WO2013002277A1 - 酵素機能改変方法及びその変異体 - Google Patents
酵素機能改変方法及びその変異体 Download PDFInfo
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
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- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
- C07H19/16—Purine radicals
- C07H19/20—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
- C07H19/207—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids the phosphoric or polyphosphoric acids being esterified by a further hydroxylic compound, e.g. flavine adenine dinucleotide or nicotinamide-adenine dinucleotide
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- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
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- C12P19/36—Dinucleotides, e.g. nicotineamide-adenine dinucleotide phosphate
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Definitions
- the present invention relates to a coenzyme-dependent modification method for alcohol dehydrogenase, and more particularly to a coenzyme-dependent modification method for medium-chain dehydrogenase / reductase (MDR).
- MDR medium-chain dehydrogenase / reductase
- Patent Document 1 Non-Patent Document 1
- Alcohol dehydrogenase can also be said to be a typical enzyme family in which mutants in which coenzyme dependency is converted have been obtained by this logical design.
- SDR short-chain dehydrogenase / reductase
- MDR medium-chain dehydrogenase / reductase
- the object is to optimize the dependency of alcohol dehydrogenase on either NADPH or NADH and to improve the productivity of optically active alcohols when a coenzyme regeneration system is conjugated.
- the present inventors have newly developed an enzyme modification method that converts the coenzyme dependency of enzymes belonging to the MDR (medium chain dehydrogenase / reductase) family.
- MDR medium chain dehydrogenase / reductase
- the position where mutations were introduced from 350 candidates was narrowed down to 4 positions, and the amino acid after mutation was successfully determined from 19 kinds of natural amino acids.
- NADPH / NADP + -dependent enzyme variants were obtained from NADH / NAD + -dependent MDR (medium-chain dehydrogenase / reductase) family enzymes, and NADPH / NADP + -dependent MDR (medium-chain dehydrogenase / reductase) We succeeded in obtaining NADH / NAD + dependent enzyme mutants from family enzymes.
- the present invention [1] It belongs to the medium chain oxidoreductase family, and the following (a) to (d): (A) the amino acid residue at a position that is stereologically equivalent to Asp-201 of SEQ ID NO: 1 is Ala or Ser; (B) the amino acid residue at a position that is three-dimensionally equivalent to Lys-202 of SEQ ID NO: 1 is Arg; (C) the amino acid residue at a position equivalent to the three-dimensional structure of Lys-203 of SEQ ID NO: 1 is Ser; and (D) the amino acid residue at a position equivalent to the three-dimensional structure of Ala-206 of SEQ ID NO: 1 is Lys; A protein having at least one amino acid residue selected from [2] The protein according to [1], comprising an amino acid sequence having 85% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, [3] In the amino acid sequence shown in SEQ ID NO: 1, the following (e) to (h): (E) Replacing Asp-
- GDH derived from lactic acid bacteria having excellent physical properties has been discovered as an NADP + reductase (International Publication No. 09/041415).
- MDR family enzyme RMA derived from Candida maltosa is NADH-dependent in the wild type, it is difficult to use GDH derived from lactic acid bacteria that is NADP + reductase as a coenzyme regenerating enzyme.
- the protein of the present invention obtained by coenzyme-dependent conversion, NADP + reductase GDH derived from lactic acid bacteria can be used as a coenzyme regenerating enzyme. Very large.
- the coenzyme dependency can be changed in the case of a multi-step reaction in which a reaction using an NADPH / NADP + -dependent enzyme and a reaction using an NADH / NAD + -dependent enzyme are performed in one reaction solution. Therefore, the merit of this technology on reaction process optimization is great.
- Example 4 of the present invention It is a reaction schematic diagram concerning Example 4 of the present invention. It is the graph which showed the analysis result of the conversion rate and optical purity of optically active alcohol in reaction which concerns on Example 4 of this invention.
- protein includes any molecule having a polypeptide structure, and a polypeptide chain that is fragmented or linked by peptide bonds is also referred to as the term “protein”. Is included.
- the protein of the present invention can be obtained by introducing an amino acid substitution mutation into a protein belonging to the medium chain dehydrogenase / reductase family.
- the medium-chain dehydrogenase / reductase (MDR) family is one of the families belonging to the alcohol dehydrogenase (ADH, EC 1.1.1.1.), 350 to 375 residues. It consists of a group and most contains zinc (Zn).
- the medium-chain dehydrogenase / reductase family is registered, classified, and defined in various bioinformatics databases.
- the enzymes associated with the family ID “PF00107” of Pfam all belong to the medium chain dehydrogenase / reductase family, and as of April 2011, 20 More than 1,000 sequences are registered. Enzymes belonging to the medium chain dehydrogenase / reductase family are involved in a wide range of physiological functions such as alcohol fermentation, aldehyde detoxification, lignin biosynthesis, fatty acid biosynthesis, or protection from oxidative damage.
- Proteins belonging to the medium chain dehydrogenase / reductase family require pyridine nucleotides as coenzymes.
- Pyridine nucleotide means ⁇ -nicotinamide adenine dinucleotide phosphate (reduced form is NADPH, oxidized form is shown as NADP +) or ⁇ -nicotinamide adenine dinucleotide (reduced form is shown as NADH, oxidized form is shown as NAD +) Refers to that.
- sequence homology can be determined by amino acid sequence homology analysis using the aforementioned program BLAST. When evaluating homology in the BLAST analysis, it is preferable to use a statistical value called E-value. The higher the homology is, the closer this value is to 0. To determine whether the enzyme is an MDR family enzyme from sequence homology, the E-value is preferably 1 ⁇ 10 ⁇ 5 or less with respect to a known MDR family enzyme, and is 1 ⁇ 10 ⁇ 10 or less. More preferably, it is 1 ⁇ 10 ⁇ 15 or less. Software for performing BLAST analyzes is available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).
- the medium chain dehydrogenase / reductase family enzyme to be introduced with amino acid substitution mutation is preferably an enzyme listed in the protein sequence database UniProtKB (http://www.uniprot.org/).
- Examples of NADH / NAD + -dependent medium chain dehydrogenase / reductase family enzymes to which NADPH / NADP + -dependent conversion technology can be applied include yeast-derived ADH1 (P00330), Bacillus-derived ADH-HT (P42328), and Human-derived ADH1 ⁇ (P00325) and the like can be mentioned.
- NADPH / NADP + -dependent MDR family enzymes to which technology for conversion to NADH / NAD + dependency can be applied include, for example, yeast-derived ADH6 (Q04894), American porcupine-derived synapyl ADH (Q94G59), and human-derived PIG3 ( Q53FA7) and the like. Note that the code numbers in the database are in parentheses.
- the protein before mutagenesis is an amino acid sequence having high sequence identity with an enzyme (International Publication No. 08/0666018, hereinafter abbreviated as RMA) contained in the MDR family derived from Candida maltosa.
- RMA International Publication No. 08/0666018
- a protein consisting of “Sequence identity” is the ratio of the number of amino acid residues that are completely matched in the homologous region, and can be determined by the above-mentioned BLAST analysis, and is represented by “Identities”.
- the sequence identity of the protein before mutagenesis with respect to the amino acid sequence of SEQ ID NO: 1 is preferably 85% or more, more preferably 90% or more, and further preferably 95% or more.
- the protein before mutagenesis is most preferably a protein consisting of the amino acid sequence of SEQ ID NO: 1, and this protein is an MDR family enzyme derived from Candida maltosa (International Publication No. 08/066018, the following) (Abbreviated as RMA).
- RMA catalyzes a reaction in which NADH is used as a coenzyme to reduce a ketone to produce an optically active alcohol.
- the protein of the present invention is designed by introducing an amino acid substitution mutation into a protein consisting of the amino acid sequence of SEQ ID NO: 1 (RMA), the coenzyme dependency can be changed from NADH dependency to NADPH dependency. .
- the present invention has been achieved by utilizing the modeling conformation of this RMA (SEQ ID NO: 1).
- An amino acid residue at an equivalent position in the three-dimensional structure means that the three-dimensional structure of the protein before mutagenesis is predicted based on the amino acid sequence information and compared with the three-dimensional structure of the protein consisting of the amino acid sequence of SEQ ID NO: 1.
- the amino acid residue which exists in the position equivalent in the protein which consists of an amino acid sequence of sequence number 1 is shown.
- the program ClustalX (Thompson, JD et al., “Nucleic Acid Res.” 1994, Vol. 22, pp. 4673-80) has amino acid sequence homology and three-dimensional structure is a protein. A multiple amino acid sequence alignment with an enzyme registered in a data bank (PDB) is created.
- a protein having amino acid sequence homology with RMA is a program BLAST (Altschul, SF. Et al., “Nucleic Acid Res.” 1997, 25, 3389-3402, for the amino acid sequence of a protein registered in PDB. Page), or PSI-BLAST (Shafer AA et al., “Bioinformatics”, 2000, 164, pages 88-489), and can be selected by searching for amino acid sequence homology.
- three-dimensional conformational alignments of proteins with known three-dimensional structures are obtained by using three-dimensional graphics programs such as Swiss-PDBViewer (Guex N. et al., “Electrophoresis”, 1997, Vol. 18, 2714-2723) , VAST Search (Gibrat J.F. et al., “Curr. Opin. Struct. Biol.” 1996, Vol. 6, 377-).
- a protein three-dimensional structure (PDB code) that is presumed to have a high degree of three-dimensional structure similarity based on the three-dimensional structure similarity previously corrected based on the three-dimensional structure similarity. : 1 LLU) is selected as a template protein for molecular modeling.
- This template protein is displayed by the program Swiss PDB-Viewer, and amino acid residues are substituted in accordance with the amino acid sequence of RMA (SEQ ID NO: 1) according to the sequence alignment.
- SEQ ID NO: 1 amino acid sequence of RMA (SEQ ID NO: 1) according to the sequence alignment.
- an optimal similar partial structure is searched from the PDB, and the three-dimensional structure model is constructed by substituting the partial structure.
- the coenzyme is bound to the state where the coenzyme is not bound (apo) by the molecular simulation calculation (energy minimization calculation) using the molecular force field method. It is possible to calculate a free energy difference value in the state of being in a state (holo).
- the details of this logic can be said to be an application of a method for designing a drug that binds to a target protein.
- LBDD ligand-based mutations in which this difference in free energy has a particularly strong tendency to be advantageous for apo in the case of NADH and in the case of holo in the case of NADPH compared to the wild type. It can be said that it is useful to change the dependency. Specifically, amino acid mutation candidates are narrowed down by computer screening using the program Shrike (Japanese Patent Publication No. 2001-184281).
- MDR medium chain dehydrogenase / reductase
- MDR medium chain dehydrogenase / reductase
- the equivalent position in the three-dimensional structure is obtained by using the three-dimensional structure comparison / similar structure search server such as VAST Search described above, and the three-dimensional structure known amino acid sequence (for example, the PDB code used in the present invention: the amino acid sequence of 1LLU). It can be easily identified by aligning amino acid sequences based on the three-dimensional structure.
- VAST Search is also available through the National Center for Biotechnology Information.
- the protein of the present invention belongs to the medium-chain dehydrogenase / reductase family, and the following (a) to (d): (A) the amino acid residue at a position that is stereologically equivalent to Asp-201 of SEQ ID NO: 1 is Ala or Ser; (B) the amino acid residue at a position that is three-dimensionally equivalent to Lys-202 of SEQ ID NO: 1 is Arg; (C) the amino acid residue at a position equivalent to the three-dimensional structure of Lys-203 of SEQ ID NO: 1 is Ser; and (D) the amino acid residue at a position equivalent to the three-dimensional structure of Ala-206 of SEQ ID NO: 1 is Lys; It preferably has at least one amino acid residue selected from
- the medium chain dehydrogenase / reductase family belongs to the medium chain dehydrogenase / reductase family, and the following (e) to (g): (E) the amino acid residue at a position equivalent to the three-dimensional structure of Asp-201 of SEQ ID NO: 1 is Ser; (F) the amino acid residue at a position that is stereologically equivalent to Lys-202 of SEQ ID NO: 1 is Arg; and (G) the amino acid residue at a position equivalent to the three-dimensional structure of Ala-206 of SEQ ID NO: 1 is Lys; It is more preferable that all amino acid residues are included.
- the alcohol dehydrogenase whose coenzyme dependency is NADH (or NAD +) dependency is changed to NADPH (or NADP +) dependency. It is possible to convert.
- sequence identity of the protein after mutagenesis with respect to the amino acid sequence of SEQ ID NO: 1 is preferably 85% or more, more preferably 90% or more, and further preferably 95% or more.
- the protein after mutagenesis preferably has the amino acid sequences shown in SEQ ID NOs: 2 to 8, and more preferably has the amino acid sequences shown in SEQ ID NOs: 2 to 3.
- the protein of the present invention has alcohol dehydrogenation activity, and the following (i) to (l): (I) the amino acid residue at a position equivalent to the three-dimensional structure of Asp-201 of SEQ ID NO: 1 is Asp. (J) The amino acid residue at a position equivalent to the three-dimensional structure of Lys-202 of SEQ ID NO: 1 is Lys. (K) the amino acid residue at a position equivalent to the three-dimensional structure of Lys-203 of SEQ ID NO: 1 is Lys; and (L) the amino acid residue at a position equivalent to the three-dimensional structure of Ala-206 of SEQ ID NO: 1 is Ala; It preferably has at least one amino acid residue selected from
- alcohol dehydrogenase whose coenzyme dependency is NADPH (or NADP +) dependency can be converted to NADH (or NAD +) dependency.
- the protein of the present invention preferably has a reduced oxidation / reduction activity with respect to a coenzyme that has exhibited high oxidation (or reduction, hereinafter referred to as “oxidation / reduction”) activity before mutagenesis.
- oxidation / reduction oxidation / reduction activity with respect to a coenzyme that showed low oxidation / reduction activity (or not shown at all) before mutagenesis.
- the protein of the present invention more preferably has both of these characteristics.
- the coenzyme oxidation / reduction activity ratio is the value obtained by dividing the oxidation / reduction activity of a coenzyme that showed dependency after mutation introduction by the oxidation / reduction activity of the other coenzyme that showed dependency before mutation introduction. I mean.
- the coenzyme oxidation / reduction activity ratio is represented by the oxidation activity ratio (NADPH / NADH).
- the coenzyme oxidation / reduction activity ratio is preferably 1 or more, more preferably 5 or more, and even more preferably 10 or more.
- the coenzyme oxidation / reduction activity ratio before mutation introduction is about 0.1 at the highest, and if it is larger than this, it is an enzyme that is dependent on both.
- the coenzyme oxidation / reduction activity is calculated by defining the enzyme activity that oxidizes 1 ⁇ mol of NADPH (or NADH) to NADP + (NAD +) per minute as 1 Unit.
- the reaction solution composition and enzyme concentration for calculating the coenzyme oxidation / reduction activity are the same before and after the mutation introduction.
- the measurement conditions for the coenzyme oxidation / reduction activity are preferably conditions for aeration and stirring for a certain period of time at a constant temperature of pH 4.0 to 10.0 and a temperature of 4 ° C. to 80 ° C. It may be set in consideration of the physical properties of the coenzyme regenerating enzyme to be combined, but is not limited thereto.
- the protein of the present invention maintains its original function, such as substrate specificity, except for the feature that coenzyme dependence is converted.
- the DNA of the present invention consists of a base sequence encoding the protein of the present invention. Any one can be used as long as it can express the protein of the present invention in the introduced host cell according to the method described later, and any untranslated region may be included. If the protein can be designed, the DNA of the present invention can be obtained by introducing the mutation from the organism that is the origin of the protein before the mutation is introduced by a method known to those skilled in the art and introducing the mutation. it can.
- Introduction of site-specific mutations into DNA encoding a wild-type MDR family enzyme can be performed using recombinant DNA techniques, PCR methods and the like. Introducing mutations by recombinant DNA technology, if there are appropriate restriction enzyme recognition sequences on both sides of the target site where mutations are desired in the wild-type enzyme gene, cleave them with the restriction enzymes and introduce mutations. Can be performed by a cassette mutation method in which a DNA fragment mutated only to the target site by chemical synthesis or the like is inserted after removing the region containing the desired site.
- the introduction of site-specific mutation by PCR has a mutation that includes a mutation primer in which the target mutation is introduced into the target site where the mutation is to be introduced in the wild-type coding gene and the sequence of one end site of the gene.
- Amplification primer that amplifies one side of the gene with a non-amplification primer and has a mutation that includes a sequence complementary to the mutation primer and a sequence at the other end of the gene Then, the other side is amplified, and the obtained two amplified fragments are annealed, and further PCR is performed with the two kinds of amplification primers.
- DNA encoding a mutation-introduced amino acid sequence may be obtained by chemical synthesis.
- Examples of the DNA encoding the protein of the present invention obtained in this manner include DNA having the base sequence set forth in any of SEQ ID NOs: 25 to 31.
- a protein that hybridizes under stringent conditions with a DNA containing a base sequence complementary to the base sequence shown in any of SEQ ID NOs: 25 to 31 and has oxidoreductase activity examples thereof include DNA consisting of a coding base sequence.
- DNA of the present invention a DNA comprising a base sequence encoding a protein having 85% or more sequence identity with any of the base sequences set forth in SEQ ID NOs: 25 to 31 and having oxidoreductase activity Is mentioned.
- DNA comprising a sequence refers to a colony hybridization method or plaque hybridization under stringent conditions using a DNA comprising a base sequence complementary to any one of SEQ ID NOs: 25 to 31 as a probe. DNA which is obtained by using a method or Southern hybridization method and which encodes a protein having oxidoreductase activity.
- DNA that hybridizes under stringent conditions means, for example, using a filter on which colony or plaque-derived DNA is immobilized at 65 ° C. in the presence of 0.7 to 1.0 M NaCl. After hybridization, the filter was washed under conditions of 65 ° C. using a 2 ⁇ concentration SSC solution (composition of 1 ⁇ concentration SSC solution consisting of 150 mM sodium chloride and 15 mM sodium citrate). The DNA which can be acquired can be mentioned.
- hybridization conditions have been described as described above, the conditions are not particularly limited.
- a plurality of factors such as temperature and salt concentration can be considered as factors affecting the stringency of hybridization, and those skilled in the art can realize optimum stringency by appropriately selecting these factors.
- the DNA capable of hybridizing under the above conditions is a DNA having a sequence identity of 85% or more, preferably 90% or more, more preferably 95% or more with the base sequence set forth in any of SEQ ID NOs: 25 to 31. As long as the encoded polypeptide has oxidoreductase activity, it is included in the DNA.
- sequence identity means that the two DNAs to be compared are optimally aligned, and the nucleobases (eg, A, T, C, G, U, or I) match in both sequences.
- the number of positions is divided by the total number of comparison bases, and the result is expressed by a value obtained by multiplying by 100.
- the vector of the present invention can be obtained by inserting the DNA of the present invention into an appropriate vector.
- the empty vector for inserting DNA is not particularly limited as long as it can autonomously replicate in the host cell, and plasmid DNA or phage DNA can be used as the vector.
- plasmid DNA such as pBR322, pUC18, pBluescript II, or phage DNA such as EMBL3, M13, ⁇ gt11, or the like can be used.
- yeast is used as a host cell
- YEp13, YCp50, etc. can be used.
- plant cells are used as host cells
- pBI121, pBI101 and the like can be used.
- animal cells are used as host cells, pcDNAI and the like can be used as vectors.
- the transformant of the present invention can be obtained by transforming host cells using the above vector.
- the host organism is not particularly limited as long as it is an organism that is transformed with an expression vector containing the coding DNA and can express the protein encoded by the introduced DNA.
- Examples of usable microorganisms include, for example, the genus Escherichia, the genus Bacillus, the genus Pseudomonas, the genus Serratia, the genus Brevibacterium, the genus Corynebacterium, Streptococcus genus, bacteria that have developed host vector systems such as Lactobacillus genus, Rhodococcus genus and Streptomyces genus actinomycetes that have been developed for host vector systems such as Saccharomyces (Saccharomyces), Kluyveromyces ) Genus, Schizosaccharomyces genus, Zygosaccharomyces genus, Yarrowia genus
- a method for transformation when introducing recombinant DNA into bacteria, for example, a method using calcium ions, an electroporation method and the like can be mentioned.
- the method for introducing the recombinant DNA into yeast include an electroporation method, a spheroplast method, and a lithium acetate method.
- methods for introducing recombinant DNA into plant cells include the Agrobacterium infection method, the particle gun method, and the polyethylene glycol method.
- methods for introducing recombinant DNA into animal cells include the electroporation method and the calcium phosphate method.
- the culture of the present invention can be obtained by culturing the transformant in a medium.
- a culture containing the protein of the present invention can be obtained by culturing the transformant in a medium, producing and accumulating the protein in the cultured microbial cells or the culture supernatant, and collecting the enzyme mutant.
- the transformant is cultured according to a usual method used for host culture.
- Examples of the medium for culturing transformed cells obtained using bacteria such as Escherichia coli as a host include complete medium or synthetic medium such as LB medium, TB medium, M9 medium and the like.
- the enzyme variant of the present invention is accumulated in the cells and collected by aerobic culture at a culture temperature of 20 to 40 ° C.
- the enzyme mutant is purified by centrifuging the culture obtained by the above-described culture method, and crushing the cells with a sonicator or the like, affinity chromatography, cation or anion exchange chromatography, gel filtration, etc. Can be carried out alone or in appropriate combination.
- the obtained purified substance is the target enzyme can be performed by an ordinary method such as SDS polyacrylamide gel electrophoresis, Western blotting or the like.
- the purification treatment of the culture of the transformant is to remove impurities other than the target enzyme without losing the enzyme activity, and the purified treatment product is an enzyme-containing product obtained by the above treatment.
- the purified product include a cell-free extract obtained by disrupting cells, an enzyme solution obtained by purification, or a freeze-dried product of the enzyme solution.
- the protein obtained by introducing at least one amino acid substitution mutation selected from the above (a) to (d) into a protein having alcohol dehydrogenase activity is more dependent on NADPH than the protein before substitution mutation introduction. And the dependence on NADH is reduced. Therefore, oxidized nicotinamide adenine dinucleotide phosphate (NADP +) can be obtained by converting reduced nicotinamide adenine dinucleotide phosphate (NADPH) using this protein.
- NADP + oxidized nicotinamide adenine dinucleotide phosphate
- NADPH reduced nicotinamide adenine dinucleotide phosphate
- the protein obtained by introducing at least one amino acid substitution mutation selected from the above (i) to (l) into a protein having alcohol dehydrogenase activity is more dependent on NADH than the protein before substitution mutation introduction. And the dependence on NADPH is reduced. Therefore, oxidized nicotinamide adenine dinucleotide (NAD +) can be obtained by converting reduced nicotinamide adenine dinucleotide (NADH) using the protein of the present invention.
- NADH reduced nicotinamide adenine dinucleotide
- NAD + oxidized nicotinamide adenine dinucleotide
- Reduced nicotinamide adenine is produced by allowing a coenzyme regeneration system to act on oxidized nicotinamide adenine dinucleotide phosphate (NADP +) or oxidized nicotinamide adenine dinucleotide (NAD +) produced using the protein of the present invention.
- Dinucleotide phosphate (NADPH) or reduced nicotinamide adenine dinucleotide (NADH) can be produced.
- Such a coenzyme regeneration system is an enzyme having an activity to reduce oxidized coenzyme. Specific examples thereof include glucose dehydrogenase (GDH), formate dehydrogenase (FDH), and lactate dehydrogenase (LDH). And malate dehydrogenase (MDH).
- oxidized nicotine is produced by allowing a coenzyme regeneration system to act on reduced nicotinamide adenine dinucleotide phosphate (NADPH) or reduced nicotinamide adenine dinucleotide (NADH) produced using the protein of the present invention.
- Amide adenine dinucleotide phosphate (NADP +) or oxidized nicotinamide adenine dinucleotide (NAD +) can be produced.
- Such a coenzyme regeneration system is an enzyme having an activity to oxidize a reduced coenzyme. Specific examples thereof include glucose dehydrogenase (GDH), formate dehydrogenase (FDH), and lactate dehydrogenase (LDH). And malate dehydrogenase (MDH).
- NADPH reduced nicotinamide adenine dinucleotide phosphate
- NADH reduced nicotinamide adenine dinucleotide
- NADP + oxidized nicotinamide adenine dinucleotide phosphate
- NADPH oxidized nicotinamide adenine dinucleotide NAD +
- the amino acid of a wild type or a non-mutated type is attached
- the mutated amino acid is attached
- D201A a mutation that replaces Asp at position 201 with Ala.
- Example 1 Production of Recombinant Vector Containing RMA Mutant Gene and Production of Recombinant Escherichia coli MDR (Medium Chain Dehydrogenase / Reductase) Family Enzyme from Candida maltosa (International Publication)
- RMA Random Access to E. coli
- expression plasmids of various mutants were prepared using the pNCM vector (RMA wild type expression plasmid) described in the same document. .
- recombinant plasmids containing various RMA mutant genes were obtained by a quick change method using two kinds of synthetic primers designed so that mutations can be introduced at the intended site using the pNCM vector as a template.
- the quick change method was performed using QuickChange Site-Directed Mutagenesis Kit (manufactured by Stratagene) according to the attached protocol.
- an RMA mutant expression plasmid into which mutation A206K was introduced by carrying out a quick change method using pNCM (SEQ ID NO: 9) as a template DNA and the two synthetic primers used in SEQ ID NOs: 10 to 11 was prepared.
- the coding DNA sequence of the resulting expression plasmid is shown in SEQ ID NO: 12.
- the quick change method is a mutagenesis method including transformation in the protocol.
- Escherichia coli HB101 manufactured by Takara
- the expression plasmid containing the coding DNA of SEQ ID NO: 9 (wild type) or SEQ ID NO: 12 (mutant K206R) was used as a template.
- SEQ ID NO: 9 wild type
- SEQ ID NO: 12 mutant K206R
- Each of the expression plasmids encoding the amino acid sequences of SEQ ID NOs: 2 to 8 and transformed recombinant Escherichia coli was prepared (the coding DNA sequence of each expression plasmid is SEQ ID NO: 25).
- Table 1 shows the correspondence between amino acid / coding DNA sequences of various mutant expression plasmids and template DNA plasmid / mutagenesis primer sequence numbers used in the preparation in this experiment.
- Example 2 Expression of RMA mutant by recombinant Escherichia coli / Preparation of cell-free extract
- Each recombinant Escherichia coli HB101 obtained in Example 1 was mixed with semi-synthetic medium (glycerin 1.5% (w / v), Yeast extract 0.3% (w / v), Na 2 HPO 4 0.6% (w / v), KH 2 HPO 4 0.3% (w / v), NaCl 0.2% (w / v) , MgSO 4 ⁇ 7H 2 O 0.5% (w / v), 100 ⁇ g / ml ampicillin, pH 7.2), and cultured at 30 ° C. for 60 hours. After removing the supernatant, the suspension is suspended in a buffer solution (100 mM potassium phosphate, pH 7.0) equivalent to the medium, disrupted by ultrasonic disruption, and the supernatant is recovered again by centrifugation.
- a buffer solution 100 mM potassium phosphat
- Example 3 Oxidation activity measurement of pyridine nucleotides To the following solution to which a ketone compound was added as a reaction substrate, various RMA mutant-containing cell-free extracts obtained in Example 2 were added, and their NADPH oxidation activity, And NADH oxidation activity was measured.
- 0.05 mL of the enzyme solution was added to 0.95 mL of a reaction solution consisting of 100 mM potassium phosphate buffer (pH 7.0), 5 mM NADPH, 0.6 M 2-butanone (methyl ethyl ketone, MEK), and a constant temperature (25 ° C.).
- the decrease in absorbance at a wavelength of 340 nm was measured.
- the enzyme activity that oxidizes 1 ⁇ mol of NADPH to NADP + per minute was defined as 1 Unit.
- the enzyme activity was calculated in consideration of the dilution factor. Further, in the above reaction, only the NADPH in the reaction solution was changed to NADH, and thereafter the enzyme activity for oxidizing NADH to NAD + was also determined by the same measurement.
- the oxidation activity value was expressed as a relative value (%) with respect to wild type RMA.
- Various types of RMA mutants had NADPH that exhibited a low oxidative activity before mutagenesis, and the oxidative activity against NADH that had exhibited high oxidative activity before mutagenesis was reduced to about 1/50 to 1/500.
- Oxidizing activity against the urine increased by 10 to 50 times.
- the oxidation activity ratio (NADPH / NADH) which was about 0.003 before the mutagenesis, increased about 100 to 2000 times after the mutagenesis.
- Example 4 Construction of NADPH regeneration cycle in ketone reduction reaction by RMA mutant Using the RMA mutant (D201S / K202R / A206K, SEQ ID NO: 2), 2-butanone was converted into (S) -2-butanol (S- Reaction to reduce to MEH) was performed.
- NADPH is oxidized to NADP +.
- NADP + -dependent GDH GDHLP, WO 09/041415
- the reaction formula of this elementary reaction (NADPH regeneration cycle) is shown in FIG.
- An enzyme solution of NADP + -dependent GDH derived from lactic acid bacteria was prepared by a method similar to the method described in International Publication No. 09/041415.
- the composition of the reaction solution is as follows.
- the conditions of the analysis system are as follows. [Conversion rate to S-MEH: Gas chromatography analysis conditions] Column: TC-WAX (60m x 0.25mm) Detection: FID Hydrogen: 50kPa Column temperature: 50 ° C Injection temperature: 200 ° C Detection temperature: 200 ° C Carrier gas: helium (300 kPa) Elution time: 2-butanone (MEK) 4.8 minutes 2-butanol (MEH) 7.1 minutes
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Abstract
Description
[1]中鎖酸化還元酵素ファミリーに属し、下記の(a)~(d):
(a)配列番号1のAsp-201と立体構造上同等な位置のアミノ酸残基が、Ala、又はSerである、
(b)配列番号1のLys-202と立体構造上同等な位置のアミノ酸残基が、Argである、
(c)配列番号1のLys-203と立体構造上同等な位置のアミノ酸残基が、Serである、及び、
(d)配列番号1のAla-206と立体構造上同等な位置のアミノ酸残基が、Lysである、
から選択される少なくとも1つのアミノ酸残基を有するタンパク質、
[2]配列番号1に示されるアミノ酸配列と85%以上の配列同一性を有するアミノ酸配列からなる、[1]に記載のタンパク質、
[3]配列番号1に示すアミノ酸配列において、下記の(e)~(h):
(e)Asp-201を、Ala、又はSerに置換、
(f)Lys-202を、Argに置換、
(g)Lys-203を、Serに置換、及び、
(h)Ala-206を、Lysに置換、
から選択される少なくとも1つの変異が導入されたアミノ酸配列からなる、[1]又は[2]に記載のタンパク質、
[4]配列番号2~8のいずれかに記載のアミノ酸配列からなる、[1]~[3]のいずれかに記載のタンパク質、
[5][1]~[4]のいずれかに記載のタンパク質をコードする塩基配列からなるDNA、
[6]以下の(A)、(B)及び(C):
(A)配列番号25~31のいずれかに記載の塩基配列からなるDNA:
(B)配列番号25~31のいずれかに記載の塩基配列と相補的な塩基配列を含むDNAとストリンジェントな条件下でハイブリダイズし、かつ、酸化還元酵素活性を有するタンパク質をコードする塩基配列からなるDNA:
(C)配列番号25~31のいずれかに記載の塩基配列と85%以上の配列同一性を有し、かつ、酸化還元酵素活性を有するタンパク質をコードする塩基配列からなるDNA、
からなる群から選択されるDNA、
[7][6]に記載のDNAを含むベクター、
[8][7]に記載のベクターを用いて宿主細胞を形質転換して得られる形質転換体、
[9][8]に記載の形質転換体の培養物、
[10][1]~[5]のいずれかに記載のタンパク質を用いて、還元型ニコチンアミドアデニンジヌクレオチドリン酸を変換させて、酸化型ニコチンアミドアデニンジヌクレオチドリン酸を得る工程を含む、酸化型ニコチンアミドアデニンジヌクレオチドリン酸の製造方法、
[11][1]又は[2]に記載のタンパク質を用いて、酸化型ニコチンアミドアデニンジヌクレオチドリン酸を変換させて、還元型ニコチンアミドアデニンジヌクレオチドリン酸を得る工程を含む、還元型ニコチンアミドアデニンジヌクレオチドリン酸の製造方法、
[12][10]に記載の方法で得られる酸化型ニコチンアミドアデニンジヌクレオチドリン酸に、還元酵素を作用させることによる、還元型ニコチンアミドアデニンジヌクレオチドリン酸の製造方法、
[13][11]に記載の方法で得られる還元型ニコチンアミドアデニンジヌクレオチドリン酸に、酸化酵素を作用させることによる、酸化型ニコチンアミドアデニンジヌクレオチドリン酸の製造方法、
[14][8]に記載の形質転換体、または、[9]に記載の培養物を利用することを特徴とする、[10]~[13]のいずれかに記載の製造方法、
[15][10]~[14]のいずれかに記載の製造方法によって製造された化合物、
に関する。
(a)配列番号1のAsp-201と立体構造上同等な位置のアミノ酸残基が、Ala、又はSerである、
(b)配列番号1のLys-202と立体構造上同等な位置のアミノ酸残基が、Argである、
(c)配列番号1のLys-203と立体構造上同等な位置のアミノ酸残基が、Serである、及び、
(d)配列番号1のAla-206と立体構造上同等な位置のアミノ酸残基が、Lysである、
から選択される少なくとも1つのアミノ酸残基を有することが好ましい。
(e)配列番号1のAsp-201と立体構造上同等な位置のアミノ酸残基が、Serである、
(f)配列番号1のLys-202と立体構造上同等な位置のアミノ酸残基が、Argである、及び、
(g)配列番号1のAla-206と立体構造上同等な位置のアミノ酸残基が、Lysである、
のアミノ酸残基を全て含むことがより好ましい。
(i)配列番号1のAsp-201と立体構造上同等な位置のアミノ酸残基がAspである、
(j)配列番号1のLys-202と立体構造上同等な位置のアミノ酸残基がLysである、
(k)配列番号1のLys-203と立体構造上同等な位置のアミノ酸残基がLysである、及び、
(l)配列番号1のAla-206と立体構造上同等な位置のアミノ酸残基がAlaである、
から選択される少なくとも1つのアミノ酸残基を有することが好ましい。
本発明のタンパク質を用いて製造した酸化型ニコチンアミドアデニンジヌクレオチドリン酸(NADP+)、又は酸化型ニコチンアミドアデニンジヌクレオチド(NAD+)に、補酵素再生系を作用させることにより、還元型ニコチンアミドアデニンジヌクレオチドリン酸(NADPH)、又は還元型ニコチンアミドアデニンジヌクレオチド(NADH)を製造することが可能である。このような補酵素再生系は、酸化型補酵素を還元する活性を有する酵素であり、具体例として、グルコース脱水素酵素(GDH)、ギ酸脱水素酵素(FDH)、乳酸脱水素酵素(LDH)、及びリンゴ酸脱水素酵素(MDH)が挙げられる。
キャンディダ・マルトーサ(Candida maltosa)由来のMDR(中鎖脱水素酵素/還元酵素)ファミリー酵素(国際公開第08/066018号パンフレット、以下RMAと略す)の変異体を大腸菌において発現させるために、同文献に記載のpNCMベクター(RMA野生型発現プラスミド)を用いて、各種変異体の発現プラスミドを調製した。
実施例1で得たそれぞれの組換え大腸菌HB101を、半合成培地(グリセリン1.5%(w/v)、イーストエキス 0.3%(w/v)、Na2HPO4 0.6%(w/v)、KH2HPO4 0.3%(w/v)、NaCl 0.2%(w/v)、MgSO4・7H2O 0.5%(w/v)、100μg/ml アンピシリン、pH7.2)に植菌し、30℃にて60時間培養し、それぞれの培養液を集菌し培養上清を除いた後、培地と等量の緩衝液(100mM リン酸カリウム、pH7.0)に懸濁し、超音波破砕法により破砕し、再度遠心によって上清を回収することで、無細胞抽出液を得た。
反応基質としてケトン化合物を加えた下記の溶液に、実施例2で得られた各種RMA変異体含有無細胞抽出液を添加し、それらのNADPH酸化活性、および、NADH酸化活性を測定した。
RMA変異体(D201S/K202R/A206K、配列番号2)を用いて、2-ブタノンを(S)-2-ブタノール(S-MEH)へ還元する反応を行った。この反応系では、NADPHがNADP+に酸化されるが、この反応系に、乳酸菌由来NADP+依存性GDH(GDHLP、国際公開第09/041415号パンフレット)とその基質であるグルコースを加え、NADPHを再生するサイクルを同時に反応させた。この素反応の反応式(NADPH再生サイクル)を、図1に示した。乳酸菌由来NADP+依存性GDHの酵素液は、国際公開第09/041415号パンフレットに記載されている手法と同様の手法にて調製した。
[S-MEHへの変換率:ガスクロマトグラフィー分析条件]
カラム:TC-WAX(60m×0.25mm)
検出:FID
水素:50kPa
カラム温度:50℃
注入温度:200℃
検出温度:200℃
キャリアーガス:ヘリウム(300kPa)
溶出時間:2-ブタノン(MEK) 4.8分
2-ブタノール(MEH) 7.1分
カラム:Cyclodex-β(60m×0.25mm)
検出:FID
水素:50kPa
カラム温度:35℃
注入温度:150℃
検出温度:150℃
キャリアーガス:ヘリウム(300kPa)
溶出時間: 2-ブタノンMEK 6.5分
(R)-2-ブタノール(R-MEH) 11.0分
(S)-2-ブタノール(S-MEH) 11.4分
野生型RMA(配列番号1)については、鋳型に用いた発現プラスミド(pNCMベクター)を用いて、大腸菌HB101(Takara社製)を形質転換し、組換え大腸菌を作製した。実施例2と同様の手法にて無細胞抽出液を調製し、実施例3に倣って、ピリジンヌクレオチドの酸化活性測定を実施した。測定結果については、表2に併せて記載している。
Claims (15)
- 中鎖脱水素酵素/還元酵素ファミリーに属し、下記の(a)~(d):
(a)配列番号1のAsp-201と立体構造上同等な位置のアミノ酸残基が、Ala、又はSerである、
(b)配列番号1のLys-202と立体構造上同等な位置のアミノ酸残基が、Argである、
(c)配列番号1のLys-203と立体構造上同等な位置のアミノ酸残基が、Serである、及び、
(d)配列番号1のAla-206と立体構造上同等な位置のアミノ酸残基が、Lysである、
から選択される少なくとも1つのアミノ酸残基を有するタンパク質。 - 配列番号1に示されるアミノ酸配列と85%以上の配列同一性を有するアミノ酸配列からなる、請求項1に記載のタンパク質。
- 配列番号1に示すアミノ酸配列において、下記の(e)~(h):
(e)Asp-201を、Ala、又はSerに置換、
(f)Lys-202を、Argに置換、
(g)Lys-203を、Serに置換、及び、
(h)Ala-206を、Lysに置換、
から選択される少なくとも1つの変異が導入されたアミノ酸配列からなる、請求項1又は2に記載のタンパク質。 - 配列番号2~8のいずれかに記載のアミノ酸配列からなる、請求項1~3のいずれかに記載のタンパク質。
- 請求項1~4のいずれかに記載のタンパク質をコードする塩基配列からなるDNA。
- 以下の(A)、(B)及び(C):
(A)配列番号25~31のいずれかに記載の塩基配列からなるDNA:
(B)配列番号25~31のいずれかに記載の塩基配列と相補的な塩基配列を含むDNAとストリンジェントな条件下でハイブリダイズし、かつ、酸化還元酵素活性を有するタンパク質をコードする塩基配列からなるDNA:
(C)配列番号25~31のいずれかに記載の塩基配列と85%以上の配列同一性を有し、かつ、酸化還元酵素活性を有するタンパク質をコードする塩基配列からなるDNA、
からなる群から選択されるDNA。 - 請求項5又は6に記載のDNAを含むベクター。
- 請求項7に記載のベクターを用いて宿主細胞を形質転換して得られる形質転換体。
- 請求項8に記載の形質転換体の培養物。
- 請求項1~4のいずれかに記載のタンパク質を用いて、還元型ニコチンアミドアデニンジヌクレオチドリン酸を変換させて、酸化型ニコチンアミドアデニンジヌクレオチドリン酸を得る工程を含む、酸化型ニコチンアミドアデニンジヌクレオチドリン酸の製造方法。
- 請求項1又は2に記載のタンパク質を用いて、酸化型ニコチンアミドアデニンジヌクレオチドリン酸を変換させて、還元型ニコチンアミドアデニンジヌクレオチドリン酸を得る工程を含む、還元型ニコチンアミドアデニンジヌクレオチドリン酸の製造方法。
- 請求項10に記載の方法で得られる酸化型ニコチンアミドアデニンジヌクレオチドリン酸に、還元酵素を作用させることによる、還元型ニコチンアミドアデニンジヌクレオチドリン酸の製造方法。
- 請求項11に記載の方法で得られる還元型ニコチンアミドアデニンジヌクレオチドリン酸に、酸化酵素を作用させることによる、酸化型ニコチンアミドアデニンジヌクレオチドリン酸の製造方法。
- 請求項8に記載の形質転換体、または、請求項9に記載の培養物を利用することを特徴とする、請求項10~13のいずれかに記載の製造方法。
- 請求項10~14のいずれかに記載の製造方法によって製造された化合物。
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JP2020535810A (ja) * | 2017-10-01 | 2020-12-10 | エンザイマスター(ニングボ) バイオ−エンジニアリング カンパニー リミテッド | 改変デカルボキシラーゼポリペプチドおよびβ−アラニンの調製におけるそれらの用途 |
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US20140256930A1 (en) | 2014-09-11 |
EP2728001A1 (en) | 2014-05-07 |
CN103717734A (zh) | 2014-04-09 |
JPWO2013002277A1 (ja) | 2015-02-23 |
EP2728001A4 (en) | 2015-05-27 |
US9416350B2 (en) | 2016-08-16 |
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