WO2019192505A1 - 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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WO2019192505A1
WO2019192505A1 PCT/CN2019/081172 CN2019081172W WO2019192505A1 WO 2019192505 A1 WO2019192505 A1 WO 2019192505A1 CN 2019081172 W CN2019081172 W CN 2019081172W WO 2019192505 A1 WO2019192505 A1 WO 2019192505A1
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seq
glufosinate
dehydrogenase
reaction
amino acid
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PCT/CN2019/081172
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English (en)
French (fr)
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田振华
程占冰
丁少南
徐文选
王茹茹
焦琦
黄瑶
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上海弈柯莱生物医药科技有限公司
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Priority to US17/043,450 priority Critical patent/US11634693B2/en
Priority to EP19780685.4A priority patent/EP3783101A4/en
Priority to JP2021503191A priority patent/JP7436450B2/ja
Publication of WO2019192505A1 publication Critical patent/WO2019192505A1/zh
Priority to US18/183,191 priority patent/US20230340427A1/en

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    • 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)
    • C12N9/0026—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)
    • 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)
    • C12Y104/01004—Glutamate dehydrogenase (NADP+) (1.4.1.4)
    • 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
    • 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

Definitions

  • the invention belongs to the field of biotechnology, and particularly relates to an L-glutamic acid dehydrogenase mutant and application thereof.
  • Glufosinate 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 properties and less side effects. There are two optical isomers of glufosinate, D-glufosinate and L-glufosinate, but only L-glufosinate has herbicidal activity, so the method of developing L-glufosinate is to improve atomic economy. It is of great significance to reduce the cost of use and reduce environmental pressure.
  • the methods for preparing L-glufosinate mainly include chiral separation, chemical synthesis and biocatalysis.
  • the chiral resolution method such as CN1053669C, discloses the use of quinine alkaloids as a resolving agent, recrystallizing the L- glufosinate quinine salt, and then neutralizing the salt with an acid to obtain L-glufosinate. Simultaneously using 5-nitrosalicylaldehyde or 3,5-dinitrosalicylaldehyde as a racemizing agent to racer unreacted D-glufosinate to obtain DL-glufosinate and continue to be used for the resolution reaction. .
  • this method requires expensive chiral resolving reagents, as well as multi-step recrystallization, which is cumbersome to operate and is not an ideal method.
  • the chemical synthesis method and the biocatalytic method have the advantages of strong specificity and mild reaction conditions, and are an advantageous method for producing L-glufosinate.
  • EP0382113A describes a process for the catalytic cracking of a carboxylic acid ester of N-acetyl- glufosinate using an acyltransferase to give L-glufosinate, but the enzyme in this method is not specific for free N-acetyl-glyphosate. Therefore, it is necessary to esterify N-acetyl-glyphosate, increasing the reaction step and correspondingly increasing the production cost.
  • Another part is a method for preparing L-glufosinate by transaminase using 2-oxo-4-(hydroxymethylphosphinyl)butyric acid (PPO) as a substrate, wherein US5221737A and EP0344683A describe the use of glutamine.
  • the acid is used as an amino donor, and the method for obtaining L-glufosinate from the corresponding keto acid 4-(hydroxymethylphosphinyl)-2-oxobutyric acid by aminotransferase derived from Escherichia coli needs to be waited in the reaction system.
  • the amount or excess of the amino donor glutamic acid makes the product difficult to purify.
  • CN1284858C The above method is improved by using aspartic acid as an amino donor and obtaining L-by the corresponding keto acid 4-(hydroxymethylphosphinyl)-2-oxobutanoic acid by aspartate aminotransferase.
  • the reaction requires only equimolar amounts of amino donor and amino acceptor.
  • the amino donor used in the method using the transaminase is mostly an amino acid, and the cost is high.
  • 2-oxo-4-(hydroxymethylphosphinyl)butyric acid is used as a substrate to prepare L-glufosinate by catalysis of amino acid dehydrogenase, such as CN106978453A, which uses inorganic
  • amino acid dehydrogenase such as CN106978453A
  • CN106978453A which uses inorganic
  • the amino donor allows for simple product separation and reduced cost.
  • the substrate concentration of the enzyme catalyzed in CN106978453A is only 10-100 mM, and the catalytic efficiency of the enzyme is not high.
  • the technical problem to be solved by the present invention is that the existing L-glutamic acid dehydrogenase has defects such as low catalytic efficiency in the preparation of L-glufosinate, and thus the present invention provides an L-glutamic acid dehydrogenase mutant and Its use in the preparation of L-glufosinate or a salt thereof.
  • L-glufosinate is prepared by using the L-glutamic acid dehydrogenase mutant of the present invention, the substrate concentration can be catalyzed higher than that of the wild-type L-glutamate dehydrogenase, and the efficiency of the enzyme is improved. , reducing the cost of the reaction and facilitating industrial production.
  • the wild-type L-glutamic acid dehydrogenase used in the present invention is Corynebacterium glutamicum, and the PDB number is 5IJZ.
  • the wild-type L-glutamate dehydrogenase consists of 447 amino acid residues, and the substrate 2-oxo-4-(hydroxymethylphosphinyl)butyric acid (PPO) catalyzes a very low enzyme activity, which is not suitable.
  • Industrial production The present inventors screened the different amino acid positions of the above-mentioned wild type enzyme against the substrate PPO, and found that some mutants of A166, V376 and T196 sites significantly improved the enzyme activity of the new substrate PPO. Further, these sites were subjected to combinatorial mutation and a mutant library was constructed, from which the L-glutamic acid dehydrogenase mutant of the present invention was screened.
  • an L-glutamic acid dehydrogenase mutant and the sequence of the L-glutamic acid dehydrogenase mutant includes the sequence shown in SEQ ID NO. a sequence in which amino acid residue A at position 166, and/or amino acid residue at position 376 is mutated to a basic, hydrophilic or small sterically hindered amino acid residue,
  • sequence of the L-glutamate dehydrogenase mutant is not the sequence shown in SEQ ID NO. 8, and is not the sequence shown in SEQ ID NO.
  • the L-glutamic acid dehydrogenase mutant has an activity of catalyzing 2-oxo-4-(hydroxymethylphosphinyl)butyric acid or a salt thereof.
  • the basic, hydrophilic or small steric hindrance means that the amino acid residue after the mutation and the amino acid residue in the wild sequence are more alkaline, more hydrophilic or less sterically hindered than the amino acid residue in the wild sequence.
  • the amino acid residue after the other point mutation is not necessarily strictly limited to have the above characteristics.
  • the amino acid may be a modified or unmodified natural amino acid; the present invention exemplifies a natural amino acid.
  • the amino acid residue A at position 166 can be mutated to G, C, E, H or T, and/or the amino acid residue at position 376 can be mutated to A, E, G, P, Q or S. .
  • the mutant of the present invention further comprises mutating the amino acid residue T at position 196 shown in SEQ ID NO. 1 to V, S or C.
  • amino acid residue A at position 166 is mutated to G, H or T
  • amino acid residue at position 376 is mutated to E, G, Q or S
  • amino acid residue at position 196 is T mutation.
  • S or C amino acid residue A at position 166 is mutated to G, H or T
  • amino acid residue at position 376 is mutated to E, G, Q or S
  • amino acid residue at position 196 is T mutation.
  • the amino acid residue A at position 166 is mutated to T, or the amino acid residue at position 376 is mutated to G.
  • amino acid sequence of the L-glutamate dehydrogenase mutant is SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. SEQ ID NO. 20, SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36 or SEQ ID Shown in NO.38.
  • the nucleotide sequence of the L-glutamate dehydrogenase mutant is SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. SEQ ID NO. 35, SEQ ID NO. 37 or SEQ ID NO.
  • the second aspect of the present invention to solve the above technical problems is: an isolated nucleic acid encoding the L-glutamic acid dehydrogenase mutant according to any one of claims 1 to 4.
  • the nucleotide sequence encoding the nucleic acid is SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. SEQ ID NO. 21, SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, SEQ ID NO. NO.37 or SEQ ID NO.39.
  • the third technical solution of the present invention to solve the above technical problems is: a recombinant expression vector comprising the nucleic acid.
  • the fourth aspect of the present invention to solve the above technical problems is: a transformant comprising the nucleic acid or the recombinant expression vector.
  • the fifth technical solution of the present invention to solve the above technical problem is: a preparation method of L-glufosinate salt, which comprises the following steps: in a reaction solvent, an L-glutamic acid dehydrogenase mutant, an inorganic amino donor, and 2-Amino-4-(hydroxymethylphosphinyl)butanoate is subjected to amination reaction in the presence of reduced coenzyme NADPH (nicotinamide adenine dinucleotide phosphate) to obtain L-glufosinate salt.
  • NADPH nicotinamide adenine dinucleotide phosphate
  • the L-glutamic acid dehydrogenase mutant is obtained by the present invention, and the remaining raw materials, reaction steps and conditions can be conventional in the art.
  • the remaining raw materials, reaction steps and conditions can be conventional in the art.
  • CN106978453A and the applicant's application number CN201810162629.
  • the patent application of .4 is incorporated herein by reference in its entirety.
  • the method for preparing the L-glufosinate salt may further comprise the step of: oxidizing the D-glufosinate salt in the presence of D-amino acid oxidase (DAAO) to obtain the 2-oxygen Derived to 4-(hydroxymethylphosphinyl) butyrate.
  • DAAO D-amino acid oxidase
  • the cation of the D-glufosinate salt may be a conventional cation such as ammonium ion, sodium ion and/or potassium ion or the like. It can also be the cation of the buffer used.
  • the D-glufosinate salt may be present alone or in combination with the L-glufosinate salt (in this case, the L-glufosinate may not react), for example: D type Enriched glufosinate (ie, where the content of the D-enantiomer is >50%, or even pure D-glufosinate), L-enriched glufosinate (ie, L-glufosinate) The content is >50%, excluding the case of pure L-glufosinate salt) or the racemic glufosinate salt.
  • D type Enriched glufosinate ie, where the content of the D-enantiomer is >50%, or even pure D-glufosinate
  • L-enriched glufosinate ie, L-glufosinate
  • the content is >50%, excluding the case of pure L-glufosinate salt) or the racemic glufosinate salt.
  • the concentration of the D amino acid oxidase (DAAO) may be conventional in the art, preferably 0.6-6 U/mL, more preferably 3.6 U/mL.
  • the concentration of the D-glufosinate salt may be conventional in the art, preferably from 100 to 600 mM, more preferably 300 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 venting is preferably air or oxygen; the rate of venting is preferably 0.5 VVM-1 VVM.
  • the oxidation reaction can also be carried out in the presence of an antifoaming agent.
  • the air may be conventional air in the art, generally containing oxygen, and the content of oxygen contained therein is also conventional in the art. It is the oxygen in the air that participates in the reaction.
  • the pH of the reaction system is preferably from 7 to 9, more preferably 8.
  • the pH can be achieved by using a buffer.
  • the pH can also be achieved by adjustment using an alkali (or alkaline solution).
  • the buffer solution is preferably a phosphate buffer solution or a Tris-HCl buffer solution, and the phosphate buffer solution is preferably sodium hydrogen phosphate-sodium dihydrogen phosphate buffer or dipotassium hydrogen phosphate-phosphate. Potassium hydrogen buffer.
  • the alkali solution is preferably ammonia water.
  • the temperature of the reaction system may be conventional in the art, preferably 20 to 50 ° C, more preferably 37 ° C.
  • the oxidation reaction and the amination reaction may be carried out separately or simultaneously (same reaction system). Simultaneously performing the oxidation of the D-glufosinate salt in the presence of, for example, D-amino acid oxidase (DAAO), L-glutamate dehydrogenase mutant, inorganic amino donor, and reduced coenzyme NADPH.
  • DAAO D-amino acid oxidase
  • L-glutamate dehydrogenase mutant L-glutamate dehydrogenase mutant
  • inorganic amino donor for example, L-glutamate dehydrogenase mutant
  • reduced coenzyme NADPH reduced coenzyme
  • the cation of the L-glufosinate salt may be a conventional cation such as ammonium ion, sodium ion and/or potassium ion or the like. It can also be the cation of the buffer used.
  • the cation of the 2-oxo-4-(hydroxymethylphosphinyl)butanoate 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 amount of the L-glutamic acid dehydrogenase mutant can be routinely used in the art, and the concentration of the L-glutamic acid dehydrogenase mutant is 0.09-3 U/ml. It is preferably from 0.3 to 1.5 U/ml, more preferably 0.9 U/ml.
  • the amount of the inorganic amino donor may be conventional in the art, and the concentration of the inorganic amino donor is from 100 to 2000 mM, preferably 600 mM.
  • the concentration of the 2-oxo-4-(hydroxymethylphosphinyl)butanoate is from 100 to 600 mM, preferably 300 mM.
  • the amount of the 2-oxo-4-(hydroxymethylphosphinyl)butanoate may be conventional in the art, and the reduced coenzyme NADPH and the 2-
  • the molar ratio of oxo-4-(hydroxymethylphosphinyl)butanoate is 1:30000-1:1000, preferably 1:20000-1:5000, more preferably 1:10000.
  • the inorganic amino donor is one or more of ammonia gas, ammonium sulfate, ammonium chloride, diammonium hydrogen phosphate, ammonium acetate, ammonium formate, and ammonium hydrogencarbonate.
  • the temperature of the reaction may be conventional in the art, and in order to ensure the catalytic efficiency of the L-glutamic acid dehydrogenase mutant, the temperature at which the amination reaction is carried out is preferably 20-50 ° C, more preferably 37 ° C, when the temperature of the amination reaction is lower than 20 ° C, the ammoniation reaction is slow; when the temperature of the amination reaction is higher than 50 ° C, the enzyme will be irreversible Degeneration is inactivated.
  • reaction solvent is water.
  • the pH of the amination reaction is preferably from 7 to 9, more preferably 8.
  • the pH can be achieved by using a buffer.
  • the pH can also be achieved by adjustment using an alkali (or alkaline solution).
  • the buffer solution is preferably a phosphate buffer solution or a Tris-HCl buffer solution, etc.
  • the phosphate buffer solution is preferably sodium hydrogen phosphate-sodium dihydrogen phosphate buffer or dipotassium hydrogen phosphate-phosphoric acid. Dihydrogen potassium buffer and the like.
  • the alkali solution is preferably ammonia water.
  • the method for preparing the L-glufosinate salt further comprises the steps of: dehydrogenase (such as glucose dehydrogenase, alcohol dehydrogenase or formate dehydrogenase, etc.) and hydrogen donor (glucose, isopropanol or In the presence of a formate or the like, the oxidized coenzyme NADP + may be subjected to a reduction reaction to obtain the reduced coenzyme NADPH.
  • dehydrogenase such as glucose dehydrogenase, alcohol dehydrogenase or formate dehydrogenase, etc.
  • hydrogen donor glucose, isopropanol or In the presence of a formate or the like, the oxidized coenzyme NADP + may be subjected to a reduction reaction to obtain the reduced coenzyme NADPH.
  • the dehydrogenase is in one-to-one correspondence with the hydrogen donor, for example:
  • the hydrogen donor is isopropanol
  • the hydrogen donor is glucose
  • the hydrogen donor is a formate.
  • the concentration of the dehydrogenase may be conventional in the art, preferably 0.6-6 U/mL, more preferably 2 U/mL.
  • the concentration of the hydrogen donor may be conventional in the art, preferably from 100 to 1000 mM, more preferably 360 mM.
  • the concentration of the oxidized coenzyme NADP + may be conventional in the art.
  • the pH of the reduction reaction is preferably from 7 to 9, more preferably 8.
  • the pH can be achieved by using a buffer.
  • the pH can also be achieved by adjustment using an alkali (or alkaline solution).
  • the buffer solution is preferably a phosphate buffer solution or a Tris-HCl buffer solution, etc.
  • the phosphate buffer solution is preferably sodium hydrogen phosphate-sodium dihydrogen phosphate buffer or dipotassium hydrogen phosphate-phosphoric acid. Dihydrogen potassium buffer and the like.
  • the alkali solution is preferably ammonia water.
  • the temperature of the reaction system may be conventional in the art, preferably 20 to 50 ° C, more preferably 37 ° C.
  • the reduction reaction and the amination reaction may be carried out separately or simultaneously (same reaction system). Simultaneously performing the same as shown in the preferred embodiment of the invention: in the presence of glucose dehydrogenase, glucose, oxidized coenzyme NADP + , L-glutamate dehydrogenase mutant, inorganic amino donor, The 2-oxo-4-(hydroxymethylphosphinyl)butyrate is subjected to an amination reaction (the simultaneous reduction reaction of NADP + ) to obtain an L- glufosinate salt.
  • the NADPH used for the amination reaction can be generated by the reduction reaction cycle.
  • the concentration of the oxidized coenzyme NADP + may be conventional in the art, and in order to ensure that the reaction proceeds normally, it is preferably 0.02 to 0.1 mM, more preferably 0.03 mM.
  • the reduction reaction, the oxidation reaction, and the amination reaction may be carried out separately or simultaneously (same reaction system). Simultaneously performing the same as shown in the preferred embodiment of the invention: 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 the D-glufosinate salt may be subjected to an oxidation reaction and an amination reaction (there is also a reduction reaction of NADP + present) to obtain an L- glufosinate salt.
  • the NADPH used in the amination reaction can be generated by the reduction reaction cycle.
  • the concentration of the oxidized coenzyme NADP + may be conventional in the art, and in order to ensure that the reaction proceeds normally, it is preferably 0.02 to 0.1 mM, more preferably 0.03 mM.
  • the reaction time of the preparation method can be stopped at the final concentration of the raw material or the final concentration or conversion rate of the product in the case of detection by a conventional method; the conventional method includes pre-column derivatization high-performance liquid phase Chromatography or ion pair chromatography.
  • the sixth technical solution of the present invention to solve the above technical problem is: a method for preparing L-glufosinate, which comprises the following steps:
  • the L-glufosinate salt obtained in the step (1) is subjected to an acidification reaction to obtain L- glufosinate.
  • the seventh aspect of the present invention to solve the above technical problems is: an application of an L-glutamic acid dehydrogenase mutant in the preparation of L-glufosinate or a salt thereof.
  • the use of the L-glufosinate salt can include the following steps: 2-oxo-4-(hydroxyl) in the presence of L-amino acid dehydrogenase, inorganic amino donor and reduced coenzyme
  • the phosphinyl)butyrate is reacted, wherein the L-glutamic acid dehydrogenase mutant is the L-glutamic acid dehydrogenase mutant prepared above.
  • the use of the L-glufosinate can include the following steps: 2-oxo-4-(hydroxymethyl) in the presence of an L-amino acid dehydrogenase, an inorganic amino donor, and a reduced coenzyme
  • the phosphinyl)butyric acid is reacted to obtain L-glufosinate; wherein the L-glutamic acid dehydrogenase mutant is the L-glutamic acid dehydrogenase mutant prepared above.
  • the concentrations of the above compounds are the concentrations of the compounds before the reaction.
  • the reagents and starting materials used in the present invention are commercially available.
  • the L-glutamic acid dehydrogenase mutant of the present invention is capable of catalyzing a higher substrate concentration than the wild-type L-glutamate dehydrogenase when preparing L-glufosinate.
  • 10 mL of L-glutamate dehydrogenase is capable of catalyzing a substrate concentration of 10-100 mM, whereas in a preferred embodiment of the invention 15 mL of L-glutamate dehydrogenase mutant The catalyzed substrate concentration has reached 300 mM.
  • the L-glutamic acid dehydrogenase mutant of the present invention reduces the cost of the reaction and facilitates industrial production.
  • Fig. 1 shows the results of pre-column derivatization HPLC analysis of Marfey reagent of D-glyphos-phosphine and L-glufosinate in the obtained product when L-glutamic acid dehydrogenase mutant 14 is used in the reaction.
  • Figure 2 shows the results of pre-column derivatization HPLC analysis of the Marfey reagent of the racemic glufosinate standard, wherein the last two peaks are peaks of the Marfey reagent blank.
  • Fig. 3 is a result of ion-pair HPLC analysis of the obtained L-glufosinate prepared by the reaction of L-glutamic acid dehydrogenase mutant 14 in the reaction.
  • Figure 4 shows the results of ion-pair HPLC analysis of PPO standards.
  • Figure 5 is a result of ion pair HPLC analysis of racemic glufosinate standards.
  • Figure 6 is a mass spectrum of the PPO standard.
  • the experimental methods in the present invention are conventional methods unless otherwise specified, and the gene cloning operation can specifically participate in the "Molecular Cloning Experiment Guide" edited by J. Sambrook et al.
  • codons corresponding to the amino acids are also conventional in the art, and the correspondence between specific amino acids and codons is shown in Table 2.
  • pET28a, pET21a and bugbuster protein extraction reagent were purchased from Novagen; DpnI enzyme was purchased from Infineon (Shanghai) Trading Co., Ltd.; NdeI enzyme, HindIII enzyme purchased from Thermo Fisher, E.coli BL21 (DE3) competent cell purchase Since Beijing Dingguo Changsheng Biotechnology Co., Ltd.; catalase was purchased from Shandong Fengtai Biotechnology Co., Ltd.
  • N represents any one of A, G, C, and T
  • M represents A or C
  • K represents G or T; which is selected based on the coding nucleotide of the amino acid to be mutated at the site.
  • the NNK in the A166-forward primer can represent AAG (lysine), AAT (aspartic acid), AGG (arginine) or AGT (serine), etc.
  • the nucleotide corresponding to the specific amino acid can be referred to Table 2.
  • the gene cgGLUDH (Corynebacterium glutamicum) was synthesized according to the sequence of SEQ ID NO. 1 in the sequence listing.
  • the gene synthesis company is Suzhou Jinweizhi Biotechnology Co., Ltd. (C3 floor, Bio-Nano Science and Technology Park, No. 218, Xinghu Street, Suzhou Industrial Park), PDB of cgGLUDH For 5IJZ.
  • Plasmid pET21a was then introduced into the NdeI and HindIII cleavage sites to construct plasmid pET21a-cgGLUDH.
  • the target band was amplified by PCR using the plasmid pET21a-cgGLUDH as a template.
  • the PCR amplification system is:
  • the PCR amplification procedure is as follows:
  • the PCR product was subjected to DpnI digestion at 37 ° C for 2 hr.
  • the reaction was completed and transformed into E. coli BL21 (DE3) competent cells, and plated in LB medium containing 100 ⁇ g/mL ampicillin, and cultured at 37 ° C overnight to collect a transformant to obtain a transformant containing a mutant library.
  • the transformants were inoculated into 96-well plates and induced by IPTG at 30 ° C overnight. After collecting bacteria, add the bugbuster protein extraction reagent to lyse and centrifuge to obtain the enzyme solution.
  • the selected positive clones are cultured as follows:
  • LB liquid medium composition peptone 10g / L, yeast powder 5g / L, NaCl 10g / L, dissolved in deionized water, constant volume, sterilization at 121 ° C for 20min, set aside.
  • Monoclonal inoculation was carried out in 5 ml of LB liquid medium containing 100 ⁇ g/ml ampicillin, and cultured at 37 ° C for 12 h with shaking. Transfer to 2 ml of fresh LB liquid medium containing 100 ⁇ g/ml ampicillin at 2% inoculum, shake at 37 ° C until the OD 600 value reaches 0.8, add IPTG to its final concentration of 0.5 mM, induce at 18 ° C Cultured for 16h. After the completion of the culture, the culture solution was centrifuged at 10,000 rpm for 10 min, the supernatant was discarded, and the cells were collected and stored in a -20 ° C ultra-low temperature freezer for use.
  • the cells collected after the completion of the culture were washed twice with 50 mM pH 8.0 phosphate buffer, and then the body weight was suspended in a phosphate buffer of pH 8.0, homogenized and disrupted by low temperature and high pressure, and the precipitate was centrifuged to remove the precipitate.
  • the supernatant is a crude enzyme solution containing a recombinant L-glutamate dehydrogenase mutant.
  • Enzyme activity detection method 25g/L wet cell (homogeneous machine crushed), 10mM PPO, 20mM coenzyme (NADPH), 750mM NH 4 Cl, total system is 400 ⁇ L, reaction medium is disodium hydrogen phosphate-phosphate with pH 8.0 Sodium dihydrogen buffer. The reaction was carried out in a 30 ° C metal bath shaking reactor for 6 h, and the reaction was terminated by adding 2 volumes of acetonitrile. After the sample was diluted by a certain multiple, the concentration of L-glufosinate was measured by pre-column derivatization high-performance liquid phase, and the enzyme activity was calculated. The results are shown in Table 4.
  • unit enzyme activity The amount of enzyme required to produce 1 ⁇ mol of L-glufosinate per minute under specific reaction conditions (30 ° C).
  • * represents 1U/ml or less, ** represents enzyme activity between 3-5U/ml; *** represents enzyme activity between 5-10U/ml; **** represents enzyme activity above 10U/ml .
  • the method for preparing the L-glutamic acid dehydrogenase crude enzyme solution used in the following examples employs the above methods.
  • the DAAO enzyme gene was fully synthesized according to the gene sequence of the AC302 DAAO enzyme described in the patent US Pat. No. 9834802B2.
  • the synthesis company is Suzhou Jinweizhi Biotechnology Co., Ltd., No. 211, Pubin Road, Yanchuang Park, Jiangbei New District, Nanjing, Jiangsu province.
  • LB liquid medium composition peptone 10g / L, yeast powder 5g / L, NaCl 10g / L, dissolved in deionized water, constant volume, sterilization at 121 ° C for 20min, set aside.
  • the DAAO enzyme gene synthesized in Example 3 was ligated into pET28a, the restriction enzyme site NdeI & HindIII, and the enzyme-ligated vector was transformed into host E. coli BL21 (DE3) competent cells to obtain an engineered strain containing DAAO enzyme.
  • Example 4 The cells collected in Example 4 were washed twice with 50 mM pH 8.0 phosphate buffer, and then the body weight was suspended in a phosphate buffer of pH 8.0, homogenized and disrupted by low temperature and high pressure, and the precipitate was centrifuged to remove the precipitate. The resulting supernatant was 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 50 mmol/L and peroxidase 0.1 mg/mL), and adding 50 ⁇ L of display agent (60 ⁇ g/mL 2 , 4,6-tribromo-3-hydroxybenzoic acid and 1 mg/mL 4-aminoantipyrine), 50 ⁇ L of DAAO enzyme, UV absorbance measurement H 2 O 2 concentration at 510 nm, calculate PPO concentration, and calculate Enzyme activity.
  • display agent 60 ⁇ g/mL 2 , 4,6-tribromo-3-hydroxybenzoic acid and 1 mg/mL 4-aminoantipyrine
  • unit enzyme activity The amount of enzyme required to produce 1 ⁇ mol of PPO per minute under specific reaction conditions (30 ° C).
  • the DAAO enzyme crude enzyme solution used in the following examples was prepared by the above method.
  • the glucose dehydrogenase gene was fully synthesized based on the glucose dehydrogenase gene sequence derived from Bacillus subtilis 168 (NCBI Accession No. NP_388275.1).
  • LB liquid medium composition peptone 10g / L, yeast powder 5g / L, NaCl 10g / L, dissolved in deionized water, constant volume, sterilization at 121 ° C for 20min, set aside.
  • the glucose dehydrogenase gene is linked to pET28a, and the enzyme cleavage site NdeI & HindIII is used to transform the enzyme-ligated vector into host E. coli BL21 (DE3) competent cells to obtain an engineered strain containing the glucose dehydrogenase gene.
  • E. coli BL21 DE3 competent cells
  • single colonies were inoculated into 5 ml of LB liquid medium containing 50 ⁇ g/ml kanamycin, and cultured at 37 ° C for 12 h with shaking.
  • Example 6 The cells collected in Example 6 were washed twice with 50 mM pH 8.0 phosphate buffer, and then the body weight was suspended in a phosphate buffer of pH 8.0, homogenized and disrupted by low temperature and high pressure, and the precipitate was centrifuged to remove the precipitate. The resulting supernatant was a crude enzyme solution containing recombinant glucose dehydrogenase.
  • Enzyme activity detection method 1mL reaction system, add 980 ⁇ L pH 7.0 50mM sodium phosphate dibasic sodium phosphate monobasic buffer (containing 400mM glucose), add 10 ⁇ L NADP + (25mM), and finally add 10 at 25 °C. An appropriate amount of ⁇ L of the enzyme solution and an ultraviolet spectrophotometer were used to measure the OD value at 340 nm.
  • Unit Enzyme Activity Definition The amount of enzyme required to produce 1 ⁇ mol of NADPH per minute under specific reaction conditions (30 ° C).
  • the method for preparing the glucose dehydrogenase crude enzyme solution used in the following examples employs the above methods.
  • the alcohol dehydrogenase gene was synthesized from the whole gene according to the Cyclopentanol dehydrogenase gene sequence derived from Lactobacillus brevis KB290 (Genbank Accession No. BAN05992.1).
  • the alcohol dehydrogenase gene is ligated to the pET28a vector, and the enzyme cleavage site NdeI & HindIII is used to transform the enzyme-ligated vector into the competent E. coli BL21 (DE3) competent cell to obtain an engineered strain containing the alcohol dehydrogenase gene.
  • the engineered bacteria containing the alcohol dehydrogenase gene were activated by plate scribing, single colonies were inoculated into 5 ml of LB liquid medium containing 50 ⁇ g/ml kanamycin, and cultured at 37 ° C for 12 h with shaking.
  • Enzyme activity detection method 3ml reaction system, at 25 ° C, first add 2850 ⁇ L pH 8.0 400 mM isopropanol (100 mM phosphate buffer), add 50 ⁇ L NADP + (25 mM), UV spectrophotometer zero, plus 100 ⁇ L of the enzyme solution diluted 100 times, and the OD value at 340 nm was measured by an ultraviolet spectrophotometer.
  • Unit Enzyme Activity Definition The amount of enzyme required to produce 1 ⁇ mol of NADPH per minute under specific reaction conditions (25 ° C, pH 8.0), defined as 1 U.
  • the pH was controlled to 8.0 with ammonia water, and the magnetic resonance reaction was carried out in a 37 °C water bath for 10 h.
  • the residual concentration of PPO was detected by ion-pair HPLC.
  • the yield and ee value of L-glufosinate were detected by pre-column derivatization high performance liquid chromatography. .
  • reaction end data is shown in Table 5.
  • 10 mL of L-glutamate dehydrogenase is capable of catalyzing a substrate concentration of 10-100 mM, while the substrate concentration catalyzed by 15 mL of L-glutamate dehydrogenase mutant in this example It has reached 300 mM.
  • the ion pair HPLC analysis results of the prepared PPO are shown in Fig. 3, wherein 10.121 min is the peak position of PPO, and 3.833 min is the peak position of L-glufosinate.
  • the ion-pair HPLC chromatogram of the PPO standard (this standard is laboratory-made, preparation method reference US8017797B, Figure 6 is its corresponding mass spectrum) is shown in Figure 4, wherein the retention time of the PPO standard is 9.520 min.
  • the ion-pair HPLC chromatogram of the racemic glufosinate standard (purchased from Shanghai Aladdin Biotechnology Co., Ltd.) is shown in Figure 5, wherein the racemic glufosinate standard has a retention time of 3.829 min. It can be seen that the peak time of the PPO and the product glufosinate in this example is substantially consistent with the peak time of the respective standard.

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Abstract

提供了一种L-谷氨酸脱氢酶突变体及其应用，所述突变体将SEQ ID NO.1所示的第166位氨基酸残基A，和/或第376位氨基酸残基V突变为亲水性或小位阻氨基酸残基，其应用为在L-氨基酸脱氢酶突变体、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，获得的L-草铵膦盐进行酸化反应制得L-草铵膦。所述L-谷氨酸脱氢酶突变体与野生型L-谷氨酸脱氢酶相比，在制备L-草铵膦时能够催化的底物浓度更高，从而提高酶的作用效率，降低反应的成本。

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2018年4月3日的中国专利申请CN201810291900.4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 PCTCN2019081172-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 PCTCN2019081172-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 PCTCN2019081172-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 PCTCN2019081172-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 PCTCN2019081172-appb-000005
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-草铵膦时与野生型L-谷氨酸脱氢酶相比，能够催化的底物浓度更高，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Corynebacterium glutamicum，PDB号为5IJZ。该野生型L-谷氨酸脱氢酶由447个氨基酸残基组成，催化底物2-氧代-4-(羟基甲基氧膦基)丁酸(PPO)时酶活很低，不适合工业化生产。本发明人针对该底物PPO对上述野生型酶的不同氨基酸位点进行了饱和突变筛选，发现A166，V376，T196位点的部分突变体对新底物PPO的酶活有明显提升。进而对这些位点进行组合突变并构建突变体文库，从中筛选出本发明所述的L-谷氨酸脱氢酶突变体。
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包括将SEQ ID NO.1所示的第166位氨基酸残基A，和/或第376位氨基酸残基V突变为碱性、亲水性或小位阻氨基酸残基后的序列，
其中，所述L-谷氨酸脱氢酶突变体的序列不为SEQ ID NO.8所示的序列，且不为SEQ ID NO.22所示的序列。
较佳地，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。
根据本发明，所述的碱性、亲水性或小位阻是指突变后的氨基酸残基和野生序列中氨基酸残基相比碱性更强、更亲水或空间位阻更小。一般来说，只要166或376位任一位点突变后的氨基酸残基已经符合上述特性的情况下，另一位点突变后的氨基酸残基不必严格限制需具有上述特性。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。
较佳地，所述第166位氨基酸残基A可以突变为G、C、E、H或T，和/或第376位氨基酸残基V可以突变为A、E、G、P、Q或S。
更佳地，本发明突变体还包括将SEQ ID NO.1所示的第196位氨基酸残基T突变为V、S或C。
进一步更佳地，所述第166位氨基酸残基A突变为G、H或T，第376位氨基酸残基V突变为E、G、Q或S，和/或第196位氨基酸残基T突变为S或C。
在本发明一较佳实施例中，所述第166位氨基酸残基A突变为T，或第376位氨基 酸残基V突变为G。
上述大写英文单字母代表如本领域技术人员熟知的氨基酸，根据本发明，在此代表的是相应的氨基酸残基。
较佳地，所述L-谷氨酸脱氢酶突变体的氨基酸序列如SEQ ID NO.10，SEQ ID NO.12，SEQ ID NO.14，SEQ ID NO.16，SEQ ID NO.18，SEQ ID NO.20，SEQ ID NO.24，SEQ ID NO.26，SEQ ID NO.28，SEQ ID NO.30，SEQ ID NO.32，SEQ ID NO.34，SEQ ID NO.36或SEQ ID NO.38所示。
较佳地，所述L-谷氨酸脱氢酶突变体的核苷酸序列如SEQ ID NO.9，SEQ ID NO.11，SEQ ID NO.13，SEQ ID NO.15，SEQ ID NO.17，SEQ ID NO.19，SEQ ID NO.21，SEQ ID NO.23，SEQ ID NO.25，SEQ ID NO.27，SEQ ID NO.29，SEQ ID NO.31，SEQ ID NO.33，SEQ ID NO.35，SEQ ID NO.37或SEQ ID NO.39所示。
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，所述核酸编码如权利要求1～4任一项所述的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.9，SEQ ID NO.11，SEQ ID NO.13，SEQ ID NO.15，SEQ ID NO.17，SEQ ID NO.19，SEQ ID NO.21，SEQ ID NO.23，SEQ ID NO.25，SEQ ID NO.27，SEQ ID NO.29，SEQ ID NO.31，SEQ ID NO.33，SEQ ID NO.35，SEQ ID NO.37或SEQ ID NO.39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，本申请在此引用这些文献的全文。
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3.6U/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300mM。
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0.5VVM-1VVM。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。
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。
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37℃。
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为0.09-3U/ml，较佳地为0.3-1.5U/ml，更佳地为0.9U/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为100-2000mM，较佳地为600mM。
在所述的氨化反应中，所述的2-氧代-4-(羟基甲基氧膦基)丁酸盐的浓度为100-600mM，较佳地为300mM。
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)丁酸盐的摩尔比为1:30000-1:1000，较佳地为1:20000-1:5000，更佳地为1:10000。
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。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(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360mM。
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。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，较佳地为0.02-0.1mM，更佳地为0.03mM。
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，较佳地为0.02-0.1mM，更佳地为0.03mM。
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。
所述的在制备L-草铵膦盐中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，即可，其中，所述L-谷氨酸脱氢酶突变体为上述制得的L-谷氨酸脱氢酶突变体。
所述的在制备L-草铵膦中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，制得L-草铵膦；其中，所述L-谷氨酸脱氢酶突变体为上述制得的L-谷氨酸脱氢酶突变体。
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-草铵膦时与野生型L-谷氨酸脱氢酶相比，能够催化的底物浓度更高。相关专利CN106978453A的最佳实施例中10mL L-谷氨酸脱氢酶能够催化的底物浓度为10-100mM，而本发明的最佳实施例中15mL L-谷氨酸脱氢酶突变体所能催化的底物浓度已经达到300mM了。本发明的L-谷氨酸脱氢酶突变体其降低了反应的成本，利于工业化生产。
附图说明
图1为利用L-谷氨酸脱氢酶突变体14参与反应时，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2为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试剂空白样的峰。
图3为利用L-谷氨酸脱氢酶突变体14参与反应时，制备所得L-草铵膦的离子对HPLC分析结果。
图4为PPO标准品离子对HPLC分析结果。
图5为外消旋草铵膦标准品的离子对HPLC分析结果。
图6为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 PCTCN2019081172-appb-000006
Figure PCTCN2019081172-appb-000007
pET28a、pET21a和bugbuster protein extraction reagent购买自Novagen公司；DpnI酶购买自英潍捷基(上海)贸易有限公司；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。
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℃。
实施例1 L-谷氨酸脱氢酶突变体文库的构建
针对序列表中SEQ ID NO.1的第166、376、196位点进行突变的突变体文库构建所设计的引物序列如表3所示。
表3
Figure PCTCN2019081172-appb-000008
Figure PCTCN2019081172-appb-000009
其中，N代表A、G、C、T中任何一种核苷酸，M代表A或C，K代表G或T；其根据所述位点需突变成的氨基酸的编码核苷酸来选择，如A166-正向引物中的NNK可以代表AAG(赖氨酸)、AAT(天冬氨酸)、AGG(精氨酸)或AGT(丝氨酸)等，具体氨基酸所对应的核苷酸可参见表2。
根据序列表中SEQ ID NO.1的序列合成基因cgGLUDH(Corynebacterium glutamicum)，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)，cgGLUDH的PDB号为5IJZ。然后以NdeI和HindIII酶切位点引入质粒pET21a，构建质粒pET21a-cgGLUDH。以质粒pET21a-cgGLUDH为模板，进行PCR扩增目的条带。
PCR扩增体系为：
试剂 用量(μL)
2XPCR buffer(含高保真酶) 25
引物F 1
引物R 1
模板 1
去离子水 22
PCR扩增程序如下：
Figure PCTCN2019081172-appb-000010
对PCR产物进行DpnI消化，37℃，2hr。反应完成转化至E.coli BL21(DE3)感受态细胞，涂布在含有100μg/mL氨苄青霉素的LB培养基，37℃培养过夜，收菌，得到包含突变体文库的转化子。
实施例2高通量筛选突变体文库
按照如下实验步骤进行筛选：
将转化子接种96孔板培养，加IPTG 30℃过夜诱导。之后收菌，加bugbuster protein extraction reagent裂解，离心得酶液。
配制终浓度分别为20mM的PPO、200mM的NH 4Cl与0.37mM的NADPH的反应液，取上述反应液180μL于酶标板然后加入20μL酶液，总体系200μL，在酶标仪中读取OD 340时的数值。以野生型为参照系，选择阳性克隆子，测序并检测其酶活。测序公司为生工生物工程(上海)股份有限公司，上海市松江区香闵路698号。
将所选阳性克隆子进行培养，方法如下：
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。
挑单克隆接种至含100μg/ml氨苄霉素的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100μg/ml氨苄霉素的新鲜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。
将培养结束后收集到的菌体，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L-谷氨酸脱氢酶突变体粗酶液。
酶活检测方法：25g/L湿菌体(均质机破碎)、10mM PPO、20mM辅酶(NADPH)、750mM NH 4Cl，总体系为400μL，反应介质为pH为8.0的磷酸氢二钠-磷酸二氢钠缓冲液。于30℃金属浴振荡反应器内反应6h，反应结束后加入2倍体积的乙腈终止反应。样品稀释一定倍数后利用柱前衍生化高效液相检测其L-草铵膦的浓度，并计算酶活。结果如表4所示。
单位酶活的定义：在特定反应条件(30℃)下，每分钟生成1μmol L-草铵膦所需要的酶量。
表4
Figure PCTCN2019081172-appb-000011
Figure PCTCN2019081172-appb-000012
其中，*代表1U/ml以下，**代表酶活在3-5U/ml之间；***代表酶活在5-10U/ml之间；****代表酶活在10U/ml以上。
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AC302 DAAO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葡萄糖脱氢酶基因的获取和表达
根据来源于枯草芽胞杆菌(Bacillus subtilis)168(NCBI登录号为NP_388275.1)的葡萄糖脱氢酶基因序列，全合成葡萄糖脱氢酶基因。
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。
葡萄糖脱氢酶基因连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，得到含有葡萄糖脱氢酶基因的工程菌株。将含有葡萄糖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葡萄糖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，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葡萄糖脱氢酶粗酶液。
酶活检测方法：1mL反应体系，25℃条件下，先加980μL的pH 7.0 50mM磷酸氢二钠-磷酸二氢钠缓冲液(含葡萄糖400mM)，再加10μL NADP +(25mM)，最后加10 μL适量的酶液，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(30℃)下，每分钟产生1μmol NADPH所需要的酶量。
以下实施例中所用到的葡萄糖脱氢酶粗酶液的制备方法均采用以上方法。
实施例8醇脱氢酶粗酶液的制备及酶活测定
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，全基因合成醇脱氢酶基因。
醇脱氢酶基因连接pET28a载体，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0.1mM，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。
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 8.0)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9 L-谷氨酸脱氢酶突变体催化制备L-草铵膦
称取PPO、NADP +、NH 4Cl和葡萄糖到反应瓶中，50mM pH为8.0磷酸氢二钠-磷酸二氢钠缓冲液完全溶解，用25％浓氨水调pH至8.0，加入15mL根据实施例2方法制得的L-谷氨酸脱氢酶突变体1(1U/mL)、2(1.3U/mL)、8(2.1U/mL)、14(3U/mL)、15(2.8U/mL)和16(2.5U/mL)的粗酶液和1mL根据实施例7方法制得的葡萄糖脱氢酶粗酶液(100U/mL)，用50mM pH为8.0磷酸氢二钠-磷酸二氢钠缓冲液定容至50mL，使PPO的终浓度为300mM，氯化铵终浓度为600mM，葡萄糖终浓度360mM，NADP +终浓度为0.03mM。反应过程中用氨水控制pH为8.0，37℃水浴磁力搅拌反应10h后利 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L-草铵膦的生成量和ee值。
反应结束数据如表5所示。CN106978453A的最佳实施例中10mL L-谷氨酸脱氢酶能够催化的底物浓度为10-100mM，而该实施例中15mL L-谷氨酸脱氢酶突变体所能催化的底物浓度已经达到300mM了。
产物中D-草铵膦与L-草铵膦的HPLC分析结果见图1(附图中均以L-谷氨酸脱氢酶突变体14为例进行说明)，其中，保留时间为13.735min的为L-草铵膦，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2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，说明该实施例制备得到L-草铵膦。
制备所得PPO的离子对HPLC分析结果见图3，其中，10.121min为PPO的出峰位置，3.833min为L-草铵膦的出峰位置。PPO标准品(本标准品为实验室自制，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5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与产物草铵膦的出峰时间与各自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14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
突变体酶编号 PPO转化率 ee值
1 10％ 90％
2 15％ 92％
8 45％ 95％
14 99％ >99％
15 98％ >99％
16 97％ >99％
实施例10 DAAO酶和L-谷氨酸脱氢酶突变体催化制备L-草铵膦
称取D,L-草铵膦、NADP +、NH4Cl和葡萄糖到反应瓶中，50mM pH为8.0磷酸氢二钠-磷酸二氢钠缓冲液完全溶解，用25％浓氨水调pH至8.0，加入15mL根据实施例5方法制备的DAAO酶粗酶液(12U/mL)，0.2g 20万U/g过氧化氢酶，15mL根据实施例2中制得的L-谷氨酸脱氢酶突变体1(1U/mL)或14的粗酶液(3U/mL)和1mL根据实施例7方法制得的葡萄糖脱氢酶粗酶液(100U/mL)，用50mM pH为8.0磷酸氢二钠-磷酸二氢钠缓冲液定容至50mL，使草铵膦的终浓度为600mM，氯化铵终浓度为600mM，葡萄糖终浓度360mM，NADP +终浓度为0.03mM。反应过程中用氨水控制pH为8.0，37℃水浴磁力搅拌，按照1VVM通入空气(每分钟通入1倍反应体积的空气)，加入200μL消泡剂防止起泡，反应24h后利用离子对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L-草铵膦的生成量和ee值。反应结束数据表6所示。
表6
突变体酶编号 转化率 ee值
1 90％ 90％
14 99％ >99％
实施例11 DAAO酶和L-谷氨酸脱氢酶突变体分步催化制备L-草铵膦
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5g 20万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0.5VVM通入氧气(每分钟通入0.5倍反应体积的氧气)，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L-草铵膦的生成量和ee值，当ee值大于99％时停止反应。
取2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8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L-草铵膦的生成量和ee值。反应结束数据表7所示。
表7
突变体酶编号 转化率 ee值
1 92％ 90％
14 99％ >99％
虽然以上描述了本发明的具体实施方式，但是本领域的技术人员应当理解，这些仅是举例说明，在不背离本发明的原理和实质的前提下，可以对这些实施方式做出多种变更或修改。因此，本发明的保护范围由所附权利要求书限定。

Claims (14)

  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包括将SEQ ID NO.1所示的第166位氨基酸残基A，
    和/或第376位氨基酸残基V突变为碱性、亲水性或小位阻氨基酸残基后的序列；
    其中，所述L-谷氨酸脱氢酶突变体的序列不为SEQ ID NO.8或SEQ ID NO.22所示的序列。
  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具有催化2-氧代-4-(羟基甲基氧膦基)丁酸或其盐的活性。
  3. 如权利要求1或2所述的L-谷氨酸脱氢酶突变体，其特征在于，所述第166位氨基酸残基A突变为G、C、E、H或T，
    和/或第376位氨基酸残基V突变为A、E、G、P、Q或S；
    较佳地，所述L-谷氨酸脱氢酶突变体的序列还包括将SEQ ID NO.1所示的第196位氨基酸残基T突变为V、S或C；
    更佳地，所述第166位氨基酸残基A突变为G、H或T，
    和/或第376位氨基酸残基V突变为E、G、Q或S，
    和/或第196位氨基酸残基T突变为S或C。
  4. 如权利要求1～3任一项所述的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SEQ ID NO.10，SEQ ID NO.12，SEQ ID NO.14，SEQ ID NO.16，SEQ ID NO.18，SEQ ID NO.20，SEQ ID NO.24，SEQ ID NO.26，SEQ ID NO.28，SEQ ID NO.30，SEQ ID NO.32，SEQ ID NO.34，SEQ ID NO.36或SEQ ID NO.38所示；较佳地，所述L-谷氨酸脱氢酶突变体的核苷酸序列如SEQ ID NO.9，SEQ ID NO.11，SEQ ID NO.13，SEQ ID NO.15，SEQ ID NO.17，SEQ ID NO.19，SEQ ID NO.21，SEQ ID NO.23，SEQ ID NO.25，SEQ ID NO.27，SEQ ID NO.29，SEQ ID NO.31，SEQ ID NO.33，SEQ ID NO.35，SEQ ID NO.37或SEQ ID NO.39所示。
  5. 一种分离的核酸，其特征在于，所述核酸编码如权利要求1～4任一项所述的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.9，SEQ ID NO.11，SEQ ID NO.13，SEQ ID NO.15，SEQ ID NO.17，SEQ ID NO.19，SEQ ID NO.21，SEQ ID NO.23，SEQ ID NO.25，SEQ ID NO.27，SEQ ID NO.29，SEQ ID NO.31，SEQ ID NO.33，SEQ ID NO.35，SEQ ID NO.37或SEQ ID NO.39所示。
  6. 一种包含如权利要求5所述的核酸的重组表达载体。
  7. 一种包含如权利要求5所述的核酸或如权利要求6所述的重组表达载体的转化体。
  8. 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1～4任一项所述的L-谷氨酸脱氢酶突变体。
  9. 如权利要求8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，较佳地为3.6U/mL；
    和/或，所述的氧化反应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0.5VVM-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，较佳地为300mM；
    和/或，所述的氧化反应的反应体系的pH为7-9，较佳地为8；
    和/或，所述的氧化反应的反应体系的温度为20-50℃，较佳地为37℃。
  10. 如权利要求8或9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0.09-3U/ml，较佳地为0.3-1.5U/ml，更佳地为0.9U/ml；
    和/或，所述的无机氨基供体的浓度为100-2000mM，较佳地为600mM；
    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300mM；
    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)丁酸盐的摩尔比为1:30000-1:1000，较佳地为1:20000-1:5000，更佳地为1:10000；
    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；
    和/或，所述氨化反应的反应体系的温度为20-50℃，较佳地为37℃。
  11. 如权利要求8～10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。
  12. 如权利要求11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 +的浓度为0.02-0.1mM，较佳地为0.03mM；
    和/或，所述的供氢体的浓度为100-1000mM，较佳地为360mM；
    和/或，所述还原反应的反应体系的pH为7-9，较佳地为8；
    和/或，所述还原反应的反应体系的温度为20-50℃，较佳地为37℃。
  13. 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8～12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。
  14. 一种如权利要求1～4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。
PCT/CN2019/081172 2018-04-03 2019-04-03 一种l-谷氨酸脱氢酶突变体及其应用 WO2019192505A1 (zh)

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