WO2020103928A1 - 一种d-氨基酸氧化酶突变体及其应用 - Google Patents

一种d-氨基酸氧化酶突变体及其应用

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Publication number
WO2020103928A1
WO2020103928A1 PCT/CN2019/120249 CN2019120249W WO2020103928A1 WO 2020103928 A1 WO2020103928 A1 WO 2020103928A1 CN 2019120249 W CN2019120249 W CN 2019120249W WO 2020103928 A1 WO2020103928 A1 WO 2020103928A1
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Prior art keywords
seq
amino acid
acid residue
mutated
sequence
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PCT/CN2019/120249
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English (en)
French (fr)
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田振华
程占冰
徐艳冰
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上海弈柯莱生物医药科技有限公司
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Application filed by 上海弈柯莱生物医药科技有限公司 filed Critical 上海弈柯莱生物医药科技有限公司
Priority to US17/295,676 priority Critical patent/US11667897B2/en
Priority to EP19887398.6A priority patent/EP3885435A4/en
Publication of WO2020103928A1 publication Critical patent/WO2020103928A1/zh

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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)
    • C12N9/0014—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
    • 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, in particular to a D-amino acid oxidase mutant and its application in the preparation of 2-oxo-4- (hydroxymethylphosphinyl) butanoic acid (PPO).
  • PPO 2-oxo-4- (hydroxymethylphosphinyl) butanoic acid
  • Glufosinate is a broad-spectrum contact herbicide developed by Hearst Corporation in the 1980s. At present, the three major herbicides in the world are glyphosate, glufosinate, and paraquat. Compared with glyphosate and paraquat, glufosinate has excellent herbicidal performance and minor side effects. There are two optical isomers of glufosinate, namely D-glufosinate and L-glufosinate, but only L-glufosinate has herbicidal activity, so the method of developing L-glufosinate is to improve the economical efficiency It is of great significance to reduce the use cost and reduce the environmental pressure.
  • a method for preparing L-glufosinate by transaminase-catalyzed 2-oxo-4- (hydroxymethylphosphinyl) butanoic acid (PPO) is generally used for preparing L-glufosinate.
  • PPO hydroxymethylphosphinyl butanoic acid
  • US5221737A and EP0344683A describe the use of glutamic acid as the amino donor to obtain L-grass from the corresponding keto acid 4- (hydroxymethylphosphinyl) -2-oxobutanoic acid by the aminotransferase derived from E. coli Ammonium phosphine method.
  • the reaction system requires an equal or excess amount of amino donor glutamic acid, making the product difficult to purify.
  • CN1284858C improves the above method, adopts aspartic acid as the amino donor, and obtains L- from the corresponding keto acid 4- (hydroxymethylphosphinyl) -2-oxobutanoic acid through the action of aspartate aminotransferase
  • the method of glufosinate In this method, aspartic acid is converted to oxaloacetic acid, and oxaloacetic acid is unstable in an aqueous medium, and spontaneously decarboxylated to pyruvate; pyruvate can be removed by an enzymatic reaction, making the reverse reaction impossible
  • the reaction requires only equimolar amino donors and amino acceptors.
  • most of the amino donors used in the method using aminotransferase are amino acids, and the cost is relatively high.
  • US9834802B discloses a method for preparing L-glufosinate by using D, L-glufosinate as a raw material.
  • D-amino acid oxidase DAAO
  • PPO is catalyzed by transaminase. L-glufosinate.
  • the technical problem to be solved by the present invention is to overcome the defects of the prior art that the enzyme activity of D-amino acid oxidase is not high, the stability of enzyme activity is poor, or the ammonium resistance is poor.
  • the present invention provides a mutant of D-amino acid oxidase and Its application in the preparation of 2-oxo-4- (hydroxymethylphosphinyl) butyric acid.
  • the D-amino acid oxidase mutant of the present invention has high enzyme activity, improved enzyme activity stability and / or enhanced ammonium resistance, thereby reducing costs and facilitating industrial production.
  • the inventor discovered at the beginning of the experiment that high concentrations of ammonium ions have a great inhibitory effect on enzyme activity, screened the mutants at position 54, 58 and 213 of the prior art DAAO amino acid sequence and found The 76% -derived N 2 DAAO sequence has the weakest inhibition, so the N 2 DAAO sequence was selected for further mutation. After extensive screening, the inventors found that when introducing mutations at the C211 site, the resulting mutants had increased enzyme activity under the action of ammonium ions, and then combined mutations at sites including C211 and constructed mutant libraries , From which the D-amino acid oxidase mutants of the present invention were screened.
  • the present invention aims to provide a D-amino acid oxidase mutant whose sequence includes the sequence shown in SEQ ID NO. 1 or at least the sequence shown in SEQ ID NO. 1.
  • the at least 76% identity is preferably at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity Sex, at least 98% identity, or at least 99% identity.
  • the ammonium resistance of the present invention refers to the increased tolerance of the mutant of the present invention to ammonium ions during the reaction.
  • the enhanced tolerance is mainly reflected in that the mutant of the present invention remains stable when the concentration of ammonium ions is high That is, there is little loss of enzyme activity), the enzyme activity is still high.
  • sequence having at least 76% identity with SEQ ID NO. 1 is shown in SEQ ID NO. 3.
  • the sequence of the D-amino acid oxidase mutant includes mutating the amino acid residue N at position 54 shown in SEQ ID NO. 1 to A, C, G, S, T or V; and / or, Amino acid residue F at position 58 is mutated to a hydrophilic or slightly hindered amino acid residue; and / or, amino acid residue C at position 211 is mutated to A, D, G, H, L, M, S, or Y; And / or, the amino acid residue M at position 213 is mutated to the sequence of A, C, F, L, R, S, T, V or W.
  • the hydrophilicity or small steric hindrance means that the mutated amino acid residue is more hydrophilic or has less steric hindrance than the amino acid residue in the wild sequence.
  • the amino acids may be modified or unmodified natural amino acids; the present invention takes natural amino acids as an example.
  • the amino acid residue N at position 54 is mutated to V
  • the amino acid residue F at position 58 is mutated to Q
  • the amino acid residue C at position 211 is mutated to A, D, G, H, L, M, S Or Y
  • the amino acid residue M at position 213 is mutated to A, C, F, L, R, S, T, V, or W
  • the amino acid residue C at position 211 is mutated to A, G, M, S, Y, or L
  • the amino acid residue M at position 213 is mutated to F, L, R, T, or W
  • the amino acid residue C at position 211 is mutated to L Or M, and / or, the amino acid residue M at position 213 is mutated to T.
  • the amino acid residue C at position 211 is mutated to L
  • the amino acid residue M at position 213 is mutated to T
  • the amino acid residue N at position 54 is mutated to A, C, G, S, T or V
  • Amino acid residue F at position 58 is mutated to A, G, H, K or Q
  • amino acid residue N at position 54 is mutated to A, G, S or T, and / or, the 58th Amino acid residue F is mutated to H, K or Q
  • the amino acid residue N at position 54 is mutated to A, and / or, the amino acid residue F at position 58 is mutated to H or K
  • the sequence of the D-amino acid oxidase mutant further includes mutating the amino acid residue T at position 56 shown in SEQ ID NO. 1 to N, S, or L, and the amino acid residue at position 56 T is preferably mutated to N.
  • the amino acid sequence of the D-amino acid oxidase mutant is SEQ ID NO. 5, SEQ ID NO. 7, 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 SEQ ID NO. 39, SEQ ID NO. 41, SEQ ID NO. 43, SEQ ID NO. 45, SEQ ID NO.
  • SEQ ID NO.49 SEQ ID NO.51, SEQ ID NO.53, SEQ ID NO.55, SEQ ID NO.57, SEQ ID NO.59, SEQ ID NO.61, SEQ ID NO.63, SEQ ID NO.65 , SEQ ID NO. 68, SEQ ID NO. 70, SEQ ID NO. 72, SEQ ID NO. 75, SEQ ID NO. 79, SEQ ID NO. 82, SEQ ID NO. 90, SEQ ID NO. 100, or SEQ ID NO.102.
  • the nucleotide sequence encoding the D-amino acid oxidase mutant is SEQ ID NO. 6, SEQ ID NO. 8, 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. 22, 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, SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID NO. 42, SEQ ID NO. 44, SEQ ID NO. 46, SEQ ID NO. 48.
  • the present invention aims to provide an isolated nucleic acid encoding the above-mentioned D-amino acid oxidase mutant.
  • the present invention aims to provide a recombinant expression vector containing the above nucleic acid.
  • the present invention aims to provide a transformant comprising the above nucleic acid or the above recombinant expression vector.
  • the present invention aims to provide an application of the above-mentioned D-amino acid oxidase mutant in the preparation of 2-oxo-4- (hydroxymethylphosphinyl) butyric acid.
  • the enzyme activity of the present invention includes specific enzyme activity and enzyme activity characteristics of the enzyme.
  • the reagents and raw materials used in the present invention are commercially available.
  • the positive progress effect of the present invention is that the D-amino acid oxidase mutant of the present invention has high enzyme activity, improved enzyme activity stability and / or enhanced ammonium resistance, thereby reducing costs and facilitating industrial production.
  • the experimental methods in the present invention are conventional methods, and the gene cloning operation can be specifically participated in "Molecular Cloning Experimental Guide” edited by J. Sambrook and others.
  • Amino acid name Three letter symbol Single letter symbol Amino acid name Three letter symbol Single letter symbol Amino acid name Three letter symbol Single letter symbol Alanine Ala A Leucine Leu L Arginine Arg R Lysine Lys K Asparagine Asn N Methionine Met M Aspartic acid Asp D Phenylalanine Phe F Cysteine Cys C Proline Pro P Glutamine (glutanine) Gln Q Serine Ser S Glutamic acid Glu E Threonine Thr T Glicine Gly G Tryptophan Trp W Histidine His H Tyrosine Tyr Y Isoleucine Ile I Valine Val V
  • 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.
  • Pet28a and bugbuster protein were purchased from Novagen; NdeI and HindIII enzymes were purchased from Thermo Fisher; BL21 competent cells were purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd .; catalase was purchased from Shandong Fengtai Biotechnology Co., Ltd. the company.
  • the synthesized wtN 2 DAAO enzyme gene was ligated into plasmid pET28a (for specific methods, see J. Am. Chem. Soc., 2017, 139 (32), 11241-11247), and the enzyme cleavage sites were NdeI and HindIII.
  • the enzyme-linked vector was transformed into host E. coli BL21 competent cells. It was inoculated with LB liquid medium at 37 ° C and cultured on a 200 rpm shaker. When the OD 600 until to 0.8, was added to a final concentration of bacteria take 25% sterile glycerol number, placed in a deep freezer -80 °C preservation backup.
  • Composition of LB liquid culture medium peptone 10g / L, yeast powder 5g / L, NaCl 10g / L, dissolved in deionized water, set the volume, sterilized at 121 ° C for 20min, and ready for use.
  • the cells collected at the end of the culture were washed with 50 mM pH 8.0 phosphate buffer, and washed twice. Afterwards, it was resuspended in 50 mL of phosphate buffer at pH 8.0 and homogenized. The crushed liquid was centrifuged to remove the precipitate to obtain a crude enzyme liquid containing recombinant wtN 2 DAAO enzyme.
  • the mutant D-amino acid oxidase sequence was obtained, and the gene N 2 DAAO was synthesized according to the sequence (N54V, F58Q), the gene synthesis company is Suzhou Jinweizhi Biological Technology Co., Ltd. (Floor C3, Bio-Nano Technology Park, 218 Xinghu Street, Suzhou Industrial Park). Then, NdeI and HindIII were used for double digestion, and the mutant gene was introduced into plasmid pET28a to construct plasmid pET28a-N 2 DAAO (see J. Am. Chem. Soc., 2017, 139 (32), 11241-11247 for plasmid construction methods). Using the plasmid pET28a-N 2 DAAO as a template, the target band was amplified by PCR.
  • the primer sequences of the designed PCR were constructed based on the mutant library of the mutated D-amino acid oxidase sequence (N54V, F58Q) at positions 211 and 213, as shown in Table 3:
  • 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 encoding nucleotide of the amino acid to be mutated at the site ,
  • NNK in the A166-forward primer can represent AAG (lysine), AAT (aspartic acid), AGG (arginine) or AGT (serine), etc.
  • the nucleotides corresponding to specific amino acids can be found in Table 2.
  • the PCR amplification system is:
  • Reagent Dosage ( ⁇ L) 2 ⁇ PCR buffer (including high fidelity enzyme) 25 Forward primer 1 Reverse primer 1 template 1
  • the PCR amplification procedure is as follows:
  • the PCR product was digested with DpnI at 37 ° C for 2h. After the reaction was completed, the cells were transformed into BL21 competent cells, spread on LB medium containing 50 ⁇ g / mL kanamycin, and cultured overnight at 37 ° C. Collect bacteria to obtain transformants containing the mutant library.
  • Example 2 The transformants obtained in Example 2 were inoculated into 96-well plates and cultured, and IPTG was added at 30 ° C overnight for induction. After collecting bacteria, add bugbuster protein extraction reagent to lyse and centrifuge to obtain DAAO mutant enzyme solution.
  • Detection method of microplate reader take 100 ⁇ L of 100 mM substrate with pH 8.0 (racemic glufosinate, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.), add 50 ⁇ L of color developing solution (containing 60 mg / mL TBHBA (3- Hydroxy-2,4,6-tribromobenzoic acid), 100 mg / mL of 4-AAP (4-aminoantipyrine)) and 25 ⁇ L of HRP (horseradish peroxidase, 0.1 mg / mL), and finally add 25 ⁇ L of the DAAO mutant enzyme solution was used to obtain a 200 ⁇ L reaction system of an enzyme-labeled plate. It was analyzed at 30 ° C and pH 8.0. The absorbance at 510 nm was recorded at 0 min and 20 min, respectively, and the difference was taken. The wild type was used as a reference line to screen positive clones.
  • Composition of LB liquid culture medium peptone 10g / L, yeast powder 5g / L, NaCl 10g / L, dissolved in deionized water, set the volume, sterilized at 121 ° C for 20min, and ready for use.
  • the method of enzyme activity detection for mutant rescreening is:
  • reaction medium is disodium hydrogen phosphate-sodium dihydrogen phosphate buffer with pH 8.0.
  • the reaction was shaken on a shaker at 30 ° C, and every 2 minutes, the absorbance value at 510 nm was scanned from the reaction solution to make an enzyme reaction kinetic curve of absorbance and time (min), and the enzyme activity was calculated according to the slope of the curve.
  • the results are shown in Table 4.
  • unit enzyme activity Under specific reaction conditions (30 ° C), the amount of enzyme required to generate 1 ⁇ mol H 2 O 2 per minute, the unit of enzyme activity is U.
  • Specific enzyme activity is the unit of activity contained per milligram of enzyme protein.
  • Example 4 Construction of a mutant library with mutations at positions 54, 56 and 58 of the DAAO oxidase mutant 11 in Example 3
  • the primer sequences designed for the DAAO oxidase mutant 11 at the 54th, 56th, and 58th mutant library construction are shown in Table 5.
  • 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 encoding nucleotide of the amino acid to be mutated at the site ,
  • NNK in the A166-forward primer can represent AAG (lysine), AAT (aspartic acid), AGG (arginine) or AGT (serine), etc.
  • the nucleotides corresponding to specific amino acids can be found in Table 2.
  • the plasmid template pET28a-DAAO oxidase mutant 12 was constructed according to the method disclosed in the literature J. Am. Chem. Soc., 2017, 139 (32), 11241-11247, using pET28a-DAAO oxidase mutant 11 as a template, Perform PCR to amplify the band of interest.
  • the amplification reaction system is:
  • the amplification procedure is as follows:
  • the PCR product was digested with DpnI at 37 ° C for 2h. After the reaction was completed, the cells were transformed into BL21 competent cells and plated on LB medium containing 50 ⁇ g / mL kanamycin. After culturing overnight at 37 ° C, the bacteria were harvested to obtain transformants containing the mutant library.
  • Example 4 The transformants obtained in Example 4 were inoculated into 96-well plates and cultured, and IPTG was added at 30 ° C overnight to induce. After collecting bacteria, add bugbuster protein extraction reagent to lyse and centrifuge to obtain the mutant enzyme solution.
  • the mutant enzyme solution was treated at 50 ° C for 2h. According to the detection method of the microplate reader described in Example 3, the heat-stable mutation effect of the positive clones was analyzed, and the positive clones were screened.
  • Composition of LB liquid culture medium peptone 10g / L, yeast powder 5g / L, NaCl 10g / L, dissolved in deionized water, set the volume, sterilized at 121 ° C for 20min, and ready for use.
  • the obtained DAAO mutant crude enzyme solution was subjected to enzyme activity detection of mutant rescreening, and the method was the same as that in Example 3.
  • the results obtained are shown in Table 6, where the remaining enzyme activity value after 2h reflects the stability of the mutant enzyme activity.
  • # represents the remaining enzyme activity between 0-50% after 2h
  • ## represents the remaining enzyme activity between 50-80% after 2h
  • ### represents the remaining enzyme activity after 2h Between 80-100%.
  • Mutants 39 and 40 have the same amino acid sequence, but the DNA sequence is different, and the codons are different.
  • the sequence table see the sequence table.
  • mutant 34 the enzyme activity and enzyme activity stability of the other mutants are higher than those of the wild type.
  • the enzyme activities of Mutant 36, Mutant 41, Mutant 42, Mutant 43, Mutant 45, Mutant 46, Mutant 49, Mutant 50 were greatly improved compared with Mutant 11, and the enzyme activity was stable Sex has also been greatly improved.
  • the above mutants have the same amino acid sequence, and when the nucleotide sequence is different, their tolerance to ammonium ions is different.
  • mutants have higher tolerance to ammonium ions than the wild type.
  • mutant 42 and mutant 49 have greater enzyme activity, enzyme activity stability, and tolerance to ammonium ions than mutant 11.
  • the above mutants have the same amino acid sequence, and when the nucleotide sequence is different, their tolerance to ammonium ions is different.
  • Example 6 Other DAAO enzymes 211 and 213 combined mutation enzyme activity
  • the rtDAAO enzyme with 76% identity with the sequence of GenBank accession number KWU45700 (SEQ ID NO. 1) is derived from Rhodosporidium toruloides UniProtKB / Swiss-Prot P80324, and the sequence is shown in SEQ ID NO.3. After mutating the 54th, 58th and 213th positions (specifically N54V, F58Q, M213S) based on this sequence, the mutant D-amino acid oxidase sequence was obtained, and the gene rtDAAO (N54V-F58Q-M213S) was synthesized according to this sequence. Gene Synthesis Company Ibid.
  • the 54th position was A
  • the 56th position was N
  • the 58th position was H
  • the 211th position was L
  • the 213th position was T.
  • the mutants shown in Table 8 were obtained.
  • the obtained mutants were subjected to enzyme activity detection of mutant rescreening in the same manner as in Example 3, and the calculated enzyme activity and specific enzyme activity data are shown in Table 8.
  • the ammonium resistance of the obtained mutant was tested according to the 200 ⁇ L reaction system of the microplate reader in the microplate reader detection method as described in Example 3, the final concentration of 2M NH 4 Cl was added, and the enzyme activity before and after the addition of NH 4 Cl was analyzed. To confirm the effect of NH 4 Cl concentration on enzyme activity.
  • the obtained enzyme activity data is shown in Table 9.
  • mutant 56 N54A-F58H-C211L-M213T
  • mutant 57 N54A-T56N-F58H-C211L-M213T
  • Improved ammonium resistance N54A-T56N-F58H-C211L-M213T

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Abstract

提供了一种D-氨基酸氧化酶突变体，其序列包括将SEQ ID NO.1所示序列或与SEQ ID NO.1至少有76％同一性的序列的第54位氨基酸残基N、第58位氨基酸残基F、第211位氨基酸残基C和第213位氨基酸残基M进行突变后的序列；所述D-氨基酸氧化酶突变体具有比野生型D-氨基酸氧化酶高的酶活性、酶活稳定性和/或耐铵性。还提供了所述D-氨基酸氧化酶突变体在制备2-氧代-4-(羟基甲基氧膦基)丁酸中的应用。

Description

一种D-氨基酸氧化酶突变体及其应用
本申请要求申请日为2018/11/23的中国专利申请2018114080851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-氨基酸氧化酶突变体及其在制备2-氧代-4-(羟基甲基氧膦基)丁酸(PPO)中的应用。
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-草铵膦多采用转氨酶催化2-氧代-4-(羟基甲基氧膦基)丁酸(PPO)来制备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，自相应的酮酸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。其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，因此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 PCTCN2019120249-appb-000001
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。
Figure PCTCN2019120249-appb-000002
以上都采用PPO为反应原料，但是PPO成本很高，从而导致生产L-草铵膦的成本很高。因此，US9834802B公开了以D,L-草铵膦为原料制备L-草铵膦的方法，先由D-氨基酸氧化酶(DAAO)氧化D-草铵膦得到PPO，再由PPO经转氨酶催化得到L-草铵膦。
Figure PCTCN2019120249-appb-000003
但是以D,L-草铵膦为原料，仍然存在一个问题，因为市场上一般采用Strecker方法生产D,L-草铵膦，需要使用过量的氯化铵，所以生产的D,L-草铵膦都为铵盐，同时还有过量的氯化铵存在。铵离子的存在对DAAO的酶活有抑制作用，从而降低了酶活。例如本发明人发现D,L-草铵膦铵盐投料浓度高时，高浓度的铵离子对上述US9834802B中效果最佳的突变体的酶活有很大的抑制作用。
因此迫切需要寻找一种不受铵离子抑制的DAAO，其具有较高的酶活稳定性，且能够耐受较高的D,L-草铵膦铵盐投料浓度。
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、酶活稳定性差或耐铵性差等缺陷，本发明提供了一种D-氨基酸氧化酶突变体以及其在制备2-氧代-4-(羟基甲基氧膦基)丁酸中的应用。本发明的D-氨基酸氧化酶突变体酶活性很高、酶活稳定性提高和/或耐铵性增强，进而降低成本，利于工业化生产。
本发明人在实验初期发现高浓度的铵离子对酶活有很大的抑制作用，筛选了在现有技术DAAO氨基酸序列第54、58、213位发生突变的突变体，并发现铵离子对同源性为76％的N 2DAAO序列的抑制作用最弱，因此选用N 2DAAO序列进行进一步突变。本发明人经过大量筛选后发现，引入C211位点的突变时，所得的突变体在铵离子的作用下酶活性有所提高，进而对包括C211在内的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D-氨基酸氧化酶突变体。后续对其它现有技术的DAAO氨基酸序列同样进行包括C211位点在内的组合突变，并惊奇地发现所得突变体确实对铵离子的耐受性有 所提高，从而验证了C211位点对不同D-氨基酸氧化酶的耐铵性均有很大的影响，且C211位连同54、58、213位的突变可显著提高D-氨基酸氧化酶在铵离子存在环境下的酶活和酶活稳定性。
为解决上述技术问题，本发明旨在提供一种D-氨基酸氧化酶突变体，所述D-氨基酸氧化酶突变体的序列包括将SEQ ID NO.1所示序列或与SEQ ID NO.1至少有76％同一性的序列的第54位氨基酸残基N、第58位氨基酸残基F、第211位氨基酸残基C和第213位氨基酸残基M进行突变后的序列；所述D-氨基酸氧化酶突变体具有比野生型D-氨基酸氧化酶高的酶活性、酶活稳定性和/或耐铵性。所述至少有76％同一性优选为至少有80％同一性、至少有85％同一性、至少有90％同一性、至少有95％同一性、至少有96％同一性、至少有97％同一性、至少有98％同一性或至少有99％同一性。
本发明所述耐铵性是指反应过程中本发明所述突变体对铵离子的耐受性增强，所述耐受性增强主要体现在本发明突变体在铵离子浓度较高时保持稳定(即酶活损失少)，酶活仍然较高。
较佳地，所述与SEQ ID NO.1至少有76％同一性的序列如SEQ ID NO.3所示。
较佳地，所述D-氨基酸氧化酶突变体的序列包括将SEQ ID NO.1所示的第54位氨基酸残基N突变为A、C、G、S、T或V；和/或，第58位氨基酸残基F突变为亲水性或小位阻的氨基酸残基；和/或，第211位氨基酸残基C突变为A、D、G、H、L、M、S或Y；和/或，第213位氨基酸残基M突变为A、C、F、L、R、S、T、V或W的序列。
根据本发明，所述的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更亲水或空间位阻更小。所述的氨基酸可以为修饰或未修饰的天然氨基酸；本发明以天然氨基酸为例。
较佳地，所述第54位氨基酸残基N突变为V、第58位氨基酸残基F突变为Q、第211位氨基酸残基C突变为A、D、G、H、L、M、S或Y，和第213位氨基酸残基M突变为A、C、F、L、R、S、T、V或W；更佳地，所述第211位氨基酸残基C突变为A、G、M、S、Y或L，和/或，所述第213位氨基酸残基M突变为F、L、R、T或W；进一步更佳地，所述第211位氨基酸残基C突变为L或M，和/或，所述第213位氨基酸残基M突变为T。
较佳地，所述第211位氨基酸残基C突变为L，第213位氨基酸残基M突变为T，第54位氨基酸残基N突变为A、C、G、S、T或V，和第58位氨基酸残基F突变为A、G、H、K或Q；更佳地，所述第54位氨基酸残基N突变为A、G、S或T，和/或，所述第58位氨基酸残基F突变为H、K或Q；进一步更佳地，所述第54位氨基酸残基N突 变为A，和/或，所述第58位氨基酸残基F突变为H或K；进一步更佳地，所述D-氨基酸氧化酶突变体的序列还包括将SEQ ID NO.1所示的第56位氨基酸残基T突变为N、S或L，所述第56位氨基酸残基T优选突变为N。
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D-氨基酸氧化酶突变体的氨基酸序列如SEQ ID NO.5、SEQ ID NO.7、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.37SEQ ID NO.39、SEQ ID NO.41、SEQ ID NO.43、SEQ ID NO.45、SEQ ID NO.47、SEQ ID NO.49、SEQ ID NO.51、SEQ ID NO.53、SEQ ID NO.55、SEQ ID NO.57、SEQ ID NO.59、SEQ ID NO.61、SEQ ID NO.63、SEQ ID NO.65、SEQ ID NO.68、SEQ ID NO.70、SEQ ID NO.72、SEQ ID NO.75、SEQ ID NO.79、SEQ ID NO.82、SEQ ID NO.90、SEQ ID NO.100、或SEQ ID NO.102所示。
较佳地，编码所述D-氨基酸氧化酶突变体的核苷酸序列如SEQ ID NO.6、SEQ ID NO.8、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.22、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、SEQ ID NO.38、SEQ ID NO.40、SEQ ID NO.42、SEQ ID NO.44、SEQ ID NO.46、SEQ ID NO.48、SEQ ID NO.50、SEQ ID NO.52、SEQ ID NO.54、SEQ ID NO.56、SEQ ID NO.58、SEQ ID NO.60、SEQ ID NO.62、SEQ ID NO.64、SEQ ID NO.66、SEQ ID NO.67、SEQ ID NO.69、SEQ ID NO.71、SEQ ID NO.73、SEQ ID NO.74、SEQ ID NO.76、SEQ ID NO.77、SEQ ID NO.78、SEQ ID NO.80、SEQ ID NO.81、SEQ ID NO.83、SEQ ID NO.84、SEQ ID NO.85、SEQ ID NO.86、SEQ ID NO.87、SEQ ID NO.88、SEQ ID NO.89、SEQ ID NO.91、SEQ ID NO.101、或SEQ ID NO.103所示。
为解决上述技术问题，本发明旨在提供一种分离的核酸，所述核酸编码上述的D-氨基酸氧化酶突变体。
为解决上述技术问题，本发明旨在提供一种包含上述核酸的重组表达载体。
为解决上述技术问题，本发明旨在提供一种包含上述核酸或上述重组表达载体的转化体。
为解决上述技术问题，本发明旨在提供一种上述的D-氨基酸氧化酶突变体在制备2- 氧代-4-(羟基甲基氧膦基)丁酸中的应用。
本发明所述酶活性包括酶的比酶活和酶活的特性。
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D-氨基酸氧化酶突变体酶活性很高、酶活稳定性提高和/或耐铵性增强，进而降低成本，利于工业化生产。
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 PCTCN2019120249-appb-000004
Pet28a和bugbuster protein extraction reagent购买自Novagen公司；NdeI酶、HindIII酶购买自Thermo Fisher公司，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 野生型D-氨基酸氧化酶(DAAO)的制备
全合成野生型(wt)N 2DAAO酶基因(GenBank登录号为KWU45700，来源于Rhodotorula sp.JG-1b)，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)。
将合成得到的wtN 2DAAO酶基因连接质粒pET28a(具体方法参见J.Am.Chem.Soc.,2017,139(32),11241-11247)，酶切位点为NdeI和HindIII。将酶连好的载体，转化至宿主大肠杆菌BL21感受态细胞。将其接种LB液体培养基于37℃下，200rpm摇床培养。待OD 600至0.8左右时，取菌液加入终浓度为25％的无菌甘油编号后，置于-80℃低温冰箱保藏备用。
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。
将上述置于-80℃低温冰箱保藏备用的含有酶基因的工程菌在经平皿划线活化后，挑单菌落接种至含50μg/ml卡那霉素的5ml LB液体培养基中，37℃震荡培养12h。按2％ 接种量转接至150ml同样含50μg/ml卡那霉素的新鲜LB液体培养基中，37℃震荡。至OD 600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。
用50mM pH8.0磷酸缓冲液洗涤培养结束后收集到的菌体，共洗涤两次。之后重悬于50mLpH8.0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wtN 2DAAO酶的粗酶液。
实施例2 D-氨基酸氧化酶(DAAO)突变体文库(211位、213位)的构建
在实施例1中所述的wtN 2DAAO序列的基础上突变了第54、58位(具体为N54V、F58Q)之后，得到了突变的D-氨基酸氧化酶序列，根据该序列合成基因N 2DAAO(N54V、F58Q)，基因合成公司为苏州金唯智生物科技有限公司(苏州工业园区星湖街218号生物纳米科技园C3楼)。然后以NdeI和HindIII双酶切，将突变基因引入质粒pET28a，构建质粒pET28a-N 2DAAO(质粒构建方法参见J.Am.Chem.Soc.,2017,139(32),11241-11247)。以质粒pET28a-N 2DAAO为模板，PCR扩增目的条带。
其中，针对突变的D-氨基酸氧化酶序列(N54V、F58Q)的第211位、213位进行突变的突变体文库构建设计PCR的引物序列，具体如表3所示：
表3
Figure PCTCN2019120249-appb-000005
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。
PCR扩增体系为：
试剂 用量(μL)
2×PCR buffer(含高保真酶) 25
正向引物 1
反向引物 1
模板 1
去离子水 22
PCR扩增程序如下：
Figure PCTCN2019120249-appb-000006
对PCR产物进行DpnI消化，37℃，2h。反应完成转化至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。
实施例3 高通量筛选突变体文库
按照如下实验步骤进行筛选：
将实施例2中所得转化子接种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DAAO突变体酶液。
酶标仪检测方法：取100μL pH为8.0的100mM底物(消旋草铵膦，购自上海阿拉丁生化科技股份有限公司)，加50μL的显色液(含60mg/mL的TBHBA(3-羟基-2,4,6-三溴苯甲酸)、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。
将所筛选出的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50μg/ml卡纳霉素的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卡纳霉素的新鲜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，30℃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。
用50mM pH 8.0的磷酸缓冲液洗涤菌体培养结束后收集到的菌体，共洗涤两次。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，得到的上清液为初步的含重组DAAO突变体粗酶液。
突变体复筛的酶活检测方法为：
5mL反应体系中，加入1mL 500mM的D,L-草铵膦铵盐，0.25mL的上述初步的含重组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，并根据曲线斜率计算酶活。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 H 2O 2所需要的酶量，酶活单位是U。
比酶活为每毫克酶蛋白所含的活力单位，计算公式：酶活/蛋白含量，单位是U/mg或U/g。
表4
Figure PCTCN2019120249-appb-000007
Figure PCTCN2019120249-appb-000008
备注：表格中*代表比酶活在0-0.10U/mg之间，**代表比酶活在0.1-1.0U/mg之间，***代表比酶活在1.0-20U/mg之间，****代表比酶活在20-50U/mg之间。
由表4可以看出，所得突变体的比酶活均高于野生型N 2DAAO的比酶活。其中比酶活最高的为DAAO氧化酶突变体11，其突变位点第54位的N突变为V，第58位的F突变为Q，第211位的C突变为L，第213位的M突变为T。
实施例4 针对实施例3中的DAAO氧化酶突变体11的第54位、56位、58位进行突变的突变体文库的构建
针对DAAO氧化酶突变体11的第54位、56位、58位突变体文库构建所设计的引物序列如表5所示。
表5
Figure PCTCN2019120249-appb-000009
Figure PCTCN2019120249-appb-000010
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。
根据文献J.Am.Chem.Soc.,2017,139(32),11241-11247中公开的方法构建了质粒模板pET28a-DAAO氧化酶突变体12，以pET28a-DAAO氧化酶突变体11为模板，进行PCR扩增目的条带。
扩增反应体系为：
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
扩增程序如下：
Figure PCTCN2019120249-appb-000011
对PCR产物进行DpnI消化，37℃，2h。反应完成转化至BL21感受态细胞，涂布在含有50μg/mL卡纳霉素的LB培养基。37℃培养过夜后收菌，得到包含突变体文库的转化子。
实施例5 酶活稳定性检测与NH 4 +离子浓度影响检测
将实施例4中所得转化子接种96孔板培养，加IPTG 30℃过夜诱导。之后收菌，加bugbuster protein extraction reagent裂解，离心得突变子酶液。
将突变子酶液在50℃处理2h。按照实施例3中所述酶标仪检测方法分析阳性克隆的热稳定突变效果，筛选阳性克隆子。
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50μg/ml卡纳霉素的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卡纳霉素的新鲜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，30℃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DAAO突变体粗酶液。
对所得的DAAO突变体粗酶液进行突变体复筛的酶活检测，方法同实施例3。所得结果如表6所示，其中2h后剩余酶活％数值反应了突变体酶活的稳定性。
表6
Figure PCTCN2019120249-appb-000012
Figure PCTCN2019120249-appb-000013
备注：表格中酶活这一列中，*代表酶活在0-0.10U/mg之间，**代表比酶活在0.1-2.0U/mg之间，***代表比酶活在2.0-3.5U/mg之间，****代表比酶活在3.5-10U/mg之间。
2h后剩余酶活这一列中，#代表2h后剩余酶活在0-50％之间，##代表2h后剩余酶活在50-80％之间，###代表2h后剩余酶活在80-100％之间。
表中有些突变体(例如突变体39和40、突变体43-45等)的氨基酸序列一样，但是 DNA序列不一样，其密码子不相同，相关的氨基酸序列和DNA序列详见序列表。
结果显示，除突变体34以外，上述其他突变体的酶活和酶活稳定性均比野生型高。其中，突变体36、突变体41、突变体42、突变体43、突变体45、突变体46、突变体49、突变体50的酶活较突变体11有较大的提高，且酶活稳定性也有较大的提高。此外由表中可以看出，上述突变体氨基酸序列相同，核苷酸序列不同时，其对铵离子的耐受性不同。
接着将上述所得突变体进行NH 4 +离子浓度影响检测，方法如下：
在如实施例3中所述的酶标仪检测方法中的酶标板200μL反应体系中，加入终浓度为2M的NH 4Cl，分析加入NH 4Cl前后的酶活，确认NH 4Cl浓度对酶活的影响。所得酶活数据如表7所示：
表7
Figure PCTCN2019120249-appb-000014
Figure PCTCN2019120249-appb-000015
备注：表格中+代表酶活残留比例在0-0.30之间，++代表酶活残留比例在0.3-0.5之间，+++代表酶活残留比例在0.5-0.6之间，++++代表酶活残留比例在0.6-1之间。
以上结果显示，上述突变体对铵离子的耐受性均比野生型高。其中，突变体42、突变体49的酶活、酶活稳定性、且对铵离子的耐受性较突变体11有较大的提高。此外由表中可以看出，上述突变体氨基酸序列相同，核苷酸序列不同时，其对铵离子的耐受性不同。
实施例6 其它DAAO酶211位和213位组合突变的酶活
与GenBank登录号为KWU45700的序列(SEQ ID NO.1)的同一性为76％的rtDAAO酶，其来源于Rhodosporidium toruloidesUniProtKB/Swiss-Prot P80324，序列如SEQ ID NO.3所示。在该序列的基础上突变了第54、58和213位(具体为N54V、F58Q、M213S)之后得到了突变的D-氨基酸氧化酶序列，根据该序列合成基因rtDAAO(N54V-F58Q-M213S)，基因合成公司同上。针对此rtDAAO(N54V-F58Q-M213S)酶定点突变54位为A，56位为N，58位为H，211位为L、213位为T，得到如表8所示的突变体。对所得突变体进行突变体复筛的酶活检测，方法同实施例3，计算酶活和比酶活数据如表8所示。
按照如实施例3中所述的酶标仪检测方法中的酶标板200μL反应体系检测所得突变体的耐铵性，加入终浓度为2M的NH 4Cl，分析加入NH 4Cl前后的酶活，确认NH 4Cl浓度对酶活的影响。所得酶活数据如表9所示。
表8
Figure PCTCN2019120249-appb-000016
表9
Figure PCTCN2019120249-appb-000017
由上表可以看出，突变体56(N54A-F58H-C211L-M213T)的耐铵性有所提高；突变体57(N54A-T56N-F58H-C211L-M213T)在酶活变化不大的情况下，提高了耐铵性。

Claims (10)

  1. 一种D-氨基酸氧化酶突变体，其特征在于，所述D-氨基酸氧化酶突变体的序列包括将SEQ ID NO.1所示序列或与SEQ ID NO.1至少有76％同一性的序列的第54位氨基酸残基N、第58位氨基酸残基F、第211位氨基酸残基C和第213位氨基酸残基M进行突变后的序列；所述D-氨基酸氧化酶突变体具有比野生型D-氨基酸氧化酶高的酶活性、酶活稳定性和/或耐铵性；
    较佳地，所述与SEQ ID NO.1至少有76％同一性的序列如SEQ ID NO.3所示。
  2. 如权利要求1所述的D-氨基酸氧化酶突变体，其特征在于，所述D-氨基酸氧化酶突变体的序列包括将SEQ ID NO.1所示的第54位氨基酸残基N突变为A、C、G、S、T或V；和/或，第58位氨基酸残基F突变为亲水性或小位阻的氨基酸残基；和/或，第211位氨基酸残基C突变为A、D、G、H、L、M、S或Y；和/或，第213位氨基酸残基M突变为A、C、F、L、R、S、T、V或W的序列。
  3. 如权利要求2所述的D-氨基酸氧化酶突变体，其特征在于，所述第54位氨基酸残基N突变为V、第58位氨基酸残基F突变为Q、第211位氨基酸残基C突变为A、D、G、H、L、M、S或Y，和第213位氨基酸残基M突变为A、C、F、L、R、S、T、V或W；
    较佳地，所述第211位氨基酸残基C突变为A、G、M、S、Y或L，和/或，所述第213位氨基酸残基M突变为F、L、R、T或W；更佳地，所述第211位氨基酸残基C突变为L或M，和/或，所述第213位氨基酸残基M突变为T。
  4. 如权利要求2所述的D-氨基酸氧化酶突变体，其特征在于，所述第211位氨基酸残基C突变为L，第213位氨基酸残基M突变为T，第54位氨基酸残基N突变为A、C、G、S、T或V，和第58位氨基酸残基F突变为A、G、H、K或Q；
    较佳地，所述第54位氨基酸残基N突变为A、G、S或T，和/或，所述第58位氨基酸残基F突变为H、K或Q；更佳地，所述第54位氨基酸残基N突变为A，和/或，所述第58位氨基酸残基F突变为H或K。
  5. 如权利要求4所述的D-氨基酸氧化酶突变体，其特征在于，所述D-氨基酸氧化酶突变体的序列还包括将SEQ ID NO.1所示的第56位氨基酸残基T突变为N、S或L；
    较佳地，所述第56位氨基酸残基T突变为N。
  6. 如权利要求1～5任一项所述的D-氨基酸氧化酶突变体，其特征在于，所述D-氨基酸氧化酶突变体的氨基酸序列如SEQ ID NO.5、SEQ ID NO.7、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.37SEQ ID NO.39、SEQ ID NO.41、SEQ ID NO.43、SEQ ID NO.45、SEQ ID NO.47、SEQ ID NO.49、SEQ ID NO.51、SEQ ID NO.53、SEQ ID NO.55、SEQ ID NO.57、SEQ ID NO.59、SEQ ID NO.61、SEQ ID NO.63、SEQ ID NO.65、SEQ ID NO.68、SEQ ID NO.70、SEQ ID NO.72、SEQ ID NO.75、SEQ ID NO.79、SEQ ID NO.82、SEQ ID NO.90、SEQ ID NO.100，或SEQ ID NO.102所示；较佳地，编码所述D-氨基酸氧化酶突变体的核苷酸序列如SEQ ID NO.6、SEQ ID NO.8、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.22、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、SEQ ID NO.38、SEQ ID NO.40、SEQ ID NO.42、SEQ ID NO.44、SEQ ID NO.46、SEQ ID NO.48、SEQ ID NO.50、SEQ ID NO.52、SEQ ID NO.54、SEQ ID NO.56、SEQ ID NO.58、SEQ ID NO.60、SEQ ID NO.62、SEQ ID NO.64、SEQ ID NO.66、SEQ ID NO.67、SEQ ID NO.69、SEQ ID NO.71、SEQ ID NO.73、SEQ ID NO.74、SEQ ID NO.76、SEQ ID NO.77、SEQ ID NO.78、SEQ ID NO.80、SEQ ID NO.81、SEQ ID NO.83、SEQ ID NO.84、SEQ ID NO.85、SEQ ID NO.86、SEQ ID NO.87、SEQ ID NO.88、SEQ ID NO.89、SEQ ID NO.91、SEQ ID NO.101，或SEQ ID NO.103所示。
  7. 一种分离的核酸，其特征在于，所述核酸编码如权利要求1～6任一项所述的D-氨基酸氧化酶突变体。
  8. 一种包含如权利要求7所述的核酸的重组表达载体。
  9. 一种包含如权利要求7所述的核酸或如权利要求8所述的重组表达载体的转化体。
  10. 一种如权利要求1～6任一项所述的D-氨基酸氧化酶突变体在制备2-氧代-4-(羟基甲基氧膦基)丁酸中的应用。
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111909907A (zh) * 2020-07-08 2020-11-10 浙江工业大学 天冬氨酸氧化酶突变体、工程菌及其在氧化-还原偶联制备精草铵膦中的应用
CN116064446A (zh) * 2022-12-02 2023-05-05 中南大学 一种d-氨基酸氧化酶的突变体、表达载体、基因工程菌及其构建方法和应用
WO2023143621A1 (zh) * 2022-01-30 2023-08-03 弈柯莱生物科技(上海)股份有限公司 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

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2023504869A (ja) * 2019-12-09 2023-02-07 フーナン リアー バイオテック カンパニー リミテッド 改変ｄａａｏ及びその使用
CN113969269B (zh) * 2021-04-29 2024-05-03 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

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0344683A2 (de) 1988-06-03 1989-12-06 Hoechst Aktiengesellschaft Neue Transaminase, ihre Herstellung und ihre Verwendung
US5221737A (en) 1988-12-15 1993-06-22 Hoechst Aktiengesellschaft Gene and gene structure coding for an aminotransferase, and microorganisms which express this gene
CN1680558A (zh) * 2004-04-08 2005-10-12 百瑞全球有限公司 重组d－氨基酸氧化酶
CN1284858C (zh) 1999-04-30 2006-11-15 阿温提斯作物科学有限公司 用天冬氨酸通过酶促的转氨基作用生产l－膦丝菌素的方法
CN103103167A (zh) * 2013-01-29 2013-05-15 河北师范大学 一种d-氨基酸氧化酶的突变体酶蛋白及其制备方法
CN106978453A (zh) 2017-03-31 2017-07-25 浙江大学 一种利用氨基酸脱氢酶制备l‑草铵膦的方法
US20170253897A1 (en) * 2016-03-02 2017-09-07 Agrimetis, Llc Methods for making l-glufosinate

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107636155B (zh) * 2015-04-03 2022-01-07 淡马锡生命科学研究院有限公司 红冬孢酵母属和红酵母属的d-氨基酸诱导型基因表达系统

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0344683A2 (de) 1988-06-03 1989-12-06 Hoechst Aktiengesellschaft Neue Transaminase, ihre Herstellung und ihre Verwendung
US5221737A (en) 1988-12-15 1993-06-22 Hoechst Aktiengesellschaft Gene and gene structure coding for an aminotransferase, and microorganisms which express this gene
CN1284858C (zh) 1999-04-30 2006-11-15 阿温提斯作物科学有限公司 用天冬氨酸通过酶促的转氨基作用生产l－膦丝菌素的方法
CN1680558A (zh) * 2004-04-08 2005-10-12 百瑞全球有限公司 重组d－氨基酸氧化酶
CN103103167A (zh) * 2013-01-29 2013-05-15 河北师范大学 一种d-氨基酸氧化酶的突变体酶蛋白及其制备方法
US20170253897A1 (en) * 2016-03-02 2017-09-07 Agrimetis, Llc Methods for making l-glufosinate
US9834802B2 (en) 2016-03-02 2017-12-05 Agrimetis, Llc Methods for making L-glufosinate
CN106978453A (zh) 2017-03-31 2017-07-25 浙江大学 一种利用氨基酸脱氢酶制备l‑草铵膦的方法

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
"GenBank", Database accession no. KWU45700
J. AM. CHEM. SOC., vol. 139, no. 32, 2017, pages 11241 - 11247
J.AM.CHEM.SOC, vol. 139, no. 32, 2017, pages 11241 - 11247
See also references of EP3885435A4

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111909907A (zh) * 2020-07-08 2020-11-10 浙江工业大学 天冬氨酸氧化酶突变体、工程菌及其在氧化-还原偶联制备精草铵膦中的应用
CN111909907B (zh) * 2020-07-08 2022-05-24 浙江工业大学 天冬氨酸氧化酶突变体、工程菌及其在氧化-还原偶联制备精草铵膦中的应用
WO2023143621A1 (zh) * 2022-01-30 2023-08-03 弈柯莱生物科技(上海)股份有限公司 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
CN116064446A (zh) * 2022-12-02 2023-05-05 中南大学 一种d-氨基酸氧化酶的突变体、表达载体、基因工程菌及其构建方法和应用
CN116064446B (zh) * 2022-12-02 2023-11-14 湖南麦肯伟科技有限公司 一种d-氨基酸氧化酶的突变体、表达载体、基因工程菌及其构建方法和应用

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