WO2024119940A1 - D-氨基酸氧化酶突变体及其在制备l-草铵膦中的应用 - Google Patents

D-氨基酸氧化酶突变体及其在制备l-草铵膦中的应用 Download PDF

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WO2024119940A1
WO2024119940A1 PCT/CN2023/118314 CN2023118314W WO2024119940A1 WO 2024119940 A1 WO2024119940 A1 WO 2024119940A1 CN 2023118314 W CN2023118314 W CN 2023118314W WO 2024119940 A1 WO2024119940 A1 WO 2024119940A1
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amino acid
acid residue
mutates
mutant
mutated
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薛亚平
王柳玉
汤恒
郑裕国
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浙江工业大学
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    • 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)
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    • 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)
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    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli
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Definitions

  • the present invention relates to a D-amino acid oxidase mutant (DAAO), a coding gene, a vector containing the coding gene, a genetic engineering bacterium, and application of the mutant in preparing L-glufosinate ammonium by microbial catalysis.
  • DAAO D-amino acid oxidase mutant
  • Glufosinate also known as Phosphinothricin (PPT) in English and 2-amino-4-[hydroxy(methyl)phosphonyl]butyric acid in chemical name, is the world's second largest genetically modified crop-tolerant herbicide with excellent weed control performance and less phytotoxic side effects, and has huge market potential in the future.
  • PPT Phosphinothricin
  • PPT has two optical isomers, L-PPT and D-PPT; but only the L-type has herbicidal activity.
  • PPT sold on the market is generally a racemic mixture. If PPT products can be used in the form of pure optical isomers of L-configuration, the amount of PPT used can be significantly reduced, which is of great significance for improving atom economy, reducing use costs, and alleviating environmental pressure.
  • the most widely used method for removing D-PPT is to use D, L-PPT as raw materials, catalyze D-PPT with DAAO to obtain L-PPT precursor 2-carbonyl-4-[hydroxy(methyl)phosphonyl]butyric acid (PPO), and then catalyze amino acid dehydrogenase or transaminase to obtain L-PPT.
  • this method can not only effectively remove D-PPT, but its key intermediate PPO can also be further converted into L-PPT, effectively improving the atomic utilization rate. Therefore, it is particularly important to screen high-yield PPO mutants.
  • the purpose of the present invention is to overcome the shortcomings of the prior art such as low enzyme activity and poor enzyme thermal stability of DAAO, and to provide a D-AAO mutant with significantly improved catalytic performance, an encoding gene, a vector containing the encoding gene, a genetically engineered bacterium, and the use thereof in the microbial catalytic preparation of L-PPT.
  • a DAAO mutant is obtained by single-point mutation or multi-point combined mutation at positions 54, 58, 213, 239, 73, 77, 79, 147, 185, 43, 45, 206, 207, 215, 122, 132, 195 and 234 of the amino acid sequence shown in SEQ ID NO.1.
  • the amino acid sequence of D-AAO comprising the sequence shown in SEQ ID NO.1 may be referred to as the wild-type enzyme of DAAO in this application.
  • the nucleotide sequence of the wild-type enzyme may be the nucleotide sequence shown in SEQ ID NO.8.
  • SEQ ID NO.1 is the amino acid sequence of DAAO annotated from Rhodotorula taiwanensis.
  • SEQ ID NO.8 is the nucleotide sequence of DAAO from Rhodotorula taiwanensis.
  • the mutation is one of the following: (1) the amino acid residue N at position 54 mutates to V, D or T, the amino acid residue F at position 58 mutates to H, R, K or Q, and the amino acid residue M at position 213 mutates to S, N or R; (2) the amino acid residue N at position 54 mutates to V, D or T, the amino acid residue F at position 58 mutates to H, R, K or Q, the amino acid residue M at position 213 mutates to S, N or R, and the amino acid residue L at position 239 mutates to E, D, G or Q; (3) the amino acid residue N at position 54 mutates to V, D or T, the amino acid residue F at position 58 mutates to H, R, K or Q, the amino acid residue M at position 213 mutates to S, N or R, the amino acid residue L at position 239 mutates to E, D, G or Q, the amino acid residue A at position 73 mutates to L, the amino acid residue Q at position
  • amino acid sequence of the mutant is shown in one of SEQ ID NOs. 2 to 7.
  • SEQ ID NO.2 is the amino acid sequence of DAAO mutant I (M213S/F58H/N54V).
  • SEQ ID NO.3 is the amino acid sequence of DAAO mutant II (M213S/F58H/N54V/L239G).
  • SEQ ID NO.4 is the amino acid sequence of DAAO mutant III (M213S/F58H/N54V/L239G/A73L/Q77W/V79M/Q147M/S185M).
  • SEQ ID NO.5 is the amino acid sequence of DAAO mutant IV (M213S/F58H/N54V/L239G/A43S/T45M/S206A/D207P/S215F).
  • SEQ ID NO.6 is the amino acid sequence of DAAO mutant V (M213S/F58H/N54V/L239G/E122P/Y132F/E195Y/C234L).
  • SEQ ID NO.7 is the amino acid sequence of DAAO mutant VI (M213S/F58H/N54V/L239G/A73L/Q77W/V79M/Q147M/S185M/A43S/T45M/S206A/D207P/S215FE122P/Y132F/E195Y/C234L).
  • the present invention also relates to a gene encoding the DAAO mutant and a recombinant vector.
  • the recombinant expression vector can be constructed by connecting the nucleic acid encoding the DAAO mutant gene of the present invention to various suitable vectors by conventional methods in the art.
  • the vector can be various conventional vectors in the art, such as commercially available plasmids, cosmids, phages or viral vectors, as long as the recombinant expression vector can be normally replicated in the corresponding expression host and express the DAAO.
  • the vector is preferably a plasmid, more preferably plasmid pET28a.
  • the present invention also relates to a genetically engineered bacterium containing a gene encoding the DAAO mutant.
  • the recombinant expression transformant can be prepared by transforming the constructed recombinant expression vector into a host cell.
  • the host cell is any conventional host cell in the art, as long as the recombinant expression vector can stably replicate itself and can effectively express the target protein after being induced by an inducer.
  • Escherichia coli is preferably used as a host cell
  • Escherichia coli BL21 (DE3) is more preferably used for efficiently expressing the DAAO mutant of the present invention.
  • the present invention also relates to the application of the DAAO mutant in the preparation of L-PPT by microbial catalysis.
  • the beneficial effects of the present invention are mainly reflected in: the present invention provides a DAAO mutant with improved enzyme activity and enzyme thermal stability, which can be used for preparing L-PPT by microbial catalysis, is conducive to industrial production, and has good application prospects.
  • the plasmid extraction kit and DNA purification and recovery kit used in the examples were purchased from Hangzhou Qingke Zixi Biotechnology Co., Ltd.; the one-step cloning kit was purchased from Novazon Co., Ltd.; E. coli BL21 (DE3), plasmid pET-24a (+) and whole gene synthesis were completed by Sangon Biotechnology (Shanghai) Co., Ltd.; DNA markers, low molecular weight standard proteins, and protein precast gels were purchased from Beijing GenStar Co., Ltd.; ClonExpress II OneStep Cloning Kit seamless cloning kit was purchased from Nanjing Novazon Biotechnology Co., Ltd.; pfu DNA polymerase and DpnI endonuclease were purchased from Thermo Fisher Scientific (China) Co., Ltd.; primer synthesis and sequence sequencing were completed by Hangzhou Qingke Zixi Biotechnology Co., Ltd.
  • the use of the above reagents refers to the product instructions.
  • D and L-PPT The reagents used in the downstream catalytic process, D and L-PPT, were purchased from Sigma-Aldrich; 2,4-dinitrophenylhydrazine (DNPH) was purchased from Aladdin Reagents (Shanghai, China), and commercial micrococcal catalase was purchased from Sigma Aldrich (Shanghai, China). Other commonly used reagents were purchased from Sinopharm Chemical Reagent Co., Ltd.
  • HPLC analysis method is: chromatographic column/Welchrom®C18; column temperature/30°C; flow rate/1mL/min; detection wavelength/232nm; mobile phase: 50mM (NH2)HPO4, add 1% of 10% tetrabutylammonium bromide aqueous solution, adjust the pH to 3.8 with phosphoric acid, and add 12% acetonitrile.
  • the two configuration contents of PPT were checked by chiral HPLC analysis method.
  • Derivatization reagent Weigh 0.1g of o-phthalaldehyde and 0.12g of N-acetyl-L-cysteine respectively, dissolve with 10ml of ethanol, and then add 40ml of 0.1M boric acid buffer (pH 9.8). Shake to fully dissolve, and store in a refrigerator at 4°C for later use (no more than 3 days).
  • Derivatization reaction and determination Take 200 ⁇ L of sample and add 400 ⁇ L of derivatization reagent, mix well and keep warm at 30°C for 5min, add 400 ⁇ L of ultrapure water and mix, and inject 10 ⁇ L for analysis.
  • DAAO Rhodotorula taiwanensis
  • GenBank No.: POY70719.1 the amino acid sequence is shown in SEQ ID NO.1
  • nucleotide sequence is shown in SEQ ID NO.8
  • pET-24a (+) DAAO was transferred into the expression host Escherichia coli BL21 (DE3) for subsequent expression of the recombinase.
  • LB liquid culture medium composition peptone 10g/L, yeast powder 5g/L, NaCl 10g/L, dissolved in water and made up to volume, sterilized at 121°C for 20min, and set aside.
  • the culture solution was centrifuged at 8000 rpm for 10 minutes, the supernatant was discarded, the bacteria were collected, and stored in a -20°C refrigerator for standby use.
  • the bacteria collected after the culture was washed twice with 50 mM pH 8.0 phosphate buffer, then resuspended in 50 mL pH 8.0 phosphate buffer, homogenized and broken, and the broken solution was centrifuged to remove the precipitate to obtain a crude enzyme solution containing the recombinant wtDAAO enzyme.
  • positions 213, 58, and 54 were mutated.
  • the PCR primer sequences were designed for the mutants with mutations at positions 213, 58, and 54 of the mutated DAAO sequence, and the design was performed according to the mutation order of the mutation sites as shown in Tables 1, 2, and 3:
  • the PCR was completed, 5 ⁇ L of the amplified product was analyzed by nucleic acid gel electrophoresis, and 0.5 ⁇ L of Dpn I endonuclease was added to the PCR product with a clear target band, and the template was digested at 37°C for 1 hour. After the reaction was completed, it was transformed into BL21 competent cells, coated in LB medium containing 50 ⁇ g/mL kanamycin, cultured at 37°C overnight, and the bacteria were harvested to obtain transformants containing mutants. The bacterial cells were obtained as described in Example 1.
  • the transformants obtained in Example 2 were inoculated into a 96-well plate and cultured in a 37°C constant temperature shaker for 12-16 hours at a shaker speed of 200 rpm.
  • the cultured 96-well fermentation broth was centrifuged at 4000 rpm for 10 minutes, the supernatant was discarded, and the cells were collected.
  • the collected cells were reacted with a certain concentration of D, L-PPT in a 96-well plate for 1 hour and then centrifuged at 4000 rpm for 10 minutes.
  • Color development reaction Use a pistol to draw the supernatant of the reaction solution after centrifugation into a 96-well transparent plate, add 2mM 2,4-dinitrophenylhydrazine reagent, blow evenly with a pistol, place in a microplate reader thermostat at 37°C for 20 minutes, add 1M NaOH 100 ⁇ L after the reaction, mix for 30s to generate a reddish-brown compound, and use a microplate reader to detect the absorbance at 380nm.
  • the positive clones (M213N, M213R, M213S), (M213S/F58H, M213S/F58R, M213S/F58K, M213S/F58Q), (M213S/F58H/N54V, M213S/F58H/N54D, M213S/F58H/N54T) obtained by screening in Example 3 were rescreened, and the rescreening reaction was carried out by detecting the catalytic efficiency of the mutants. Specifically, the catalytic efficiency of DAAO and its mutants was compared by measuring the amount of PPO produced by HPLC.
  • the 1 ml reaction system includes: 50 mM racemic PPT ammonium salt, 50 mM pH 8.0 phosphate buffer, 8000 U/L catalase, and 50 g/L DAAO or its mutant frozen cells. After 2 h of reaction, the reaction solution sample was taken for treatment, the PPO concentration was determined and the conversion rate was calculated (product PPO concentration/initial substrate D,L PPT concentration ⁇ 100%), as shown in Table 4.
  • Error-prone PCR (epPCR) mutation was performed based on the mutant sequence (N54V/F58Q/M213S) described in Example 4. Primer sequences were designed for error-prone PCR, as shown in Table 5:
  • the epPCR (30 ⁇ L) amplification system was as follows: using a transient error-prone PCR kit, 1 ⁇ L of the template plasmid and 0.5 ⁇ L of each of the upstream and downstream primers were used in a 30 ⁇ L reaction mixture in 10 mM Tris-HCl pH 8.3, 50 mM KCl, 2 mM MgCl2, 0.25 mM MnCl2.
  • the PCR amplification conditions were: 95°C for 5 min, (90°C for 30 s, 55°C-65°C for 30 s, 72°C for 5 min) ⁇ 30 cycles, 72°C for 10 min.
  • Example 3 After PCR, gel electrophoresis analysis, template digestion, and transformation were performed as described in Example 2. Then, bacteria were obtained as described in Example 1. Finally, positive clones were screened as described in Example 3. The mutant at position 239 (specifically L239G) was obtained.
  • Example 6 As described in Example 4, the positive clones screened in Example 5 were combined and screened again, and the catalytic efficiency between the mutants was compared, as shown in Table 6:
  • mutant II M213S/F58H/N54V/L239G
  • thermal stability mutation modification will be further carried out based on mutant II.
  • Example 7 Construction of DAAO mutant III and comparison of enzyme activity and thermal stability
  • positions 73, 77, 79, 147, and 185 were mutated, specifically III (M213S/F58H/N54V/L239G/A73L/Q77W/V79M/Q147M/S185M), and primer sequences were designed for mutant PCR at the sites of mutant III, as shown in Table 7:
  • the PCR amplification system and conditions are the same as those described in Example 2, and the bacterial cells are obtained as described in Example 1.
  • the catalytic efficiency of the mutant is detected as described in Example 4.
  • the thermal stability detection of the mutant is to measure the residual enzyme activity after incubation at a certain temperature for a certain period of time. Specifically, the residual enzyme activity is detected after incubation at 45°C for 30 minutes and then reacting at 30°C for 1 hour.
  • the catalytic efficiency and thermal stability effects between the single point mutation and the combined mutant are shown in Table 8:
  • Example 8 Construction of DAAO mutant IV and comparison of enzyme activity and thermal stability
  • the PCR amplification system and conditions are the same as those described in Example 2, and the bacterial cells are obtained as described in Example 1.
  • the catalytic efficiency of the mutant is detected as described in Example 4.
  • the thermal stability detection of the mutant is to measure the residual enzyme activity after incubation at a certain temperature for a certain period of time. Specifically, the residual enzyme activity is detected after incubation at 45°C for 30 minutes and then reacting at 30°C for 1 hour.
  • the catalytic efficiency and thermal stability effects between the single point mutation and the combination mutant are shown in Table 10:
  • Example 9 Construction of DAAO mutant V and comparison of enzyme activity and thermal stability
  • positions 122, 132, 195 and 234 were mutated, specifically (M213S/F58H/N54V/L239G/E122P/Y132F/E195Y/C234L), and primer sequences were designed for mutant PCR at the sites of mutant V, as shown in Table 11:
  • the PCR amplification system and conditions are the same as those described in Example 2, and the bacterial cells are obtained as described in Example 1.
  • the catalytic efficiency of the mutant is detected as described in Example 4.
  • the thermal stability detection of the mutant is to measure the residual enzyme activity after incubation at a certain temperature for a certain period of time. Specifically, the residual enzyme activity is detected after incubation at 45°C for 30 minutes and then reacting at 30°C for 1 hour.
  • the catalytic efficiency and thermal stability effects between the single point mutation and the combined mutant are shown in Table 12:
  • Example 10 Construction of DAAO mutant VI and comparison of enzyme activity and thermal stability
  • mutant VI M213S/F58H/N54V/L239G/A73L/Q77W/V79M/Q147M/S185M/A43S/T45M/S206A/D207P/S215F/E122P/Y132F/E195Y/C234L.
  • the PCR amplification system and conditions were the same as those described in Example 2, and the bacteria were obtained as described in Example 1.
  • the catalytic efficiency of the combined mutants II, III, IV, V, and VI was detected as described in Example 4.
  • the thermal stability test of the mutants is to measure the residual enzyme activity after being incubated at a certain temperature for a certain period of time. Specifically, the residual enzyme activity is detected after being incubated at 50°C for 15 minutes and 55°C for 15 minutes, and then reacted at 30°C for 1h.
  • the catalytic efficiency and thermal stability effects between the combined mutants are as described in Table 13:

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Abstract

一种催化性能明显改善的D-氨基酸氧化酶突变体、编码基因，含有编码基因的载体、基因工程菌，以及所述突变体在微生物催化制备L-草铵膦应用。D-氨基酸氧化酶突变体由SEQ ID NO.1所示氨基酸经单点突变或多点联合突变获得。有益效果主要体现在：提供了酶活性和酶热稳定性均有所提高的D-氨基酸氧化酶突变体，可用于微生物催化制备L-草铵膦，利于工业化生产，具有较好应用前景。

Description

D-氨基酸氧化酶突变体及其在制备L-草铵膦中的应用 技术领域
本发明涉及一种D-氨基酸氧化酶突变体（DAAO）、编码基因，含有编码基因的载体、基因工程菌，以及其在微生物催化制备L-草铵膦中的应用。
 背景技术
草铵膦，英文名为Phosphinothricin（PPT），化学名为2-氨基-4-[羟基（甲基）膦酰基]丁酸，是世界第二大转基因作物耐受除草剂，具有优异的除草性能和较小的药害副作用，在未来一段时间内拥有巨大的市场潜力。
PPT有两种光学异构体，分别为L-PPT和D-PPT；但只有L-型具有除草活性。市场上销售的PPT一般都是外消旋混合物。若PPT产品能以L-构型的纯光学异构体形式使用，可显著降低PPT的使用量，这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，去除D-PPT最广泛的方法是以D,L-PPT为原料，经DAAO催化D-PPT得到L-PPT前体2-羰基-4-[羟基（甲基）膦酰基]丁酸（PPO），再经氨基酸脱氢酶或转氨酶催化得到L-PPT。因为DAAO的高度选择特异性，此法不仅可以有效去除D-PPT，其关键中间体PPO还可以进一步被转化为L-PPT，有效提高原子利用率。因此，筛选高产PPO突变株就显得尤为重要。
 发明内容
本发明目的是克服现有技术中DAAO的酶活性不高和酶热稳定性差等不足，提供一种催化性能明显改善的D-AAO突变体、编码基因，含有编码基因的载体、基因工程菌，以及其在微生物催化制备L-PPT中的应用。
本发明采用的技术方案是：
一种DAAO突变体，由SEQ ID NO.1所示氨基酸序列第54位、第58位、第213位、第239位、第73位、第77位、第79位、第147位、第185位、第43位、第45位、第206位、第207位、第215位、第122位、第132位、第195位、第234位经单点突变或多点联合突变获得。
包含SEQ ID NO.1所示序列的D-AAO的氨基酸序列在本申请中可以被称为DAAO的野生型酶。所述野生型酶的核苷酸序列可以为SEQ ID NO.8 所示的核苷酸序列。SEQ ID NO.1是来源于Rhodotorula taiwanensis的注释为DAAO的氨基酸序列。 SEQ ID NO.8是来源于Rhodotorula taiwanensis的DAAO的核苷酸序列。
优选的，所述突变为下列之一：（1）第54位氨基酸残基N突变为V、D或T，第58位氨基酸残基F突变为H、R、K或Q，第213位氨基酸残基M突变为S、N或R；（2）第54位氨基酸残基N突变为V、D或T，第58位氨基酸残基F突变为H、R、K或Q，第213位氨基酸残基M突变为S、N或R，第239位氨基酸残基L突变为E、D、G或Q；（3）第54位氨基酸残基N突变为V、D或T，第58位氨基酸残基F突变为H、R、K或Q，第213位氨基酸残基M突变为S、N或R，第239位氨基酸残基L突变为E、D、G或Q，第73位氨基酸残基A突变为L，第77位氨基酸残基Q突变为W，第79位氨基酸残基V突变为M，第147位氨基酸残基Q突变为M，第185位氨基酸残基S突变为M；（5）第54位氨基酸残基N突变为V、D或T，第58位氨基酸残基F突变为H、R、K或Q，第213位氨基酸残基M突变为S、N或R，第239位氨基酸残基L突变为E、D、G或Q，第43位氨基酸残基A突变为S，第45位氨基酸残基T突变为M，第206位氨基酸残基S突变为A，第207位氨基酸残基D突变为P，第215位氨基酸残基S突变为F；（6）第54位氨基酸残基N突变为V、D或T，第58位氨基酸残基F突变为H、R、K或Q，第213位氨基酸残基M突变为S、N或R，第239位氨基酸残基L突变为E、D、G或Q，第122位氨基酸残基E突变为P，第132位氨基酸残基Y突变为F，第195位氨基酸残基E突变为Y，第234位氨基酸残基C突变为L。
更为优选的，所述突变体氨基酸序列如SEQ ID NO.2~7之一所示。
SEQ ID NO.2是DAAO突变体I （M213S/F58H/N54V）的氨基酸序列。  
SEQ ID NO.3是DAAO突变体II （M213S/F58H/N54V/L239G）的氨基酸序列。  
SEQ ID NO.4是DAAO突变体III （M213S/F58H/N54V/L239G /A73L/Q77W/V79M/Q147M/S185M）的氨基酸序列。  
SEQ ID NO.5是DAAO突变体IV （M213S/F58H/N54V/L239G /A43S/T45M/S206A/D207P/S215F）的氨基酸序列。  
SEQ ID NO.6是DAAO突变体V （M213S/F58H/N54V/L239G /E122P/Y132F/E195Y/C234L）的氨基酸序列。  
SEQ ID NO.7是DAAO突变体VI （M213S/F58H/N54V/L239G /A73L/Q77W/V79M/Q147M/S185M/A43S/T45M/S206A/D207P/S215FE122P/Y132F/E195Y/C234L）的氨基酸序列。
本发明还涉及编码所述的DAAO突变体的基因以及重组载体。
所述重组表达载体可通过本领域常规方法将编码本发明所述DAAO突变体基因的核酸连接于各种合适载体上构建而成。所述载体可以是本领域的各种常规载体，如市售的质粒、粘粒、噬菌体或病毒载体等，只要所述重组表达载体可以在相应的表达宿主中正常复制，并表达所述的DAAO即可。所述载体优选质粒，更优选质粒pET28a。
本发明还涉及含有编码所述的DAAO突变体的基因的基因工程菌。
可通过将已经构建好的重组表达载体转化至宿主细胞来制备重组表达转化体。所述宿主细胞为本领域的各种常规宿主细胞，只要所述的重组表达载体能够稳定地自行复制并能通过诱导剂诱导后有效表达目标蛋白质即可。本发明首选大肠杆菌作为宿主细胞，更优选大肠杆菌E.coli BL21（DE3）用于高效表达本发明所述的DAAO突变体。
本发明还涉及所述DAAO突变体在微生物催化制备L-PPT中的应用。
涉及的反应式如下：
本发明的有益效果主要体现在：本发明提供了酶活性和酶热稳定性均有所提高的DAAO突变体，可用于微生物催化制备L-PPT，利于工业化生产，具有较好应用前景。
具体实施方式
下面结合具体实施例对本发明做进一步详细地说明，但本发明并不限于以下实施例：
实施例中使用的质粒提取试剂盒、DNA纯化回收试剂盒购自杭州擎科梓熙生物技术有限公司；一步克隆试剂盒购自诺唯赞有限公司；E.coli BL21（DE3）、质粒pET-24a（+）和全基因合成等由生工生物工程（上海）股份有限公司完成；DNA标记、低分子量标准蛋白、蛋白预制胶购自北京GenStar有限公司；ClonExpress II OneStep Cloning Kit无缝克隆试剂盒购自南京诺唯赞生物科技股份有限公司；pfu DNA聚合酶和DpnI内切酶购自赛默飞世尔科技（中国）有限公司；引物合成，序列测序工作由杭州擎科梓熙生物技术有限公司完成。以上试剂使用方法参考商品说明书。
下游催化工艺所用试剂D，L-PPT购自Sigma-Aldrich公司；2 4-二硝基苯肼（DNPH）购自阿拉丁试剂（中国上海），商品化的微球菌过氧化氢酶购自Sigma Aldrich （上海，中国）。其他常用试剂购自国药集团化学试剂有限公司。
实施例中其他未注明具体条件的实验方法，均按照常规方法和条件，或按照商品说明书选择。
下列实施例通过高效液相色谱（HPLC）进行产物的检测，并对产物进行分析。
HPLC分析方法为：色谱柱/Welchrom®C18；柱温/30℃；流速/1mL/min；检测波长/232nm；流动相：50mM（NH2）HPO4，加入1%的10%的四丁基溴化铵水溶液，用磷酸调pH至3.8，加入12%的乙腈。
通过手性HPLC分析方法检查PPT的两个构型含量。具体的，手性HPLC分析方法为：色谱柱/Pntulips QS-C18；流动相/50mM乙酸铵溶液：甲醇=9:1；检测波长/338nm；流速/1mL/min；柱温/30℃。
衍生化试剂：分别称取0.1g邻苯二甲醛与0.12gN-乙酰-L-半胱氨酸，用10ml乙醇助溶，在加入40ml 0.1M硼酸缓冲液（pH）9.8。振荡使其充分溶解，4℃冰箱保存备用（不超过3天）。衍生化反应与测定：取200μL样品加入400μL衍生化试剂，混匀至30℃保温5min，加入400μL超纯水混合，进样10μL进行分析。
实施例1：基因工程菌的制备
   将来源于Rhodotorula taiwanensis的野生型DAAO（wtDAAO），GenBank号：POY70719.1，氨基酸序列为SEQ ID NO.1所示，核苷酸序列为SEQ ID NO.8所示的基因序列进行全基因合成后，插入表达质粒pET-24a（+），得到pET-24a（+）-DAAO。测序验证无误后将pET-24a（+）DAAO转入表达宿主大肠杆菌E.coli BL21（DE3）中用于后续重组酶的表达。
   LB液体培养基组成：蛋白胨10g/L，酵母粉5g/L，NaCl 10g/L，用水溶解后定容，121℃灭菌20min，待用。
将上述测序无误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10ml LB液体培养基中，37℃震荡培养10-12h，按2％接种量转接至100ml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8000rpm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 pH8.0磷酸缓冲液洗涤两次，之后重悬于50mL pH8.0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wtDAAO酶的粗酶液。
实施例2 ：DAAO突变体I（54位、58位、213位）的构建
在实施例1中所述野生型DAAO序列的基础上突变了第213位、58位、54位。针对突变的DAAO序列的第213位、第58位、第54位进行突变的突变体设计PCR的引物序列，按突变位点的突变顺序设计如表1，2，3所示：
表1
表2
表3
PCR （25μL） 扩增体系为：
   2×PCR buffer缓冲液12 .5μL，上下游引物各0.5μL，模板质粒0.5μL，dNTP 0.5μL，高保真酶0.5μL，加入ddH2 O补足至25μL。
   PCR扩增程序为：  
（1）95℃预变性5min，（2）95℃变性30秒，（3）60℃退火30秒，（4）72℃延伸5min，30个循环，（5）72℃延伸10min，（6）4℃保存。
PCR结束后取5μL对扩增产物进行核酸凝胶电泳分析，将得到的目标条带清晰的PCR产物加入0.5μL Dpn I内切酶，放于37℃下消化模板1h。反应完成后转化至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，得到包含突变体的转化子。按照实施例1所述获得菌体。
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，在37℃恒温摇床中培养12-16小时，摇床转速200rpm。然后将96孔板的种子培养液转接至96孔板发酵培养基中，并在OD600 = 0.4~0.7时加入IPTG诱导剂，在28℃恒温摇床中培养12-16小时，摇床转速200rpm。将培养好的96孔发酵液4000rpm离心10分钟，弃上清，收集菌体。将收集的菌体与一定浓度的D，L-PPT在96孔板中反应1小时后4000rpm离心10分钟。
显色反应：用排枪吸取离心后的反应液上清于96孔透明板中，加入2mM的2，4-二硝基苯肼试剂，用排枪吹打均匀，置于酶标仪恒温槽中37℃保温20分钟，反应结束后加入1M的NaOH 100μL，混匀30s，生成红棕色的化合物，利用酶标仪高通量检测380nm吸光值。以wtDAAO为对照，筛选得到阳性克隆子（M213N，M213R，M213S），（M213S/F58H， M213S/F58R， M213S/F58K， M213S/F58Q），（M213S/F58H/N54V， M213S/F58H/N54D， M213S/F58H/N54T）。
实施例4 ：DAAO突变体I酶活的比较  
对实施例3筛选得到的阳性克隆子（M213N，M213R，M213S），（M213S/F58H， M213S/F58R， M213S/F58K， M213S/F58Q），（M213S/F58H/N54V， M213S/F58H/N54D， M213S/F58H/N54T）进行复筛，复筛反应通过检测突变体催化效率进行。具体是通过HPLC方法测定PPO的生成量来比较DAAO与其突变体的催化效率。1ml反应体系包括：50mM外消旋PPT铵盐，50mM pH8.0磷酸盐缓冲液，8000U/L过氧化氢酶，50g/L DAAO或其突变体冻干细胞。反应2h后，取反应液样品进行处理，测定PPO的浓度并计算转化率（产物PPO浓度/初始底物D,L PPT浓度×100％），具体如表4所示。
表4
实施例5： DAAO突变体（239位）的构建
在实施例4中所述突变序列（N54V/F58Q/M213S）的基础上进行了易错PCR（epPCR）的突变。针对易错PCR设计引物序列，具体如表5所示：
表5
epPCR（30μL）扩增体系如下：使用瞬时易错PCR试剂盒在，在10 mM Tris-HCl pH 8.3， 50 mM KCl， 2 mM MgCl2， 0.25 mM MnCl2的条件下，用模板质粒1μL和上下游引物各0.5μL在30μL反应混合物中进行。PCR扩增条件为：95°C 5 min，（90°C 30 s，55°C-65°C 30 s，72°C 5 min） × 30个循环，72°C 10 min。
   PCR结束后参照实施例2所述进行凝胶电泳分析，消化模板，转化。然后参照实施例1所述获得菌体。最后参照实施例3所述筛选阳性克隆子。得到了第239位（具体为L239G）的突变体。
实施例6： DAAO突变体II酶活的比较
如实施例4所述对实施例5筛选得到的阳性克隆子进行组合复筛并比较突变体之间的催化效率，具体如表6所述：
表6
由上表可以看出，突变体II （M213S/F58H/N54V/L239G）的活力明显提高，接下来将在突变体II的基础上进一步进行热稳定性的突变改造。
实施例7： DAAO突变体III的构建及酶活和热稳定性比较
在实施例6中所述突变体II序列的基础上突变了第73，77，79，147，185位，具体为III（M213S/F58H/N54V/L239G/A73L/Q77W/V79M/Q147M/S185M），针对突变体III所述位点进行突变体PCR设计引物序列，具体如表7所示：
表7
PCR扩增体系及条件同实施例2所述步骤，如实施例1所述获得菌体。如实施例4所述检测突变体的催化效率。突变体的热稳定性检测是在一定温度下保温一定时间后测定残余酶活。具体是在45℃下保温30分钟后在30℃反应1h检测残余酶活。所述单点突变及组合突变体之间的催化效率及热稳定性效果如表8所述：
表8
实施例8 ：DAAO突变体IV的构建及酶活和热稳定性比较
在实施例6中所述突变体II序列的基础上突变了第43，45，112，206，207和215位，具体为（M213S/F58H/N54V/L239G/A43S/T45M/S206A/D207P/S215F），针对突变体IV所述位点进行突变体PCR设计引物序列，具体如表9所示：
表9
PCR扩增体系及条件同实施例2所述步骤，如实施例1所述获得菌体。如实施例4所述检测突变体的催化效率。突变体的热稳定性检测是在一定温度下保温一定时间后测定残余酶活。具体是在45℃下保温30分钟后在30℃反应1h检测残余酶活。所述单点突变及组合突变体之间的催化效率及热稳定性效果如表10所述：
表10
实施例9 ：DAAO突变体V的构建及酶活和热稳定性比较
在实施例6中所述突变体II序列的基础上突变了第122，132，195和234位，具体为（M213S/F58H/N54V/L239G/E122P/Y132F/E195Y/C234L），针对突变体V所述位点进行突变体PCR设计引物序列，具体如表11所示：
表11
PCR扩增体系及条件同实施例2所述步骤，如实施例1所述获得菌体。如实施例4所述检测突变体的催化效率。突变体的热稳定性检测是在一定温度下保温一定时间后测定残余酶活。具体是在45℃下保温30分钟后在30℃反应1h检测残余酶活。所述单点突变及组合突变体之间的催化效率及热稳定性效果如表12所述：
表12
实施例10： DAAO突变体VI的构建及酶活和热稳定性比较
将上述DAAO突变体I-V进行组合突变得到突变体VI （M213S/F58H/N54V/L239G/A73L/Q77W/V79M/Q147M/S185M/A43S/T45M/S206A/D207P/S215F/E122P/Y132F/E195Y/C234L）。PCR扩增体系及条件同实施例2所述步骤，如实施例1所述获得菌体。如实施例4所述检测组合突变体II，III，IV，V，VI的催化效率。突变体的热稳定性检测是在一定温度下保温一定时间后测定残余酶活。具体是分别在50℃保温15分钟，55℃保温15分钟后在30℃反应1h检测残余酶活。所述组合突变体之间的催化效率及热稳定性效果如表13所述：
表13
由上表可以看出，突变体VI在酶活力不受影响的情况下热稳定性明显提高。显然，上述实施例仅仅是为清楚地说明所作的举例，而并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸出的显而易见的变化或变动仍处于本发明创造的保护范围之中。

Claims (7)

  1. 一种D-氨基酸氧化酶突变体，由SEQ ID NO.1所示氨基酸序列第54位、第58位、第213位、第239位、第73位、第77位、第79位、第147位、第185位、第43位、第45位、第206位、第207位、第215位、第122位、第132位、第195位、第234位经单点突变或多点联合突变获得。
  2. 如权利要求1所述的D-氨基酸氧化酶突变体，其特征在于所述突变为下列之一：（1）第54位氨基酸残基N突变为V、D或T，第58位氨基酸残基F突变为H、R、K或Q，第213位氨基酸残基M突变为S、N或R；（2）第54位氨基酸残基N突变为V、D或T，第58位氨基酸残基F突变为H、R、K或Q，第213位氨基酸残基M突变为S、N或R，第239位氨基酸残基L突变为E、D、G或Q；（3）第54位氨基酸残基N突变为V、D或T，第58位氨基酸残基F突变为H、R、K或Q，第213位氨基酸残基M突变为S、N或R，第239位氨基酸残基L突变为E、D、G或Q，第73位氨基酸残基A突变为L，第77位氨基酸残基Q突变为W，第79位氨基酸残基V突变为M，第147位氨基酸残基Q突变为M，第185位氨基酸残基S突变为M；（4）第54位氨基酸残基N突变为V、D或T，第58位氨基酸残基F突变为H、R、K或Q，第213位氨基酸残基M突变为S、N或R，第239位氨基酸残基L突变为E、D、G或Q，第43位氨基酸残基A突变为S，第45位氨基酸残基T突变为M，第206位氨基酸残基S突变为A，第207位氨基酸残基D突变为P，第215位氨基酸残基S突变为F；（5）第54位氨基酸残基N突变为V、D或T，第58位氨基酸残基F突变为H、R、K或Q，第213位氨基酸残基M突变为S、N或R，第239位氨基酸残基L突变为E、D、G或Q，第122位氨基酸残基E突变为P，第132位氨基酸残基Y突变为F，第195位氨基酸残基E突变为Y，第234位氨基酸残基C突变为L。
  3. 如权利要求2所述的D-氨基酸氧化酶突变体，其特征在于所述突变体氨基酸序列如SEQ ID NO.2~7之一所示。
  4. 编码权利要求1所述的D-氨基酸氧化酶突变体的基因。
  5. 含有编码权利要求1所述的D-氨基酸氧化酶突变体的基因的重组载体。
  6. 含有编码权利要求1所述的D-氨基酸氧化酶突变体的基因的基因工程菌。
  7. 权利要求1所述D-氨基酸氧化酶突变体在微生物催化制备L-草铵膦中的应用。
PCT/CN2023/118314 2022-12-05 2023-09-12 D-氨基酸氧化酶突变体及其在制备l-草铵膦中的应用 WO2024119940A1 (zh)

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