WO2017167250A1 - 一种酶及其应用 - Google Patents

一种酶及其应用 Download PDF

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WO2017167250A1
WO2017167250A1 PCT/CN2017/078912 CN2017078912W WO2017167250A1 WO 2017167250 A1 WO2017167250 A1 WO 2017167250A1 CN 2017078912 W CN2017078912 W CN 2017078912W WO 2017167250 A1 WO2017167250 A1 WO 2017167250A1
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amino acid
enzyme
nicotine
nica2
seq
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PCT/CN2017/078912
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French (fr)
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许平
唐鸿志
张坤智
吴更
陶飞
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上海交通大学
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Priority to US16/090,535 priority Critical patent/US11279916B2/en
Publication of WO2017167250A1 publication Critical patent/WO2017167250A1/zh
Priority to US17/700,237 priority patent/US20220282225A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
    • C12N9/0022—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with oxygen as acceptor (1.4.3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/44—Oxidoreductases (1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/30—Drugs for disorders of the nervous system for treating abuse or dependence
    • A61P25/34—Tobacco-abuse
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y104/00—Oxidoreductases acting on the CH-NH2 group of donors (1.4)
    • C12Y104/03—Oxidoreductases acting on the CH-NH2 group of donors (1.4) with oxygen as acceptor (1.4.3)
    • C12Y104/03004—Monoamine oxidase (1.4.3.4)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y105/00—Oxidoreductases acting on the CH-NH group of donors (1.5)
    • C12Y105/99—Oxidoreductases acting on the CH-NH group of donors (1.5) with other acceptors (1.5.99)
    • C12Y105/99004—Nicotine dehydrogenase (1.5.99.4)

Definitions

  • the invention belongs to the field of protein engineering, and relates to a protein mutant, in particular to a nicotine dehydrogenase mutant, and the application thereof in developing a protein drug for treating nicotine addiction.
  • Nicotine is one of the main harmful components in tobacco leaves, cigarette smoke and tobacco waste. It is not only directly harmful to health, but also the main carcinogen in tobacco. An important precursor of the constituent tobacco-specific nitrosamines (TSNA). A large number of high-nicotine tobacco leaves are produced every year in the production of tobacco leaves in China, which not only seriously affects the sucking quality of tobacco leaves, but also increases the harmfulness of tobacco leaves. Reducing the content of nicotine in tobacco and the environment is extremely important for maintaining human health and protecting the ecological environment.
  • Pseudomonas and Arthrobacter can decompose nicotine to produce the carbon necessary for its growth. Source, nitrogen source and energy.
  • the nicotine-reducing bacteria that have been found are Pseudomonas (such as Pseudomonas sp. No. 41, P. convexa PC1, P. putida), Arthrobacter (such as Arthobacter oxidans P-34, and later re-identified as A. Ureafaciens; A.
  • the fungi mainly include Cunninghamella echinulata, Microsporum gypseum, Streptomyces griseus, S. platenses, and Pelliculariafilamentosa. .
  • the 3-carboxylic acid pyridinium salt is formed, and the 6' position of the pyridine ring is rehydroxylated to form a 6-hydroxy, 3-carboxylic acid pyridinium salt, and the side chain is removed to form a 2,5-dihydroxy-pyridinium salt and a succinate salt.
  • This metabolic pathway can also start with 6-hydroxy-pseudo-oxidized nicotine, which eventually still produces 2,5-dihydroxy-pyridinium salts and succinates.
  • the nicotine dehydrogenase NicA2 is a nicotine-degrading enzyme that has recently been isolated from Pseudomonas putida S16.
  • a report on nicotine dehydrogenase NicA2 can be found in: PLoS Genet. 2013Oct;9(10):e1003923.doi:10.1371/journal.pgen.1003923.Epub 2013Oct 24.Systematic unraveling of the unsolved pathway of nicotine degradation in Pseudomonas.Tang H,Wang L,Wang W,Yu H,Zhang K, Yao Y, Xu P.
  • Nicotine dehydrogenase NicA2 catalyzes the dehydrogenation reaction of the first step of nicotine degradation, converting nicotine (I) to N-methymyosmine (II), and N-methyl hydramine can be further Spontaneous hydration produces pseudooxynicotine (III).
  • NicA2 is encoded by the nicA2 gene and plays a crucial role in the degradation of nicotine by Pseudomonas putida S16. When the nicA2 gene is knocked out, Pseudomonas putida S16 cannot use nicotine as the sole carbon. Nitrogen source growth (PLoS Genet. 10.1371/journal.pgen. 1003923).
  • NicA2 belongs to the family of monoamine oxidase (MAO).
  • MAO monoamine oxidase
  • MAO is mainly found in the outer membrane of mitochondria. Its main role in the body is to catalyze the metabolism of endogenous and exogenous monoamines. The amine species are oxidized to produce deamination.
  • MAO can be divided into two categories, namely MAO-A and MAO-B, depending on the substrate, distribution location and selective inhibitor of MAO.
  • MAO-A has high affinity for serotonin (5-HT), norepinephrine (NE), dopamine (DA) (J. Mol. Biol. 338, 103–114);
  • MAO-B is phenethyl Amines (PEA), benzylamine, and the like have high affinity (Proc. Natl. Acad. Sci. USA 100, 9750-9575).
  • those skilled in the art are directed to developing a high rate of nicotine dehydrogenase, and its use in biocatalysis, metabolic engineering, and protein drug development.
  • the technical problem to be solved by the present invention is to improve nicotine dehydrogenation with high catalytic rate and its application by improving nicotine dehydrogenase.
  • an aspect of the present invention provides an artificial enzyme.
  • the artificial enzyme is produced based on a sequence modification of a natural enzyme having an activity of catalyzing a reaction of converting from Compound I to Compound II as shown below.
  • the natural enzyme is a nicotine dehydrogenase
  • the above modifications include replacing at least one of the amino acids that hinder the release of the product with an amino acid having a smaller side chain.
  • the above natural enzyme also has one or more of the following characteristics:
  • the amino acid sequence of the natural enzyme includes an amino acid sequence having 85% or more, or 90% or more, or 95% or more, or 98% or more, or 99% or more homology with the amino acid sequence shown by SEQ ID NO: 1.
  • the amino acid sequence of the native enzyme comprises the amino acid sequence shown in SEQ ID NO: 1; or the amino acid sequence of the native enzyme is shown in SEQ ID NO: 1;
  • the native enzyme is encoded by a nucleic acid that hybridizes under high stringency conditions to a complementary strand of a nucleic acid encoding a protein of amino acid sequence SEQ ID NO: 1;
  • the native enzyme is present in a microorganism of the genus Pseudomonas.
  • the native enzyme is present in Pseudomonas putida S16.
  • the native enzyme has a product release channel for product release
  • the amino acids whose side chains are located in the product release channel of the native enzyme include tryptophan, tyrosine, phenylalanine, glutamic acid, and methotrexate. At least one of the acids; the improvement comprising: replacing at least one of the above-mentioned tryptophan, tyrosine, phenylalanine, glutamic acid, methionine having a side chain in a product release channel of the native enzyme with Smaller amino acids in the side chain.
  • amino acid of the side chain located in the product release channel of the native enzyme comprises at least 9 amino acids selected from the group consisting of tryptophan, tyrosine, phenylalanine, glutamic acid and methionine.
  • the three-dimensional structure of the natural enzyme includes at least one ⁇ -sheet and one ⁇ -helix located at the release channel, and three of the nine amino acids are located in the ⁇ -sheet, and the relative position is 353-position of phenylalanine Acid, 355 phenylalanine and 364 tryptophan; 6 of the 9 amino acids are located in the alpha helix, calculated as relative position, 163 phenylalanine, 214 tyrosine Acid, tyrosine at position 218, tyrosine at position 242, methionine at position 246, and glutamic acid at position 249.
  • the above alternative is that if the amino acid to be replaced is located on the ⁇ -sheet, it is replaced with a proline; if the amino acid to be replaced is located on the ⁇ -helix, it is replaced with alanine.
  • the above improvement comprises replacing all of the amino acids having a molecular weight of 120 or more which hinder the release of the product with amino acids having a smaller side chain.
  • Another aspect of the invention provides an enzyme, in one embodiment, the enzyme has one or more of the following characteristics:
  • the amino acid sequence of the enzyme includes an amino acid sequence having 85% or more, or 90% or more, or 95% or more, or 98% or more, or 99% or more homology with the amino acid sequence shown by SEQ ID NO: 2.
  • the amino acid sequence of the enzyme comprises the amino acid sequence shown in SEQ ID NO: 2; or the amino acid sequence of the enzyme is shown in SEQ ID NO: 2;
  • the enzyme is encoded by a nucleic acid which hybridizes under high stringency conditions to a complementary strand of a nucleic acid encoding a protein of the amino acid sequence of SEQ ID NO: 2;
  • the amino acid sequence of the enzyme comprises an amino acid sequence in which the amino acid sequence shown in SEQ ID NO: 2 is conservatively substituted;
  • At least one of the 353th amino acid, the 355th amino acid and the 364th amino acid of the enzyme is the first small molecular weight amino acid; the 163th amino acid, the 214th amino acid, the 218th of the enzyme At least one of the amino acid, the 242th amino acid, the 246th amino acid, and the 249th amino acid is a second small molecular weight amino acid, the first small molecular weight amino acid has a molecular weight of 140 or less, and the second small molecular weight amino acid has a molecular weight of 110 or less.
  • the enzyme has an activity of catalyzing a reaction of converting from Compound I to Compound II as follows:
  • the enzyme is a nicotine dehydrogenase.
  • amino acid 353, amino acid 355 and amino acid 364 are located in the ⁇ -sheet; amino acid 163, amino acid 214, amino acid 218, amino acid 242, amino acid 246, and 249th The amino acid is located in the alpha helix.
  • first small molecular weight amino acid is proline and the second small molecular weight amino acid is alanine.
  • amino acid at position 353, the amino acid at position 355 and the amino acid at position 364 are all first small molecular weight amino acids; amino acid 163, amino acid 214, amino acid 218, amino acid 242, amino acid 246 and The 249th amino acid is all the second small molecular weight amino acid.
  • Another aspect of the present invention provides an enzyme which, in a specific embodiment, has an activity of catalyzing a reaction of converting from Compound I to Compound II as shown below,
  • the enzyme is a nicotine dehydrogenase
  • the enzyme has a product release channel for product release, and the amino acid having a side chain at the narrowest point of the product release channel has a molecular weight below 140.
  • the amino acid of the side chain located at the narrowest point of the product release channel has a molecular weight below 120.
  • the amino acid at the narrowest point of the product release channel of the product release channel is valine or alanine.
  • Another aspect of the invention provides a nucleotide sequence encoding the artificial enzyme or enzyme described above.
  • nucleotide sequence has one or more of the following characteristics:
  • the nucleotide sequence comprises 85% or more, or 90% or more, or 95% or more, or 98% or more, or 99% or more homology to the nucleotide sequence shown by SEQ ID NO: a nucleotide sequence; or the nucleotide sequence comprises the nucleotide sequence shown in SEQ ID NO: 3; or the nucleotide sequence is shown in SEQ ID NO: 3;
  • nucleotide sequence hybridizes to the complementary strand of the nucleotide sequence shown by SEQ ID NO: 3 under high stringency conditions.
  • a further aspect of the invention provides an expression vector or host cell comprising the above nucleotide sequence.
  • a further aspect of the invention provides the use of the above-described artificial enzyme or enzyme for the degradation of nicotine.
  • the invention also provides the use of the above artificial enzyme or enzyme in the preparation of a medicament for treating nicotine addiction.
  • the invention also provides the use of the above-described artificial enzyme or enzyme for the preparation of an enzyme preparation for biocatalytic conversion.
  • the invention also provides the use of the above artificial enzymes or enzymes in metabolic engineering and synthetic biology.
  • a method of improving an enzyme of the family of monoamine oxidases comprising: replacing at least one amino acid that inhibits product release with an amino acid having a smaller side chain.
  • the invention adopts the method of protein engineering to start from the wild-type nicotine dehydrogenase, and obtains the relevant structure which affects the release of the product through crystal structure analysis, and eliminates the influence of the related amino acid on the product release by means of amino acid substitution, thereby improving the mutant enzyme.
  • Catalytic rate The nicotine dehydrogenase mutant of the preferred embodiment of the present invention, although slightly less affinity than the wild-type nicotine dehydrogenase, has a rate of catalytic substrate nicotine that is much greater than that of the wild-type nicotine dehydrogenase, which is 3.67 times.
  • the present invention firstly analyzes the structure of nicotine dehydrogenase and obtains a structure in which nicotine dehydrogenase and substrate nicotine are combined.
  • nine large amino acid residues hindering the release of the product were obtained, which laid a good foundation for the subsequent mutation to obtain high catalytic efficiency nicotine dehydrogenase.
  • the above 9 large amino acid residues that hinder the release of the product are somewhat conserved among other members of the monoamine oxidase family, which also provides a good basis for improving the members of the monoamine oxidase family.
  • the nicotine dehydrogenase mutant of the invention can be applied at least in biocatalysis, metabolic engineering and synthetic biology, and development of protein drugs, and has great commercial application prospects:
  • the nicotine dehydrogenase mutant obtained by the present invention has superior catalytic properties to natural nicotine dehydrogenase and can be used for obtaining higher catalytic properties of nicotine as a product, and thus can be used for developing enzyme preparations. Catalytic conversion of organisms;
  • Figure 1 is a polyacrylamide gel electrophoresis pattern of purified NicA2 protein of a preferred embodiment of the present invention.
  • Lane 1 lane-clear supernatant
  • lane 2 pellet after lysis
  • lane 3 column effluent
  • lane 4 20 mM imidazole wash
  • lane 5 50 mM imidazole wash
  • lane 6 80 mM imidazole Eluent
  • Lane 7 170 mM imidazole eluate
  • Lane 8 300 mM imidazole eluate
  • M protein molecular weight marker.
  • FIG. 2 is a series of optimized crystals of a NicA2 protein according to a preferred embodiment of the present invention, such as crystal primary screening, precipitant optimization, seed addition, amino acid sequence truncation, and ratio of protein to crystallization reagent.
  • the presence of FAD gives the crystal a bright yellow color.
  • Figure 3 is a structural view of a NicA2 ⁇ 20-nicotine crystal according to a preferred embodiment of the present invention.
  • a Looking down at the product release channel, no product was observed from the outer surface of the crystal structure, indicating that the product was completely embedded by NicA2;
  • b adjusting the gray scale to transparent, overlooking the product release channel, and the product release channel was 9 large amino acids. Blocking;
  • c cross section of the product release channel.
  • Figure 4 is a polyacrylamide gel electrophoresis pattern of the purified NicA2-M9 protein of a preferred embodiment of the present invention.
  • Lane 1 Lane supernatant after lysis; Lane 2: pellet after lysis; Lane 3: effluent from column; Lane 4: 20 mM imidazole lotion; Lane 5: 50 mM imidazole deproteinization; Lane 6: 170mM Imidazole elutes the protein of interest; M: protein molecular weight marker.
  • Figure 5 is a graph showing the Michaelis curve of NicA2 wild type catalyzed nicotine formation of N-methyl phthymidine according to a preferred embodiment of the present invention.
  • Figure 6 is a graph showing the Michaelis equation for the formation of pseudo-oxidized nicotine by NicA2 wild type catalyzed nicotine in accordance with a preferred embodiment of the present invention.
  • Figure 7 is a graph showing the Michaelis equation for the formation of N-methylpyramine by nicotine by NicA2-M9 in accordance with a preferred embodiment of the present invention.
  • Figure 8 is a graph showing the Michaelis equation for the formation of pseudo-oxidized nicotine by nicotine by NicA2-M9 in accordance with a preferred embodiment of the present invention.
  • Figure 9 is a graph showing the amount of pseudo-oxidized nicotine released by GC using a preferred embodiment of the present invention.
  • Figure 10 is a cross-sectional view of the product release channels of the five NicA2 mutants produced in a particular embodiment of the invention and the NicA2 wild type.
  • A NicA2 wild type
  • B NicA2-M3V mutant
  • C NicA2-M3A mutant
  • D NicA2-M5 mutant
  • E NicA2-M7 mutant
  • F NicA2-M9 mutant.
  • Figure 11 shows the results of alignment of the amino acid sequence of NicA2 with proteins in the monoamine oxidase family.
  • One aspect of the present invention provides an artificial enzyme which is produced based on a sequence modification of a natural enzyme.
  • the artificial enzyme referred to herein should be understood as any enzyme that has been modified by natural enzymes, including genetic engineering improvements such as enzyme mutants.
  • This natural enzyme has an activity of catalyzing a reaction of converting from Compound I to Compound II as shown below.
  • the native enzyme is a nicotine dehydrogenase.
  • the enzymes described herein having the activity of catalyzing the above reactions should be understood to mean not only the enzymes which specifically catalyze the above reactions, but also those which do not specifically catalyze the above reactions, for example, in addition to catalyzing the above reactions, An enzyme that catalyzes the dehydrogenation of 6-hydroxy nicotine (including D and L).
  • nicotine dehydrogenase refers to an enzyme that catalyzes the dehydrogenation of a pyrrole ring of a nicotine or nicotine analog such as 6-hydroxy nicotine.
  • the improvement comprises replacing at least one of the amino acids that hinder the release of the product with an amino acid having a smaller side chain.
  • amino acid hindering product release shall be understood to mean that any Its amino acid is an amino acid that increases product release efficiency or enzyme catalytic efficiency. For example, if the side chain of an amino acid is located in the channel or pathway of product release, if the amino acid is replaced with a smaller side chain amino acid, the product release efficiency or enzyme catalytic efficiency can be increased, then the amino acid is "blocking product release. Amino acid”.
  • the native enzyme further has one or more of the following characteristics:
  • the amino acid sequence of the native enzyme includes an amino acid having 85% or more, or 90% or more, or 95% or more, or 98% or more, or 99% or more homology with the amino acid sequence shown by SEQ ID NO: 1.
  • a sequence; or the amino acid sequence of the native enzyme comprises the amino acid sequence set forth in SEQ ID NO: 1; or the amino acid sequence of the native enzyme is set forth in SEQ ID NO: 1;
  • the native enzyme is encoded by a nucleic acid that hybridizes under high stringency conditions to a complementary strand of a nucleic acid encoding a protein of amino acid sequence SEQ ID NO: 1;
  • the natural enzyme is present in a microorganism of the genus Pseudomonas.
  • homology can refer to an optimal alignment of sequences (nucleotides or amino acids) that can be performed using a computerized implementation of the algorithm.
  • the "homology” associated with a polynucleotide can be determined using BLASTN version 2.0 using the default parameters for analysis.
  • the "homology” associated with a polypeptide (ie, an amino acid) can be determined using a program (such as BLASTP version 2.2.2) using default parameters that align the polypeptide or fragment being compared (and can also align the nucleotide fragment) And then determine the degree of amino acid identity or similarity of the comparator.
  • Hybridization and washing conditions are described herein as "hybridization in conditions of low stringency, medium stringency, high stringency, or very high stringency.”
  • Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Both aqueous and non-aqueous methods are described in this document, either of which is useful.
  • Specific hybridization conditions herein are as follows: 1) Low stringency hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45 ° C, followed by at least 50 ° C in 0.2X SSC, 0.1% SDS (for Low stringency conditions, wash temperature should be increased to 55 ° C) wash twice; 2) medium stringency hybridization conditions are hybridization in 6X SSC at about 45 ° C, followed by 0.2 ° SSC, 0.1% SDS at 60 ° C Washing one or more times; 3) High stringency hybridization conditions are hybridization in 6X SSC at about 45 ° C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 65 ° C; 4) Very high stringency hybridization The conditions were hybridization at 65 ° C in 0.5 M sodium phosphate, 7% SDS, followed by one or more washes at 65 ° C in 0.2X SSC, 1% SDS. High stringency conditions (3) are preferred and should be used unless otherwise stated.
  • the native enzyme is present in Pseudomonas putida S16.
  • Pseudomonas putida S16 has the accession number CCTCC NO.M205038, which was deposited with the China Center for Type Culture Collection on April 18, 2005.
  • the strain is also deposited with the German National Collection (DSMZ), the deposit number. For DSM 28022.
  • the native enzyme has a product release channel
  • the amino acids whose side chains are located in the product release channel of the native enzyme include tryptophan, tyrosine, phenylalanine, glutamic acid, A At least one of methionines; the improvement comprising: at least one of the tryptophan, tyrosine, phenylalanine, glutamic acid, methionine in which the side chain is located in the release channel of the native enzyme One is replaced by an amino acid with a smaller side chain.
  • product release channel is understood to mean the path that a product needs to pass from the beginning of the reactive center of the enzyme to the position at which it does not affect the catalytic reaction at all.
  • the reactive center of the enzyme is in the cavity of the three-dimensional structure of the enzyme.
  • the artificial enzyme or enzyme disclosed in the present invention is also the same, and the product starts from the reactive center of the enzyme to the position that does not affect the enzyme catalytic reaction at all.
  • the path that needs to pass is at least partially wrapped by some structure, and the degree of encapsulation is different for different enzymes. Therefore, the path that the product needs to pass from the reactive center that starts to leave the enzyme to the position that does not affect the enzyme catalytic reaction at all can be It is understood as the form of "channel”.
  • the amino acid of the side chain located in the product release channel of the native enzyme comprises at least 9 amino acids, and the 9 amino acids are selected from the group consisting of tryptophan, tyrosine and phenylalanine. , glutamic acid and methionine.
  • the three-dimensional structure of the native enzyme includes at least one ⁇ -sheet and one ⁇ -helix located at the release channel, and 3 of the 9 amino acids are located in the ⁇ Folding, calculated as relative position, 353 phenylalanine, 355 phenylalanine, and 364 tryptophan; 3 of the 9 amino acids are located in the alpha helix, Calculated by relative position, 163 phenylalanine, 214 tyrosine, 218 tyrosine, 242 tyrosine, 246 methionine and 249 glutamic acid .
  • relative position for an amino acid sequence is understood to mean that the position number of an amino acid in the sequence only represents the relative position of the amino acid.
  • 353-position phenylalanine and 355-position phenylalanine only the position of the former and the latter differs by 2; if the entire protein sequence has a sequence of amino acid number x at the N-terminus, From the absolute position calculation, the former position number is 353+x, and the latter position number is 355+x; if the entire protein sequence is reduced at the N-terminus by a sequence of amino acid number x, the position number of the former is calculated from the absolute position. Is 353-x, the latter is numbered 355-x; if calculated by relative position, "353 phenylalanine" and “355 phenylalanine” should also be understood to include both cases .
  • the amino acid sequence of the enzyme includes an amino acid having 85% or more, or 90% or more, or 95% or more, or 98% or more, or 99% or more homology with the amino acid sequence shown by SEQ ID NO: 2.
  • a sequence; or the amino acid sequence of the enzyme comprises the amino acid sequence set forth in SEQ ID NO: 2; or the ammonia of the enzyme The acid sequence is shown in SEQ ID NO: 2;
  • the enzyme is encoded by a nucleic acid that hybridizes under high stringency conditions to a complementary strand of a nucleic acid encoding a protein of amino acid sequence SEQ ID NO: 2;
  • the amino acid sequence of the enzyme comprises an amino acid sequence in which the amino acid sequence shown in SEQ ID NO: 2 is conservatively substituted;
  • At least one of the 353th amino acid, the 355th amino acid and the 364th amino acid of the enzyme is a first small molecular weight amino acid; the 163th amino acid and the 214th amino acid of the enzyme, At least one of amino acid 218, amino acid 242, amino acid 246 and amino acid 249 is a second small molecular weight amino acid, and the molecular weight of the first small molecular weight amino acid is below 140, the second small The molecular weight of the molecular weight amino acid is 110 or less.
  • conservative substitution shall be taken to mean those substitutions of a given amino acid in a polypeptide with another amino acid having a similar identity. Typically, the following substitutions are considered conservative substitutions: substitution of an aliphatic amino acid such as Ala, Val, Leu, and Ile with another aliphatic amino acid; replacement of Ser with Thr, and vice versa; acidic residue such as Asp or Glu Substituting for another acidic residue; replacing an amide group-containing residue such as Asn or Gln with another amide group-containing residue; replacing a basic residue such as Lys or Arg with another base a residue; an aromatic residue such as Phe or Tyr is replaced with another aromatic residue.
  • amino acids with similar properties can be grouped as follows:
  • Another aspect of the present invention provides an enzyme having an activity of catalyzing a reaction of converting from Compound I to Compound II as shown below,
  • the enzyme is a nicotine dehydrogenase
  • the enzyme has a product release channel, characterized in that the molecular weight of the amino acid whose side chain is located at the narrowest point of the product release channel is below 140.
  • the amino acid at the narrowest point of the product release channel is understood to mean that the product is in the path that is required to pass from the reactive center of the enzyme to the position at which the enzyme is not affected at all. Block the most powerful amino acids.
  • the materials used in the present invention can be obtained by direct purchase unless otherwise specified.
  • the experimental methods used in the present invention are conventional methods in the art unless otherwise specified.
  • Example 1 Cloning, expression and purification of wild-type nicotine dehydrogenase (NicA2) and NicA2 nitrogen-terminal truncation (NicA2 ⁇ 20)
  • Cloning of NicA2 The full-length gene sequence of the NicA2 protein was obtained by pre-sequencing of the Xuping group, as shown in SEQ ID NO: 4.
  • the full-length sequence of NicA2 was amplified from Pseudomonas putida S16 (CCTCC No.M 205038) by PCR using primers NicA2-F1 and NicA2-R1, digested with NcoI and XhoI, and ligated into pET28a vector. There are six histidine tags at the carbon end of the vector. Among them, the primer sequence is:
  • NicA2-R1 5'-GTGCTCGAGGCTTAAGAGCTGCTTAACCTCCCTA-3'.
  • the target protein was subjected to deimidazole by a superdex 200 column (available from GE Corporation, model: 28-9909-44), and then centrifuged at a speed of not more than 5000 rpm using a 30 KD ultrafiltration tube (MD) to concentrate the target protein to 12 mg/ mL for use in spotting.
  • the purified protein was verified by polyacrylamide gel electrophoresis, as shown in Fig. 1, and its purity was over 90%.
  • NicA nitrogen-terminal truncation (NicA2 ⁇ 20) was obtained by cloning, expression and purification according to the above method.
  • the primers used for cloning were: NicA2-F2 and NicA2-R1, and the obtained recombinant plasmid was pET28a-NicA2 ⁇ 20.
  • Example 2 Crystallization and crystal diffraction of wild-type nicotine dehydrogenase (NicA2), NicA2 nitrogen-terminal truncation (NicA2 ⁇ 20), and NicA2 ⁇ 20-nicotine eutectic
  • Crystal Diffraction of NicA2 Crystals grown under the conditions of Index-65 crystallization have almost no diffraction points by X-ray diffraction, and no crystals grow again in repeated experiments, so the conditions are abandoned and optimized; and SaltRX2-36 crystallization conditions The crystal that grows underneath, the resolution of X-ray diffraction is about Left and right, and the diffraction point has a tail, indicating that the crystal grows under this condition, and the internal stack is irregular.
  • Purified NicA2 ⁇ 20 protein was obtained according to the method of cloning, expression and purification in Example 1, and crystal preliminary screening was carried out as described above. Crystals with improved shape still appear in SaltRX2-36: 1.4 MAmmonium tartrate dibasic, 0.1 M Tris pH 8.5. The crystal of NicA2 ⁇ 20 is then subjected to optimization steps such as the concentration of precipitant, micro-seeding, ratio of changing protein and crystallization reagent, and the X-ray diffraction resolution is obtained. Crystals. Among them, the concentration of the precipitant is preferably 1.4M Ammonium tartrate dibasic. The microscope observation of the crystal is shown in Fig. 2.
  • NicA2 ⁇ 20 is compared with the published amino acid sequence of the protein in the Protein Data Bank (PDB) with a consistency of only 28%. Simple molecular replacement cannot resolve the structure of NicA2, so it can only be purified by selenoprotein. The anomalous scattering of selenium atoms in an amino acid determines the phase and thus the structure.
  • PDB Protein Data Bank
  • the recombinant plasmid pET28a-NicA2 ⁇ 20 was transformed into the host E. coli B834 (DE3), and the selenoprotein was purified by the method of Example 1.
  • the host is a methionine-deficient strain.
  • glucose and yeast inorganic nitrogen sources are used as nutrients, and selenomethionine is added instead of normal methionine, so that seleno-NicA2 can be expressed. , that is, SeMet-NicA2 ⁇ 20.
  • the specific expression purification step of SeMet-NicA2 ⁇ 20 was as described in Example 1.
  • SeMet-NicA2 ⁇ 20 was crystallized, and crystals with excellent appearance appeared in SaltRX2-36:1.4MAmmonium tartrate dibasic, 0.1M Tris pH 8.5. X-ray diffraction was obtained after optimization of the ratio of crystallized protein and precipitant. Resolution is Crystals.
  • NicA2 ⁇ 20-nicotine eutectic was obtained by soaking a higher concentration of nicotine solution with NicA2 ⁇ 20 protein crystal. Dilute the pure nicotine with a crystal growth buffer (the composition of the buffer as described above) to form a 1 M nicotine mother solution and adjust the pH to 8.5; dissolve the insurance powder (sodium dithionite) with a crystal growth buffer to 100 mM and adjusted to pH 7.0 to form a mother liquor of the powder. In the mixed buffer of the soaked crystals, the final concentration of nicotine was 10 mM, and the final concentration of the powder was 20 mM. Among them, the insurance powder solution produces an oxygen-free environment.
  • a crystal growth buffer the composition of the buffer as described above
  • the insurance powder sodium dithionite
  • the crystal with the best crystal form quickly soak it into the nicotine + insurance powder mixture solution, soaking time is 30 seconds, and capture the instant of the combination of NicA2 and substrate nicotine.
  • the crystals after soaking the nicotine are quickly frozen in liquid nitrogen.
  • the X-ray diffraction resolution is NicA2 ⁇ 20-nicotine crystals.
  • the pseudo-oxidized nicotine was extracted with chloroform-benzyl alcohol, and the total amount of pseudo-oxidized nicotine produced by the reaction was quantitatively detected by GC.
  • the Ni-NTA column was repeatedly passed until the concentration of the effluent NicA2 was less than 0.1 mg/mL, and it was considered that NicA2 was completely bound to the nickel column.
  • the pseudo-oxidized nicotine in the effluent was extracted with chloroform-benzyl alcohol, and the amount of pseudo-oxidized nicotine released from the solution of NicA2 into the solution was quantitatively detected by GC. The results showed that approximately 75% of the pseudo-oxidized nicotine remained in the NicA2 protein.
  • the position of the nine large amino acids obtained in Example 3 on the crystal structure of NicA2 ⁇ 20 was analyzed, and the amino acid located on the ⁇ -sheet was replaced with valine (Valine, V), and the amino acids located on the ⁇ -helix were replaced with alanine ( Alanine, A).
  • the basic principle of amino acid substitution is to replace the amino acid with a large side chain group with a small amino acid with a side chain group, and it is considered that alanine is the simplest amino acid structure and a strong alpha helix former; proline
  • the structure is also very simple and is a strong beta-former.
  • the gene sequence of the above nicotine dehydrogenase mutant NicA2-M9 was synthesized by Jinweizhi Company.
  • the synthesized sequence was amplified by a PCR method, and the primers used for amplification were: NicA2-F1 and NicA2-R1.
  • the mutant gene fragment obtained by amplification and the vector pET28a were digested with NcoI and XhoI, and ligated with T4 to obtain a recombinant plasmid pET28a-NicA2-M9.
  • the recombinant plasmid was correctly sequenced and transformed into the expression host E. coli BL21 (DE3) for expression.
  • the purification method of the expressed nicotine dehydrogenase mutant NicA2-M9 refer to the NicA2 wild type protein purification method in Example 1.
  • Example 5 Determination and comparison of enzyme activity of wild-type nicotine dehydrogenase and nicotine dehydrogenase mutants
  • the enzyme activities of the NicA2 wild type and the NicA2 mutant NicA2-M9 were labeled by liquid chromatography-mass spectrometry (LC-MS) by measuring the amount of the product N-methylzincamine and pseudo-oxidized nicotine.
  • the LC-MS parameters were as follows: Agilent 1290 liquid chromatography, EC-C8 column (4.6 x 100 mm, 1.8 ⁇ m), 0.2 mL/min. In the positive ion mode, the nuclear-to-mass ratio (m/z) of N-methylpyramine is 161, and the nuclear-mass ratio (m/z) of pseudo-oxidized nicotine is 179.
  • the concentration of N-methylzincamine and pseudo-oxidized nicotine was diluted from the highest concentration of 2 ⁇ M to the lowest concentration of 31.25 nM.
  • the sample was applied to an LC-MS instrument, and the peaks of nucleoplasmic ratios 161 and 179 were selected.
  • the recording instrument automatically annotated Peak area.
  • a standard curve of the two products was drawn using the software OriginPro8.
  • the final concentration of the immobilized NicA2 wild-type protein was 10 nM
  • the concentration of the substrate nicotine was diluted from the highest concentration of 2 ⁇ M to the lowest concentration of 12.5 nM
  • the protein and substrate were incubated at 30 ° C for 20 minutes, and the reaction was inactivated with 2.5 volumes of acetonitrile.
  • the protein pellet was removed by centrifugation at 12,000 rpm for 2 min. Inject the sample into the LC-MS instrument, select the peaks of the nuclear-to-mass ratios 161 and 179, and record the peak area automatically annotated by the instrument.
  • Draw with OriginPro8 The Michaelis-Menten equation curve is shown in Figures 5 and 6.
  • Standard binding curves product calculate K m and k cat values of the wild-type nicotine NicA2 reaction Oh N- methyl Marks Yasmin nicotine oxide and false.
  • the nicotine dehydrogenase mutant NicA2-M9 enzyme activity was determined in the same manner as the NicA2 wild type enzyme activity assay.
  • the final concentration of the immobilized NicA2-M9 protein was 10 nM
  • the concentration of the substrate nicotine was diluted from the highest concentration of 2 ⁇ M to the lowest concentration of 12.5 nM
  • the protein and substrate were incubated at 30 ° C for 20 minutes, and the reaction was inactivated with 2.5 volumes of acetonitrile.
  • the protein pellet was removed by centrifugation at 12,000 rpm for 2 min. Inject the sample into the LC-MS instrument, select the peaks of the nuclear-to-mass ratios 161 and 179, and record the peak area automatically annotated by the instrument. Use the OriginPro8 to plot the Michaelis-Menten equation curve, as shown in Figures 7 and 8.
  • Standard binding curves product calculate K m and k cat values
  • NicA2-M9 catalytic reaction of N- methyl nicotine Marks Oh Yasmin nicotine oxide and false.
  • Table 1 shows the K m and k cat values of the wild-type and NicA2 NicA2-M9 on the substrate to yield N- methyl-catalyzed oxidation and false Marks Oh Yasmin nicotine.
  • NicA2-M9 catalyze the formation of methyl N- Marks Oh Yasmin K m is the wild-type NicA2 1.96 times, the catalytic oxidation of nicotine false NicA2 K m is 2.5 times the wild-type; however NicA2-M9 catalyze the formation of N
  • the k cat of methyl hydrazine is 3.76 times that of the wild type of NicA2, and the k cat catalyzing the pseudo-oxidation of nicotine is 3.67 times that of the wild type of NicA2.
  • Example 6 Comparative analysis of nicotine dehydrogenase mutant properties and blood concentration of nicotine
  • Example 5 The results of Example 5 indicate that the rate of nicotine catalyzed by NicA2-M9 is much greater than that of NicA2 wild-type protein, while the affinity of NicA2-M9 for nicotine is slightly weaker than that of NicA2 wild-type protein. Subsequently, the affinity of NicA2-M9 for nicotine (K m ) was compared with the concentration of nicotine in the blood, as shown in Table 3, and it was found that even NicA2-M9 had a slightly weaker affinity for nicotine than NicA2 wild. type proteins, K m NicA2-M9 catalytic nicotine concentration range lower than most of the nicotine in the blood.
  • the NicA2 wild type and NicA2-M9 gene sequences were ligated into the shuttle plasmid pME6032 to obtain the pME6032-NicA2 plasmid and the pME6032-NicA2-M9 plasmid.
  • the pME6032 plasmid can respond well to the induction of isopropylthiogalactoside.
  • Recombinant plasmid was electroporated into Pseudomonas putida S16, and electroporated pME6032 empty plasmid, coupled with NicA2-M9 gene sequence and downstream catalyzed pseudo-oxidized nicotine protein pseudo-oxidized nicotine AO gene sequence recombinant fragment (false nicotine AO can efficiently catalyze false
  • the pME6032-NicA2-M9+pNAO plasmid of nicotine oxide was used as two sets of controls, respectively.
  • Pseudomonas putida S16 transformed with the above four plasmids was cultured in an inorganic salt medium containing nicotine as the sole carbon and nitrogen source.
  • OD 600 value was 0.6
  • 3 mg/mL nicotine and 0.8 mM isopropyl sulfide were added.
  • the galactosides induce protein expression.
  • the experimental results confirmed that the pME6032-NicA2-M9 plasmid was electroporated (pME6032-NicA2-M9 plasmid) by electroporation into the pME6032 empty plasmid and the pME6032-NicA2 plasmid (i.e., the legend s16 (NicA2-WT) of Fig. 9). That is, after the legend s16 (NicA2-9MT) of Fig. 9 is shown, the release amount of pseudo-oxidized nicotine is remarkably improved.
  • the product release channel of wild-type NicA2 is blocked by the nine large amino acids described above. From the mutations of the various combinations of the nine large amino acids, it can be seen from Fig. 10 that the NicA2-M3V mutant (B), the NicA2-M3A mutant (C), the NicA2-M5 mutant (D), The product release channels of the NicA2-M7 mutant (E) and the NicA2-M9 mutant (F) were broadened to varying degrees. In combination with the above related activity experiments against the NicA2-M9 mutant, those skilled in the art will appreciate that the above mutations can improve the product release and improve the reaction efficiency after replacing the large amino acid released by the product with a smaller amino acid. effect.
  • Nicotine dehydrogenase contains 9 large amino acids that hinder the release of substrates. This phenomenon is common among members of the monoamine oxidase family.
  • the sequence alignment software Vector NTI was used to compare the amino acid sequence of NicA2 with the amino acid sequences of 10 proteins including monoamine oxidase A (MAO-A) and monoamine oxidase B (MAO-B) in the monoamine oxidase family. The results are shown in the figure. 11 is shown. Nine large amino acids that block NicA2 inhibition of substrate release were found to be somewhat conserved with family members (see arrows in Figure 11). The amino acid alignment revealed that the amino acids corresponding to the 9 large amino acids and family members of NicA2 hindering the release of the substrate were almost all amino acids with side chains.
  • Monoamine oxidase is a mitochondrial transmembrane protein that is responsible for the transmission of neurotransmitters.
  • An important substrate for monoamine oxidase A is serotonin, which catalyzes the further conversion of serotonin to melatonin. It is worth noting that serotonin is highly contained in the cerebral cortex and synapses, which can excite the body, while melatonin is a tranquilizer and a substance that induces sleep; the important substrate of monoamine oxidase B is tetrahydropyridine, which catalyzes The product is a neurotoxin that produces Parkinson's disease.
  • the substrates of monoamine oxidase A and monoamine oxidase B, especially the products are small but functionally small molecules. It is conceivable that the protein will embed the product release channel with large amino acids, which is actually a product. Controllable release mechanism.

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Abstract

提供了一种人工酶，是对天然尼古丁脱氢酶的序列改良制得，改良包括将阻碍产物释放的氨基酸中的至少一个替换为侧链更小的氨基酸，以提高催化速率。

Description

一种酶及其应用 技术领域
本发明属于蛋白质工程领域，涉及一种蛋白质突变体，尤其涉及一种尼古丁脱氢酶突变体，及其在开发治疗尼古丁成瘾方面蛋白质药物等方面的应用。
背景技术
尼古丁(Nicotine，1-甲基-2-[3-吡啶]-吡咯烷)是烟叶、卷烟烟气及烟草废弃物中的主要有害成分之一，不仅直接有害于健康，而且是烟草中主要致癌成分烟草特有亚硝胺(TSNA)的重要前体物。我国烟叶生产中每年都有大量的高尼古丁烟叶产生，不仅严重影响了烟叶的吸昧品质，而且增加了烟叶的有害性。降低烟草和环境中尼古丁的含量，对于维护人类的健康和保护生态环境有着极为重要的意义。
利用微生物降解尼古丁是降低烟叶和环境中尼古丁含量和有害性的一条新途径，特别是假单胞菌属(Pseudomonas)和节杆菌属(Arthrobacter)能将尼古丁代谢分解以产生其生长所必需的碳源、氮源及能量。已发现的可代谢尼古丁的细菌有假单胞菌属(如Pseudomonas sp.No.41，P.convexa PC1，P.putida)、节杆菌属(如Arthobacter oxidans P-34，后来重新鉴定为A.ureafaciens；A.oxidans pAO1，后来重新鉴定为Arthrobacter nicotinoborans)、纤维单胞菌属(Cellulomonas sp.)以及苍白杆菌属(Ochrobactrum intermedium)。真菌主要有刺孢小克银汉霉(Cunninghamella echinulata)、石膏样小孢子菌(Microsporum gypseum)、灰色链霉菌(Streptomyces griseus)、钝顶螺旋藻(S.platenses)和丝核薄膜革菌(Pelliculariafilamentosa)。
已经发现微生物代谢尼古丁主要通过3种途径：吡啶环途径(Pyridinepathway)，主要以节杆菌(Arthrobacter sp.)为主；吡咯环途径(Pyrrolidine pathway)，主要以假单胞菌属(Pseudomonas sp.)为主；去甲基化途径(Me pathway)，主要存在于真菌当中。在吡咯环途径中，尼古丁的吡咯环被氧化，生成甲基麦斯明烟草碱(methylmyosmine)，当加入水后，甲基麦斯明烟草碱开环形成假氧化型尼古丁，进而去甲基化，形成3-羧酸吡啶盐，吡啶环的6’位置再羟基化，形成6-羟,3-羧酸吡啶盐，脱去侧链形成2,5-二羟基-吡啶盐和琥珀酸盐。此代谢途径也可从6-羟-假氧化型尼古丁开始，最终仍生成2,5-二羟基-吡啶盐和琥珀酸盐。
尼古丁脱氢酶NicA2是最近从恶臭假单胞菌S16(Pseudomonas putida S16)分离出的能降解尼古丁的酶。有关尼古丁脱氢酶NicA2的报道可参见：PLoS Genet. 2013Oct；9(10):e1003923.doi:10.1371/journal.pgen.1003923.Epub 2013Oct 24.Systematic unraveling of the unsolved pathway of nicotine degradation in Pseudomonas.Tang H,Wang L,Wang W,Yu H,Zhang K,Yao Y,Xu P。
尼古丁脱氢酶NicA2催化尼古丁降解第一步的脱氢反应，将尼古丁(I)转化为N-甲基唛喔思明(N-methymyosmine)(II)，N-甲基唛喔思明可进一步自发水合生成假氧化尼古丁(pseudooxynicotine)(III)。
Figure PCTCN2017078912-appb-000001
NicA2由nicA2基因编码，在恶臭假单胞菌S16(Pseudomonas putida S16)代谢尼古丁的降解途径中发挥至关重要的作用，敲除nicA2基因，则恶臭假单胞菌S16无法以尼古丁作为唯一的碳氮源生长(PLoS Genet.10.1371/journal.pgen.1003923)。
NicA2隶属于单胺氧化酶(monoamine oxidase,MAO)家族，MAO主要存在于线粒体外膜上，其在机体内的主要作用是催化内源性和外源性单胺类物质的代谢，在MAO的作用下单胺类物质被氧化而产生脱氨基作用。根据MAO的作用底物、分布位置和选择性抑制剂不同，可将MAO分成两类，即MAO-A和MAO-B。MAO-A对5-羟色胺(5-HT)、去甲肾上腺素(NE)、多巴胺(DA)具有高亲和性(J.Mol.Biol.338,103–114)；而MAO-B对苯乙基胺(PEA)、苯甲胺等具有高亲和性(Proc.Natl.Acad.Sci.USA 100,9750–9755)。
NicA2在恶臭假单胞菌降解尼古丁的起始，其催化尼古丁并生成假氧化尼古丁，为整条代谢途径的顺畅进行提供了起始的动力。许平课题组对尼古丁代谢通路的研究已经证实了后续的6-羟基-3-琥珀酰吡啶单加氧酶(HspB)和2,5-二羟基吡啶双加氧酶(HPO)可以进一步催化尼古丁脱酸、开环，进而形成简单的结构，最终进入三羧酸循环(J.Biol.Chem.42:29158–29170；Sci.Rep./3-3235/DOI:10.1038/srep03235)。所以说，NicA2对尼古丁的第一步加工，开启了后续的酶催化乃至整条代谢途径的高效运行，NicA2催化效率的高低，是决定整条尼古丁代谢效率的关键点，也是后续代谢途径构建的重要所在。2015年发表在《JACS》上的《Anewstrategy for smoking cessation:characterization of a bacterial enzyme for thedegradation of nicotine》一文首次报道了NicA2对于治疗香烟成瘾的可能性。该文指出，NicA2对于底物尼古丁的催化具有高效性和稳定性，即使在鼠血清中，37℃下也能维持较高的活性。同时，NicA2对于底物尼古丁具有高亲和性，它催化尼古丁降解的Km值为91.9nM，而血液中的尼古丁最高含量在162-370nM之间，所以理论上，如果NicA2能成功在人体内发挥降解尼古丁的作用，那么人体中的尼古 丁含量对于NicA2来说，则是饱和状态。用尼古丁脱氢酶开发蛋白质药物的关键是高效的尼古丁脱氢酶，酶的活性越高开发的药物用量就可以越低，效果也会越好，野生型存在催化速率的局限性，如果能够用蛋白质工程的方法对其进行提高效率的改造将为开发利用尼古丁脱氢酶带来极大的便利。
因此，本领域的技术人员致力于开发一种催化速率高的尼古丁脱氢酶，及其在生物催化、代谢工程改造和蛋白质药物开发方面的应用。
发明内容
有鉴于现有技术的上述缺陷，本发明所要解决的技术问题是改良尼古丁脱氢酶获得催化速率高的尼古丁脱氢及其应用。
为实现上述目的，本发明的一方面提供了一种人工酶。
在本发明的一个具体实施方式中，该人工酶基于天然酶的序列改良制得，该天然酶具有催化如下所示的从化合物Ⅰ转变成化合物Ⅱ的反应的活性，
Figure PCTCN2017078912-appb-000002
或者，该天然酶为尼古丁脱氢酶；
上述改良包括：将阻碍产物释放的氨基酸中的至少一个替换为侧链更小的氨基酸。
进一步地，上述天然酶还具有以下一种或多种特征：
1)该天然酶的氨基酸序列包括与SEQ ID NO:1所示的氨基酸序列具有85％以上、或90％以上、或95％以上、或98％以上、或99％以上同源性的氨基酸序列；或者该天然酶的氨基酸序列包括SEQ ID NO:1所示的氨基酸序列；或者该天然酶的氨基酸序列如SEQ ID NO:1所示；
2)该天然酶由在高严谨条件与编码氨基酸序列为SEQ ID NO:1所示的蛋白质的核酸的互补链杂交的核酸编码；
3)该天然酶存在于假单胞菌属(Pseudomonas)的微生物中。
进一步地，该天然酶存在于恶臭假单胞菌S16(Pseudomonas putida S16)中。
进一步地，该天然酶具有用于产物释放的产物释放通道，侧链位于该天然酶的产物释放通道中的氨基酸包括色氨酸、酪氨酸、苯丙氨酸、谷氨酸、甲硫氨酸中的至少一个；上述改良包括：将侧链位于天然酶的产物释放通道中的上述色氨酸、酪氨酸、苯丙氨酸、谷氨酸、甲硫氨酸中的至少一个替换为侧链更小的氨基酸。
进一步地，侧链位于该天然酶的产物释放通道中氨基酸至少包括9个氨基酸，该9个氨基酸选自色氨酸、酪氨酸、苯丙氨酸、谷氨酸和甲硫氨酸。
进一步地，该天然酶的三维结构至少包括位于释放通道处的一个β折叠和一个α螺旋，该9个氨基酸中的3个氨基酸位于β折叠，按相对位置算，分别为353位的苯丙氨酸、355位的苯丙氨酸和364位的色氨酸；该9个氨基酸中的6个氨基酸位于α螺旋，按相对位置算，分别为163位的苯丙氨酸、214位的酪氨酸、218位的酪氨酸、242位的酪氨酸、246位的甲硫氨酸和249位的谷氨酸。
进一步地，上述替换的方式为：如果需要替换的氨基酸位于β折叠上，则将其替换为缬氨酸；如果需要替换的氨基酸位于α螺旋上，则将其替换为丙氨酸。
优选地，上述改良包括：将阻碍产物释放的分子量为120以上的氨基酸全部替换为侧链更小的氨基酸。
本发明的另一方面提供一种酶，在一个具体实施方式中，该酶具有以下一种或多种特征：
1)该酶的氨基酸序列包括与SEQ ID NO:2所示的氨基酸序列具有85％以上、或90％以上、或95％以上、或98％以上、或99％以上的同源性的氨基酸序列；或者该酶的氨基酸序列包括SEQ ID NO:2所示的氨基酸序列；或者该酶的氨基酸序列如SEQ ID NO:2所示；
2)该酶由在高严谨条件与编码氨基酸序列为SEQ ID NO:2所示的蛋白质的核酸的互补链杂交的核酸编码；
3)该酶的氨基酸序列包括如SEQ ID NO:2所示的氨基酸序列被保守取代的氨基酸序列；
其中，按相对位置算，该酶的第353位氨基酸、第355位氨基酸和第364位氨基酸中的至少一个为第一小分子量氨基酸；该酶的第163位氨基酸、第214位氨基酸、第218位氨基酸、第242位氨基酸、第246位氨基酸和第249位氨基酸中的至少一个为第二小分子量氨基酸，第一小分子量氨基酸的分子量在140以下，第二小分子量氨基酸的分子量在110以下。
进一步地，该酶具有催化如下所示的从化合物Ⅰ转变成化合物Ⅱ的反应的活性：
Figure PCTCN2017078912-appb-000003
或者，该酶为尼古丁脱氢酶。
进一步地，第353位氨基酸、第355位氨基酸和第364位氨基酸位于β折叠中；第163位氨基酸、第214位氨基酸、第218位氨基酸、第242位氨基酸、第246位氨基酸和第249位氨基酸位于α螺旋中。
进一步地，第一小分子量氨基酸为缬氨酸，第二小分子量氨基酸为丙氨酸。
优选地，第353位氨基酸、第355位氨基酸和第364位氨基酸全部为第一小分子量氨基酸；第163位氨基酸、第214位氨基酸、第218位氨基酸、第242位氨基酸、第246位氨基酸和第249位氨基酸全部为第二小分子量氨基酸。
本发明的另一方面提供一种酶，在一个具体实施方式中，该酶具有催化如下所示的从化合物Ⅰ转变成化合物Ⅱ的反应的活性，
Figure PCTCN2017078912-appb-000004
或者该酶为尼古丁脱氢酶；
该酶具有用于产物释放的产物释放通道，侧链位于该产物释放通道的最窄处的氨基酸的分子量在140以下。
优选地，侧链位于产物释放通道的最窄处的氨基酸的分子量在120以下。
优选地，侧链位于产物释放通道的最窄处的氨基酸为缬氨酸或丙氨酸。
本发明的另一方面提供编码上述人工酶或酶的核苷酸序列。
进一步地，上述核苷酸序列具有以下一种或多种特征：
1)该核苷酸序列包括与SEQ ID NO:3所示的核苷酸序列具有85％以上、或90％以上、或95％以上、或98％以上、或99％以上的同源性的核苷酸序列；或者该核苷酸序列包括SEQ ID NO:3所示的核苷酸序列；或者该核苷酸序列如SEQ IDNO:3所示；
2)该核苷酸序列在高严谨条件能与SEQ ID NO:3所示的核苷酸序列的互补链杂交。
本发明的又一方面提供一种含有上述核苷酸序列的表达载体或宿主细胞。
本发明的再一方面提供上述人工酶或酶用于降解尼古丁的应用。
本发明还提供了上述人工酶或酶在制备治疗尼古丁成瘾的药物中的应用。
本发明还提供了上述人工酶或酶在制备用于生物催化转化的酶制剂中的用途。
本发明还提供了上述人工酶或酶在代谢工程改造和合成生物学中的用途。
本发明的还有一方面提供了一种改良单胺氧化酶家族的酶的方法，该方法包括：将阻碍产物释放的氨基酸的至少一个替换为侧链更小的氨基酸。
本发明通过蛋白质工程的方法，从野生型尼古丁脱氢酶出发，通过晶体结构分析，获得影响产物释放的相关结构，通过氨基酸置换的方式消除相关氨基酸对产物释放的影响，从而提高突变体酶的催化速率。本发明的较佳实施例的尼古丁脱氢酶突变体虽然亲和性略弱于野生型尼古丁脱氢酶，但是其催化底物尼古丁的速率远大于野生型尼古丁脱氢酶，是其3.67倍。
本发明首次解析了尼古丁脱氢酶的结构，并获得了尼古丁脱氢酶和底物尼古丁结合的结构。通过对上述结构的分析及相关实验，获得了阻碍产物释放的9个大型氨基酸残基，从而为后续突变获得高催化效率的尼古丁脱氢酶奠定了良好的基础。此外，上述阻碍产物释放的9个大型氨基酸残基，在单胺氧化酶家族的其他成员中具有一定的保守性，这也为改良单胺氧化酶家族成员提供了良好的基础。
本发明的尼古丁脱氢酶突变体至少可在生物催化、代谢工程和合成生物学以及蛋白质药物的开发等方面进行应用，具有很大的商业应用前景：
(a)生物催化：本发明获得的尼古丁脱氢酶突变体具有比天然尼古丁脱氢酶优良的催化性质，可用于获得更高的催化尼古丁成为产物的催化特性，因而可以用于开发酶制剂用于生物的催化转化；
(b)代谢工程和合成生物学：上述获得突变体和天然尼古丁脱氢酶催化的反应相同，但是具有更高催化效率，因此可以用于进行代谢工程改造和合成生物学用于开发高效的代谢途径，提高菌株的尼古丁的转化能力；
(c)开发蛋白质药物：上述获得的突变体可以用于转化尼古丁最终生成假氧化尼古丁，并且比天然蛋白具有更好的催化转化能力，因此可以用于高效的转化血液中的尼古丁，从而用于开发与治疗尼古丁成瘾相关的药物。
以下将结合附图对本发明的构思、具体步骤及产生的技术效果作进一步说明，以充分地了解本发明的目的、特征和效果。
附图说明
图1是本发明的一个较佳实施例的纯化出的NicA2蛋白的聚丙烯酰胺凝胶电泳图。其中，泳道1：破菌后的上清；泳道2：破菌后的沉淀；泳道3：过柱流出液；泳道4：20mM咪唑洗涤液；泳道5：50mM咪唑洗涤液；泳道6：80mM咪唑洗脱液；泳道7：170mM咪唑洗脱液；泳道8：300mM咪唑洗脱液；M：蛋白分子量标记。
图2是本发明的一个较佳实施例的NicA2蛋白经过晶体初筛、沉淀剂优化、添加晶种、氨基酸序列截断、调整蛋白与结晶试剂的比例等一系列优化后的晶体。FAD的存在使晶体呈现明亮的黄色。
图3是本发明的一个较佳实施例的NicA2Δ20-尼古丁晶体的结构图。a：俯看产物释放通道，从晶体结构的外表面观察不到产物，说明产物完全被NicA2包埋；b：调节灰度至透明，俯看产物释放通道，产物的释放通道被9个大型氨基酸阻挡；c：产物释放通道的横截面。
图4是本发明的一个较佳实施例的纯化出的NicA2-M9蛋白的聚丙烯酰胺凝胶电泳图。其中，泳道1：破菌后的上清；泳道2：破菌后的沉淀；泳道3：过柱流出液；泳道4：20mM咪唑润洗液；泳道5：50mM咪唑除杂蛋白；泳道6：170mM 咪唑洗脱目的蛋白；M：蛋白分子量标记。
图5是本发明的一个较佳实施例的NicA2野生型催化尼古丁生成N-甲基唛喔思明的米氏方程曲线。
图6是本发明的一个较佳实施例的NicA2野生型催化尼古丁生成假氧化尼古丁的米氏方程曲线。
图7是本发明的一个较佳实施例的NicA2-M9催化尼古丁生成N-甲基唛喔思明的米氏方程曲线。
图8是本发明的一个较佳实施例的NicA2-M9催化尼古丁生成假氧化尼古丁的米氏方程曲线。
图9是本发明的一个较佳实施例运用GC检测假氧化尼古丁释放量的图。
图10是本发明的具体实施方式中产生的5个NicA2突变体与NicA2野生型的产物释放通道的横截面图的比较。其中，A：NicA2野生型；B：NicA2-M3V突变体；C：NicA2-M3A突变体；D：NicA2-M5突变体；E：NicA2-M7突变体；F：NicA2-M9突变体。
图11是NicA2氨基酸序列与单胺氧化酶家族中的蛋白进行比对的结果。
具体实施方式
本发明的一个方面是提供了一种人工酶，所述人工酶基于天然酶的序列改良制得。
本文所说的人工酶应当被理解为任何对天然酶进行改良后的酶，包括遗传工程方面的改良，例如酶突变体。
该天然酶具有催化如下所示的从化合物Ⅰ转变成化合物Ⅱ的反应的活性。
Figure PCTCN2017078912-appb-000005
或者，所述天然酶为尼古丁脱氢酶。
本文所述的具有催化上述反应的活性的酶应当被理解为：不仅指专一催化上述反应的酶，还指那些非专一催化上述反应的酶，例如，除了能催化上述反应外，还能催化6-羟基尼古丁(包括D型和L型)的脱氢反应的酶。
本文所说的“尼古丁脱氢酶”指的是能够催化尼古丁或尼古丁类似物(例如6-羟基尼古丁)的吡咯环进行脱氢反应的酶。
该改良包括：将阻碍产物释放的氨基酸中的至少一个替换为侧链更小的氨基酸。
本文所说的“阻碍产物释放的氨基酸”应当被理解为：任何能通过替换为其 它氨基酸来提高产物释放效率或酶催化效率的氨基酸。例如，如果一种氨基酸的侧链位于产物释放的通道或路径中，如果将该氨基酸替换为侧链更小的氨基酸，产物释放效率或酶催化效率能够提高，则该氨基酸为“阻碍产物释放的氨基酸”。
作为一种优选的具体实施方式，所述天然酶还具有以下一种或多种特征：
1)所述天然酶的氨基酸序列包括与SEQ ID NO:1所示的氨基酸序列具有85％以上、或90％以上、或95％以上、或98％以上、或99％以上同源性的氨基酸序列；或者所述天然酶的氨基酸序列包括SEQ ID NO:1所示的氨基酸序列；或者所述天然酶的氨基酸序列如SEQ ID NO:1所示；
2)所述天然酶由在高严谨条件与编码氨基酸序列为SEQ ID NO:1所示的蛋白质的核酸的互补链杂交的核酸编码；
3)所述天然酶存在于假单胞菌属(Pseudomonas)的微生物中。
本文所说的“同源性”可以指序列(核苷酸或氨基酸)的最佳比对，所述比对可利用算法的计算机化实施来执行。举例来说，与多核苷酸有关的“同源性”可采用BLASTN2.0版，使用默认参数进行分析而确定。与多肽(即，氨基酸)有关的“同源性”可采用程序(比如BLASTP2.2.2版)使用默认参数确定，此程序将正被比较的多肽或片段进行比对(也可比对核苷酸片段)，继而确定比较物的氨基酸同一性或相似性程度。
本文所说的“在低严谨度、中严谨度、高严谨度、或极高严谨度条件杂交”描述了杂交和洗涤的条件。进行杂交反应的指导可见于Current Protocols inMolecular Biology，John Wiley&Sons，N.Y.(1989)，6.3.1-6.3.6。此文献中描述了水相和非水相方法，任一种都可用。本文的具体杂交条件如下：1)低严谨度杂交条件是在6X氯化钠/柠檬酸钠(SSC)中在约45℃杂交，之后在0.2X SSC，0.1％SDS中至少在50℃(对于低严谨度条件，洗涤的温度应升高到55℃)洗涤两次；2)中严谨度杂交条件是在6X SSC中在约45℃杂交，之后在0.2X SSC，0.1％SDS中在60℃洗涤一或多次；3)高严谨度杂交条件是在6X SSC中在约45℃杂交，之后在0.2X SSC，0.1％SDS中在65℃洗涤一或多次；4)极高严谨度杂交条件是在0.5M磷酸钠，7％SDS中在65℃杂交，之后在0.2X SSC，1％SDS中在65℃洗涤一或多次。高严谨度条件(3)是优选的，除特别指出之外应使用该条件。
作为一种更优选的具体实施方式，所述天然酶存在于恶臭假单胞菌S16(Pseudomonas putida S16)中。恶臭假单胞菌S16的保藏号为CCTCC NO.M205038，于2005年4月18日保藏于中国典型培养物保藏中心；另外，该菌株也保藏在德国国家菌种保藏中心(DSMZ)，保藏号为DSM 28022。
作为一种优选的具体实施方式，所述天然酶具有产物释放通道，侧链位于所述天然酶的产物释放通道中的氨基酸包括色氨酸、酪氨酸、苯丙氨酸、谷氨酸、甲 硫氨酸中的至少一个；所述改良包括：将侧链位于天然酶的释放通道中的所述色氨酸、酪氨酸、苯丙氨酸、谷氨酸、甲硫氨酸中的至少一个替换为侧链更小的氨基酸。
本文所说的“产物释放通道”应当被理解为：产物从开始离开酶的反应活性中心到完全不影响酶催化反应的位置所需要经过的路径。一般情况下，酶的反应活性中心处于酶的三维结构的凹穴中，本发明所披露的人工酶或酶亦是如此，产物从开始离开酶的反应活性中心到完全不影响酶催化反应的位置所需要经过的路径至少部分地会被一些结构包裹，对于不同的酶，包裹的程度不同，因此，产物从开始离开酶的反应活性中心到完全不影响酶催化反应的位置所需要经过的路径可以被理解为“通道”的形式。
本文所说的“氨基酸的侧链位于产物释放通道”应当被理解为：从酶的三维结构来看，氨基酸的侧链对产物的释放效率或酶催化效率或多或少会产生影响。例如，如果将该氨基酸替换为侧链更小的氨基酸，产物释放效率或酶催化效率能够提高，则该氨基酸的侧链可以被认为是位于产物释放通道。
作为一种更优选的具体实施方式，侧链位于所述天然酶的产物释放通道中的氨基酸至少包括9个氨基酸，所述的9个氨基酸选自色氨酸、酪氨酸、苯丙氨酸、谷氨酸和甲硫氨酸。
作为一种更优选的具体实施方式，所述天然酶的三维结构至少包括位于所述释放通道处的一个β折叠和一个α螺旋，所述的9个氨基酸中的3个氨基酸位于所述的β折叠，按相对位置算，分别为353位的苯丙氨酸、355位的苯丙氨酸和364位的色氨酸；所述的9个氨基酸中的3个氨基酸位于所述的α螺旋，按相对位置算，分别为163位的苯丙氨酸、214位的酪氨酸、218位的酪氨酸、242位的酪氨酸、246位的甲硫氨酸和249位的谷氨酸。
本文所说的针对氨基酸序列的“按相对位置算”应当被理解为：某个氨基酸在序列中的位置编号仅代表该氨基酸的相对位置。例如，对于上述的353位的苯丙氨酸和355位的苯丙氨酸，仅仅代表前者和后者的位置相差2位；如果整个蛋白质序列在N端增加了一段氨基酸数量为x的序列，从绝对位置计算，前者的位置编号为353+x，后者的位置编号为355+x；如果整个蛋白质序列在N端减少了一段氨基酸数量为x的序列，从绝对位置计算，前者的位置编号为353-x，后者的位置编号为355-x；如果按相对位置算，“353位的苯丙氨酸”和“355位的苯丙氨酸”也应当被理解为包括这两种情况。
本发明的另一个方面是提供了一种酶，所述酶具有以下一种或多种特征：
1)所述酶的氨基酸序列包括与SEQ ID NO:2所示的氨基酸序列具有85％以上、或90％以上、或95％以上、或98％以上、或99％以上的同源性的氨基酸序列；或者所述酶的氨基酸序列包括SEQ ID NO:2所示的氨基酸序列；或者所述酶的氨 基酸序列如SEQ ID NO:2所示；
2)所述酶由在高严谨条件与编码氨基酸序列为SEQ ID NO:2所示的蛋白质的核酸的互补链杂交的核酸编码；
3)所述酶的氨基酸序列包括如SEQ ID NO:2所示的氨基酸序列被保守取代的氨基酸序列；
其中，按相对位置算，所述酶的第353位氨基酸、第355位氨基酸和第364位氨基酸中的至少一个为第一小分子量氨基酸；所述酶的第163位氨基酸、第214位氨基酸、第218位氨基酸、第242位氨基酸、第246位氨基酸和第249位氨基酸中的至少一个为第二小分子量氨基酸，所述的第一小分子量氨基酸的分子量在140以下，所述的第二小分子量氨基酸的分子量在110以下。
本文所说的“保守取代”应当被理解为：用另一种特征相似的氨基酸取代多肽中的给定氨基酸的那些取代。典型地，把以下替换视作保守取代：将如Ala、Val、Leu和Ile的脂族氨基酸替换为另一种脂族氨基酸；用Thr替换Ser，反之亦然；将如Asp或Glu的酸性残基替换为另一种酸性残基；将如Asn或Gln的含有酰胺基的残基替换为另一种含有酰胺基的残基；将如Lys或Arg的碱性残基替换为另一种碱性残基；将如Phe或Tyr的芳族残基替换为另一种芳族残基。
功能等效的氨基酸总体上是与它们替代的这些氨基酸在大小和/或特征(例如，电荷或疏水性)方面是类似的。类似特性的氨基酸可以分组如下：
(1)疏水性：His、Trp、Trp、Tyr、Phe、Met、Leu、Ile、Val、Ala；
(2)中性疏水性：Cys、Ser、Thr；
(3)极性：Ser、Thr、Asn、Gln；
(4)酸性/带负电荷的：Asp、Glu；
(5)带电荷的：Asp、Glu、Asn、Lys、His；
(6)碱性/带正电荷的：Asn、Lys、His；
(7)碱性的：Asn、Gln、His、Lys、Arg；
(8)影响链取向的残基：Gly、Pro；以及
(9)芳香族：Trp、Tyr、Phe、His。
本发明的另一个方面是提供了一种酶，所述酶具有催化如下所示的从化合物Ⅰ转变成化合物Ⅱ的反应的活性，
Figure PCTCN2017078912-appb-000006
或者所述酶为尼古丁脱氢酶；
所述酶具有用于产物释放通道，其特征在于，侧链位于所述产物释放通道的最窄处的氨基酸的分子量在140以下。
本文所说的“侧链位于所述产物释放通道的最窄处的氨基酸”应当被理解为：产物从开始离开酶的反应活性中心到完全不影响酶催化反应的位置所需要经过的路径中被阻碍最厉害的氨基酸。
以下结合具体实施例进一步详述本发明。
本发明使用的材料，若无特别说明，均可通过直接购买获得。本发明中使用的实验方法，如无特殊说明，均为本领域的常规方法。
实施例1：野生型尼古丁脱氢酶(NicA2)和NicA2氮端截短体(NicA2Δ20)的克隆、表达及纯化
1.NicA2的克隆：NicA2蛋白的全长基因序列由许平课题组先期测序获得，如SEQ ID NO:4所示。利用PCR方法，以引物NicA2-F1和NicA2-R1从恶臭假单胞菌S16(CCTCC No.M 205038)菌液中扩增出NicA2全长序列，经NcoI和XhoI酶切后，连接到pET28a载体中，载体碳端有6个组氨酸标签。其中，引物序列为：
NicA2-F1：5’-ATACCATGGTGAGTGATAAAACAAAAACAAATGAAG-3’；
NicA2-R1：5’-GTGCTCGAGGCTTAAGAGCTGCTTAACCTCCCTA-3’。
2.NicA2的表达：重组质粒pET28a-NicA2经测序正确后，转化入表达载体大肠杆菌BL21(DE3)中，挑取单菌落进行表达检测，将确定有蛋白表达的菌落于37℃，220rpm放大培养，待菌液OD600为0.6-0.8时，用0.2mM异丙基硫代半乳糖苷在16℃诱导16小时。
3.NicA2的纯化：诱导完成的菌液离心后收集菌体并重悬，经1500bar压力破碎后高速离心，用预先填充且平衡过的Ni-NTA重力柱(购自Qiagen公司，货号：30430)收集上清中的蛋白。用20mM和50mM咪唑除去非特异性结合的杂蛋白，用80mM、170mM和300mM咪唑洗脱目的蛋白。目的蛋白经superdex200层析柱(购自GE公司，型号：28-9909-44)脱咪唑，然后使用30KD超滤管(MD公司)以不超过5000rpm的转速离心，从而将目的蛋白浓缩至12mg/mL，以备点晶使用。进行聚丙烯酰胺凝胶电泳验证纯化的蛋白，如图1所示，其纯度可达到90％以上。
4.NicA2Δ20的克隆、表达及纯化：NicA氮端截短体(NicA2Δ20)按照上述方法克隆、表达及纯化获得。
其中，克隆使用的引物为：NicA2-F2和NicA2-R1，获得的重组质粒为pET28a-NicA2Δ20。
NicA2-F2：ATACCATGGCAGTCGTAACAGCAGGTGTTGCGGGA；
NicA2-R1：GTGCTCGAGGCTTAAGAGCTGCTTAACCTCCCTA
实施例2：野生型尼古丁脱氢酶(NicA2)、NicA2氮端截短体(NicA2Δ20)以及NicA2Δ20-尼古丁共晶的结晶和晶体衍射
1.NicA2的结晶：在14℃环境下，采用坐滴法对NicA2全长蛋白进行晶体培养。采用Hampton Research(货号：HR2-109)的蛋白结晶试剂盒，在48孔板上，将1μL蛋白和1μL结晶试剂(下槽液)混合点晶。大约静置一周之后，显微镜下开始观察到有晶体的生长；静置两周之后，显微镜下观察到晶体的生长停滞。晶体生长条件分别为：Index-65：0.1M Ammonium acetate,0.1M BIS-TRIS pH 5.5,17％w/v Polyethylene glycol 10,000；SaltRX2-36：1.4M Ammonium tartrate dibasic,0.1MTris pH 8.5。
2.NicA2的晶体衍射：Index-65结晶条件下长出的晶体，经X射线衍射，几乎没有衍射点，且重复实验也再无晶体长出，故而放弃优化这个条件；而SaltRX2-36结晶条件下长出的晶体，X射线衍射的分辨率约
Figure PCTCN2017078912-appb-000007
左右，且衍射点有拖尾，说明此条件下长出的晶体，内部堆叠不规律。
NicA2全长蛋白晶体初筛后的优化经历了沉淀剂浓度的优化、添加晶种优化，但得到的晶体衍射依然不佳。
3.NicA2Δ20的结晶及晶体衍射：经过序列比对，从N端截短保守性不佳的20个氨基酸。其中，序列比对为在NCBI中，和尼古丁胺氧化酶HZN6(NCBI登录号：AGH68979.1)、(S)-6-羟基尼古丁-氧化酶(NCBI登录号：AGS16700.1)以及胺氧化酶(NCBI登录号：AEJ14619.1)等蛋白加以比对。
按照实施例1中的克隆、表达和纯化方法，获得纯化的NicA2Δ20蛋白，并按上述方法进行晶体初筛。外形有改善的晶体依然出现在SaltRX2-36：1.4MAmmonium tartrate dibasic,0.1M Tris pH 8.5此结晶试剂中。NicA2Δ20的晶体随后又经过沉淀剂浓度的摸索、微接种法(micro-seeding)、改变蛋白和结晶试剂的比例等优化步骤，得到了X-射线衍射分辨率为
Figure PCTCN2017078912-appb-000008
的晶体。其中，沉淀剂的浓度为1.4M Ammonium tartrate dibasic时最佳。该晶体的显微镜观察如图2所示。
4.NicA2Δ20的硒代以及晶体衍射：
NicA2Δ20在蛋白质数据库(Protein Data Bank，PDB)中与结构已经发表的蛋白氨基酸序列对比，一致性仅为28％，简单的分子置换无法解析NicA2的结构，故而只能通过纯化硒代蛋白，通过自身氨基酸中硒原子的反常散射来确定相位，进而解析结构。
将重组质粒pET28a-NicA2Δ20转化到宿主大肠杆菌B834(DE3)中，利用实施例1中的方法纯化硒代蛋白。该宿主为甲硫氨酸缺陷菌株，在无机盐培养基中，以葡萄糖和酵母无机氮源为营养成分，添加硒代甲硫氨酸取代正常的甲硫氨酸，故而可以表达出硒代NicA2，即SeMet-NicA2Δ20。SeMet-NicA2Δ20的具体表达纯化 步骤如实施例1中所述。SeMet-NicA2Δ20经过晶体初筛，在SaltRX2-36：1.4MAmmonium tartrate dibasic,0.1M Tris pH 8.5条件中出现外观较优的晶体，在经过点晶蛋白和沉淀剂比例的优化，得到了X-射线衍射分辨率为
Figure PCTCN2017078912-appb-000009
的晶体。
5.NicA2Δ20-尼古丁共晶的结晶和晶体衍射：运用NicA2Δ20蛋白晶体浸泡较高浓度的尼古丁溶液方法来获得NicA2Δ20-尼古丁共晶。用晶体生长的缓冲液(缓冲液的成分如前文所述)稀释纯的尼古丁，形成浓度为1M的尼古丁母液，并调pH为8.5；用晶体生长的缓冲液溶解保险粉(连二亚硫酸钠)至100mM，并调pH为7.0，形成保险粉母液。在浸泡晶体的混合缓冲液中，尼古丁终浓度为10mM，保险粉终浓度为20mM。其中，保险粉溶液制造无氧环境。取晶型最优的晶体，快速浸泡入尼古丁+保险粉混合溶液中，浸泡时间为30秒，最大限度快速捕捉到NicA2与底物尼古丁结合的瞬间。浸泡尼古丁后的晶体，快速冻存于液氮中。最终得到了X-射线衍射分辨率为
Figure PCTCN2017078912-appb-000010
的NicA2Δ20-尼古丁晶体。
实施例3：尼古丁脱氢酶NicA2结构解析及分析
1.NicAΔ20的结构解析：将实施例2中获得的SeMet-NicA2Δ20晶体的X-射线衍射数据经过HKL2000软件处理，运用phenix程序，初步解析出了SeMet-NicA2Δ20结构，以硒代结构为模板，通过同晶置换，运用程序coot和ccp4对晶体结构加以精修，最终结果解析出了NicAΔ20的结构。
2.NicA2Δ20-尼古丁晶体的结构解析：将实施例2中获得的NicA2Δ20-尼古丁晶体，以之前解析出的NicAΔ20的结构为模型，运用分子置换方法，最终解析了NicA2Δ20与底物尼古丁共晶的结构，如图3所示。
3.结构分析：
2011年发表在PNAS上的《Crystallographic snapshots of the complete reactioncycle of nicotine degradation by an amine oxidase of the monoamine oxidase(MAO)family》一文，解析了另一株可高效降解尼古丁的菌株中，代谢尼古丁途径中的第一个脱氢酶6-羟基-l-尼古丁脱氢酶(6HLNO)的结构，并分析了底物6-羟基-尼古丁进入催化位点以及产物6-羟基-N-甲基唛喔思明、6-羟基-假氧化尼古丁释放的通道。
以此发现为根据，比较了NicA2与6HLNO氨基酸序列(NCBI登录号：YP_007988777.1)的相似性。纵使氨基酸序列的相似性只有28％，NicA2Δ20的晶体结构与之前报道的节杆菌降解尼古丁途径中的6HLNO结构具有一定的相似性，同时两者之间也有显著的差异。最明显的差异是二酰基甘油磷脂结合在6HLNO中，而在NicA2Δ20上不存在这一现象。NicA2Δ20的α3a和α3b螺旋与6HLNO上的对应的螺旋相比有个45度的旋转，这两个α螺旋正对尼古丁结合区域，使得NicA2Δ20与6HLNO相比，对于底物尼古丁的包裹严密程度甚于6HLNO对其底 物6-羟基-l-尼古丁(6HLN)的包裹。也就是说，脂分子的缺失使NicA2Δ20形成一个压缩更紧密的结构，使得其降解尼古丁的产物N-甲基唛喔思明和假氧化尼古丁局促于NicA2Δ20内部一个狭窄的活性位点口袋中。
基于假氧化尼古丁PNAS上的报道(Galina Kachalova et.al,Crystallographicsnapshots of the complete reaction cycle of nicotine degradation by an amine oxidase ofthe monoamine oxidase(MAO)family,[J]PNAS,108(12):4800-4805)，在NicA2Δ20结构中也找到了对应的产物释放的通道。有趣的是，底物尼古丁完全包埋在NicA2Δ20的内部，无论是从底物进入通道还是从产物释放通道向内看都找不到尼古丁的踪影(如图3的a所示)，只有将NicA2Δ20表面灰度调至透明，才能看出尼古丁深深包埋于活性位点的口袋中(如图3的b所示)。通过对产物释放通道的仔细分析，发现这一通道被9个大型氨基酸阻挡：W364(色氨酸)、Y214(酪氨酸)、Y218(酪氨酸)、F355(苯丙氨酸)、F353(苯丙氨酸)、E249(谷氨酸)、F163(苯丙氨酸)、M246(甲硫氨酸)和Y242(酪氨酸)(如图3的b和c所示)。因此推测这个极度束缚的通道很可能有力地阻挡了产物假氧化尼古丁从NicA2Δ20的释放。
另外，也通过全波长扫描实验和气象色谱(GC)实验证明NicA2结构上“完全包埋”且“极度束缚”的特点大大阻碍了产物假氧化尼古丁的释放。其中，全波长扫描实验证明NicA2催化尼古丁脱氢可以快速进行，气象色谱(GC)实验证明产物假氧化尼古丁从NicA2中的释放非常缓慢。具体如下：
先前的实验观察到，在1mL 10mg/mL NicA2蛋白溶液中加入等摩尔的底物尼古丁，NicA2蛋白溶液明亮的黄色瞬间变成无色透明，考虑到NicA2的辅基为FAD，且NicA2的功能为催化尼古丁脱氢，可以认为：NicA2催化尼古丁脱氢，并将氢传递给FAD使其氧化成FADH2这一过程是瞬间进行的。也就是说，尼古丁脱氢变成假氧化尼古丁这一反应被NicA2快速催化。UV-扫描(UV-Scan)实验检测到在NicA2溶液中加入尼古丁，FAD特征紫外吸收峰(375mm，450mm)很快消失。
随后用气相色谱法检测了体积为1ml的10mg/mL NicA2溶液中加入等摩尔的尼古丁，反应1.5个小时后检测假氧化尼古丁的总量和释放到蛋白之外的假氧化尼古丁的量。
其中，反应1.5小时之后的样品，用氯仿-苯甲醇对假氧化尼古丁进行萃取，用GC定量检测反应生成的假氧化尼古丁总量。反复过Ni-NTA柱子，直至流出液经浓度检测NicA2浓度低于0.1mg/mL，认为NicA2已经完全结合在镍柱上。用氯仿-苯甲醇对流出液中的假氧化尼古丁进行萃取，用GC定量检测获得了从NicA2中释放到溶液中的假氧化尼古丁的量。结果显示，约有75％的假氧化尼古丁仍然保留在NicA2蛋白之中。
实施例4：尼古丁脱氢酶突变体的构建
分析实施例3中得到的九个大型氨基酸在NicA2Δ20晶体结构上的位置，将位于β折叠上的氨基酸替换成缬氨酸(Valine，V)，均位于α螺旋上的氨基酸替换成丙氨酸(Alanine，A)。氨基酸替换的基本原则是用侧链基团尽量小的氨基酸替换掉侧链基团大的氨基酸，同时考虑到，丙氨酸是结构最简单的氨基酸，也是强力的α螺旋形成者；缬氨酸结构也很简单，且是较强的β形成者。最终将F163、Y214、Y218、Y242、M246和E249突变成丙氨酸，将F353、F355V和W364V突变成缬氨酸，突变体命名为NicA2-M9。通过设计突变后的DNA序列来实现上述突变，突变后的DNA序列如SEQ ID NO:3所示。
上述尼古丁脱氢酶突变体NicA2-M9的基因序列由金唯智公司合成。合成的序列经过PCR方法扩增出，扩增使用的引物为：NicA2-F1和NicA2-R1。扩增获得的突变体基因片段和载体pET28a经过NcoI和XhoI酶切，用T4连接将二者连接，获得重组质粒pET28a-NicA2-M9。该重组质粒经测序正确后转化入表达宿主大肠杆菌BL21(DE3)中进行表达。表达的尼古丁脱氢酶突变体NicA2-M9的纯化方法参照实施例1中NicA2野生型蛋白纯化方法。
将菌体破碎后的上清、沉淀，以及纯化过程中的过柱流出液、20mM咪唑润洗、50mM咪唑除杂蛋白、170mM咪唑洗脱目的蛋白进行取样，经聚丙烯酰胺凝胶电泳胶检测，结果图4所示。NicA2-M9蛋白丰度上佳，纯度90％以上，满足后续酶活实验的要求。
实施例5：野生型尼古丁脱氢酶和尼古丁脱氢酶突变体的酶活性质测定及比较
运用液相色谱-质谱联用(LC-MS)的方法通过检测产物N-甲基唛喔思明和假氧化尼古丁的量来标记NicA2野生型和NicA2突变体NicA2-M9的酶活。LC-MS参数如下：安捷伦1290液相色谱、EC-C8column(4.6×100mm,1.8μm)、0.2mL/min。正离子模式下，N-甲基唛喔思明的核质比(m/z)为161，假氧化尼古丁的核质比(m/z)为179。
N-甲基唛喔思明和假氧化尼古丁的浓度从最高浓度2μM倍比稀释到最低浓度31.25nM，上样于LC-MS仪器中，选取核质比161和179的峰，记录仪器自动注释出的峰面积。运用软件OriginPro8绘制两个产物的标准曲线。
1.野生型尼古丁脱氢酶酶活性测定：
固定NicA2野生型蛋白终浓度为10nM，底物尼古丁的浓度从最高浓度2μM开始倍比稀释至最低浓度12.5nM，蛋白和底物在30℃孵育20分钟，用2.5倍体积的乙腈灭活反应，12000rpm离心2min，去除蛋白沉淀。进样于LC-MS仪器中，选取核质比161和179的峰，记录仪器自动注释出的峰面积。运用OriginPro8绘制 米氏方程(Michaelis-Menten equation)曲线，如图5和图6所示。
结合产物的标准曲线，计算出NicA2野生型尼古丁反应生成N-甲基唛喔思明和假氧化尼古丁的Km和kcat值。NicA野生型催化尼古丁反应生成N-甲基唛喔思明的Km＝24.24nM，kcat＝6.17×10-3/s；生成假氧化尼古丁的Km＝19.71nM，kcat＝13.75×10-3/s。
2.尼古丁脱氢酶突变体酶活性测定：
尼古丁脱氢酶突变体NicA2-M9酶活性质的测定与NicA2野生型酶活测定的方法相同。固定NicA2-M9蛋白终浓度为10nM，底物尼古丁的浓度从最高浓度2μM开始倍比稀释至最低浓度12.5nM，蛋白和底物在30℃孵育20分钟，用2.5倍体积的乙腈灭活反应，12000rpm离心2min，去除蛋白沉淀。进样于LC-MS仪器中，选取核质比161和179的峰，记录仪器自动注释出的峰面积。运用OriginPro8绘制米氏方程(Michaelis-Menten equation)曲线，如图7和图8所。
结合产物的标准曲线，计算出NicA2-M9催化尼古丁反应生成N-甲基唛喔思明和假氧化尼古丁的Km和kcat值。NicA-M9催化尼古丁反应生成N-甲基唛喔思明的Km＝46.61nM，kcat＝23.20×10-3/s。
3.对比
表1示出了NicA2野生型和NicA2-M9对底物催化生成N-甲基唛喔思明和假氧化尼古丁的Km和kcat值。通过比较可以看出，NicA2-M9催化生成N-甲基唛喔思明的Km是NicA2野生型1.96倍，催化假氧化尼古丁的Km是NicA2野生型2.5倍；但是NicA2-M9催化生成N-甲基唛喔思明的kcat是NicA2野生型3.76倍，催化假氧化尼古丁的kcat是NicA2野生型3.67倍。这进一步证明了用侧链简单的氨基酸替换体积庞大的氨基酸，可以有效加快产物假氧化尼古丁的释放，提高反应的效率。
表1 NicA2野生型和NicA2突变体催化尼古丁反应速率常数
Figure PCTCN2017078912-appb-000011
实施例6：尼古丁脱氢酶突变体性质与尼古丁血液浓度比较分析
实施例5的结果表明，NicA2-M9催化尼古丁的速率远大于NicA2野生型蛋白，同时NicA2-M9对尼古丁的亲和性略弱于NicA2野生型蛋白。随后，对NicA2-M9对尼古丁的亲和性(Km)与血液中的尼古丁的浓度进行比较，如表3所示，结果发现，即使NicA2-M9对尼古丁的亲和性略弱于NicA2野生型蛋白，NicA2-M9催 化尼古丁的Km也低于血液中尼古丁的多数浓度范围。
表2 NicA2突变体对尼古丁的亲和性与血液中尼古丁最高浓度的比较
Figure PCTCN2017078912-appb-000012
实施例7：尼古丁脱氢酶突变体改造原有代谢途径
将NicA2野生型和NicA2-M9基因序列连接到穿梭质粒pME6032上，获得pME6032-NicA2质粒和pME6032-NicA2-M9质粒。其中，pME6032质粒可以较好地响应异丙基硫代半乳糖苷的诱导。重组质粒电转进恶臭假单胞菌S16，同时电转pME6032空质粒、连有NicA2-M9基因序列与下游催化假氧化尼古丁的蛋白假氧化尼古丁AO基因序列的重组片段(假氧化尼古丁AO可以高效催化假氧化尼古丁)的pME6032-NicA2-M9+pNAO质粒，分别作为两组对照。
将转化了上述4种质粒的恶臭假单胞菌S16培养在以尼古丁为唯一碳氮源的无机盐培养基中，待OD600值为0.6时，添加3mg/mL尼古丁和0.8mM异丙基硫代半乳糖苷诱导蛋白表达。收集各个样本诱导0-5小时的菌液上清2mL，经冻干后，用苯甲醇-氯仿将假氧化尼古丁萃取，上样于气象色谱仪中，通过前期绘制的假氧化尼古丁标准曲线，检测假氧化尼古丁的量。
如图9所示，实验结果证明，相比于电转入pME6032空质粒和pME6032-NicA2质粒(即图9的图例s16(NicA2-WT)所示)，电转入pME6032-NicA2-M9质粒(即图9的图例s16(NicA2-9MT)所示)后，使假氧化尼古丁的释放量明显提升。而NicA2-M9与假氧化尼古丁AO重组(即图9的图例s16(NicA2-9MT+pNAO)所示)，假氧化尼古丁AO将假氧化尼古丁降解，则可以使假氧化尼古丁的释放量显著减少。这就进一步证实，假氧化尼古丁的释放量的增多，是由NicA2蛋白的改造带来的。
实施例8：
类似于实施例4，分析实施例3中得到的九个大型氨基酸在NicA2Δ20晶体结构上的位置，进行了如下突变：
(1)将F353、F355和W364突变成缬氨酸，突变体命名为NicA2-M3V；
(2)将F353、F355和W364突变成丙氨酸，突变体命名为NicA2-M3A；
(3)将F353、F355和W364突变成缬氨酸，将Y214和Y218突变成丙氨酸，突变体命名为NicA2-M5；
(4)将F353、F355和W364突变成缬氨酸，将Y214、Y218、F163和E249突变成丙氨酸，突变体命名为NicA2-M7。
从图10的A中可以看出，野生型NicA2的产物释放通道被前文所述的9个大型氨基酸所阻碍。通过对该9个大型氨基酸的多种不同组合的突变，从图10中可以看出，NicA2-M3V突变体(B)、NicA2-M3A突变体(C)、NicA2-M5突变体(D)、NicA2-M7突变体(E)以及NicA2-M9突变体(F)的产物释放通道均有不同程度的变宽。结合上文针对NicA2-M9突变体的相关活性实验，本领域技术人员可以了解，上述突变将阻碍产物释放的大型氨基酸替换成较小的氨基酸后，可以起到加快产物释放、提高反应效率的良好效果。
本领域的技术人员还可知，只要将阻碍产物释放的大型氨基酸部分或全部替换为较小的氨基酸，就可以实现提高NicA2的产物释放速度。实施例中列举的突变仅为举例说明。
实施例9：尼古丁脱氢酶含有阻碍底物释放的9个大型氨基酸这一现象在单胺氧化酶家族成员间的共性
使用序列比对软件Vector NTI将NicA2的氨基酸序列与单胺氧化酶家族中包括单胺氧化酶A(MAO-A)、单胺氧化酶B(MAO-B)在内的共10个蛋白的氨基酸序列进行了比对，结果如图11所示。发现NicA2阻碍底物释放的9个大型氨基酸与家族成员具有一定的保守性(参见图11中箭头所指之处)。氨基酸的比对发现，NicA2阻碍底物释放的9个大型氨基酸与家族成员间对应的氨基酸几乎也都是侧链庞大的氨基酸。
单胺氧化酶A和单胺氧化酶B的结构已经报道，结构分析推测它们的底物也包埋在芳香族氨基酸组成的“洞穴”中，这一点与NicA2极其相似(J.Mol.Biol.338,103–114；Proc.Natl.Acad.Sci.USA 100,9750–9755)，这使得我们对单胺氧化酶A和单胺氧化酶B产物通道已有大型氨基酸阻隔这个现象进行了合理的阐释。单胺氧化酶是线粒体跨膜蛋白，主要负责神经递质的传递。单胺氧化酶A的重要底物是五羟色胺，它催化五羟色胺进一步转化为褪黑素。值得注意的是，五羟色胺在大脑皮层和神经突触内含量很高，可以使机体兴奋，而褪黑素则是镇定剂和诱发睡眠的物质；单胺氧化酶B的重要底物是四氢吡啶，其催化产物是产生帕金森病的神经毒素。
与NicA2相似，单胺氧化酶A和单胺氧化酶B的底物，尤其产物都是微量但是功能显著的小分子，可以想象的是，蛋白将产物释放通道用大型的氨基酸进行包埋，实际上是一种对于产物的可控释放机制。
以上详细描述了本发明的较佳具体实施例。应当理解，本领域的普通技术无需创造性劳动就可以根据本发明的构思作出诸多修改和变化。因此，凡本技术领域中技术人员依本发明的构思在现有技术的基础上通过逻辑分析、推理或者有限的实验可以得到的技术方案，皆应在由权利要求书所确定的保护范围内。

Claims (24)

  1. 一种人工酶，所述人工酶基于天然酶的序列改良制得，所述天然酶具有催化如下所示的从化合物Ⅰ转变成化合物Ⅱ的反应的活性，
    Figure PCTCN2017078912-appb-100001
    或者，所述天然酶为尼古丁脱氢酶；
    其特征在于，所述改良包括：将阻碍产物释放的氨基酸中的至少一个替换为侧链更小的氨基酸。
  2. 如权利要求1所述的人工酶，其中，所述天然酶还具有以下一种或多种特征：
    1)所述天然酶的氨基酸序列包括与SEQ ID NO:1所示的氨基酸序列具有85％以上、或90％以上、或95％以上、或98％以上、或99％以上同源性的氨基酸序列；或者所述天然酶的氨基酸序列包括SEQ ID NO:1所示的氨基酸序列；或者所述天然酶的氨基酸序列如SEQ ID NO:1所示；
    2)所述天然酶由在高严谨条件与编码氨基酸序列为SEQ ID NO:1所示的蛋白质的核酸的互补链杂交的核酸编码；
    3)所述天然酶存在于假单胞菌属(Pseudomonas)的微生物中。
  3. 如权利要求2所述的人工酶，其中，所述天然酶存在于恶臭假单胞菌S16(Pseudomonas putida S16)中。
  4. 如权利要求1所述的人工酶，其中，所述天然酶具有用于产物释放的产物释放通道，侧链位于所述天然酶的产物释放通道中的氨基酸包括色氨酸、酪氨酸、苯丙氨酸、谷氨酸、甲硫氨酸中的至少一个；所述改良包括：将侧链位于天然酶的产物释放通道中的所述色氨酸、酪氨酸、苯丙氨酸、谷氨酸、甲硫氨酸中的至少一个替换为侧链更小的氨基酸。
  5. 如权利要求4所述的人工酶，其中，侧链位于所述天然酶的产物释放通道中氨基酸至少包括9个氨基酸，所述的9个氨基酸选自色氨酸、酪氨酸、苯丙氨酸、谷氨酸和甲硫氨酸。
  6. 如权利要求5所述的人工酶，其中，所述天然酶的三维结构至少包括位于所述释放通道处的一个β折叠和一个α螺旋，所述的9个氨基酸中的3个氨基酸位于所述的β折叠，按相对位置算，分别为353位的苯丙氨酸、355位的苯丙氨酸和364位的色氨酸；所述的9个氨基酸中的6个氨基酸位于所述的α螺旋，按相对位置算，分别为163位的苯丙氨酸、214位的酪氨酸、218位的酪氨酸、242位的酪 氨酸、246位的甲硫氨酸和249位的谷氨酸。
  7. 如权利要求1所述的人工酶，其中，所述替换的方式为：如果需要替换的氨基酸位于β折叠上，则将其替换为缬氨酸；如果需要替换的氨基酸位于α螺旋上，则将其替换为丙氨酸。
  8. 如权利要求1所述的人工酶，其中，所述改良包括：将阻碍产物释放的分子量为120以上的氨基酸全部替换为侧链更小的氨基酸。
  9. 一种酶，其特征在于，所述酶具有以下一种或多种特征：
    1)所述酶的氨基酸序列包括与SEQ ID NO:2所示的氨基酸序列具有85％以上、或90％以上、或95％以上、或98％以上、或99％以上的同源性的氨基酸序列；或者所述酶的氨基酸序列包括SEQ ID NO:2所示的氨基酸序列；或者所述酶的氨基酸序列如SEQ ID NO:2所示；
    2)所述酶由在高严谨条件与编码氨基酸序列为SEQ ID NO:2所示的蛋白质的核酸的互补链杂交的核酸编码；
    3)所述酶的氨基酸序列包括如SEQ ID NO:2所示的氨基酸序列被保守取代的氨基酸序列；
    其中，按相对位置算，所述酶的第353位氨基酸、第355位氨基酸和第364位氨基酸中的至少一个为第一小分子量氨基酸；所述酶的第163位氨基酸、第214位氨基酸、第218位氨基酸、第242位氨基酸、第246位氨基酸和第249位氨基酸中的至少一个为第二小分子量氨基酸，所述的第一小分子量氨基酸的分子量在140以下，所述的第二小分子量氨基酸的分子量在110以下。
  10. 如权利要求9所述的酶，其中，所述酶具有催化如下所示的从化合物Ⅰ转变成化合物Ⅱ的反应的活性：
    Figure PCTCN2017078912-appb-100002
    或者，所述酶为尼古丁脱氢酶。
  11. 如权利要求9所述的酶，其中，所述的第353位氨基酸、第355位氨基酸和第364位氨基酸位于β折叠中；所述的第163位氨基酸、第214位氨基酸、第218位氨基酸、第242位氨基酸、第246位氨基酸和第249位氨基酸位于α螺旋中。
  12. 如权利要求9所述的酶，其中，所述的第一小分子量氨基酸为缬氨酸，所述的第二小分子量氨基酸为丙氨酸。
  13. 如权利要求9所述的酶，其中，所述的第353位氨基酸、第355位氨基酸和第364位氨基酸全部为第一小分子量氨基酸；所述的第163位氨基酸、第214位氨基酸、第218位氨基酸、第242位氨基酸、第246位氨基酸和第249位氨基酸 全部为第二小分子量氨基酸。
  14. 一种酶，所述酶具有催化如下所示的从化合物Ⅰ转变成化合物Ⅱ的反应的活性，
    Figure PCTCN2017078912-appb-100003
    或者所述酶为尼古丁脱氢酶；
    所述酶具有用于产物释放的产物释放通道，其特征在于，侧链位于所述产物释放通道的最窄处的氨基酸的分子量在140以下。
  15. 如权利要求14所述的酶，其中，侧链位于所述产物释放通道的最窄处的氨基酸的分子量在120以下。
  16. 如权利要求14所述的酶，其中，侧链位于所述产物释放通道的最窄处的氨基酸为缬氨酸或丙氨酸。
  17. 编码如权利要求1-16中任一所述的人工酶或酶的核苷酸序列。
  18. 如权利要求17所述的核苷酸序列，其特征在于，所述核苷酸序列具有以下一种或多种特征：
    1)所述核苷酸序列包括与SEQ ID NO:3所示的核苷酸序列具有85％以上、或90％以上、或95％以上、或98％以上、或99％以上的同源性的核苷酸序列；或者所述核苷酸序列包括SEQ ID NO:3所示的核苷酸序列；或者所述核苷酸序列如SEQ ID NO:3所示；
    2)所述核苷酸序列在高严谨条件能与SEQ ID NO:3所示的核苷酸序列的互补链杂交。
  19. 含有如权利要求17所述的核苷酸序列的表达载体或宿主细胞。
  20. 如权利要求1-16中任一所述的人工酶或酶用于降解尼古丁的应用。
  21. 如权利要求1-16中任一所述的人工酶或酶在制备治疗尼古丁成瘾的药物中的应用。
  22. 如权利要求1-16中任一所述的人工酶或酶在制备用于生物催化转化的酶制剂中的用途。
  23. 如权利要求1-16中任一所述的人工酶或酶在代谢工程改造和合成生物学中的用途。
  24. 一种改良单胺氧化酶家族的酶的方法，其特征在于，所述方法包括：将阻碍产物释放的氨基酸的至少一个替换为侧链更小的氨基酸。
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