WO2024092967A1 - Rare earth-dependent alcohol dehydrogenase mutant and use thereof - Google Patents
Rare earth-dependent alcohol dehydrogenase mutant and use thereof Download PDFInfo
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- WO2024092967A1 WO2024092967A1 PCT/CN2022/138388 CN2022138388W WO2024092967A1 WO 2024092967 A1 WO2024092967 A1 WO 2024092967A1 CN 2022138388 W CN2022138388 W CN 2022138388W WO 2024092967 A1 WO2024092967 A1 WO 2024092967A1
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- alcohol dehydrogenase
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- dependent alcohol
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
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- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/02—Oxygen as only ring hetero atoms
- C12P17/04—Oxygen as only ring hetero atoms containing a five-membered hetero ring, e.g. griseofulvin, vitamin C
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- C12R2001/185—Escherichia
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Definitions
- the invention relates to the technical field of enzyme engineering, and in particular to a rare earth-dependent alcohol dehydrogenase mutant and application thereof.
- a rare earth-dependent alcohol dehydrogenase PedH from Pseudomonas putida can be expressed heterologously and soluble in Escherichia coli, which is convenient for enzyme engineering. Studies have shown that Pseudomonas putida expressing PedH can grow with ethanol as the sole carbon source. In addition, PedH can catalyze methanol to form formaldehyde, and the oxidation of methanol is a key rate-limiting step in one-carbon metabolism and an indispensable step in natural methylotrophic bacteria and artificial methylotrophic bacteria.
- PedH can catalyze 5-hydroxymethylfurfural (HMF) to generate 5-hydroxymethyl-2-furancarboxylic acid (HFCA) and further oxidize it to 5-formylfuran-2-carboxylic acid (FFCA).
- FFCA is the precursor of 2,5-furandicarboxylic acid (FDCA).
- FDCA is an important bio-based platform compound with the potential to be used as a substitute for terephthalic acid (PTA) in the synthesis of renewable polyethylene 2,5-furandicarboxylate (PEF). It is widely used in the production of a variety of bio-based polymers, such as polyamides, polyesters and polyurethanes.
- the method combines the deep learning-based MutCompute design tool and the energy function-based FoldX tool to determine the mutation sites of rare earth-dependent alcohol dehydrogenase PedH, and screens mutants with improved stability and/or increased activity for methanol, ethanol, and 5-hydroxymethylfurfural, which is of great significance. It can promote the development of one-carbon metabolism, improve resource shortages and environmental pollution problems, and advance the research on green biomanufacturing of important compounds such as FDCA.
- the present invention provides a rare earth-dependent alcohol dehydrogenase PedH mutant and its application.
- the present invention uses the MutCompute online design tool based on deep learning to predict high-performance mutants of rare earth-dependent alcohol dehydrogenase PedH (amino acid sequence as shown in SEQ ID NO.1, nucleotide sequence as shown in SEQ ID NO.2) from Pseudomonas putida, obtains a series of predicted mutants, and uses the FoldX tool based on energy function to calculate the binding free energy change ⁇ G of each predicted mutant.
- the top 10 mutants with the highest MutCompute prediction score and the 10 mutants with the lowest ⁇ G calculated by FoldX are selected for site-directed mutagenesis.
- the 20 mutants are cultured, expressed and purified, and the specific enzyme activity of the mutants to methanol, ethanol and 5-hydroxymethylfurfural is determined by an ELISA instrument, and then the dissolution temperature T m value of each mutant is determined by a protein stability analyzer, and through combined mutations, finally a mutant of rare earth-dependent alcohol dehydrogenase PedH with improved activity and/or stability is obtained.
- the mutants are Q207N, H486Y, and Q207N/H486Y.
- Q207N means that the amino acid at position 207 is mutated from glutamine to asparagine
- H486Y means that the amino acid at position 486 is mutated from histidine to tyrosine
- Q207N/H486Y means that the amino acid at position 207 is mutated from glutamine to asparagine and the amino acid at position 486 is mutated from histidine to tyrosine.
- the present invention provides a rare earth-dependent alcohol dehydrogenase mutant, which is obtained by mutating a wild-type alcohol dehydrogenase from Pseudomonas putida, wherein the amino acid sequence of the wild-type alcohol dehydrogenase is shown in SEQ ID NO.1, and the specific mutations are (1) and/or (2):
- the present invention also provides the use of the rare earth-dependent alcohol dehydrogenase mutant in catalyzing methanol to generate formaldehyde, catalyzing ethanol to generate acetaldehyde, or catalyzing 5-hydroxymethylfurfural to generate 5-hydroxymethyl-2-furancarboxylic acid.
- the invention also provides a gene encoding the rare earth-dependent alcohol dehydrogenase mutant.
- nucleotide sequence of the gene is shown as SEQ ID NO.7, SEQ ID NO.8 or SEQ ID NO.9.
- the present invention also provides the use of the gene in catalyzing methanol to generate formaldehyde, catalyzing ethanol to generate acetaldehyde, or catalyzing 5-hydroxymethylfurfural to generate 5-hydroxymethyl-2-furancarboxylic acid.
- the present invention also provides an expression vector comprising the gene.
- the original expression vector of the recombinant vector used in the present invention is pET28a.
- the present invention also provides a genetically engineered bacterium that expresses the rare earth-dependent alcohol dehydrogenase mutant.
- the host cell of the genetically engineered bacterium used in the present invention is E. coli BL21 (DE3).
- the present invention also provides the use of the genetically engineered bacteria in catalyzing methanol to generate formaldehyde, catalyzing ethanol to generate acetaldehyde, or catalyzing 5-hydroxymethylfurfural to generate 5-hydroxymethyl-2-furancarboxylic acid.
- the present invention also provides a method for preparing formaldehyde, acetaldehyde or 5-hydroxymethyl-2-furancarboxylic acid, using the rare earth-dependent alcohol dehydrogenase mutant to catalyze the oxidation reaction of methanol, ethanol or 5-hydroxymethylfurfural to prepare formaldehyde, acetaldehyde or 5-hydroxymethyl-2-furancarboxylic acid.
- the present invention has the following beneficial effects:
- the present invention is based on a rare earth-dependent alcohol dehydrogenase, uses the MutCompute online design tool based on deep learning and the FoldX tool based on energy function to predict the sites to be mutated, and screens mutants with improved activity and/or stability, solving the problem that high stability and high activity cannot be taken into account at the same time.
- mutants Q207N, H486Y and combined mutants Q207N/H486Y for ethanol are 2.4, 2.6, and 3.6 times that of the original enzyme PedH, respectively, and the specific enzyme activities for methanol are 2.3, 2.8, and 3.6 times that of the original enzyme PedH, respectively, and the specific enzyme activities for 5-hydroxymethylfurfural are 2.3, 2.6, and 2.8 times that of the original enzyme PedH, respectively.
- the mutant H486Y and the combined mutant Q207N/H486Y have improved specific enzyme activities and improved stability, and the dissolution temperature T m values are increased by 2.3°C and 2.2°C respectively compared with the original enzyme PedH.
- the rational design method used in the present invention which combines the MutCompute design tool and the FoldX tool, can quickly obtain highly stable and highly active rare earth-dependent alcohol dehydrogenase mutants through screening with a smaller mutation library.
- Figure 1 is an SDS-PAGE image of the expression and purification of PedH protein; from left to right: M: Maker, 1: ultrafiltrate, 2: eluent, 3: flow-through, 4: supernatant.
- FIG2 is a graph showing the enzymatic activities of wild-type PedH and Q207N, H486Y, and Q207N/H486Y mutants against three substrates.
- FIG3 is a graph showing the T m values of the wild-type PedH and the Q207N, H486Y, and Q207N/H486Y mutants.
- E. coli BL21 (DE3), plasmid pET28a, etc. used in the embodiments of the present invention were purchased from Novagen; gene synthesis, primer synthesis and sequence sequencing of PedH were completed by Qingke Biotechnology Co., Ltd.
- Reagents used in the catalytic reaction methanol, ethanol, Tris and hydrochloric acid were purchased from Sinopharm Chemical Reagent Co., Ltd.; PQQ (pyrroloquinoline quinone) was purchased from TCI (Shanghai) Chemical Industry Development Co., Ltd.; praseodymium chloride and 5-hydroxymethylfurfural were purchased from Aladdin Reagent (Shanghai) Co., Ltd.; DCPIP (2,6-dichlorophenol indophenol) was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; PES (phenazine ethyl sulfate) was purchased from Shanghai En Chemical Technology Co., Ltd.
- enzyme activity unit (U) the amount of enzyme required to consume 1 ⁇ M DCPIP in the reaction system per minute.
- the original rare earth-dependent alcohol dehydrogenase gene sequence (amino acid sequence as shown in SEQ ID NO.1) was synthesized by Qingke Biotechnology Co., Ltd., and the sequence was shown in SEQ ID NO.2, and inserted into the restriction sites BamH I and Hind III of the vector pET28a to obtain the recombinant plasmid pET28a-PedH.
- the recombinant plasmid was transformed into E. coli BL21 (DE3) competent cells by heat shock method, and the original rare earth-dependent alcohol dehydrogenase recombinant bacteria were obtained after sequencing verification.
- the high-performance mutants of PedH were predicted using the deep learning-based MutCompute online design tool (https://mutcompute.com/). Specifically, after logging in to the website to register a user, enter the PDB number of PedH (6zcw), click Predict, and obtain a series of predicted mutants. Then, the FoldX tool based on the energy function was used to calculate the binding free energy change ⁇ G of each predicted mutant. Combining the MutCompute prediction score and the ⁇ G value calculated by FoldX, two potential mutation sites were screened.
- Primer name Primer sequence (5’ to 3’) Q207N-F TAATGCATATAATCCGGAAAATGGTGAACTGC Q207N-R CCGGATTATATGCATTAATTTTACCAACAACACCAAATTCA H486Y-F GAAGTTTGGCGTTATAAAAATTATGCACCGCTGTGG H486Y-R TTATAACGCCAAACTTCTTTACCGCTAACCGG
- mutant strain Q207N the nucleotide sequence encoding the rare earth-dependent alcohol dehydrogenase mutant is shown in SEQ ID NO.7
- mutant strain H486Y the nucleotide sequence encoding the rare earth-dependent alcohol dehydrogenase mutant is shown in SEQ ID NO.8
- mutant strain Q207N/H486Y the nucleotide sequence encoding the rare earth-dependent alcohol dehydrogenase mutant is shown in SEQ ID NO.9.
- the recombinant bacteria original rare earth-dependent alcohol dehydrogenase recombinant bacteria and rare earth-dependent alcohol dehydrogenase mutant strains sequenced correctly in Example 3 were selected and placed in 5 mL LB liquid medium (containing 50 ⁇ g/mL kanamycin) and cultured at 37°C for 12 h.
- the inoculum was transferred to 50 mL LB liquid medium containing 50 ⁇ g/mL kanamycin and 10 g/L ⁇ -lactose at a 2% inoculum volume and cultured at 30°C for 12 h.
- the bacterial solution is centrifuged at 4000 ⁇ g for 15 minutes at 4°C, the supernatant is discarded, and the bacteria are collected. After washing the bacteria with 100mM Tris-HCl buffer, pH 8.0, the bacteria are resuspended in Tris-HCl buffer and placed in an ice water bath for ultrasonic cell lysis until clarified. The cell lysis solution is centrifuged at 8000 ⁇ g for 20 minutes at 4°C, and the supernatant is subjected to affinity chromatography on a nickel column.
- the impurities are washed with a washing buffer (100mM Tris-HCl buffer, 150mM NaCl, 50mM imidazole, pH 8.0), and the target protein is eluted with an elution buffer (100mM Tris-HCl buffer, 150mM NaCl, 250mM imidazole, pH 8.0).
- a washing buffer 100mM Tris-HCl buffer, 150mM NaCl, 50mM imidazole, pH 8.0
- an elution buffer 100mM Tris-HCl buffer, 150mM NaCl, 250mM imidazole, pH 8.0.
- the amount of DCPIP consumed in the reaction system was quantitatively analyzed by an ELISA instrument, and the specific enzyme activities of the original rare earth-dependent alcohol dehydrogenase and the rare earth-dependent alcohol dehydrogenase mutants on the substrates methanol, ethanol and 5-hydroxymethylfurfural were calculated.
- the reaction system contained an appropriate amount of enzyme solution, 1.5 ⁇ M PQQ, 1.5 ⁇ M PrCl 3 , 15mM substrate, and the total system was 200 ⁇ L.
- the reaction medium was Tris-HCl buffer (100mM, pH 8.0), which contained 1mM PES and 150 ⁇ M DCPIP. The results of the specific enzyme activity determination are shown in Figure 2.
- the specific enzyme activities of the mutants Q207N, H486Y and the combined mutant Q207N/H486Y on the three substrates were significantly improved compared with the original enzyme PedH.
- the specific enzyme activities of the original enzyme PedH on ethanol, methanol and 5-hydroxymethylfurfural were 0.433U/mg, 0.088U/mg and 0.053U/mg, respectively.
- mutants Q207N, H486Y and combined mutant Q207N/H486Y towards ethanol were 2.4, 2.6 and 3.6 times that of the original enzyme PedH, respectively; the specific enzyme activities towards methanol were 2.3, 2.8 and 3.6 times that of the original enzyme PedH, respectively; and the specific enzyme activities towards 5-hydroxymethylfurfural were 2.3, 2.6 and 2.8 times that of the original enzyme PedH, respectively.
- the enzyme concentrations of the original rare earth-dependent alcohol dehydrogenase and the rare earth-dependent alcohol dehydrogenase mutants were uniformly adjusted to 10 ⁇ M and added to a high-precision quartz glass capillary set.
- the T m value was determined using a protein stability analyzer (Prometheus NT.Plex). The measurement results are shown in Figure 3. It can be seen that there is no obvious difference in the dissolution temperature T m value between the mutant Q207N and the wild-type PedH, but the stability of the mutant H486Y and the combined mutant Q207N/H486Y is improved, and the dissolution temperature T m values are increased by 2.3°C and 2.2°C respectively compared with the wild-type PedH (63.7°C).
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Abstract
The present invention relates to the technical field of enzyme engineering, and in particular, to a rare earth-dependent alcohol dehydrogenase mutant and use thereof. According to the present invention, a method combining the MutCompute design tool based on deep learning and a FoldX tool based on an energy function is adopted to determine a to-be-mutated site of a rare earth-dependent alcohol dehydrogenase PedH. Mutants with an increase of 130% to 260% in the activity for methanol, ethanol, or 5-hydroxymethylfurfural were obtained by means of screening, among which two mutants also demonstrated improved stability. The present invention has important prospects in the construction of artificial methylotrophic bacteria and the production of important compounds such as FDCA in an environment-friendly manner, and solves the contradiction between stability and activity of enzymes.
Description
本发明涉及酶工程技术领域，具体涉及一种稀土依赖型醇脱氢酶突变体及其应用。The invention relates to the technical field of enzyme engineering, and in particular to a rare earth-dependent alcohol dehydrogenase mutant and application thereof.
为了获得性能优异的酶，人类不断探索发展改造蛋白质的方法，例如初级理性设计、定向进化以及半理性设计。近年来，随着计算机运算能力大幅提升、结构生物学、计算生物学和人工智能技术的飞速发展，计算机辅助蛋白质设计受到了极大的关注，已成为蛋白质工程新的重要方向。基于结构模拟与能量计算(如Rosetta、FoldX等)和基于机器学习(如MLDE、Mutcompute等)的蛋白质计算设计已取得瞩目的成绩。In order to obtain enzymes with excellent performance, humans continue to explore and develop methods to transform proteins, such as primary rational design, directed evolution, and semi-rational design. In recent years, with the substantial improvement of computer computing power and the rapid development of structural biology, computational biology, and artificial intelligence technology, computer-aided protein design has received great attention and has become an important new direction in protein engineering. Computational protein design based on structural simulation and energy calculation (such as Rosetta, FoldX, etc.) and machine learning (such as MLDE, Mutcompute, etc.) has achieved remarkable results.
醇氧化生成酮或醛是有机合成中的重要反应。与化学方法相比，醇的酶氧化是在温和条件下进行的，不使用任何有毒试剂，且往往具有较高的催化特异性和选择性，产生的副产物较少，因此一直是醇类绿色氧化的首选。醇脱氢酶(ADHs)是常用的氧化酶。2012年，首个稀土依赖型醇脱氢酶XoxF被发掘报道，相比于同样以PQQ为辅酶的钙依赖型醇脱氢酶，XoxF表现出更高的催化活性。不同于普通的金属离子，稀土元素独特的4f价电子结构，使其具有优异的光、电、磁与催化等性能。稀土离子电子跃迁、自旋耦合与轨道杂化等特性，可以为酶工程提供更多可能性。The oxidation of alcohols to form ketones or aldehydes is an important reaction in organic synthesis. Compared with chemical methods, enzymatic oxidation of alcohols is carried out under mild conditions, without the use of any toxic reagents, and often has higher catalytic specificity and selectivity, with fewer by-products. Therefore, it has always been the first choice for the green oxidation of alcohols. Alcohol dehydrogenases (ADHs) are commonly used oxidases. In 2012, the first rare earth-dependent alcohol dehydrogenase XoxF was discovered and reported. Compared with the calcium-dependent alcohol dehydrogenase that also uses PQQ as a coenzyme, XoxF exhibits higher catalytic activity. Unlike ordinary metal ions, the unique 4f valence electron structure of rare earth elements gives them excellent optical, electrical, magnetic and catalytic properties. The characteristics of rare earth ion electronic transitions, spin coupling and orbital hybridization can provide more possibilities for enzyme engineering.
一种来源于恶臭假单胞菌的稀土依赖型醇脱氢酶PedH，可在大肠杆菌中实现异源可溶表达，便于进行酶工程改造。有研究表明表达PedH的恶臭假单胞菌能够以乙醇作为唯一碳源生长。此外，PedH可以催化甲醇生成甲醛，而甲醇的氧化是一碳代谢的关键限速步骤，是天然甲基营养菌和人工甲基营养菌中不可或缺的一步。随着世界人口的快速增长和工业的迅速发展，资源短缺和环境污染问题已成为人类面临的巨大挑战，以一碳代谢为代表的绿色生物制造愈发受到关注。甲醇和甲烷等一碳化合物由于原料来源广泛，价格低廉，还原力高等优势，成为绿色生物制 造大宗化学品的理想原料。A rare earth-dependent alcohol dehydrogenase PedH from Pseudomonas putida can be expressed heterologously and soluble in Escherichia coli, which is convenient for enzyme engineering. Studies have shown that Pseudomonas putida expressing PedH can grow with ethanol as the sole carbon source. In addition, PedH can catalyze methanol to form formaldehyde, and the oxidation of methanol is a key rate-limiting step in one-carbon metabolism and an indispensable step in natural methylotrophic bacteria and artificial methylotrophic bacteria. With the rapid growth of the world's population and the rapid development of industry, resource shortages and environmental pollution have become huge challenges facing mankind, and green biomanufacturing represented by one-carbon metabolism has received increasing attention. One-carbon compounds such as methanol and methane have become ideal raw materials for green biomanufacturing of bulk chemicals due to their wide sources of raw materials, low prices, and high reducing power.
PedH能够催化5-羟甲基糠醛(HMF)生成5-羟甲基-2-呋喃甲酸(HFCA)并进一步氧化成为5-甲醛基呋喃-2-羧酸(FFCA)。FFCA是2,5-呋喃二甲酸(FDCA)的前体。FDCA是一种重要的生物基平台化合物，具有作为对苯二甲酸(PTA)的替代品用于合成可再生的聚2,5-呋喃二甲酸乙二醇酯(PEF)的潜力，被广泛应用于生产多种生物基高分子聚合物，例如聚酰胺、聚酯和聚氨酯等。PedH can catalyze 5-hydroxymethylfurfural (HMF) to generate 5-hydroxymethyl-2-furancarboxylic acid (HFCA) and further oxidize it to 5-formylfuran-2-carboxylic acid (FFCA). FFCA is the precursor of 2,5-furandicarboxylic acid (FDCA). FDCA is an important bio-based platform compound with the potential to be used as a substitute for terephthalic acid (PTA) in the synthesis of renewable polyethylene 2,5-furandicarboxylate (PEF). It is widely used in the production of a variety of bio-based polymers, such as polyamides, polyesters and polyurethanes.
然而，与大多数醇脱氢酶一样，PedH在催化氧化的过程中存在活性和稳定性较低的问题，这限制了其工业应用。因此，通过基于深度学习的MutCompute设计工具以及基于能量函数的FoldX工具相结合的方法，确定稀土依赖型醇脱氢酶PedH的待突变位点，并通过筛选得到稳定性提高和/或对甲醇、乙醇、5-羟甲基糠醛活性提高的突变体具有重要意义。可促进一碳代谢的发展，改善资源短缺和环境污染问题，推进绿色生物制造FDCA等重要化合物的研究。However, like most alcohol dehydrogenases, PedH has low activity and stability during catalytic oxidation, which limits its industrial application. Therefore, the method combines the deep learning-based MutCompute design tool and the energy function-based FoldX tool to determine the mutation sites of rare earth-dependent alcohol dehydrogenase PedH, and screens mutants with improved stability and/or increased activity for methanol, ethanol, and 5-hydroxymethylfurfural, which is of great significance. It can promote the development of one-carbon metabolism, improve resource shortages and environmental pollution problems, and advance the research on green biomanufacturing of important compounds such as FDCA.
发明内容Summary of the invention
为了解决来源于恶臭假单胞菌Pseudomonas putida的原始稀土依赖型醇脱氢酶PedH的活性和稳定性较低的问题，本发明提供了一种稀土依赖型醇脱氢酶PedH突变体及其应用。In order to solve the problem of low activity and stability of the original rare earth-dependent alcohol dehydrogenase PedH derived from Pseudomonas putida, the present invention provides a rare earth-dependent alcohol dehydrogenase PedH mutant and its application.
本发明使用基于深度学习的MutCompute在线设计工具对来源于Pseudomonas putida的稀土依赖型醇脱氢酶PedH(氨基酸序列如SEQ ID NO.1所示，核苷酸序列如SEQ ID NO.2所示)的高性能突变体进行预测，获得一系列预测突变体，并使用基于能量函数的FoldX工具计算各预测突变体的结合自由能变ΔΔG。选取MutCompute预测得分最高的前10个突变体以及FoldX计算的ΔΔG最低的10个突变体进行定点突变。对该20种突变体进行培养表达和纯化，并使用酶标仪测定突变体对甲醇、乙醇和5-羟甲基糠醛的比酶活，然后使用蛋白质稳定性分析仪测定各突变体的溶解温度T
m值，并通过组合突变，最终获得活性和/或稳定性提高的稀土依赖型醇脱氢酶PedH的突变体。
The present invention uses the MutCompute online design tool based on deep learning to predict high-performance mutants of rare earth-dependent alcohol dehydrogenase PedH (amino acid sequence as shown in SEQ ID NO.1, nucleotide sequence as shown in SEQ ID NO.2) from Pseudomonas putida, obtains a series of predicted mutants, and uses the FoldX tool based on energy function to calculate the binding free energy change ΔΔG of each predicted mutant. The top 10 mutants with the highest MutCompute prediction score and the 10 mutants with the lowest ΔΔG calculated by FoldX are selected for site-directed mutagenesis. The 20 mutants are cultured, expressed and purified, and the specific enzyme activity of the mutants to methanol, ethanol and 5-hydroxymethylfurfural is determined by an ELISA instrument, and then the dissolution temperature T m value of each mutant is determined by a protein stability analyzer, and through combined mutations, finally a mutant of rare earth-dependent alcohol dehydrogenase PedH with improved activity and/or stability is obtained.
所述突变体为Q207N、H486Y、Q207N/H486Y。其中，Q207N表示：第207位的氨基酸由谷氨酰胺突变为天冬酰胺；H486Y表示：第486位的 氨基酸由组氨酸突变为酪氨酸；Q207N/H486Y表示：第207位的氨基酸由谷氨酰胺突变为天冬酰胺且第486位的氨基酸由组氨酸突变为酪氨酸。The mutants are Q207N, H486Y, and Q207N/H486Y. Among them, Q207N means that the amino acid at position 207 is mutated from glutamine to asparagine; H486Y means that the amino acid at position 486 is mutated from histidine to tyrosine; Q207N/H486Y means that the amino acid at position 207 is mutated from glutamine to asparagine and the amino acid at position 486 is mutated from histidine to tyrosine.
具体的技术方案如下：The specific technical solutions are as follows:
本发明提供了一种稀土依赖型醇脱氢酶突变体，是由来自恶臭假单胞菌(Pseudomonas putida)的野生型醇脱氢酶进行突变而得，所述野生型醇脱氢酶的氨基酸序列如SEQ ID NO.1所示，具体突变为(1)和/或(2)：The present invention provides a rare earth-dependent alcohol dehydrogenase mutant, which is obtained by mutating a wild-type alcohol dehydrogenase from Pseudomonas putida, wherein the amino acid sequence of the wild-type alcohol dehydrogenase is shown in SEQ ID NO.1, and the specific mutations are (1) and/or (2):
(1)第207位的氨基酸由谷氨酰胺突变为天冬酰胺；(1) The amino acid at position 207 was mutated from glutamine to asparagine;
(2)第486位的氨基酸由组氨酸突变为酪氨酸。(2) The amino acid at position 486 mutated from histidine to tyrosine.
本发明还提供了所述的稀土依赖型醇脱氢酶突变体在催化甲醇生成甲醛、催化乙醇生成乙醛、或者催化5-羟甲基糠醛生成5-羟甲基-2-呋喃甲酸中的应用。The present invention also provides the use of the rare earth-dependent alcohol dehydrogenase mutant in catalyzing methanol to generate formaldehyde, catalyzing ethanol to generate acetaldehyde, or catalyzing 5-hydroxymethylfurfural to generate 5-hydroxymethyl-2-furancarboxylic acid.
本发明还提供了编码所述稀土依赖型醇脱氢酶突变体的基因。The invention also provides a gene encoding the rare earth-dependent alcohol dehydrogenase mutant.
优选的，所述基因的核苷酸序列如SEQ ID NO.7、SEQ ID NO.8或SEQ ID NO.9所示。Preferably, the nucleotide sequence of the gene is shown as SEQ ID NO.7, SEQ ID NO.8 or SEQ ID NO.9.
本发明还提供了所述的基因在催化甲醇生成甲醛、催化乙醇生成乙醛、或者催化5-羟甲基糠醛生成5-羟甲基-2-呋喃甲酸中的应用。The present invention also provides the use of the gene in catalyzing methanol to generate formaldehyde, catalyzing ethanol to generate acetaldehyde, or catalyzing 5-hydroxymethylfurfural to generate 5-hydroxymethyl-2-furancarboxylic acid.
本发明还提供了一种包含所述基因的表达载体。本发明使用的重组载体的原始表达载体为pET28a。The present invention also provides an expression vector comprising the gene. The original expression vector of the recombinant vector used in the present invention is pET28a.
本发明还提供了一种表达所述稀土依赖型醇脱氢酶突变体的基因工程菌。本发明使用的基因工程菌的宿主细胞为E.coli BL21(DE3)。The present invention also provides a genetically engineered bacterium that expresses the rare earth-dependent alcohol dehydrogenase mutant. The host cell of the genetically engineered bacterium used in the present invention is E. coli BL21 (DE3).
本发明还提供了所述的基因工程菌在催化甲醇生成甲醛、催化乙醇生成乙醛、或者催化5-羟甲基糠醛生成5-羟甲基-2-呋喃甲酸中的应用。The present invention also provides the use of the genetically engineered bacteria in catalyzing methanol to generate formaldehyde, catalyzing ethanol to generate acetaldehyde, or catalyzing 5-hydroxymethylfurfural to generate 5-hydroxymethyl-2-furancarboxylic acid.
本发明还提供了一种甲醛、乙醛、或者5-羟甲基-2-呋喃甲酸的制备方法，使用所述稀土依赖型醇脱氢酶突变体催化甲醇、乙醇或者5-羟甲基糠醛发生氧化反应，制备得到甲醛、乙醛、或者5-羟甲基-2-呋喃甲酸。The present invention also provides a method for preparing formaldehyde, acetaldehyde or 5-hydroxymethyl-2-furancarboxylic acid, using the rare earth-dependent alcohol dehydrogenase mutant to catalyze the oxidation reaction of methanol, ethanol or 5-hydroxymethylfurfural to prepare formaldehyde, acetaldehyde or 5-hydroxymethyl-2-furancarboxylic acid.
与现有技术相比，本发明具有以下有益效果：Compared with the prior art, the present invention has the following beneficial effects:
(1)本发明以一种稀土依赖型醇脱氢酶为基础，使用基于深度学习的MutCompute在线设计工具以及基于能量函数的FoldX工具预测待突变位点，筛选得到活性和/或稳定性提高的突变体，解决了高稳定性和高活性无法兼顾的问题。其中，突变体Q207N、H486Y和组合突变体Q207N/H486Y对乙醇的比酶活分别是原始酶PedH的2.4、2.6、3.6倍，对甲醇的 比酶活分别是原始酶PedH的2.3、2.8、3.6倍，对5-羟甲基糠醛的比酶活分别是原始酶PedH的2.3、2.6、2.8倍。其中，突变体H486Y和组合突变体Q207N/H486Y在比酶活提高的同时，稳定性也有所提高，溶解温度T
m值相比原始酶PedH分别提高2.3℃、2.2℃。
(1) The present invention is based on a rare earth-dependent alcohol dehydrogenase, uses the MutCompute online design tool based on deep learning and the FoldX tool based on energy function to predict the sites to be mutated, and screens mutants with improved activity and/or stability, solving the problem that high stability and high activity cannot be taken into account at the same time. Among them, the specific enzyme activities of mutants Q207N, H486Y and combined mutants Q207N/H486Y for ethanol are 2.4, 2.6, and 3.6 times that of the original enzyme PedH, respectively, and the specific enzyme activities for methanol are 2.3, 2.8, and 3.6 times that of the original enzyme PedH, respectively, and the specific enzyme activities for 5-hydroxymethylfurfural are 2.3, 2.6, and 2.8 times that of the original enzyme PedH, respectively. Among them, the mutant H486Y and the combined mutant Q207N/H486Y have improved specific enzyme activities and improved stability, and the dissolution temperature T m values are increased by 2.3°C and 2.2°C respectively compared with the original enzyme PedH.
(2)本发明所使用的将MutCompute设计工具和FoldX工具相结合的理性设计方法，能以较小的突变文库，通过筛选快速获得高稳定性和高活性的稀土依赖型醇脱氢酶突变体。(2) The rational design method used in the present invention, which combines the MutCompute design tool and the FoldX tool, can quickly obtain highly stable and highly active rare earth-dependent alcohol dehydrogenase mutants through screening with a smaller mutation library.
图1为PedH蛋白表达纯化SDS-PAGE图；从左至右依次是M：Maker，1：超滤液，2：洗脱液，3：流穿，4：上清。Figure 1 is an SDS-PAGE image of the expression and purification of PedH protein; from left to right: M: Maker, 1: ultrafiltrate, 2: eluent, 3: flow-through, 4: supernatant.
图2为PedH野生型及Q207N、H486Y、Q207N/H486Y突变体对三种底物的比酶活图。FIG2 is a graph showing the enzymatic activities of wild-type PedH and Q207N, H486Y, and Q207N/H486Y mutants against three substrates.
图3为PedH野生型及Q207N、H486Y、Q207N/H486Y突变体的T
m值图。
FIG3 is a graph showing the T m values of the wild-type PedH and the Q207N, H486Y, and Q207N/H486Y mutants.
上游基因工程所用试剂：本发明实施例中使用的E.coli BL21(DE3)、质粒pET28a等购自Novagen公司；PedH的基因合成、引物合成与序列测序工作由擎科生物工程股份有限公司完成。Reagents used for upstream genetic engineering: E. coli BL21 (DE3), plasmid pET28a, etc. used in the embodiments of the present invention were purchased from Novagen; gene synthesis, primer synthesis and sequence sequencing of PedH were completed by Qingke Biotechnology Co., Ltd.
催化反应所用试剂：甲醇、乙醇、Tris和盐酸购自国药集团化学试剂有限公司；PQQ(吡咯喹啉醌)购自梯希爱(上海)化成工业发展有限公司；氯化镨和5-羟甲基糠醛购自阿拉丁试剂(上海)有限公司；DCPIP(2,6-二氯酚靛酚)购自上海麦克林生化科技有限公司；PES(吩嗪乙基硫酸盐)购自上海易恩化学技术有限公司。Reagents used in the catalytic reaction: methanol, ethanol, Tris and hydrochloric acid were purchased from Sinopharm Chemical Reagent Co., Ltd.; PQQ (pyrroloquinoline quinone) was purchased from TCI (Shanghai) Chemical Industry Development Co., Ltd.; praseodymium chloride and 5-hydroxymethylfurfural were purchased from Aladdin Reagent (Shanghai) Co., Ltd.; DCPIP (2,6-dichlorophenol indophenol) was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; PES (phenazine ethyl sulfate) was purchased from Shanghai En Chemical Technology Co., Ltd.
酶活单位(U)的定义：反应体系中每分钟消耗1μM DCPIP所需要的酶量。Definition of enzyme activity unit (U): the amount of enzyme required to consume 1 μM DCPIP in the reaction system per minute.
实施例1Example 1
原始稀土依赖型醇脱氢酶重组菌的构建。Construction of recombinant bacteria producing original rare earth-dependent alcohol dehydrogenase.
委托擎科生物工程股份有限公司合成原始稀土依赖型醇脱氢酶(氨基 酸序列如SEQ ID NO.1所示)的基因序列，序列如SEQ ID NO.2所示，并插入到载体pET28a的酶切位点BamH I和Hind III之间，获得重组质粒pET28a-PedH。采用热激法将重组质粒转化入E.coli BL21(DE3)感受态细胞中，测序验证正确后获得原始稀土依赖型醇脱氢酶重组菌。The original rare earth-dependent alcohol dehydrogenase gene sequence (amino acid sequence as shown in SEQ ID NO.1) was synthesized by Qingke Biotechnology Co., Ltd., and the sequence was shown in SEQ ID NO.2, and inserted into the restriction sites BamH I and Hind III of the vector pET28a to obtain the recombinant plasmid pET28a-PedH. The recombinant plasmid was transformed into E. coli BL21 (DE3) competent cells by heat shock method, and the original rare earth-dependent alcohol dehydrogenase recombinant bacteria were obtained after sequencing verification.
实施例2Example 2
稀土依赖型醇脱氢酶待突变位点的设计。Design of mutation sites in rare earth-dependent alcohol dehydrogenase.
使用基于深度学习的MutCompute在线设计工具(https://mutcompute.com/)对PedH的高性能突变体进行预测。具体地，登入网站注册用户后，输入PedH的PDB编号(6zcw)，点击Predict，获得一系列预测的突变体。接着使用基于能量函数的FoldX工具计算各预测突变体的结合自由能变ΔΔG。结合MutCompute预测得分以及FoldX计算的ΔΔG值，筛选得到2个潜在突变位点。The high-performance mutants of PedH were predicted using the deep learning-based MutCompute online design tool (https://mutcompute.com/). Specifically, after logging in to the website to register a user, enter the PDB number of PedH (6zcw), click Predict, and obtain a series of predicted mutants. Then, the FoldX tool based on the energy function was used to calculate the binding free energy change ΔΔG of each predicted mutant. Combining the MutCompute prediction score and the ΔΔG value calculated by FoldX, two potential mutation sites were screened.
实施例3Example 3
稀土依赖型醇脱氢酶突变体的构建。Construction of rare earth-dependent alcohol dehydrogenase mutants.
根据实施例2中所获得的突变体设计引物，对PedH进行定点突变。具体方法如下：Primers were designed based on the mutants obtained in Example 2 to perform site-directed mutagenesis on PedH. The specific method is as follows:
1、全质粒PCR1. Whole plasmid PCR
以pET28a-PedH质粒为模板，设计覆盖突变位点的上下游引物(表1)进行全质粒PCR。Using pET28a-PedH plasmid as template, upstream and downstream primers covering the mutation site were designed (Table 1) for whole plasmid PCR.
表1定点突变构建所用引物Table 1 Primers used for site-directed mutagenesis construction
| 引物名称Primer name | 引物序列(5’to 3’)Primer sequence (5’ to 3’) |
| Q207N-FQ207N-F | TAATGCATATAATCCGGAAAATGGTGAACTGCTAATGCATATAATCCGGAAAATGGTGAACTGC |
| Q207N-RQ207N-R | CCGGATTATATGCATTAATTTTACCAACAACACCAAATTCACCGGATTATATGCATTAATTTTACCAACAACACCAAATTCA |
| H486Y-FH486Y-F | GAAGTTTGGCGTTATAAAAATTATGCACCGCTGTGGGAAGTTTGGCGTTATAAAAATTATGCACCGCTGTGG |
| H486Y-RH486Y-R | TTATAACGCCAAACTTCTTTACCGCTAACCGGTTATAACGCCAAACTTCTTTACCGCTAACCGG |
PCR扩增体系：PCR amplification system:
PCR扩增条件：PCR amplification conditions:
1)预变性：98℃3min；1) Pre-denaturation: 98℃ for 3min;
2)变性：98℃10s；退火：60℃15s；延伸：72℃1min；共循环33次；2) Denaturation: 98°C for 10 s; annealing: 60°C for 15 s; extension: 72°C for 1 min; 33 cycles in total;
3)后延伸：72℃5min；3) Post-extension: 72°C for 5 min;
4)4℃保存。4) Store at 4°C.
2、转化及验证2. Conversion and verification
采用热激法将上述PCR产物直接转化入E.coli BL21(DE3)感受态细胞中，测序验证正确后获得稀土依赖型醇脱氢酶突变菌株，命名为突变菌株Q207N(编码稀土依赖型醇脱氢酶突变体的核苷酸序列如SEQ ID NO.7所示)、突变菌株H486Y(编码稀土依赖型醇脱氢酶突变体的核苷酸序列如SEQ ID NO.8所示)和突变菌株Q207N/H486Y(编码稀土依赖型醇脱氢酶突变体的核苷酸序列如SEQ ID NO.9所示)。The PCR products were directly transformed into E. coli BL21 (DE3) competent cells using the heat shock method. After sequencing verification, rare earth-dependent alcohol dehydrogenase mutant strains were obtained, named mutant strain Q207N (the nucleotide sequence encoding the rare earth-dependent alcohol dehydrogenase mutant is shown in SEQ ID NO.7), mutant strain H486Y (the nucleotide sequence encoding the rare earth-dependent alcohol dehydrogenase mutant is shown in SEQ ID NO.8) and mutant strain Q207N/H486Y (the nucleotide sequence encoding the rare earth-dependent alcohol dehydrogenase mutant is shown in SEQ ID NO.9).
实施例4Example 4
菌株培养及蛋白表达与纯化。Strain culture, protein expression and purification.
挑取实施例3中测序正确的重组菌(原始稀土依赖型醇脱氢酶重组菌和稀土依赖型醇脱氢酶突变菌株)单菌落于5mL LB液体培养基中(含50μg/mL卡那霉素)，37℃振荡培养12h。按2％的接种量转接至50mL含50μg/mL卡那霉素和10g/Lα-乳糖的LB液体培养基中，30℃振荡培养12h。The recombinant bacteria (original rare earth-dependent alcohol dehydrogenase recombinant bacteria and rare earth-dependent alcohol dehydrogenase mutant strains) sequenced correctly in Example 3 were selected and placed in 5 mL LB liquid medium (containing 50 μg/mL kanamycin) and cultured at 37°C for 12 h. The inoculum was transferred to 50 mL LB liquid medium containing 50 μg/mL kanamycin and 10 g/L α-lactose at a 2% inoculum volume and cultured at 30°C for 12 h.
培养结束后，将菌液4000×g 4℃离心15min，弃上清，收集菌体。用100mM pH 8.0的Tris-HCl缓冲液洗涤菌体后，重悬于Tris-HCl缓冲液中，置于冰水浴中进行超声破胞至澄清。对破胞液8000×g 4℃离心20min，取上清液用镍柱进行亲和层析，用洗杂缓冲液(100mM Tris-HCl缓冲液，150mM NaCl，50mM咪唑，pH 8.0)洗去杂质，用洗脱缓冲液(100mM Tris-HCl缓冲液，150mM NaCl，250mM咪唑，pH 8.0)洗脱目的蛋白，将分离出来的目标蛋白置于超滤离心管中进行充分的脱盐浓缩，得到纯的目标蛋白，SDS-PAGE检测，结果如图1所示。第1泳道(超滤液)、第2泳道(洗脱液)和第4泳道(上清)在55kDa～70kDa之间存在明显的蛋 白条带，这与PedH的理论蛋白分子量(63.0kDa)相符，表明PedH的纯化成功。After the culture is completed, the bacterial solution is centrifuged at 4000×g for 15 minutes at 4°C, the supernatant is discarded, and the bacteria are collected. After washing the bacteria with 100mM Tris-HCl buffer, pH 8.0, the bacteria are resuspended in Tris-HCl buffer and placed in an ice water bath for ultrasonic cell lysis until clarified. The cell lysis solution is centrifuged at 8000×g for 20 minutes at 4°C, and the supernatant is subjected to affinity chromatography on a nickel column. The impurities are washed with a washing buffer (100mM Tris-HCl buffer, 150mM NaCl, 50mM imidazole, pH 8.0), and the target protein is eluted with an elution buffer (100mM Tris-HCl buffer, 150mM NaCl, 250mM imidazole, pH 8.0). The separated target protein is placed in an ultrafiltration centrifuge tube for sufficient desalting and concentration to obtain a pure target protein. SDS-PAGE detection is shown in Figure 1. There are obvious protein bands between 55kDa and 70kDa in lane 1 (ultrafiltrate), lane 2 (eluate) and lane 4 (supernatant), which is consistent with the theoretical protein molecular weight of PedH (63.0kDa), indicating that PedH was successfully purified.
实施例5Example 5
稀土依赖型醇脱氢酶的酶活测定。Enzyme activity assay of rare earth-dependent alcohol dehydrogenase.
用酶标仪定量分析反应体系中消耗的DCPIP的量，通过计算得到原始稀土依赖型醇脱氢酶和稀土依赖型醇脱氢酶突变体对底物甲醇、乙醇和5-羟甲基糠醛的比酶活。反应体系包含适量的酶液、1.5μM PQQ、1.5μM PrCl
3、15mM底物，总体系为200μL。反应介质为Tris-HCl缓冲液(100mM，pH 8.0)，其中含有1mM PES和150μM DCPIP。比酶活测定结果见图2，突变体Q207N、H486Y和组合突变体Q207N/H486Y对三种底物的比酶活相比原始酶PedH明显提升。原始酶PedH对乙醇、甲醇和5-羟甲基糠醛的比酶活分别是0.433U/mg、0.088U/mg、0.053U/mg。而突变体Q207N、H486Y和组合突变体Q207N/H486Y对乙醇的比酶活分别是原始酶PedH的2.4、2.6、3.6倍，对甲醇的比酶活分别是原始酶PedH的2.3、2.8、3.6倍，对5-羟甲基糠醛的比酶活分别是原始酶PedH的2.3、2.6、2.8倍。
The amount of DCPIP consumed in the reaction system was quantitatively analyzed by an ELISA instrument, and the specific enzyme activities of the original rare earth-dependent alcohol dehydrogenase and the rare earth-dependent alcohol dehydrogenase mutants on the substrates methanol, ethanol and 5-hydroxymethylfurfural were calculated. The reaction system contained an appropriate amount of enzyme solution, 1.5μM PQQ, 1.5μM PrCl 3 , 15mM substrate, and the total system was 200μL. The reaction medium was Tris-HCl buffer (100mM, pH 8.0), which contained 1mM PES and 150μM DCPIP. The results of the specific enzyme activity determination are shown in Figure 2. The specific enzyme activities of the mutants Q207N, H486Y and the combined mutant Q207N/H486Y on the three substrates were significantly improved compared with the original enzyme PedH. The specific enzyme activities of the original enzyme PedH on ethanol, methanol and 5-hydroxymethylfurfural were 0.433U/mg, 0.088U/mg and 0.053U/mg, respectively. The specific enzyme activities of mutants Q207N, H486Y and combined mutant Q207N/H486Y towards ethanol were 2.4, 2.6 and 3.6 times that of the original enzyme PedH, respectively; the specific enzyme activities towards methanol were 2.3, 2.8 and 3.6 times that of the original enzyme PedH, respectively; and the specific enzyme activities towards 5-hydroxymethylfurfural were 2.3, 2.6 and 2.8 times that of the original enzyme PedH, respectively.
实施例6Example 6
稀土依赖型醇脱氢酶的T
m值测定。
Determination of T m value of rare earth-dependent alcohol dehydrogenase.
将原始稀土依赖型醇脱氢酶和稀土依赖型醇脱氢酶突变体的酶液浓度统一调整至10μM，并添加至高精度石英玻璃毛细管组中，使用蛋白质稳定性分析仪(Prometheus NT.Plex)测定T
m值，测定结果见图3，可以看到，突变体Q207N与野生型PedH的溶解温度T
m值没有明显区别，但突变体H486Y和组合突变体Q207N/H486Y的稳定性有所提高，溶解温度T
m值相比野生型PedH(63.7℃)分别提高2.3℃、2.2℃。
The enzyme concentrations of the original rare earth-dependent alcohol dehydrogenase and the rare earth-dependent alcohol dehydrogenase mutants were uniformly adjusted to 10 μM and added to a high-precision quartz glass capillary set. The T m value was determined using a protein stability analyzer (Prometheus NT.Plex). The measurement results are shown in Figure 3. It can be seen that there is no obvious difference in the dissolution temperature T m value between the mutant Q207N and the wild-type PedH, but the stability of the mutant H486Y and the combined mutant Q207N/H486Y is improved, and the dissolution temperature T m values are increased by 2.3°C and 2.2°C respectively compared with the wild-type PedH (63.7°C).
Claims (9)
- 一种稀土依赖型醇脱氢酶突变体，其特征在于，是由来自恶臭假单胞菌(Pseudomonas putida)的野生型醇脱氢酶进行突变而得，所述野生型醇脱氢酶的氨基酸序列如SEQ ID NO.1所示，具体突变为(1)和/或(2)：A rare earth-dependent alcohol dehydrogenase mutant, characterized in that it is obtained by mutating a wild-type alcohol dehydrogenase from Pseudomonas putida, wherein the amino acid sequence of the wild-type alcohol dehydrogenase is shown in SEQ ID NO.1, and the specific mutations are (1) and/or (2):(1)第207位的氨基酸由谷氨酰胺突变为天冬酰胺；(1) The amino acid at position 207 was mutated from glutamine to asparagine;(2)第486位的氨基酸由组氨酸突变为酪氨酸。(2) The amino acid at position 486 mutated from histidine to tyrosine.
- 如权利要求1所述的稀土依赖型醇脱氢酶突变体在催化甲醇生成甲醛、催化乙醇生成乙醛、或者催化5-羟甲基糠醛生成5-羟甲基-2-呋喃甲酸中的应用。Use of the rare earth-dependent alcohol dehydrogenase mutant as claimed in claim 1 in catalyzing methanol to formaldehyde, catalyzing ethanol to form acetaldehyde, or catalyzing 5-hydroxymethylfurfural to form 5-hydroxymethyl-2-furancarboxylic acid.
- 编码权利要求1所述稀土依赖型醇脱氢酶突变体的基因。A gene encoding the rare earth-dependent alcohol dehydrogenase mutant according to claim 1.
- 如权利要求3所述的基因，其特征在于，所述基因的核苷酸序列如SEQ ID NO.7、SEQ ID NO.8或SEQ ID NO.9所示。The gene as described in claim 3 is characterized in that the nucleotide sequence of the gene is shown as SEQ ID NO.7, SEQ ID NO.8 or SEQ ID NO.9.
- 如权利要求3所述的基因在催化甲醇生成甲醛、催化乙醇生成乙醛、或者催化5-羟甲基糠醛生成5-羟甲基-2-呋喃甲酸中的应用。Use of the gene as claimed in claim 3 in catalyzing methanol to produce formaldehyde, catalyzing ethanol to produce acetaldehyde, or catalyzing 5-hydroxymethylfurfural to produce 5-hydroxymethyl-2-furancarboxylic acid.
- 一种包含权利要求3所述基因的表达载体。An expression vector comprising the gene according to claim 3.
- 一种表达权利要求1所述稀土依赖型醇脱氢酶突变体的基因工程菌。A genetically engineered bacterium expressing the rare earth-dependent alcohol dehydrogenase mutant of claim 1.
- 如权利要求7所述的基因工程菌在催化甲醇生成甲醛、催化乙醇生成乙醛、或者催化5-羟甲基糠醛生成5-羟甲基-2-呋喃甲酸中的应用。Use of the genetically engineered bacteria as claimed in claim 7 in catalyzing methanol to produce formaldehyde, catalyzing ethanol to produce acetaldehyde, or catalyzing 5-hydroxymethylfurfural to produce 5-hydroxymethyl-2-furancarboxylic acid.
- 一种甲醛、乙醛、或者5-羟甲基-2-呋喃甲酸的制备方法，其特征在于，使用权利要求1所述稀土依赖型醇脱氢酶突变体催化甲醇、乙醇或者5-羟甲基糠醛发生氧化反应，制备得到甲醛、乙醛、或者5-羟甲基-2-呋喃甲酸。A method for preparing formaldehyde, acetaldehyde, or 5-hydroxymethyl-2-furancarboxylic acid, characterized in that the rare earth-dependent alcohol dehydrogenase mutant according to claim 1 is used to catalyze the oxidation reaction of methanol, ethanol or 5-hydroxymethylfurfural to prepare formaldehyde, acetaldehyde, or 5-hydroxymethyl-2-furancarboxylic acid.
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| US20140377817A1 (en) * | 2013-06-25 | 2014-12-25 | Ut-Battelle, Llc | Heat stable, fe dependent alcohol dehydrogenase for aldehyde detoxification |
| CN106211783A (en) * | 2013-10-04 | 2016-12-07 | 基因组股份公司 | Alcoholdehydrogenase variant |
| EP3222712A1 (en) * | 2016-03-22 | 2017-09-27 | Universität zu Köln | Alcohol dehydrogenase from pichia pastoris and use thereof |
| CN109402076A (en) * | 2018-10-30 | 2019-03-01 | 江南大学 | A kind of Alcohol dehydrogenase mutant and its application in regenerating coenzyme |
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| US20140377817A1 (en) * | 2013-06-25 | 2014-12-25 | Ut-Battelle, Llc | Heat stable, fe dependent alcohol dehydrogenase for aldehyde detoxification |
| CN106211783A (en) * | 2013-10-04 | 2016-12-07 | 基因组股份公司 | Alcoholdehydrogenase variant |
| EP3222712A1 (en) * | 2016-03-22 | 2017-09-27 | Universität zu Köln | Alcohol dehydrogenase from pichia pastoris and use thereof |
| CN109402076A (en) * | 2018-10-30 | 2019-03-01 | 江南大学 | A kind of Alcohol dehydrogenase mutant and its application in regenerating coenzyme |
Non-Patent Citations (3)
| Title |
|---|
| CHEN FANG; XU GANG; YANG LI-RONG; WU JIAN-PING: "Enhancing the Activity of LkTADH by Site-Directed Mutagenesis to Prepare Key Chiral Block of Statins", ZHONGGUO SHENGWU GONGCHENG ZAZHI - CHINA BIOTECHNOLOGY, ZHONGGUO KEXUEYUAN WENXIAN QINGBAO ZHONGXIN,CHINESE ACADEMY OF SCIENCES, DOCUMENTATION AND INFORMATION CENTER, CN, vol. 38, no. 9, 15 September 2018 (2018-09-15), CN, pages 59 - 64, XP009554571, ISSN: 1671-8135, DOI: 10.13523/j.cb.20180909 * |
| DATABASE PROTEIN 1 June 2021 (2021-06-01), ANONYMOUS: "MULTISPECIES: PQQ-dependent alcohol dehydrogenase PedH [Pseudomonas]", XP093169118, Database accession no. WP_019436699.1 * |
| MATTHIAS WEHRMANN: "Functional Role of Lanthanides in Enzymatic Activity and Transcriptional Regulation of Pyrroloquinoline Quinone-Dependent Alcohol Dehydrogenases inPseudomonas putidaKT2440", MBIO, AMERICAN SOCIETY FOR MICROBIOLOGY, US, vol. 8, no. 3, 27 June 2017 (2017-06-27), US , pages e00570, XP093169125, ISSN: 2161-2129, DOI: 10.1128/mbio.00570-17 * |
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