constructed strains were inoculated with TB medium at 37° C., 200 rpm shaker, and IPTG concentration of 0.1 mM was induced overnight.
  • the bacteria were harvested to obtain engineered bacteria containing glutamate dehydrogenase gene.
  • Substrate solution configuration add 355 ⁇ L 2.25M PPO (final concentration 20mM) (made by the inventor, preparation method reference US8017797B, Figure 6 is the corresponding mass spectrum) and 0.4g NH 4 Cl (final concentration 200mM), adjusted with ammonia pH to 8.5, dilute to 40ml with 50mM Tris-HCl buffer of pH8.5.
  • the total reaction system is 1ml, and the absorbance is measured at OD340nm.
  • 940 ⁇ L substrate solution is added to a 1ml cuvette, adjusted to zero, then 10 ⁇ L 25mM NADPH is added, and finally 50 ⁇ l crude enzyme solution is added, and the value change from 0-10min is recorded.
  • Take a value every 30s use the reaction time as the abscissa and the absorption value at the 340nm wavelength as the ordinate to make a curve, take the slope, calculate the rate of decrease of NADPH, and calculate the enzyme activity.
  • Specific enzyme activity is the unit of activity per milligram of enzyme protein.
  • the DAAO enzyme gene was fully synthesized according to the AC302DAAO gene sequence described in the patent US9834802B2.
  • the synthetic company is Suzhou Jinweizhi Biological Technology Co., Ltd., No. 211 Pubin Road, Yanchuang Park, Jiangbei New District, Nanjing City, Jiangsu province.
  • composition of LB liquid medium peptone 10g/L, yeast powder 5g/L, NaCl 10g/L, dissolved in deionized water and constant volume, sterilized at 121°C for 20 minutes, set aside.
  • the DAAO enzyme gene synthesized in Example 3 was linked to pET28a, and the restriction sites were NdeI&HindIII, and the vector linked to the enzyme was transformed into host E. coli BL21 (DE3) competent cells to obtain an engineered strain containing DAAO enzyme.
  • Example 4 The bacteria collected in Example 4 were washed twice with 50mM pH 8.0 phosphate buffer solution, and then the bacteria body was suspended in pH 8.0 phosphate buffer solution, homogenized at low temperature and high pressure, and the crushed liquid was centrifuged to remove the precipitate.
  • the obtained supernatant is a crude enzyme solution containing recombinant DAAO enzyme.
  • Enzyme activity detection method 100 ⁇ L pH 8.0 disodium hydrogen phosphate-sodium dihydrogen phosphate buffer (containing D- glufosinate-ammonium 50mmol/L and peroxidase 0.1mg/mL), add 50 ⁇ L of indicator (60 ⁇ g/mL 2, 4,6-Tribromo-3-hydroxybenzoic acid and 1mg/mL 4-aminoantipyrine), 50 ⁇ L of DAAO enzyme, UV absorption at 510nm to determine the H 2 O 2 concentration, calculate the PPO concentration, and calculate Enzyme activity.
  • indicator 60 ⁇ g/mL 2, 4,6-Tribromo-3-hydroxybenzoic acid and 1mg/mL 4-aminoantipyrine
  • unit enzyme activity under specific reaction conditions (30°C), the amount of enzyme required to produce 1 ⁇ mol PPO per minute.
  • composition of LB liquid medium peptone 10g/L, yeast powder 5g/L, NaCl 10g/L, dissolved in deionized water and constant volume, sterilized at 121°C for 20 minutes, set aside.
  • the alcohol dehydrogenase gene is linked to pET28a, and the enzyme cut sites are NdeI&HindIII, and the vector linked to the enzyme is transformed into host E.coli BL21(DE3) competent cells to obtain an engineered strain containing the alcohol dehydrogenase gene. After the engineered bacteria containing the alcohol dehydrogenase gene are activated by streaking on the plate, a single colony is inoculated into 5ml LB liquid medium containing 50 ⁇ g/ml kanamycin, and cultured with shaking at 37°C for 12h.
  • Example 6 Take the bacteria collected in Example 6, take 10g of bacteria mud and add 50ml 100mM pH7.5 ammonium phosphate buffer solution, stir evenly, 500bar homogeneously broken, as a crude enzyme solution, add 10% flocculant dropwise under stirring conditions (Final concentration 2-2.5 ⁇ ), stir for 5 minutes, centrifuge at 4000 rpm for 10 minutes to obtain a clear enzyme solution, and take the supernatant to measure the enzyme activity.
  • Enzyme activity detection method 3ml reaction system, at 25°C, add 2850 ⁇ L pH8.0 400mM isopropanol (prepared with 100mM phosphate buffer), then add 50 ⁇ L NADP + (25mM), adjust the UV spectrophotometer to zero, and then Add 100 ⁇ L of enzyme solution diluted 100 times, and measure the OD value at 340nm by UV spectrophotometer.
  • unit enzyme activity Under specific reaction conditions (25°C, pH 7.0), the amount of enzyme required to produce 1 ⁇ mol NADPH per minute is defined as 1U.
  • L-glutamate dehydrogenase mutant cells prepared according to Example 1 were resuspended in 50mM phosphate buffer with pH 8.0, and the volume was adjusted to 1L. The mixture was homogenized under low temperature and high pressure, and the precipitate was discarded by centrifugation. The supernatant was used to obtain the crude enzyme solution of L-glutamate dehydrogenase mutant.
  • the reaction was mechanically stirred in a water bath at 20°C, air was blown in according to 1VVM (1 times the reaction volume of air per minute), 1mL of antifoaming agent was added to prevent foaming, the concentration of PPO was detected by ion-pair HPLC, and the pre-column Derivatization high performance liquid chromatography detects the amount of remaining L-glufosinate-ammonium and ee value, and the reaction is stopped when the ee value is greater than 99%.
  • the ion-pair HPLC analysis result of the reaction solution is shown in Fig. 5.
  • the peak position of PPO has no peak
  • 3.828min is the peak position of glufosinate-ammonium.
  • the ion-pair HPLC spectrum of the PPO standard (this standard is made by the inventor, and the preparation method refers to US8017797B, Figure 6 is the corresponding mass spectrum) is shown in Figure 4, where the retention time of the PPO standard is 9.520 min.
  • Example 2 the mutant enzyme of Pseudomonas putida (Genbank accession number: NP_742836.1) glutamate dehydrogenase (hereinafter referred to as PpGluDH) disclosed in CN108588045A was obtained, and it was described in Example 2.
  • PpGluDH glutamate dehydrogenase

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Abstract

本发明公开了一种L-谷氨酸脱氢酶突变体，该L-谷氨酸脱氢酶突变体的序列为将SEQ ID NO.1的第175位氨基酸残基A突变为G，和，第386位氨基酸残基V突变为空间位阻更小的氨基酸残基后的序列。本发明还公开了该L-氨基酸脱氢酶突变体在制备L-草铵膦或其盐中的应用。本发明的L-谷氨酸脱氢酶突变体制备L-草铵膦或其盐时，与仅在175位或者仅在386突变的L-谷氨酸脱氢酶突变体相比，比酶活更高，从而酶的作用效率提高，降低了反应的成本，利于工业化生产。

Description

一种L-谷氨酸脱氢酶突变体及其应用
本申请要求申请日为2019年5月23日的中国专利申请CN201910434350.1的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明属于生物技术领域，具体涉及一种L-谷氨酸脱氢酶突变体及其应用。
背景技术
草铵膦(2-氨基-4-[羟基(甲基)膦酰基]丁酸)是由赫斯特公司80年代开发的广谱触杀型除草剂。目前世界上的三大除草剂是草甘膦、草铵膦、百草枯，相对于草甘膦和百草枯，草铵膦具有优异的除草性能及较小的副作用。草铵膦有两种光学异构体，分别为D-草铵膦和L-草铵膦，但是只有L-草铵膦具有除草活性，因此发展L-草铵膦的方法对于提高原子经济性、降低使用成本、减轻环境压力具有重要意义。
目前，制备L-草铵膦的方法主要有手性拆分法，化学合成法和生物催化法。
手性拆分法如CN1053669C公开了利用奎宁生物碱作为拆分剂的方法，重结晶出L-草铵膦奎宁盐，然后用酸中和盐可以得到L-草铵膦。同时利用5-硝基水杨醛或3,5-二硝基水杨醛作为消旋化试剂消旋化未反应的D-草铵膦，得到DL-草铵膦，继续用于拆分反应。但是这种方法需要昂贵的手性拆分试剂，以及多步重结晶，操作繁琐，还不是一种理想的方法。
Figure PCTCN2020089775-appb-000001
化学合成法如US6936444公开了钌催化剂不对称氢化2-乙酰氨基-4-(羟甲基氧膦基)-2-丁烯酸得到L-2-乙酰氨基-4-(羟甲基氧膦基)-2-丁酸，再经脱乙酰可以得到L-草铵膦。此方法需要昂贵的金属催化剂，成本较高，以及重金属残留，对环境污染严重。
Figure PCTCN2020089775-appb-000002
相对于手性拆分法，化学合成法，生物催化法具有专一性强，反应条件温和等优点，是生产L-草铵膦的优势方法。
目前有US4389488(A)记载的用N-苯乙酰-DL-草铵膦为底物，来源于大肠杆菌的青霉素-G-酰基转移酶为催化剂，得到L-草铵膦的方法，但是苯乙酰草铵膦的合成成本比较高，且反应结束后得到L-草胺膦，N-苯乙酰-D-草铵膦和苯乙酸的混合溶液，需要采用强酸阳离子交换树脂分离出L-草铵膦，操作比较复杂。
Figure PCTCN2020089775-appb-000003
EP0382113A记载了利用酰基转移酶对N-乙酰-草铵膦的羧酸酯进行催化裂解得到L-草铵膦的方法，但是此方法中的酶对游离的N-乙酰-草铵膦没有特异性，因此必须对N-乙酰-草铵膦进行酯化，增加了反应的步骤，且相应的增加了生产成本。
另有一部分是采用2-氧代-4-(羟基甲基氧膦基)丁酸(PPO)为底物，经转氨酶催化制备L-草铵膦的方法，其中US5221737A和EP0344683A记载了用谷氨酸作为氨基供体，自相应酮酸4-(羟甲基氧膦基)-2-氧代丁酸通过来源于大肠杆菌的氨基转氨酶作用得到L-草铵膦的方法，反应体系中需要等量或过量的氨基供体谷氨酸，使产物难以纯化。CN1284858C对上述方法进行了改进，采用天冬氨酸作为氨基供体，自相应的酮酸4-(羟甲基氧膦基)-2-氧代丁酸通过天冬氨酸转氨酶作用得到L-草铵膦的方法，此方法中天冬氨酸转变为草酰乙酸，而草酰乙酸在含水介质中不稳定，并自发的脱羧为丙酮酸，丙酮酸可通过酶促反应除去，使得逆反应不能进行，反应只需要等摩尔的氨基供体和氨基受体。但是使用转氨酶的方法中所用的氨基供体多为氨基酸，成本较高。
Figure PCTCN2020089775-appb-000004
另外还有采用2-氧代-4-(羟基甲基氧膦基)丁酸(PPO)为底物，经氨基酸脱氢酶催化，制备L-草铵膦的方法，如CN106978453A，其采用无机氨基供体，使得产物分离简单，成本降低。但是CN106978453A中酶催化的底物浓度范围仅为10-100mM，酶的催化效率不高。
Figure PCTCN2020089775-appb-000005
CN108588045A公开了多个谷氨酸脱氢酶突变体在制备L-草铵膦中的应用，发现来 源于pseudomonas putida的谷氨酸脱氢酶(NCBI登录号：NP_742836.1)，将其167位丙氨酸突变为甘氨酸，或者将378位缬氨酸突变为丙氨酸，都提高了该酶对PPO的催化能力，继而研究了其它来源的谷氨酸脱氢酶的第167和378位同源位点的突变体，发现这样的突变体同样可以提高谷氨酸脱氢酶对PPO的催化能力；但是并没有对这些其它来源的谷氨酸脱氢酶的同源位点的突变体的组合突变进行研究。此外，虽然该专利申请中将pseudomonas putida的谷氨酸脱氢酶的这两个位点同时进行了突变，所得双突变体的酶活提高的倍数只是与单突变的突变体相当，并且由于生物领域的不可预见性，双突变位点的突变体的效果并不一定优于其各自的单突变的突变体。
发明内容
本发明所要解决的技术问题是现有的L-谷氨酸脱氢酶在制备L-草铵膦或其盐时催化效率低等缺陷，因此本发明提供一种L-谷氨酸脱氢酶突变体以及其在制备L-草铵膦或其盐中的应用。利用本发明的L-谷氨酸脱氢酶突变体制备L-草铵膦或其盐时与单突变位点(175或386位)的谷氨酸脱氢酶突变体相比，比酶活更高，从而酶的作用效率提高，降低了反应的成本，利于工业化生产。
本发明所用的野生型L-谷氨酸脱氢酶来源是Lysinibacillus sphaericus的谷氨酸脱氢酶，氨基酸序列如SEQ ID NO.1所示，Genbank登录号WP_012293812.1。现有技术中并没有将该谷氨酸脱氢酶同时进行两个位点的双突变，并且由于生物领域的不可预见性，双突变位点的突变体的效果并不一定优于其各自的单突变的突变体。而本发明人针对该底物2-氧代-4-(羟基甲基氧膦基)丁酸(PPO)对上述野生型酶的175位和386位进行了组合突变，意外发现当第175位氨基酸残基A突变为G，并且第386位氨基酸残基V突变为空间位阻更小的氨基酸残基后，所得的突变体对底物PPO的比酶活有明显提升。
本发明解决上述技术问题的技术方案之一是：一种L-谷氨酸脱氢酶突变体，所述L-谷氨酸脱氢酶突变体的序列为将如SEQ ID NO.1所述序列的第175位氨基酸残基A突变为G，和，第386位氨基酸残基V突变为空间位阻更小的氨基酸残基后的序列，所述L-谷氨酸脱氢酶突变体具有催化2-氧代-4-(羟基甲基氧膦基)丁酸或其盐的活性。
较佳地，所述L-谷氨酸脱氢酶突变体的氨基酸序列如序列表中SEQ ID NO.7或SEQ ID NO.9所示。
较佳地，所述L-谷氨酸脱氢酶突变体的核苷酸序列如序列表中SEQ ID NO.8或SEQ ID NO.10所示。
根据本发明，所述的空间位阻更小是指突变后的氨基酸残基和野生序列中氨基酸残 基相比空间位阻更小。所述的氨基酸可以为修饰或未修饰的天然氨基酸；本发明以天然氨基酸为例。
本发明解决上述技术问题的技术方案之二是：一种分离的核酸，所述核酸编码上述的L-谷氨酸脱氢酶突变体。
较佳地，编码所述核酸的核苷酸序列如SEQ ID NO.8或SEQ ID NO.10所示。
本发明解决上述技术问题的技术方案之三是：一种包含所述核酸的重组表达载体。
本发明解决上述技术问题的技术方案之四是：一种包含所述核酸或所述重组表达载体的转化体。
本发明解决上述技术问题的技术方案之五是：一种L-草铵膦盐的制备方法，其包括以下步骤：在反应溶剂、L-谷氨酸脱氢酶突变体、无机氨基供体和还原型辅酶NADPH(烟酰胺腺嘌呤二核苷酸磷酸)的存在下，将2-氧代-4-(羟基甲基氧膦基)丁酸盐进行氨化反应，即得L-草铵膦盐。
所述制备方法中，除L-谷氨酸脱氢酶突变体为本发明所得，其余原料、反应步骤及条件均可为本领域常规，具体可参见上述CN106978453A以及本申请人的申请号为CN201810162629.4的专利申请。
所述的L-草铵膦盐的制备方法还可进一步包括以下步骤：在D-氨基酸氧化酶(DAAO)的存在下，将D-草铵膦盐进行氧化反应，得到所述的2-氧代-4-(羟基甲基氧膦基)丁酸盐即可。
在所述的氧化反应中，所述的D-草铵膦盐的阳离子可为本领域常规的阳离子，例如铵离子、钠离子和/或钾离子等。又可为所使用的缓冲液的阳离子。
在所述的氧化反应中，所述的D-草铵膦盐可单独存在、或、与L-草铵膦盐共同存在(此时L-草铵膦盐可不发生反应)，例如：D型富集的草铵膦盐(即其中D型对映体的含量＞50％，乃至于纯D-草铵膦盐)、L型富集的草铵膦盐(即其中L-草铵膦的含量＞50％，不包括纯L-草铵膦盐的情况)或消旋体草铵膦盐等。
在所述的氧化反应中，所述的D氨基酸氧化酶(DAAO)的浓度可为本领域常规，较佳地为0.6-6U/mL，更佳地为1.8U/mL。
在所述的氧化反应中，所述D-草铵膦盐的浓度可为本领域常规，较佳地为100-600mM，更佳地为200mM。
所述的氧化反应还可在过氧化氢酶的存在下进行。
所述的氧化反应还可在通气的条件下进行。所述通气较佳地为通入空气或氧气。所述通气的速率较佳地为0.5-1VVM。
本发明中，所述空气可为本领域常规的空气，一般都是含有氧气的，所含氧气的含量也是本领域常规的。在反应时参与反应的是空气中的氧气。
当所述的氧化反应还可在通气的条件下进行时，所述的氧化反应还可在消泡剂的存在下进行。
在所述的氧化反应中，所述反应体系的pH较佳地为7-9，更佳地为8。所述的pH可通过使用缓冲液来实现。所述的pH还可通过使用碱(或碱溶液)调节来实现。所述的缓冲液较佳地为磷酸盐缓冲液或者Tris-HCl缓冲液，所述的磷酸盐缓冲液较佳地为磷酸氢二钠-磷酸二氢钠缓冲液或者磷酸氢二钾-磷酸二氢钾缓冲液。所述的碱溶液较佳地为氨水。
在所述的氧化反应中，所述反应体系的温度可为本领域常规，较佳地为20-50℃，更佳地为20℃。
所述的氧化反应和所述的氨化反应可分开进行，也可同时(同一反应体系)进行。所述的同时进行例如：在D-氨基酸氧化酶(DAAO)、L-谷氨酸脱氢酶突变体、无机氨基供体和还原型辅酶NADPH的存在下，将D-草铵膦盐进行氧化反应和氨化反应，得到L-草铵膦盐即可。
在所述的氨化反应中，所述的L-草铵膦盐的阳离子可为本领域常规的阳离子，例如铵离子、钠离子和/或钾离子等。又可为所使用的缓冲液的阳离子。
在所述的氨化反应中，所述的2-氧代-4-(羟基甲基氧膦基)丁酸盐的阳离子可为本领域常规的阳离子，例如铵离子、钠离子和/或钾离子等。又可为所使用的缓冲液的阳离子。
在所述的氨化反应中，所述的L-谷氨酸脱氢酶突变体的用量可为本领域常规，所述L-谷氨酸脱氢酶突变体的浓度较佳地为0.05-2.3U/ml，更佳地为0.1-1U/ml，例如为0.23U/ml。
在所述的氨化反应中，所述的无机氨基供体的用量可为本领域常规，所述的无机氨基供体的浓度较佳地为100-2000mM，更佳地为200mM。
在所述的氨化反应中，所述的2-氧代-4-(羟基甲基氧膦基)丁酸盐的浓度较佳地为100-600mM，更佳地为200mM。
在所述的氨化反应中，所述的2-氧代-4-(羟基甲基氧膦基)丁酸盐的用量可为本领域常规，所述还原型辅酶NADPH与所述的2-氧代-4-(羟基甲基氧膦基)丁酸盐的质量比较佳地为1:100-1:20000，更佳地为1:1000-1:15000，进一步更佳地为1:5000。
在所述的氨化反应中，所述的无机氨基供体为氨气、硫酸铵、氯化铵、磷酸氢二铵、乙酸铵、甲酸铵和碳酸氢铵中的一种或多种。
在所述的氨化反应中，所述反应的温度可为本领域常规，为了保证所述的L-谷氨酸脱氢酶突变体的催化效率，进行所述氨化反应的温度较佳地为20-50℃，更佳地为37℃，当所述氨化反应的温度低于20℃时，氨化反应较慢；当所述氨化反应的温度高于50℃时，酶将不可逆变性失活。
在所述的氨化反应中，所述的反应溶剂为水。
在所述的制备方法中，进行所述氨化反应的pH较佳地为7-9，更佳地为8.5。所述的pH可通过使用缓冲液来实现。所述的pH还可通过使用碱(或碱溶液)调节来实现。所述的缓冲液较佳地为磷酸盐缓冲液或者Tris-HCl缓冲液等，所述的磷酸盐缓冲液较佳地为磷酸氢二钠-磷酸二氢钠缓冲液或者磷酸氢二钾-磷酸二氢钾缓冲液等。所述的碱溶液较佳地为氨水。
所述的L-草铵膦盐的制备方法还包括以下步骤：在脱氢酶(例如葡萄糖脱氢酶、醇脱氢酶或甲酸脱氢酶等)以及供氢体(例如葡萄糖、异丙醇或甲酸盐等)的存在下，将氧化型辅酶NADP +进行还原反应，得到所述的还原型辅酶NADPH即可。
在所述的还原反应中，所述的脱氢酶与所述的供氢体一一对应，例如：
当所述的脱氢酶为醇脱氢酶时，所述的供氢体为异丙醇；
当所述的脱氢酶为葡萄糖脱氢酶时，所述的供氢体为葡萄糖；
当所述的脱氢酶为甲酸脱氢酶时，所述的供氢体为甲酸盐。
在所述的还原反应中，所述的脱氢酶的浓度可为本领域常规，较佳地为0.6-6U/mL，更佳地为2U/mL。
在所述的还原反应中，所述的供氢体的浓度可为本领域常规，较佳地为100-1000mM，更佳地为240mM。
在所述的还原反应中，所述的氧化型辅酶NADP +的浓度可为本领域常规。
在所述的还原反应中，进行所述还原反应的pH较佳地为7-9，更佳地为8.5。所述的pH可通过使用缓冲液来实现。所述的pH还可通过使用碱(或碱溶液)调节来实现。所述的缓冲液较佳地为磷酸盐缓冲液或者Tris-HCl缓冲液等，所述的磷酸盐缓冲液较佳地为磷酸氢二钠-磷酸二氢钠缓冲液或者磷酸氢二钾-磷酸二氢钾缓冲液等。所述的碱溶液较佳地为氨水。
在所述的还原反应中，所述反应体系的温度可为本领域常规，较佳地为20-50℃，更佳地为37℃。
所述的还原反应和所述的氨化反应可分开进行，也可同时(同一反应体系)进行。所述的同时进行例如本发明较佳实施例中所示：在葡萄糖脱氢酶、葡萄糖、氧化型辅酶 NADP +、L-谷氨酸脱氢酶突变体、无机氨基供体的存在下，将2-氧代-4-(羟基甲基氧膦基)丁酸盐进行氨化反应(同时存在着的NADP +的还原反应)，得到L-草铵膦盐即可。
当所述的还原反应和所述的氨化反应同时进行时，所述的氨化反应所用的NADPH可通过所述的还原反应循环生成。所述的氧化型辅酶NADP +的浓度可为本领域常规，为保证所述反应能够正常进行，其与所述的2-氧代-4-(羟基甲基氧膦基)丁酸盐的质量比为1:100-1:20000，较佳地为1:1000-1:15000，更佳地为1:5000。
所述的还原反应、所述的氧化反应和所述的氨化反应可分开进行，也可同时(同一反应体系)进行。所述的同时进行例如本发明较佳实施例中所示：在D-氨基酸氧化酶(DAAO)、脱氢酶、氢供体、氧化型辅酶NADP +、L-谷氨酸脱氢酶突变体、无机氨基供体的存在下，将D-草铵膦盐进行氧化反应和氨化反应(同时存在着的NADP +的还原反应)，得到L-草铵膦盐即可。
当所述的还原反应、所述的氧化反应和所述的氨化反应同时进行时，所述的氨化反应所用的NADPH可通过所述的还原反应循环生成。所述的氧化型辅酶NADP +的浓度可为本领域常规，为保证所述反应能够正常进行，NADP +与所述的2-氧代-4-(羟基甲基氧膦基)丁酸盐的质量比为1:100-1:20000，较佳地为1:1000-1:15000，更佳地为1:5000。
所述制备方法的反应时间在用常规方法进行检测的情况下，以原料的终浓度或产物的终浓度或转化率达到所需目的即可停止；所述常规方法包括柱前衍生化高效液相色谱或离子对色谱等。
本发明解决上述技术问题的技术方案之六是：一种L-草铵膦的制备方法，其包括下述步骤：
(1)按照上述的L-草铵膦盐的制备方法，制得L-草铵膦盐；
(2)将步骤(1)制得的L-草铵膦盐进行酸化反应，得到L-草铵膦。
本发明解决上述技术问题的技术方案之七是：一种上述制得的L-谷氨酸脱氢酶突变体在制备L-草铵膦或其盐中的应用。
所述的应用可包括以下步骤：在L-氨基酸脱氢酶、无机氨基供体和还原型辅酶的存在下，将2-氧代-4-(羟基甲基氧膦基)丁酸盐进行反应，即得L-草铵膦盐。
或者，所述的应用可包括以下步骤：在L-氨基酸脱氢酶、无机氨基供体和还原型辅酶的存在下，将2-氧代-4-(羟基甲基氧膦基)丁酸进行反应，即得L-草铵膦。
或者，所述的应用可包括以下步骤：在L-氨基酸脱氢酶、无机氨基供体和还原型辅酶的存在下，将2-氧代-4-(羟基甲基氧膦基)丁酸盐进行反应，即得L-草铵膦盐，再进行酸化反应，得到L-草铵膦。
本发明中，所述L-草铵膦盐一般可以是以L-草铵膦铵盐的形式存在。
以上化合物所述浓度若无特殊说明，均为反应前所述化合物占整个反应体系的浓度。
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于：
本发明的L-谷氨酸脱氢酶突变体制备L-草铵膦或其盐时与单突变位点(第175位或者第386位)的谷氨酸脱氢酶突变体相比，比酶活更高，从而酶的作用效率提高(例如参与反应时的转化率更高、立体选择性更强)，降低了反应的成本，利于工业化生产。
附图说明
图1：外消旋草铵膦标准品的Marfey试剂柱前衍生化HPLC分析结果，其中最后两个峰是Marfey试剂自身的峰。
图2：制备所得产物中D-草铵膦与L-草铵膦的Marfey试剂柱前衍生化HPLC分析结果。
图3：外消旋草铵膦标准品的离子对HPLC分析结果。
图4：PPO标准品离子对HPLC分析结果。
图5：反应完后反应液的离子对HPLC分析结果。
图6：PPO标准品的质谱图。
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
本发明中的实验方法如无特别说明均为常规方法，基因克隆操作具体可参加J.萨姆布鲁克等编的《分子克隆实验指南》。
本发明中的氨基酸简写符号如无特殊说明均为本领域常规，具体简写符号对应的氨基酸如表1所示。
表1
氨基酸名称 三字母符号 单字母符号 氨基酸名称 三字母符号 单字母符号
丙氨酸(alanine) Ala A 亮氨酸(leucine) Leu L
精氨酸(arginine) Arg R 赖氨酸(lysine) Lys K
天冬酰胺(asparagine) Asn N 甲硫氨酸(methionine) Met M
天冬氨酸(aspartic acid) Asp D 笨丙氨酸(phenylalanine) Phe F
半胱氨酸(cysteine) Cys C 脯氨酸(proline) Pro P
谷氨酰胺(glutanine) Gln Q 丝胺酸(serine) Ser S
谷氨酸(glutamic acid) Glu E 苏氨酸(threonine) Thr T
甘氨酸(Glicine) Gly G 色氨酸(tryptophan) Trp W
组氨酸(histidine) His H 酪氨酸(tyrosine) Tyr Y
异亮氨酸(isoleucine) Ile I 颉氨酸(valine) Val V
所述氨基酸对应的密码子也为本领域常规，具体氨基酸与密码子的对应关系如表2所示。
表2
Figure PCTCN2020089775-appb-000006
pET28a购买自Novagen公司；NdeI酶、HindIII酶购买自Thermo Fisher公司，E.coli BL21(DE3)感受态细胞购买自北京鼎国昌盛生物技术有限责任公司；过氧化氢酶购买自山东丰泰生物科技有限公司；NADPH购买自深圳邦泰生物工程有限公司；NH 4Cl购买自上海泰坦科技股份有限。
产物的手性分析通过柱前衍生化高效液相色谱(High Performance Liquid Chromatography，HPLC)进行，具体的分析方法为：
(1)色谱条件：Agilent ZORBAX Eclipse plus C18，3.5μm，150*4.6mm。流动相A：0.1％TFA+H 2O，流动相B：0.1％TFA+CAN。检测波长：340nm，流速：1.0mL/min，柱温：30℃。
(2)衍生化试剂：Marfey试剂。准确称取50mg的N-α-(2,4-二硝基-5-氟苯基)-L-丙氨酰胺，用乙腈溶解配置成25ml溶液，备用。
(3)衍生反应：取反应液稀释100倍，加等体积的Marfey试剂衍生。进样10μl进行分析。
转化率＝(反应物-剩余反应物)/反应物×100％
2-氧代-4-(羟基甲基氧膦基)丁酸(简称PPO)通过离子对高效液相色谱(High Performance Liquid Chromatography，HPLC)分析，具体的分析方法为：
色谱条件：ULtimate AQ-C18，5μm，4.6*250mm；流动相：0.05mol/L磷酸氢二铵PH＝3.6:10％四丁基氢氧化铵水溶液：乙腈＝91:1:8；检测波长：205nm；流速：1.0ml/min；柱温：25℃。
以下实施例中，所述均为“草铵膦”，但由于“草铵膦”是在反应体系中，技术人员默认将“草铵膦铵盐”称为“草铵膦”，因此实则“草铵膦”是指“草铵膦铵盐”，相应的草铵膦标准品也均是指草铵膦铵盐标准品，相应的生成的PPO也即是PPO铵盐。在检测所得产物草铵膦的旋光度时，则是将草铵膦铵盐进行酸化反应后，得到草铵膦再进行的检测。
实施例1 L-谷氨酸脱氢酶突变体酶的获得
从NCBI检索到来源于Lysinibacillus sphaericus的谷氨酸脱氢酶(以下简称为LsGluDH)序列SEQ ID NO.1，Genbank登录号WP_012293812.1，根据表3中突变体基因的核苷酸序列SEQ ID NO.4，SEQ ID NO.6，SEQ ID NO.8，SEQ ID NO.10合成基因，基因合成公司为苏州金唯智生物科技有限公司(苏州工业园区星湖街218号生物纳米科技园C3楼)。
然后将突变体基因分别酶连pET28a，酶切位点为NdeI&HindIII。将酶连好的载体，转化宿主大肠杆菌BL21感受态细胞。将构建好的菌种接种TB培养基于37℃下，200rpm摇床，IPTG浓度0.1mM诱导过夜，收菌，得到含有谷氨酸脱氢酶基因的工程菌。
将含有谷氨酸脱氢酶基因的工程菌在经平皿划线活化后，挑单菌落接种至含50μg/ml卡那霉素的5ml LB液体培养基中，37℃震荡培养12h。按2％接种量转接至150ml同样含50μg/ml卡那霉素的新鲜LB液体培养基中，37℃震荡至OD600达到0.8左右时，加入IPTG至其终浓度为0.5mM，18℃诱导培养16h。培养结束后，将培养液10000rpm离心10min，弃上清液，收集菌体，置于-80℃超低温冰箱中保存，待用。
将上述收集到的菌体5g，用50mM pH8.5的Tris-HCl缓冲液洗涤两次，之后重悬于30mL pH8.5的Tris-HCl缓冲液中，均质破碎，破碎液12000rpm离心10min去除沉淀，得到含重组谷氨酸脱氢酶的上清液粗酶液。
表3
Figure PCTCN2020089775-appb-000007
实施例2 突变体酶的比酶活检测
底物溶液配置：加入355μL 2.25M PPO(终浓度20mM)(为发明人自制，制备方法参考文献US8017797B，图6为其对应的质谱图)与0.4g NH 4Cl(终浓度200mM)，氨水调pH至8.5，用50mM pH8.5的Tris-HCl缓冲液定容至40ml。
酶活检测方法：
反应总体系为1ml，OD340nm处测定吸光值，依次在1ml比色皿中加入940μL底物溶液，调零，然后加入10μL 25mM NADPH，最后加入50μl粗酶液，记录0-10min的数值变化，每隔30s取一个值，以反应时间为横坐标，340nm波长处吸收值为纵坐标做曲线，取斜率，计算NADPH的减少速率，并计算酶活。
单位酶活的定义：在特定反应条件(30℃)下，每分钟减少1μmol NADPH所需要的酶量。
比酶活为每毫克酶蛋白所含的活力单位，计算公式：酶活/蛋白含量，单位是U/mg或U/g。结果如表4所示。
从CN108588045A中可以得知野生型LsGluDH-WT(WP_012293812.1)的酶活比单位点突变体的酶活低很多，本领域技术人员能够得出野生型LsGluDH-WT(WP_012293812.1)的比酶活也比突变体低很多，故本发明中未对野生型LsGluDH-WT(WP_012293812.1)进行检测。
表4
Figure PCTCN2020089775-appb-000008
以下实施例中所用到的L-谷氨酸脱氢酶粗酶液的制备方法均采用上述方法。
实施例3 D氨基酸氧化酶(DAAO)基因的获取
根据专利US9834802B2中记载的AC302DAAO酶的基因序列全合成DAAO酶基因。合成公司为苏州金唯智生物科技有限公司，江苏省南京市江北新区研创园浦滨路211号。
实施例4 D氨基酸氧化酶(DAAO)基因的表达
LB液体培养基组成：蛋白胨10g/L，酵母粉5g/L，NaCl 10g/L，用去离子水溶解后定容，121℃灭菌20min，待用。
将实施例3合成的DAAO酶基因连pET28a，酶切位点NdeI&HindIII，将酶连好的载体转化至宿主E.coli BL21(DE3)感受态细胞，得到含有DAAO酶的工程菌株。
将含有DAAO酶基因的工程菌株在经平皿划线活化后，挑单菌落接种至含50μg/ml卡那霉素的5ml LB液体培养基中，37℃震荡培养12h。按2％接种量转接至50ml同样含50μg/ml卡那霉素的新鲜LB液体培养基中，37℃震荡至OD 600值达到0.8左右时，加入IPTG至其终浓度为0.5mM，18℃诱导培养16h。培养结束后，将培养液10000rpm 离心10min，弃上清液，收集菌体，置于-20℃超低温冰箱中保存，待用。
实施例5 D氨基酸氧化酶(DAAO)粗酶液的制备及酶活测定
将实施例4中收集到的菌体，用50mM pH 8.0磷酸缓冲液洗涤菌体两次，之后将菌体重悬于pH 8.0的磷酸缓冲液中，低温高压均质破碎，破碎液离心去除沉淀，得到的上清液为含重组DAAO酶粗酶液。
酶活检测方法：100μL pH 8.0磷酸氢二钠-磷酸二氢钠缓冲液(含D-草铵膦50mmol/L和过氧化物酶0.1mg/mL)，加入50μL显示剂(60μg/mL 2,4,6-三溴-3-羟基苯甲酸和1mg/mL 4-氨基安替比林)，50μL的DAAO酶，510nm处检测紫外吸收测定H 2O 2浓度，计算出PPO的浓度，并计算酶活。
单位酶活的定义：在特定反应条件(30℃)下，每分钟生成1μmol PPO所需要的酶量。
以下实施例中所用到的DAAO酶粗酶液的制备方法均采用以上方法。
实施例6 醇脱氢酶基因的获取和表达
根据来源于枯草芽胞杆菌(Lactobacillus brevis KB290)(Genbank登录号为BAN05992.1)的Cyclopentanol dehydrogenase基因序列，全合成醇脱氢酶基因。
LB液体培养基组成：蛋白胨10g/L，酵母粉5g/L，NaCl 10g/L，用去离子水溶解后定容，121℃灭菌20min，待用。
醇脱氢酶基因连pET28a，酶切位点NdeI&HindIII，将酶连好的载体转化至宿主E.coli BL21(DE3)感受态细胞，得到含有醇脱氢酶基因的工程菌株。将含有醇脱氢酶基因的工程菌在经平皿划线活化后，挑单菌落接种至含50μg/ml卡那霉素的5ml LB液体培养基中，37℃震荡培养12h。按2％接种量转接至50ml同样含50μg/ml卡那霉素的新鲜LB液体培养基中，37℃震荡至OD 600达到0.8左右时，加入IPTG至其终浓度为0.5mM，18℃诱导培养16h。培养结束后，将培养液10000rpm离心10min，弃上清液，收集菌体，置于-20℃超低温冰箱中保存，待用。
实施例7 醇脱氢酶粗酶液的制备及酶活测定
将实施例6中收集到的菌体，取10g菌泥加50ml 100mM pH7.5磷酸铵缓冲液，搅匀，500bar均质破碎，作为粗酶液，在搅拌条件下滴加10％的絮凝剂(终浓度2-2.5‰)，搅拌5min后，4000rpm离心10min得澄清酶液，取上清测酶活。
酶活检测方法：3ml反应体系，25℃条件下，先加2850μL pH8.0的400mM异丙醇(100mM磷酸缓冲液配制)，再加50μL NADP +(25mM)，紫外分光光度计调零，再加100μL稀释100倍的酶液，紫外分光光度计测定340nm处OD值。
单位酶活定义：在特定反应条件(25℃，pH 7.0)下，每分钟产生1μmol NADPH所需要的酶量，定义为1U。
以下实施例中所用到的醇脱氢酶粗酶液的制备方法均采用以上方法。
实施例8 DAAO酶和L-谷氨酸脱氢酶突变体催化制备L-草铵膦
200g L-谷氨酸脱氢酶突变体菌体(根据实施例1制备得到)用pH为8.0的50mM磷酸盐缓冲液重悬，定容至1L，低温高压均质破碎，离心弃沉淀，留上清，得L-谷氨酸脱氢酶突变体粗酶液。
称取D,L-草铵膦80g，用50mM pH为8.0的磷酸氢二钠-磷酸二氢钠缓冲液完全溶解，加入2.5g 40万U/g的过氧化氢酶，加入150mL根据实施例5方法制备的DAAO酶粗酶液(12U/mL)，氨水调节pH为8.0，50mM pH为8.0的磷酸氢二钠-磷酸二氢钠缓冲液定容至1L。20℃水浴锅中机械搅拌反应，按照1VVM通入空气(每分钟通入1倍反应体积的空气)，加入1mL消泡剂防止起泡，利用离子对HPLC检测PPO的生成浓度，同时利用柱前衍生化高效液相色谱检测剩余L-草铵膦的量和ee值，当ee值大于99％时停止反应。
取4等份50mL上述反应液，分别加入氯化铵0.54g，NADP +0.4mg和异丙醇0.73g，加入1mL实施例7方法制备的醇脱氢酶(300U/mL)，分别加入L-谷氨酸脱氢酶突变体粗酶液1mL，氨水调节pH至8.5，水浴锅磁力搅拌控制反应温度37℃，利用离子对HPLC检测PPO的残余浓度，同时利用柱前衍生化高效液相色谱检测体系中L-草铵膦的量和ee值。反应结束数据表5所示。
反应完后(18h)产物中D-草铵膦与L-草铵膦的HPLC分析结果见图2(附图中均以L-谷氨酸脱氢酶突变体1-4(LsGluDH-A166G-V376A)为例进行说明)，其中，保留时间为13.820min的为L-草铵膦，保留时间为12.469min的D-草铵膦几乎检测不到；外消旋草铵膦标准品(购买自上海阿拉丁生化科技股份有限公司)的Marfey试剂柱前衍生HPLC图谱如图1所示(L-草铵膦的保留时间为13.683min，D-草铵膦的保留时间为12.016min)。该实施例所制备得到的产物的成分与标准品中L-草铵膦的出峰时间基本一致。另外，对得到的产品经酸化处理，浓缩，柱纯化，重结晶得到纯的L-草铵膦，经旋光度测定[a]D 25＝+28.2°(C＝1,1N HCl)(现有技术中US4389488已有L-草铵膦旋光度的记载)， 说明该实施例制备得到L-草铵膦。
反应完后(18h)反应液的离子对HPLC分析结果见图5，其中，PPO的出峰位置无出峰，3.828min为草铵膦的出峰位置。PPO标准品(本标准品为发明人自制，制备方法参考文献US8017797B，图6为其对应的质谱图)的离子对HPLC图谱如图4所示，其中，PPO标准品的保留时间为9.520min。外消旋草铵膦标准品(购买自上海阿拉丁生化科技股份有限公司)的离子对HPLC图谱如图3所示，其中，外消旋草铵膦标准品的保留时间为3.829min。可见，该实施例中PPO最终被反应转化完全，其产物草铵膦的出峰时间与标准品的出峰时间基本一致。
虽然上述结果图均以L-谷氨酸脱氢酶突变体1-4(LsGluDH-A166G-V376A)为例，但是发明人进行了所有其他突变的实验，也均验证了本发明的这些突变在参与上述反应时能够催化底物，并且均生成了正确的产物。
表5
Figure PCTCN2020089775-appb-000009
对比例：
采用与实施例1同样的方法，获得CN108588045A中公开的Pseudomonas putida(Genbank登录号：NP_742836.1)谷氨酸脱氢酶(以下简称为PpGluDH)的突变体酶，并按实施例2中所述的方法测定比酶活，结果如表6所示：
表6
Figure PCTCN2020089775-appb-000010
Figure PCTCN2020089775-appb-000011
由表6可见，Pseudomonas putida来源的谷氨酸脱氢酶在其同源位点进行突变的突变体的比酶活显著低于本发明所得突变体，并且可见，并不是所有的双位点突变体的效果都比单突变的突变体的效果好。

Claims (12)

  1. 一种L-谷氨酸脱氢酶突变体，其特征在于，所述L-谷氨酸脱氢酶突变体的序列为将如SEQ ID NO.1所述序列的第175位氨基酸残基A突变为G，和，第386位氨基酸残基V突变为空间位阻更小的氨基酸残基后的序列，所述L-谷氨酸脱氢酶突变体具有催化2-氧代-4-(羟基甲基氧膦基)丁酸或其盐的活性。
  2. 如权利要求1所述的L-谷氨酸脱氢酶突变体，其特征在于，所述L-谷氨酸脱氢酶突变体的氨基酸序列如序列表中SEQ ID NO.7或SEQ ID NO.9所示；较佳地，所述L-谷氨酸脱氢酶突变体的核苷酸序列如序列表中SEQ ID NO.8或SEQ ID NO.10所示。
  3. 一种分离的核酸，其特征在于，所述核酸编码如权利要求1～2任一项所述的L-谷氨酸脱氢酶突变体。
  4. 一种包含如权利要求3所述的核酸的重组表达载体。
  5. 一种包含如权利要求3所述的核酸或如权利要求4所述的重组表达载体的转化体。
  6. 一种L-草铵膦盐的制备方法，其特征在于，所述制备方法包括以下步骤：在反应溶剂、L-谷氨酸脱氢酶突变体、无机氨基供体和还原型辅酶NADPH的存在下，将2-氧代-4-(羟基甲基氧膦基)丁酸盐进行氨化反应，即得L-草铵膦盐；其中，所述L-谷氨酸脱氢酶突变体为如权利要求1～2任一项所述的L-谷氨酸脱氢酶突变体。
  7. 如权利要求6所述的制备方法，其特征在于，所述的制备方法还包括以下步骤：在D-氨基酸氧化酶的存在下，将D-草铵膦盐进行氧化反应，得到所述的2-氧代-4-(羟基甲基氧膦基)丁酸盐即可；
    较佳地：
    所述的D-草铵膦盐单独存在、或、与L-草铵膦盐共同存在；所述的与L-草铵膦盐共同存在的形式为D型富集的草铵膦盐、L型富集的草铵膦盐或消旋体草铵膦盐；
    和/或，所述的D-氨基酸氧化酶的浓度为0.6-6U/mL，优选为1.8U/mL；
    和/或，所述的氧化反应在通气的条件下进行，所述通气优选为通入空气或氧气；所述通气的速率优选为0.5-1VVM；
    和/或，所述的氧化反应在过氧化氢酶的存在下进行；
    和/或，所述的D-草铵膦盐的浓度为100-600mM，优选为200mM；
    和/或，所述的氧化反应的反应体系的pH为7-9，优选为8.0；
    和/或，所述的氧化反应的反应体系的温度为20-50℃，优选为20℃。
  8. 如权利要求6或7所述的制备方法，其特征在于，所述的L-谷氨酸脱氢酶突变体 的浓度为0.05-3U/ml，较佳地为0.1-1U/ml，例如为0.23U/ml；
    和/或，所述的无机氨基供体的浓度为100-2000mM，较佳地为200mM；
    和/或，所述的2-氧代-4-(羟基甲基氧膦基)丁酸盐的浓度为100-600mM，较佳地为200mM；
    和/或，所述还原型辅酶NADPH与所述的2-氧代-4-(羟基甲基氧膦基)丁酸盐的质量比为1:100-1:20000，较佳地为1:1000-1:15000，更佳地为1:5000；
    和/或，所述的无机氨基供体为氨气、硫酸铵、氯化铵、磷酸氢二铵、乙酸铵、甲酸铵和碳酸氢铵中的一种或多种，所述氨气的使用形式较佳地为氨水；
    和/或，所述的反应溶剂为水；
    和/或，所述氨化反应的反应体系的pH为7-9，较佳地为8.5；
    和/或，所述氨化反应的反应体系的温度为20-50℃，较佳地为37℃。
  9. 如权利要求6～8任一项所述的制备方法，其特征在于，所述的制备方法还包括以下步骤：在脱氢酶以及供氢体的存在下，将氧化型辅酶NADP+进行还原反应，得到所述的还原型辅酶NADPH即可；
    较佳地，所述的脱氢酶为葡萄糖脱氢酶、醇脱氢酶或甲酸脱氢酶；和/或，所述的供氢体为葡萄糖、异丙醇或甲酸盐；
    更佳地，当所述的脱氢酶为醇脱氢酶时，所述的供氢体为异丙醇；当所述的脱氢酶为葡萄糖脱氢酶时，所述的供氢体为葡萄糖；当所述的脱氢酶为甲酸脱氢酶时，所述的供氢体为甲酸盐。
  10. 如权利要求9所述的制备方法，其特征在于，所述的脱氢酶的浓度为0.6-6U/mL，较佳地为2U/mL；
    和/或，所述氧化型辅酶NADP+与所述的2-氧代-4-(羟基甲基氧膦基)丁酸盐的质量比为1:100-1:20000，较佳地为1:1000-1:15000，更佳地为1:5000；
    和/或，所述的供氢体的浓度为100-1000mM，较佳地为240mM；
    和/或，所述还原反应的反应体系的pH为7-9，较佳地为8.5；
    和/或，所述还原反应的反应体系的温度为20-50℃，较佳地为37℃。
  11. 一种L-草铵膦的制备方法，其特征在于，所述制备方法包括下述步骤：
    (1)按照权利要求6～10任一项所述的制备方法，制得L-草铵膦盐；
    (2)将步骤(1)制得的L-草铵膦盐进行酸化反应，得到L-草铵膦。
  12. 一种如权利要求1～2任一项所述的L-谷氨酸脱氢酶突变体在制备L-草铵膦或其盐中的应用。
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EP3974522A1 (en) 2022-03-30
CN111979208A (zh) 2020-11-24
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