WO2023143621A1 - 一种d-氨基酸氧化酶及其在制备l-草铵膦或其中间体中的应用 - Google Patents

一种d-氨基酸氧化酶及其在制备l-草铵膦或其中间体中的应用 Download PDF

Info

Publication number
WO2023143621A1
WO2023143621A1 PCT/CN2023/073897 CN2023073897W WO2023143621A1 WO 2023143621 A1 WO2023143621 A1 WO 2023143621A1 CN 2023073897 W CN2023073897 W CN 2023073897W WO 2023143621 A1 WO2023143621 A1 WO 2023143621A1
Authority
WO
WIPO (PCT)
Prior art keywords
amino acid
ammonium
acid oxidase
salt
glufosinate
Prior art date
Application number
PCT/CN2023/073897
Other languages
English (en)
French (fr)
Inventor
焦琦
王舒
田振华
程占冰
马帅
成静
Original Assignee
弈柯莱生物科技(上海)股份有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 弈柯莱生物科技(上海)股份有限公司 filed Critical 弈柯莱生物科技(上海)股份有限公司
Publication of WO2023143621A1 publication Critical patent/WO2023143621A1/zh

Links

Classifications

    • 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)
    • C12N9/0022—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 oxygen as acceptor (1.4.3)
    • C12N9/0024—D-Amino acid oxidase (1.4.3.3)
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • 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.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P9/00—Preparation of organic compounds containing a metal or atom other than H, N, C, O, S or halogen
    • 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)
    • C12Y104/03—Oxidoreductases acting on the CH-NH2 group of donors (1.4) with oxygen as acceptor (1.4.3)
    • C12Y104/03003—D-Amino-acid oxidase (1.4.3.3)

Definitions

  • the invention belongs to the field of biotechnology, and in particular relates to a D-amino acid oxidase and its application in preparing L-glufosinate-ammonium or its intermediate.
  • Glufosinate-ammonium, glyphosate, and paraquat are the world's three major herbicides, all of which are non-selective. Among them, due to the strong toxicity of paraquat, its use has been strictly restricted or banned in many countries in recent years; glyphosate is widely used, but it has been removed from the approved list of A-level green food in my country. Glufosinate-ammonium has the characteristics of high activity, wide herbicidal spectrum, and good environmental compatibility, and its herbicidal activity is faster than glyphosate, and it can also be used in areas where weeds are resistant to glyphosate. Great market development potential and broad application prospects.
  • the commercially used glufosinate-ammonium is a mixture of racemates. There are two isomers, D and L, but only the L form is active, and it is easy to decompose in soil and has low toxicity.
  • Chemical production of glufosinate-ammonium is mainly Strecker process and pyrolysis-ACA process. Wherein the Srecker process is complex, involving inflammable and explosive raw materials, and the yield of the product is relatively low.
  • the thermal cracking-ACA process is complicated, and methyl phosphorus dichloride (MDP) is the key intermediate at the front end, and it is also a technical barrier of the process.
  • the biological method has mild reaction conditions, simple production process, and easy separation of products, but its yield is relatively low and its cost is high, so it has not yet been industrialized.
  • CN105603015A 2-oxo-4-(hydroxy(methyl)phosphono)butyric acid (PPO) and its salts are used as substrate, alanine is used as amino donor, and transaminase is expressed in vitro or in vitro
  • CN106978453B has invented a kind of method utilizing amino acid dehydrogenase to prepare L-glufosinate-ammonium, and this method uses 2-carbonyl-4 -(Hydroxymethylphosphono)butyric acid or its salt is used as a substrate for transamination reaction to obtain L-glufosinate-ammonium.
  • This method has a high conversion rate of raw materials, but the substrate synthesis process is complicated and the cost is high.
  • L-glufosinate-ammonium is used as raw material to generate intermediate product PPO under the catalysis of D-amino acid oxidase mutant, and PPO generates L-glufosinate-ammonium under the catalysis of amino acid transaminase.
  • the composition obtained by the method reaction contains L-glufosinate-ammonium, D-glufosinate-ammonium, and PPO, wherein the content of L-glufosinate-ammonium reaches more than 80%, the content of D-glufosinate-ammonium is less than 10%, and the content of PPO is less than 20%. %.
  • its enzyme activity stability still needs to be improved.
  • the technical problem to be solved by the present invention is to overcome the defects of low enzymatic activity, poor stability of enzymatic activity and low yield of L-glufosinate-ammonium in the prior art of D-amino acid oxidase.
  • the present invention provides a D-amino acid oxidase and its application in the preparation of L-glufosinate-ammonium or its intermediate.
  • the D-amino acid oxidase with higher thermostability not only improves the enzyme activity, but also expands the temperature range of the enzyme, and it can be used at a lower temperature to prolong the enzyme activity.
  • the service life of the enzyme; use at a higher temperature can improve the catalytic efficiency of the enzyme.
  • Enzymes with high thermal stability have the advantages of increasing chemical reaction rate, improving product quality, stable activity, and storage resistance.
  • a first aspect of the present invention provides a D-amino acid oxidase whose amino acid sequence comprises amino acid residue differences selected from one or more of the following compared to SEQ ID NO: 1:
  • thermostability not lower than the D-amino acid oxidase shown in the amino acid sequence of SEQ ID NO:1.
  • the "/" between the amino acid residues represents the selection that the positions corresponding to the amino acid residues have different amino acid residue differences.
  • E195N/Y/Q means that the 195th difference can be N, Y or Q.
  • the amino acid sequence of the D-amino acid oxidase comprises amino acid residue differences selected from the following two or more compared with SEQ ID NO: 1: V42Y, E195Y, V326W, C234L .
  • the D-amino acid oxidase of the present invention comprises the amino acid residue difference of C234L compared with SEQ ID NO: 1, and further comprises one or two of the amino acid residue differences of V42Y, E195Y and V326W.
  • the D-amino acid oxidase has amino acid residue differences selected from any of the following groups compared with SEQ ID NO:1:
  • the D-amino acid oxidase comprises an amino acid difference of E195Y compared with SEQ ID NO: 1, and further comprises a difference of amino acid residues of V42Y or V326W;
  • the D-amino acid oxidase has amino acid residue differences selected from any of the following groups compared with SEQ ID NO: 1: E195Y and V42Y;
  • the D-amino acid oxidase has amino acid residue differences selected from any of the following groups compared with SEQ ID NO:1:
  • the second aspect of the present invention provides an isolated nucleic acid encoding the D-amino acid oxidase according to the first aspect of the present invention.
  • the third aspect of the present invention provides a recombinant expression vector comprising the nucleic acid according to the second aspect of the present invention.
  • the fourth aspect of the present invention provides a transformant comprising the nucleic acid according to the second aspect of the present invention or the recombinant expression vector according to the third aspect of the present invention.
  • the host cell of the transformant described in the present invention can be conventional in the art, preferably Escherichia coli (Escherichia coli), such as E.coli BL21(DE3).
  • Escherichia coli Escherichia coli
  • E.coli BL21(DE3) E.coli BL21(DE3).
  • the fifth aspect of the present invention provides a method for preparing the D-amino acid oxidase according to the present invention, the method comprising culturing the D-amino acid oxidase as described in the fourth aspect of the present invention under conditions suitable for expressing the D-amino acid oxidase. described transformants.
  • a sixth aspect of the present invention provides a method for preparing 2-oxo-4-(hydroxymethylphosphinyl) butyric acid or a salt thereof, the preparation method comprising the following steps: as described in the first aspect of the present invention In the presence of the D-amino acid oxidase, the substrate is oxidized to obtain the 2-oxo-4-(hydroxymethylphosphinyl)butyric acid or its salt.
  • the substrate is preferably D-glufosinate-ammonium or a salt thereof, and the D-glufosinate-ammonium or a salt thereof may exist alone or together with L-glufosinate-ammonium or a salt thereof, for example, the substrate is in the form of racemic glufosinate-ammonium or its salts.
  • the oxidation reaction is preferably carried out under the condition of aeration; the aeration is preferably air or oxygen; the aeration rate is preferably 0.5VVM-1.5VVM such as 1VVM.
  • the oxidation reaction is preferably carried out in the presence of catalase.
  • the D-amino acid oxidase is preferably in the form of D-amino acid oxidase bacteria, crude enzyme, pure enzyme or immobilized enzyme.
  • the concentration of the substrate is preferably 0.1-0.5 mol/L; preferably 0.17 mol/L.
  • the pH of the reaction system of the oxidation reaction is preferably 7-9, such as 8.
  • the temperature of the reaction system of the oxidation reaction is preferably 20-50°C, such as 25°C.
  • the mass ratio of the D-amino acid oxidase cell to the substrate is 1:(0.5-3), such as 1:1.
  • the mass ratio of the catalase to the D-amino acid oxidase cell is 1:(20-60); for example, 1:40.
  • the reaction system of the oxidation reaction preferably also includes a buffer, which can be conventional in the art, preferably a phosphate buffer, such as ammonium dihydrogen phosphate and diammonium hydrogen phosphate, disodium hydrogen phosphate and dihydrogen phosphate sodium.
  • a buffer which can be conventional in the art, preferably a phosphate buffer, such as ammonium dihydrogen phosphate and diammonium hydrogen phosphate, disodium hydrogen phosphate and dihydrogen phosphate sodium.
  • the buffer is used to regulate the pH of the reaction system, preferably, the pH of the buffer is 8.0.
  • the seventh aspect of the present invention provides a kind of preparation method of L-glufosinate-ammonium or its salt, it comprises the steps:
  • step (1) In the presence of glutamate dehydrogenase, inorganic amino donor and reduced coenzyme, the 2-oxo-4-(hydroxymethylphosphinyl) butyric acid or its salt obtained in step (1)
  • the ammoniation reaction is carried out to obtain the L-glufosinate-ammonium or its salt.
  • the reduced coenzyme is preferably NADPH or NADH.
  • described inorganic amino donor is preferably ammonia gas, ammonium sulfate, ammonium chloride, diammonium hydrogen phosphate, ammonium acetate, ammonium formate And one or more in ammonium bicarbonate, the use form of described ammonia is preferably ammonia water.
  • the pH of the reaction system of the ammoniation reaction is preferably 7-10, more preferably 8.4-8.6.
  • the reaction temperature of the ammoniation reaction is preferably 28-35°C, more preferably 30-33°C.
  • the glutamate dehydrogenase is preferably mutant 1-4 in CN201910434350.1.
  • the mass ratio of described glutamate dehydrogenase and described substrate D-glufosinate-ammonium is preferably 1:(0.5-3) , such as 1:1.25.
  • the molar ratio of the substrate D-glufosinate-ammonium to the inorganic amino donor is preferably 1: (1-1.5), for example 1:1.
  • the preparation method of L-glufosinate-ammonium or its salt according to the present invention preferably further comprises the following steps: in the presence of dehydrogenase and hydrogen donor, reducing the oxidized coenzyme to obtain the reduced coenzyme.
  • the dehydrogenase is preferably glucose dehydrogenase, alcohol dehydrogenase or formate dehydrogenase.
  • the hydrogen donor is preferably glucose, isopropanol or formate.
  • the hydrogen donor when the dehydrogenase is alcohol dehydrogenase, the hydrogen donor is isopropanol; when the dehydrogenase is glucose dehydrogenase, the The hydrogen donor is glucose; when the dehydrogenase is formate dehydrogenase, the hydrogen donor is formate.
  • the preparation method of L-glufosinate-ammonium or its salt of the present invention can be carried out step by step, such as carrying out oxidation reaction first Prepare to obtain 2-oxo-4-(hydroxymethylphosphinyl) butyric acid or its salt, and then carry out ammoniation reaction to prepare L-glufosinate-ammonium or its salt; it can also be carried out by the method of "one pot method” , For example, L-glufosinate-ammonium or its salt is prepared by mixing all raw materials.
  • the eighth aspect of the present invention provides a D-amino acid oxidase as described in the first aspect of the present invention in the preparation of L-glufosinate-ammonium or its salt, 2-oxo-4-(hydroxymethylphosphinyl) butane acid or its salts.
  • DAAO enzyme and D-amino acid oxidase mentioned in the present invention are used interchangeably.
  • the amount of D-amino acid oxidase substrate is calculated by D-glufosinate-ammonium or its salt.
  • the reagents and raw materials used in the present invention are all commercially available.
  • the positive progress effect of the present invention is: a D-amino acid oxidase with higher thermostability is provided, while improving the enzyme activity, it also expands the use temperature range of the enzyme, and when used at a lower temperature, it can prolong The service life of the enzyme; use at a higher temperature can improve the catalytic efficiency of the enzyme.
  • the chiral analysis and concentration analysis of the product L-glufosinate-ammonium are carried out by pre-column derivatization high performance liquid chromatography, and the specific analysis method is:
  • Chromatographic conditions Agilent ZORBAX Eclipse plus C18, 3.5 ⁇ m, 150*4.6mm.
  • Mobile phase A 0.1% TFA+H2O
  • mobile phase B 0.1% TFA+CAN.
  • Detection wavelength 340nm
  • flow rate 1.0mL/min
  • column temperature 30°C.
  • PPO is analyzed by ion-pair chromatography, and the specific analysis method is:
  • Sample 5 mg/mL H 2 O solution. Inject 10 ⁇ L for analysis.
  • codons corresponding to the amino acids are also conventional in the art, and the corresponding relationship between specific amino acids and codons is shown in Table 2.
  • the pET28a plasmid and bugbuster protein extraction reagent were purchased from Novagen; NdeI and HindIII enzymes were purchased from Thermo Fisher; E.coli BL21 competent cells were purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.; catalase was purchased from Shandong Feng Thai Biotechnology Co., Ltd.
  • the DAAO enzyme engineering bacteria is derived from the engineering strain containing seq.79 disclosed in the patent CN111019916B, the amino acid sequence is shown in SEQ ID NO:1, and the nucleotide sequence is shown in SEQ ID NO:12.
  • the composition of LB liquid medium peptone 10g/L, yeast powder 5g/L, NaCl 10g/L, dissolved in deionized water, constant volume, sterilized at 121°C for 20min, ready for use.
  • the solid medium is LB medium with 2% agar added.
  • DAAO enzyme engineering bacteria were activated by streaking on a plate, single colonies were inoculated into 5 mL LB liquid medium containing 50 ⁇ g/mL kanamycin, cultured with shaking at 37 ° C for 12 hours, and transferred to 150 mL with 2% inoculum size.
  • fresh LB liquid medium containing 50 ⁇ g/mL kanamycin shake at 37°C until the OD 600 reaches about 0.8, cool down to 30°C, add IPTG to a final concentration of 0.5mM, and induce culture for 16 hours.
  • the culture solution was centrifuged at 10,000 rpm for 10 min, the supernatant was discarded, and the bacterial cells were collected and stored in a -20°C refrigerator until use.
  • the bacteria collected after the culture was finished were washed twice with 50mM pH 8.0 phosphate buffer, then resuspended in 50mL pH 8.0 phosphate buffer, homogeneously broken, and the broken solution was centrifuged to remove the precipitate to obtain recombinant DAAO enzyme crude enzyme solution.
  • Embodiment 2 Construction of D-amino acid oxidase (DAAO) mutant
  • Example 2 Insert the activated DAAO enzyme engineering bacteria described in Example 1 into a test tube containing 5 mL of LB medium, and cultivate at 37° C. and 200 rpm for 8-12 hours. After the cultured cells were obtained, the plasmid was extracted according to the operating instructions of the Sangon plasmid extraction kit. The obtained plasmids can be directly used for point mutation, or placed in a -80°C refrigerator for long-term storage.
  • mutant library Two, the construction of mutant library (K29 position, V42 position, E195 position, C234 position, V326 position)
  • the gene mutation adopts the method of whole plasmid PCR to obtain the mutant gene.
  • N represents any nucleotide in A, G, C, T, M represents A or C, and K represents G or T; it is selected according to the coding nucleotide of the amino acid to be mutated into at the site ,
  • NNK in the K29-forward primer can represent AAG (lysine), AAT (aspartic acid), AGG (arginine) or AGT (serine), etc.
  • the PCR amplification system is:
  • the PCR amplification procedure is as follows:
  • the screened out beneficial mutations are combined by overlapping PCR to form new mutant transformants.
  • Example 2 The transformant obtained in Example 2 was inoculated into a 96-well plate for culture, added with IPTG to a final concentration of 0.5 mM, and induced overnight at 30°C. Afterwards, the bacteria were harvested, cracked with bugbuster protein extraction reagent, centrifuged and the supernatant was taken to obtain the DAAO mutant enzyme solution.
  • Enzyme activity detection and analysis take 100 ⁇ L of 100 mM substrate (D,L-glufosinate-ammonium, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) with a pH of 8.0, and add 50 ⁇ L of chromogenic solution (containing 60 mg/mL of TBHBA ( 3-hydroxy-2,4,6-tribromobenzoic acid) and 100 mg/mL of 4-AAP (4-aminoantipyrine)) and 25 ⁇ L of HRP (horseradish peroxidase, 0.1 mg/mL), Finally, 25 ⁇ L of the above-mentioned DAAO mutant enzyme solution was added to obtain a 200 ⁇ L reaction system of the microtiter plate, which was analyzed at 30 °C and pH 8.0.
  • chromogenic solution containing 60 mg/mL of TBHBA ( 3-hydroxy-2,4,6-tribromobenzoic acid) and 100 mg/mL of 4-AAP (4-aminoantipyrine)
  • Embodiment 4 The mutant rescreening that thermostability improves
  • reaction medium is disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution with a pH of 8.0, shake the reaction at 30°C , every 2 minutes, take the reaction solution to scan the absorbance value at 510nm, make the enzyme reaction kinetic curve of absorbance and time (min), and calculate the enzyme activity according to the slope of the curve.
  • unit enzyme activity under specific reaction conditions (30°C), the amount of enzyme required to generate 1 ⁇ mol H 2 O 2 per minute, and the unit of enzyme activity is U.
  • Alcohol dehydrogenase gene is fully synthesized according to the Cyclopentanol dehydrogenase gene sequence derived from Lactobacillus brevis KB290 (GenBank accession number: BAN05992.1).
  • LB liquid medium composition peptone 10g/L, yeast powder 5g/L, NaCl 10g/L, dissolved in deionized water Dilute to volume, sterilize at 121°C for 20 minutes, and set aside.
  • Alcohol dehydrogenase gene is connected with pET28a, enzyme cutting sites NdeI&HindIII, and the recombinant vector is transformed into host E.coli BL21(DE3) competent cells to obtain engineering strains containing alcohol dehydrogenase gene. After the engineering bacteria containing the alcohol dehydrogenase gene were activated by streaking on a plate, a single colony was picked and inoculated into 5 mL LB liquid medium containing 50 ⁇ g/mL kanamycin, and cultured with shaking at 37°C for 12 hours.
  • Enzyme liquid preparation the thallus with high enzymatic activity screened out in embodiment 4 (enzyme numbering is Enz.27, Enz.29, Enz.32) is placed in the 50mmol/L ammonium phosphate buffer solution of pH 7.0 homogeneously, bacterium The ratio of solid to buffer solution is 1:5 (g:mL). After homogenization, flocculant is added to flocculate and centrifuge to obtain the supernatant.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

提供了一种D-氨基酸氧化酶及其在制备L-草铵膦或其中间体中的应用。提供了一种D-氨基酸氧化酶，其氨基酸序列与SEQ ID NO:1相比包含选自以下一个或多个的氨基酸残基差异：K29G/H/I/N/Q/W/Y/C/L；V42C/D/E/H/Y；E195N/Y/Q；C234L；V326W；并具有不低于如SEQ ID NO:1的氨基酸序列所示的D-氨基酸氧化酶的活性和/或热稳定性。提供了一种热稳定性更高的D-氨基酸氧化酶，在提高酶活性的同时，也扩大了酶的使用温度范围，且在较低温度下使用，能够延长酶的使用寿命；在较高温度下使用，可提高酶的催化效率。

Description

一种D-氨基酸氧化酶及其在制备L-草铵膦或其中间体中的应用
本申请要求申请日为2022/1/30的中国专利申请2022101146927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D-氨基酸氧化酶及其在制备L-草铵膦或其中间体中的应用。
背景技术
草铵膦与草甘膦、百草枯并称世界三大除草剂，都具有非选择性。其中，由于百草枯极强的毒性，近年来在多个国家严被格限制或禁止使用；草甘膦的应用广泛，但我国A级绿色食品已将其从准用名单中去除。草铵膦具有活性高、除草谱广、环境相容性好等特点，并且其发挥除草活性的速度优于草甘膦，也可在杂草对草甘膦产生抗性的地区使用，因此具有很大的市场开发潜力和广阔的应用前景。
2020年，全球草铵膦的市场规模达到10.5亿美元，近6年全球市场复合增长率为6.3％，属于增长最快的非选择性除草剂。因此开发或优化L-草铵膦生产工艺对于提高经济效益、降低使用成本、减轻环境压力具有重要意义。
商业化应用的草铵膦是外消旋体的混合物，有D型和L型两种异构体，但只有L型具有活性，且在土壤中易分解、毒性小。L-草铵膦的生产主要有两类方法：化学法和生物法。化学法生产草铵膦主要是Strecker工艺和热裂解-ACA工艺。其中Srecker工艺流程复杂，涉及原料易燃易爆，且产品的收率较低。热裂解-ACA工艺复杂，且甲基二氯化磷(MDP)是其前端关键中间体，也是该工艺的技术壁垒。生物法反应条件温和、生产流程简单，且产物易于分离，但其产量相对较低，成本较高，目前还未实现产业化。
CN105603015A中发明了以2-氧代-4-(羟基(甲基)膦酰基)丁酸(PPO)及其盐为底物，丙氨酸为氨基供体，利用离体的转氨酶或体外表达转氨酶的细胞催化底物，与丙氨酸发生转氨反应，得到L-草铵膦；CN106978453B中发明了一种利用氨基酸脱氢酶制备L-草铵膦的方法，该方法以2-羰基-4-(羟基甲基膦酰基)丁酸或其盐为底物进行转胺化反应，获得L-草铵膦。此方法原料转化率较高，但底物合成工艺复杂，成本较高。
US9834802B2中发明了以D,L-草铵膦为原料，在D-氨基酸氧化酶突变体的催化作用下生成中间产物PPO，PPO在氨基酸转氨酶的催化作用下生成L-草铵膦。该方法反应得到的组合物含L-草铵膦、D-草铵膦、PPO，其中L-草铵膦含量达到80％以上，D-草铵膦的含量小于10％、PPO含量为小于20％。但是其酶活稳定性仍有待提高。
因此迫切需要寻找一种具有较高的酶活稳定性且D-草铵膦转化率高，L-草铵膦ee值高的D-氨基酸氧化酶。
发明内容
本发明所要解决的技术问题是为了克服现有技术中D-氨基酸氧化酶的酶活性不高、酶活稳定性差和L-草铵膦产率低等缺陷。为解决上述技术问题，本发明提供了一种D-氨基酸氧化酶及其在制备L-草铵膦或其中间体中的应用。热稳定性更高的D-氨基酸氧化酶，在提高酶活性的同时，也扩大了酶的使用温度范围，且在较低温度下使用，能够延长酶 的使用寿命；在较高温度下使用，可提高酶的催化效率。热稳定性高的酶具有提高化学反应速率、提高产品质量、活性稳定、耐储藏等优点。
本发明的第一方面提供一种D-氨基酸氧化酶，其氨基酸序列与SEQ ID NO:1相比包含选自以下一个或多个的氨基酸残基差异：
K29G/H/I/N/Q/W/Y/C/L；V42C/D/E/H/Y；E195N/Y/Q；C234L；V326W；
并具有不低于如SEQ ID NO:1的氨基酸序列所示的D-氨基酸氧化酶的活性和/或热稳定性。
本发明中，氨基酸残基间的“/”代表该氨基酸残基对应的位点具有不同的氨基酸残基差异的选择。例如，E195N/Y/Q代表第195位的差异可以是N、Y或Q。
本发明的一些优选的实施例中，所述D-氨基酸氧化酶的氨基酸序列与SEQ ID NO:1相比包含选自以下两个或多个的氨基酸残基差异：V42Y、E195Y、V326W、C234L。
较佳地，本发明所述D-氨基酸氧化酶与SEQ ID NO:1相比包含C234L的氨基酸残基差异，并进一步包含V42Y、E195Y和V326W中的一个或两个的氨基酸残基差异。
更佳地，所述D-氨基酸氧化酶与SEQ ID NO:1相比具有选自以下任一组的氨基酸残基差异：
C234L和V42Y；
C234L和E195Y；
C234L和V326W；
C234L、K29C和V42Y；
C234L、K29G和V42Y；
C234L、K29L和V42Y；
C234L、V326W和V42Y；
C234L、V42Y和E195Y；
C234L、E195Y和V326W。
本发明的另一些优选的实施例中，所述D-氨基酸氧化酶与SEQ ID NO:1相比包含E195Y的氨基酸差异，并进一步包含V42Y或V326W的氨基酸残基差异；
较佳地，所述D-氨基酸氧化酶与SEQ ID NO:1相比具有选自以下任一组的氨基酸残基差异：E195Y和V42Y；
E195Y和V326W；
E195Y、K29Q和V42Y；
E195Y、K29W和V42Y；
E195Y、K29Y和V42Y。
本发明的另一些优选的实施例中，所述的D-氨基酸氧化酶与SEQ ID NO:1相比具有选自以下任一组的氨基酸残基差异：
K29G；K29H；K29I；K29N；K29Q；K29W；K29Y；V42C；V42D；V42E；V42H；V42P；V42Y；E195H；E195N；E195Y；E195Q；C234L；V326W。
本发明的第二方面提供一种分离的核酸，所述核酸编码如本发明第一方面所述的D-氨基酸氧化酶。
本发明的第三方面提供一种重组表达载体，其包含如本发明第二方面所述的核酸。
本发明的第四方面提供一种转化体，其包含如本发明第二方面所述的核酸或如本发明第三方面所述的重组表达载体。
本发明所述的转化体的宿主细胞可为本领域常规，较佳地为埃希氏大肠杆菌(Escherichia coli)，例如E.coli BL21(DE3)。
本发明的第五方面提供一种制备如本发明所述的D-氨基酸氧化酶的方法，所述方法包括在适于表达所述D-氨基酸氧化酶的条件下培养如本发明第四方面所述的转化体。
本发明的第六方面提供一种2-氧代-4-(羟基甲基氧膦基)丁酸或其盐的制备方法，所述制备方法包括以下步骤：在如本发明第一方面所述的D-氨基酸氧化酶的存在下，将底物进行氧化反应，即得所述的2-氧代-4-(羟基甲基氧膦基)丁酸或其盐。
所述底物较佳地为D-草铵膦或其盐，所述D-草铵膦或其盐可单独存在，或与L-草铵膦或其盐共同存在，例如所述底物以消旋草铵膦或其盐的形式存在。
所述的氧化反应较佳地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-1.5VVM例如1VVM。
所述的氧化反应较佳地在过氧化氢酶的存在下进行。
所述D-氨基酸氧化酶较佳地以D-氨基酸氧化酶的菌体、粗酶、纯酶或固定化酶的形式存在。
所述底物的浓度较佳地为0.1～0.5mol/L；较佳地为0.17mol/L。
所述的氧化反应的反应体系的pH较佳地为7-9，例如8。
所述的氧化反应的反应体系的温度较佳地为20-50℃，例如25℃。
在一些优选实施例中，所述D-氨基酸氧化酶的菌体与所述底物的质量比为1:(0.5-3)，例如1:1。
在一些更优选的实施例中，所述过氧化氢酶与所述D-氨基酸氧化酶的菌体的质量比为1:(20-60)；例如1:40。
所述氧化反应的反应体系较佳地还包括缓冲液，所述缓冲液可为本领域常规，优选为磷酸缓冲液，例如磷酸二氢铵和磷酸氢二铵、磷酸氢二钠和磷酸二氢钠。
所述缓冲液用于调控反应体系的pH，较佳地，所述缓冲液的pH为8.0。
本发明的第七方面提供一种L-草铵膦或其盐的制备方法，其包括如下步骤：
(1)通过如本发明第六方面所述的制备方法获得2-氧代-4-(羟基甲基氧膦基)丁酸或其盐；
(2)在谷氨酸脱氢酶、无机氨基供体和还原型辅酶的存在下，将步骤(1)获得的2-氧代-4-(羟基甲基氧膦基)丁酸或其盐进行氨化反应，即得所述L-草铵膦或其盐。
在本发明所述的L-草铵膦或其盐的制备方法中，所述还原型辅酶较佳地为NADPH或NADH。
在本发明所述的L-草铵膦或其盐的制备方法中，所述的无机氨基供体较佳地为氨气、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。
在本发明所述的L-草铵膦或其盐的制备方法中，所述氨化反应的反应体系的pH较佳地为7-10，更佳地为8.4-8.6。
在本发明所述的L-草铵膦或其盐的制备方法中，所述氨化反应的反应温度较佳地为28-35℃，更佳地为30-33℃。
在本发明所述的L-草铵膦或其盐的制备方法中，所述谷氨酸脱氢酶较佳地为CN201910434350.1中的突变体1-4。
在本发明所述的L-草铵膦或其盐的制备方法中，所述谷氨酸脱氢酶与所述底物D-草铵膦的质量比较佳地为1:(0.5-3)，例如1:1.25。
在本发明所述的L-草铵膦或其盐的制备方法中，所述底物D-草铵膦与所述无机氨基供体的摩尔比较佳地为1：(1～1.5)，例如1:1。
本发明所述的L-草铵膦或其盐的制备方法较佳地还包括以下步骤：在脱氢酶以及氢供体的存在下，将氧化型辅酶进行还原反应，得到所述的还原型辅酶即可。
所述的脱氢酶较佳地为葡萄糖脱氢酶、醇脱氢酶或甲酸脱氢酶。
所述的氢供体较佳地为葡萄糖、异丙醇或甲酸盐。
本发明一些优选实施例中，当所述的脱氢酶为醇脱氢酶时，所述的氢供体为异丙醇；当所述的脱氢酶为葡萄糖脱氢酶时，所述的氢供体为葡萄糖；当所述的脱氢酶为甲酸脱氢酶时，所述的氢供体为甲酸盐。
本发明所述的L-草铵膦或其盐的制备方法可以分步骤进行，例如先进行氧化反应制 备获得2-氧代-4-(羟基甲基氧膦基)丁酸或其盐，再进行氨化反应制备获得L-草铵膦或其盐；也可以通过“一锅法”的方法进行，例如将所有原料混合后制备获得L-草铵膦或其盐。
本发明的第八方面提供一种如本发明第一方面所述的D-氨基酸氧化酶在制备L-草铵膦或其盐、2-氧代-4-(羟基甲基氧膦基)丁酸或其盐中的应用。
本发明提及的DAAO酶与D-氨基酸氧化酶可互换地使用。
本发明中D-氨基酸氧化酶作用底物的量都以D-草铵膦或其盐来计算。
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：提供了一种热稳定性更高的D-氨基酸氧化酶，在提高酶活性的同时，也扩大了酶的使用温度范围，且在较低温度下使用，能够延长酶的使用寿命；在较高温度下使用，可提高酶的催化效率。
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。
产物L-草铵膦的手性分析及浓度分析通过柱前衍生化高效液相色谱进行，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+H2O，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
(3)衍生反应：称取50mg供试品于25ml容量瓶中，加15ml稀释液(纯水：乙腈＝50:50)并超声5min，再加纯化水稀释至刻度，混合均匀。移取上述溶液1mL于5mL容量瓶中，加入1mL的Marfey’s reagent溶液和0.1mL碳酸氢钠(1M)溶液，盖上盖子于50℃烘箱中避光加热1小时，反应结束后再加入0.1mL盐酸溶液，混合均匀。
移取1mL上述混合溶液，再加入4mL的稀释液，混合均匀即可倒入进样瓶。进样10μL进行分析。
PPO通过离子对色谱分析，具体的分析方法为：
色谱条件：ΜLtimate 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℃。
样品：5mg/mL H2O溶液。进样10μL进行分析。
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
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

pET28a质粒和bugbuster protein extraction reagent购买自Novagen公司；NdeI酶、HindIII酶购买自Thermo Fisher公司；E.coli BL21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。
实施例1 D-氨基酸氧化酶(DAAO)的制备
DAAO酶工程菌来源于专利CN111019916B所公开的含seq.79的工程菌株，氨基酸序列如SEQ ID NO:1所示，核苷酸序列如SEQ ID NO:12所示。
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。固体培养基为LB培养基加入2％的琼脂。
将上述DAAO酶工程菌经平皿划线活化后，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，降温至30℃，加入IPTG至其终浓度为0.5mM，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℃冰箱中保存，待用。
将培养结束后收集到的菌体，用50mM pH 8.0磷酸缓冲液洗涤两次，之后重悬于50mL pH 8.0的磷酸缓冲液中，均质破碎，破碎液离心去除沉淀，得到含重组DAAO酶的粗酶液。
实施例2 D-氨基酸氧化酶(DAAO)突变体的构建
一、工程菌活化和质粒提取
将实施例1所述活化的DAAO酶工程菌接入含有5mL LB培养基的试管中，37℃、200rpm培养8-12h。在获得培养菌体后，根据生工质粒提取试剂盒的操作说明书进行质粒提取。所得质粒直接用于点突变，或者置于-80℃冰箱长期保存。
二、突变体文库(K29位、V42位点、E195位点、C234位点、V326位点)的构建
基因突变采用全质粒PCR的方法，获得突变基因。
以上述步骤所提取质粒为模板，针对突变的D-氨基酸氧化酶序列的K29位、V42位点、E195位点、C234位点、V326位点进行突变的突变体文库构建设计PCR的引物序列，获得目标突变体的基因。引物序列具体如表3所示：
表3
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K29-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。
PCR扩增体系为：
表4
PCR扩增程序如下：
表5
加入DpnI酶对PCR产物进行消化，37℃，2h。反应完成转化至E.coli BL21感受态细胞，涂布在含有50μg/mL卡纳霉素的LB培养基，37℃培养过夜，收菌，得到包含突变体文库的转化子。
三、组合突变
将筛出的有益突变，通过overlap PCR的方式，实现多种不同的突变组合，形成新的突变转化子。
实施例3 高通量突变体文库初筛
将实施例2中所得转化子接种96孔板培养，加IPTG至其终浓度为0.5mM，30℃过夜诱导。之后收菌，加bugbuster protein extraction reagent裂解，离心取上清液得DAAO突变体酶液。
热稳定性筛选方法：
将上述上清酶液置于60℃水浴锅，热处理20min。再使用“酶活检测分析”，筛选阳性克隆。
酶活检测分析：取100μL pH为8.0的100mM底物(D,L-草铵膦，购自上海阿拉丁生化科技股份有限公司)，再加50μL的显色液(含60mg/mL的TBHBA(3-羟基-2,4,6-三溴苯甲酸)和100mg/mL的4-AAP(4-氨基安替比林))和25μL HRP(辣根过氧化物酶，0.1mg/mL)，最后加25μL的上述DAAO突变体酶液，得酶标板200μL反应体系，在30℃，pH 8.0的条件下对其进行分析。分别在0min和20min记录510nm处的吸光度，取差值，以野生型为参照系，筛选阳性克隆子。筛选出酶活性与Enz.01相当或更高，且热稳定增强的阳性克隆子且见表6。
表6

实施例4 热稳定性提高的突变体复筛
突变体复筛的酶活检测方法：
5mL反应体系中，加入1mL 500mM的D,L-草铵膦(铵盐)，0.25mL热处理(60℃，20min)后的DAAO突变体粗酶液，1.25mL辣根过氧化物酶(HRP)，2.5mL显色染料溶液(含60mg/mL的TBHBA和100mg/mL的4-AAP)，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℃摇床震荡反应，每隔2min，取反应液扫描510nm下的吸光值，做吸光度和时间(min)的酶反应动力学曲线，根据曲线斜率计算酶活。
单位酶活的定义：在特定反应条件(30℃)下，每分钟生成1μmol H2O2所需要的酶量，酶活单位是U。
根据以上酶活测定方法，检测热处理前和热处理后酶活，计算热稳定性相对Enz.1提高的倍数。结果见下表7。*表示热稳定性提高1～1.2倍(不含1.2)，**表示热稳定性提高1.2～2倍(不含2)，***表示热稳定性提高2倍及以上。热稳定性提高倍数计算方法：突变体热处理后酶活与处理前酶活比/Enz.01热处理后酶活与处理前酶活比。
表7

如上表所述，说明多数单点突变酶和组合突变酶热稳定的提高效果显著，Enz.27、Enz.29、Enz.32效果更佳。
实施例5 L-草铵膦的制备
本发明涉及的反应路线如下所示：
根据来源于短乳杆菌(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，将重组载体转化至宿主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-20℃超低温冰箱中保存，待用。
酶液制备：实施例4所筛选出的酶活性高的菌体(酶编号为Enz.27、Enz.29、Enz.32)置于pH 7.0的50mmol/L磷酸铵缓冲液中均质，菌体与缓冲液比例为1:5(g：mL)，均质后加絮凝剂絮凝离心得上清。
向10L夹套反应釜中加入4600g水，投入0.28g磷酸二氢铵和5.57g磷酸氢二铵搅拌溶清后向反应釜内投入400g D,L-草铵膦(铵盐)，搅拌溶解，氨水调节溶液pH 7.9-8.1，向反应釜内投入5g过氧化氢酶(酶活80万μ/g)，投入1000mL(200g菌体)突变体DAAO酶液(Enz.27、Enz.29或Enz.32)，空气鼓气，通气量控制在每分钟1反应体积，控制温度25℃，反应20h。
控制温度为30-33℃，加入0.4g NADP+、78g异丙醇、50g氯化铵，氨水调节pH 8.4-8.6，加入0.35g乙醇脱氢酶(ADH)菌体。待温度、pH正常，向釜内投入160g谷氨酸脱氢酶(即本公司CN201910434350.1中突变体1-4)菌体，开始反应。反应6h，检测PPO残留量≤2％。
表8
反应结果如表8所示；Enz.27、Enz.29和Enz.32反应24h后的ee值均在98％以上，显著优于Enz.1。

Claims (14)

  1. 一种D-氨基酸氧化酶，其氨基酸序列与SEQ ID NO:1相比包含选自以下一个或多个的氨基酸残基差异：
    K29G/H/I/N/Q/W/Y/C/L；V42C/D/E/H/Y；E195N/Y/Q；C234L；V326W；
    并具有不低于如SEQ ID NO:1的氨基酸序列所示的D-氨基酸氧化酶的活性和/或热稳定性。
  2. 如权利要求1所述的D-氨基酸氧化酶，其特征在于，所述D-氨基酸氧化酶的氨基酸序列与SEQ ID NO:1相比包含选自以下两个或多个的氨基酸残基差异：V42Y、E195Y、V326W、C234L。
  3. 如权利要求2所述的D-氨基酸氧化酶，其特征在于，所述D-氨基酸氧化酶与SEQ ID NO:1相比包含C234L的氨基酸残基差异，并进一步包含V42Y、E195Y和V326W中的一个或两个的氨基酸残基差异；
    较佳地，所述D-氨基酸氧化酶与SEQ ID NO:1相比具有选自以下任一组的氨基酸残基差异：
    C234L和V42Y；
    C234L和E195Y；
    C234L和V326W；
    C234L、K29C和V42Y；
    C234L、K29G和V42Y；
    C234L、K29L和V42Y；
    C234L、V326W和V42Y；
    C234L、V42Y和E195Y；
    C234L、E195Y和V326W。
  4. 如权利要求2所述的D-氨基酸氧化酶，其特征在于，所述D-氨基酸氧化酶与SEQ ID NO:1相比包含E195Y的氨基酸差异，并进一步包含V42Y或V326W的氨基酸残基差异；
    较佳地，所述D-氨基酸氧化酶与SEQ ID NO:1相比具有选自以下任一组的氨基酸残基差异：
    E195Y和V42Y；
    E195Y和V326W；
    E195Y、K29Q和V42Y；
    E195Y、K29W和V42Y；
    E195Y、K29Y和V42Y。
  5. 如权利要求1所述的D-氨基酸氧化酶，其特征在于，其氨基酸序列与SEQ ID NO:1相比具有选自以下任一组的氨基酸残基差异：
    K29G；K29H；K29I；K29N；K29Q；K29W；K29Y；V42C；V42D；V42E；V42H；V42P；V42Y；E195H；E195N；E195Y；E195Q；C234L；V326W。
  6. 一种分离的核酸，其特征在于，所述核酸编码如权利要求1-5任一项所述的D-氨基酸氧化酶。
  7. 一种重组表达载体，其包含如权利要求6所述的核酸。
  8. 一种转化体，其包含如权利要求6所述的核酸或如权利要求7所述的重组表达载体；较佳地，所述转化体的宿主细胞为埃希氏大肠杆菌(Escherichia coli)例如E.coli BL21(DE3)。
  9. 一种制备如权利要求1-5任一项所述的D-氨基酸氧化酶的方法，其特征在于，所述方法包括在适于表达所述D-氨基酸氧化酶的条件下培养如权利要求8所述的转化体。
  10. 一种2-氧代-4-(羟基甲基氧膦基)丁酸或其盐的制备方法，其特征在于，所述制备方法包括以下步骤：在如权利要求1-5任一项所述的D-氨基酸氧化酶的存在下，将底物进行氧化反应，即得所述的2-氧代-4-(羟基甲基氧膦基)丁酸或其盐；
    较佳地：
    所述底物为D-草铵膦或其盐，所述D-草铵膦或其盐可单独存在，或与L-草铵膦或其盐共同存在；例如所述底物以消旋草铵膦或其盐的形式存在；
    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-1.5VVM例如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D-氨基酸氧化酶以D-氨基酸氧化酶的菌体、粗酶、纯酶或固定化酶的形式存在；
    和/或，所述底物的浓度为0.1～0.5mol/L；较佳地为0.17mol/L；
    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25℃；
    更佳地，所述D-氨基酸氧化酶的菌体与所述底物的质量比为1:(0.5-3)，例如1:1；和/或，所述过氧化氢酶与所述D-氨基酸氧化酶的菌体的质量比为1:(20-60)；例如1:40。
  11. 如权利要求10所述的制备方法，其特征在于，所述氧化反应的反应体系包括缓 冲液，所述缓冲液优选为磷酸缓冲液，例如磷酸二氢铵和磷酸氢二铵、磷酸氢二钠和磷酸二氢钠。
  12. 一种L-草铵膦或其盐的制备方法，其包括如下步骤：
    (1)通过如权利要求10或11所述的制备方法获得2-氧代-4-(羟基甲基氧膦基)丁酸或其盐；
    (2)在谷氨酸脱氢酶、无机氨基供体和还原型辅酶的存在下，将步骤(1)获得的2-氧代-4-(羟基甲基氧膦基)丁酸或其盐进行氨化反应，即得所述L-草铵膦或其盐。
  13. 如权利要求12所述的制备方法，其特征在于，
    所述还原型辅酶为NADPH或NADH；
    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，所述氨化反应的反应体系的pH为7-10，较佳地为8.4-8.6；
    和/或，所述氨化反应的反应温度为28-35℃，较佳地为30-33℃。
  14. 一种如权利要求1-5任一项所述的D-氨基酸氧化酶在制备L-草铵膦或其盐、2-氧代-4-(羟基甲基氧膦基)丁酸或其盐中的应用。
PCT/CN2023/073897 2022-01-30 2023-01-30 一种d-氨基酸氧化酶及其在制备l-草铵膦或其中间体中的应用 WO2023143621A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202210114692.7A CN116555206A (zh) 2022-01-30 2022-01-30 一种d-氨基酸氧化酶及其在制备l-草铵膦或其中间体中的应用
CN202210114692.7 2022-01-30

Publications (1)

Publication Number Publication Date
WO2023143621A1 true WO2023143621A1 (zh) 2023-08-03

Family

ID=87470730

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/073897 WO2023143621A1 (zh) 2022-01-30 2023-01-30 一种d-氨基酸氧化酶及其在制备l-草铵膦或其中间体中的应用

Country Status (2)

Country Link
CN (1) CN116555206A (zh)
WO (1) WO2023143621A1 (zh)

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170253897A1 (en) * 2016-03-02 2017-09-07 Agrimetis, Llc Methods for making l-glufosinate
CN109576236A (zh) * 2018-12-28 2019-04-05 浙江工业大学 一种d-氨基酸氧化酶突变体及其应用
CN110055289A (zh) * 2018-01-19 2019-07-26 上海弈柯莱生物医药科技有限公司 一种l-草铵膦的制备方法
CN110592036A (zh) * 2019-08-30 2019-12-20 浙江工业大学 一种草铵膦脱氢酶突变体及在氧化-还原多酶偶联生产l-草铵膦中的应用
WO2020103928A1 (zh) * 2018-11-23 2020-05-28 上海弈柯莱生物医药科技有限公司 一种d-氨基酸氧化酶突变体及其应用
WO2020233451A1 (zh) * 2019-05-23 2020-11-26 弈柯莱生物科技(上海)股份有限公司 一种l-谷氨酸脱氢酶突变体及其应用
CN113969269A (zh) * 2021-04-29 2022-01-25 永农生物科学有限公司 D-氨基酸氧化酶突变体及其在制备l-草铵膦中的应用

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170253897A1 (en) * 2016-03-02 2017-09-07 Agrimetis, Llc Methods for making l-glufosinate
CN110055289A (zh) * 2018-01-19 2019-07-26 上海弈柯莱生物医药科技有限公司 一种l-草铵膦的制备方法
WO2020103928A1 (zh) * 2018-11-23 2020-05-28 上海弈柯莱生物医药科技有限公司 一种d-氨基酸氧化酶突变体及其应用
CN109576236A (zh) * 2018-12-28 2019-04-05 浙江工业大学 一种d-氨基酸氧化酶突变体及其应用
WO2020233451A1 (zh) * 2019-05-23 2020-11-26 弈柯莱生物科技(上海)股份有限公司 一种l-谷氨酸脱氢酶突变体及其应用
CN110592036A (zh) * 2019-08-30 2019-12-20 浙江工业大学 一种草铵膦脱氢酶突变体及在氧化-还原多酶偶联生产l-草铵膦中的应用
CN113969269A (zh) * 2021-04-29 2022-01-25 永农生物科学有限公司 D-氨基酸氧化酶突变体及其在制备l-草铵膦中的应用

Also Published As

Publication number Publication date
CN116555206A (zh) 2023-08-08

Similar Documents

Publication Publication Date Title
JP7436450B2 (ja) Ｌ－グルタミン酸脱水素酵素突然変異体及びその使用
WO2022001038A1 (zh) 一种草铵膦脱氢酶突变体、基因工程菌及一锅法多酶同步定向进化方法
US11339380B2 (en) Glutamate dehydrogenase mutant and application thereof
CN109609475B (zh) 草铵膦脱氢酶突变体及其合成l-草铵膦的应用
CN111321193B (zh) 一种生物多酶偶联法氧化还原不对称制备l-草铵膦的方法
CN108795835B (zh) 一种基因工程菌及其在制备l-草铵膦中的应用
CN110592036A (zh) 一种草铵膦脱氢酶突变体及在氧化-还原多酶偶联生产l-草铵膦中的应用
WO2020103928A1 (zh) 一种d-氨基酸氧化酶突变体及其应用
JP6276178B2 (ja) シス−５−ヒドロキシ−ｌ−ピペコリン酸の生物学的な製造方法
CN111454998B (zh) 一种手性羟基酸酯的生物制备方法
CN107119084B (zh) 一种利用转氨酶和乙烯合成酶生产l-草铵膦的方法
US11905534B2 (en) L-glutamate dehydrogenase mutant and application thereof
US20230024648A1 (en) Ean B Mutants and Their Uses
WO2012124513A1 (ja) Ｎ－サクシニル－ｄｌ－アミノ酸に対する向上されたｄ体選択性を有する改変型ｄ－サクシニラーゼ
CN111876396B (zh) 双辅酶依赖型草铵膦脱氢酶突变体及其在催化合成l-草铵膦中的应用
CN113322291A (zh) 一种手性氨基醇类化合物的合成方法
WO2023143621A1 (zh) 一种d-氨基酸氧化酶及其在制备l-草铵膦或其中间体中的应用
CN113897382B (zh) 辅酶自足型大肠杆菌及其构建方法与应用
CN111019982A (zh) 一种利用羟基酸脱氢酶制备l-草铵膦的方法
CN114350631B (zh) 草铵膦脱氢酶突变体、工程菌、固定化细胞及应用
US7022502B2 (en) Process for the biological production of L-pipecolic acid
CN108456668B (zh) 一种核糖体结合位点、重组表达质粒、转化子及其应用
CN111019917A (zh) 一种l-谷氨酸脱氢酶突变体及其应用
CN117402846B (zh) L-丙氨酸脱氢酶突变体及其制备方法和应用
US11781117B2 (en) Machine learning gene mining method and phosphinothricin dehydrogenase mutant for amino translocation

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23746498

Country of ref document: EP

Kind code of ref document: A1