CN116590251A - Alcohol oxidase mutant and encoding gene and application thereof - Google Patents
Alcohol oxidase mutant and encoding gene and application thereof Download PDFInfo
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- CN116590251A CN116590251A CN202310298532.7A CN202310298532A CN116590251A CN 116590251 A CN116590251 A CN 116590251A CN 202310298532 A CN202310298532 A CN 202310298532A CN 116590251 A CN116590251 A CN 116590251A
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- 108010025188 Alcohol oxidase Proteins 0.000 title claims abstract description 31
- 108090000623 proteins and genes Proteins 0.000 title claims description 27
- WVDDGKGOMKODPV-UHFFFAOYSA-N Benzyl alcohol Chemical compound OCC1=CC=CC=C1 WVDDGKGOMKODPV-UHFFFAOYSA-N 0.000 claims abstract description 44
- 239000000758 substrate Substances 0.000 claims abstract description 37
- 102220465474 Insulin-like growth factor II_R61E_mutation Human genes 0.000 claims abstract description 17
- 241000588724 Escherichia coli Species 0.000 claims abstract description 9
- 150000001413 amino acids Chemical class 0.000 claims abstract description 8
- 239000013604 expression vector Substances 0.000 claims abstract description 8
- 230000035772 mutation Effects 0.000 claims abstract description 7
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 6
- 239000000203 mixture Substances 0.000 claims abstract description 5
- 238000000034 method Methods 0.000 claims description 15
- 241000894006 Bacteria Species 0.000 claims description 12
- -1 amino, hydroxy Chemical group 0.000 claims description 6
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- 241001198387 Escherichia coli BL21(DE3) Species 0.000 claims description 2
- 125000003545 alkoxy group Chemical group 0.000 claims description 2
- 125000000217 alkyl group Chemical group 0.000 claims description 2
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- WVDDGKGOMKODPV-ZQBYOMGUSA-N phenyl(114C)methanol Chemical compound O[14CH2]C1=CC=CC=C1 WVDDGKGOMKODPV-ZQBYOMGUSA-N 0.000 abstract description 6
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- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 2
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- 239000007800 oxidant agent Substances 0.000 description 2
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- NMWCVZCSJHJYFW-UHFFFAOYSA-M sodium;3,5-dichloro-2-hydroxybenzenesulfonate Chemical compound [Na+].OC1=C(Cl)C=C(Cl)C=C1S([O-])(=O)=O NMWCVZCSJHJYFW-UHFFFAOYSA-M 0.000 description 1
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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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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C12Y—ENZYMES
- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/03—Oxidoreductases acting on the CH-OH group of donors (1.1) with a oxygen as acceptor (1.1.3)
- C12Y101/03013—Alcohol oxidase (1.1.3.13)
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Abstract
The invention relates to an alcohol oxidase mutant, which has an amino acid sequence that an amino acid composition of PcAOX generates an R61E, Y407F, W560M mutation and an N604H mutation. The invention also takes pET28a (+) as an expression vector, takes E.coli BL21 (DE 3) as an expression host to obtain mutant PcAOX, and finally obtains mutant PcAOX-EFMH with obvious benzyl alcohol substrate activity. The mutant PcAOX improves the activity of alcohol oxidase PcAOX on aromatic alcohol substrates such as benzyl alcohol, so that the alcohol oxidase PcAOX and the peroxygenase are cascaded and catalyzed to synthesize various raw materials with important application values, and a new thought and a new technical foundation are provided for green and environment-friendly synthesis of chemical raw materials.
Description
Technical Field
The invention belongs to the technical field of enzyme genetic engineering, and particularly relates to an alcohol oxidase mutant, and a coding gene and application thereof.
Background
Alcohol oxidases (AOX, EC 1.1.3.13) are a class of oxidoreductase enzymes containing flavin adenine dinucleotide (Flavin Adenin Dinucleotide, FAD) cofactors, belonging to the GMC (glucose-methanol-cholesterol) oxidoreductase superfamily. The FAD-containing oxidase has great regioselectivity and stereoselectivity in reaction because of the capability of catalyzing and oxidizing alcohols to generate corresponding aldehydes or ketones, so that the enzyme has great application value in biotechnology. The molecular oxygen required in such enzymatic processes is an inexpensive and environmentally benign oxidant that is typically oxidized to hydrogen peroxide (H 2 O 2 ). Hydrogen peroxide is also a green oxidant that can continue to be utilized by other enzymes, such as a non-specific peroxygenase cascade that catalyzes the conversion of hydrocarbons to the corresponding aldehyde or ketone or acid. The currently reported AOX has no or only low activity of aromatic alcohol substrates, which greatly limits the scope of application of AOX.
PcAOX is an alcohol oxidase derived from Phanerochaete chrysosporium. 2014 was reported for its ability to oxidize glycerol to glyceraldehyde, and then Marco w.fraaije et al have rationally engineered this property to enhance its ability to oxidize glycerol. At present, the enzyme has the capability of oxidizing cyclohexanol and phenethyl alcohol, but has weaker oxidizing capability. At a cyclohexanol substrate concentration of 50mM the reaction time was 48 hours, the conversion was only 15%. Therefore, the improvement of the oxidation capability of the PcAOX aromatic alcohol substrate has important significance for improving the industrial application potential of the enzyme, and simultaneously has important significance for the enzyme to cascade UPO to catalyze alkane to be converted into corresponding aldehyde, ketone or acid.
Disclosure of Invention
The invention aims to provide an alcohol oxidase mutant with improved aromatic alcohol oxidation catalytic activity, and a coding gene and application thereof.
The technical scheme for achieving the purpose comprises the following steps.
In a first aspect of the present invention, there is provided an alcohol oxidase mutant having an amino acid sequence in which a site mutation of R61E, Y407F, W M and N604H occurs in the amino acid composition of alcohol oxidase PcAOX.
In some of these embodiments, the amino acid sequence of the alcohol oxidase mutant is set forth in SEQ ID NO. 20.
In a second aspect of the present invention, there is provided a gene encoding the above alcohol oxidase mutant.
In some of these embodiments, the gene encoding the alcohol oxidase mutant is set forth in SEQ ID NO. 21.
In a third aspect of the present invention, there is provided a recombinant expression vector into which a gene encoding the above alcohol oxidase mutant is inserted.
In a fourth aspect of the present invention, there is provided a recombinant genetically engineered bacterium comprising a gene encoding the above alcohol oxidase mutant.
In a fifth aspect of the present invention, a method for preparing the recombinant genetically engineered bacterium is provided, wherein the encoding gene of the alcohol oxidase mutant is cloned on an expression vector, and competent cells of escherichia coli are transformed to obtain the recombinant genetically engineered bacterium.
In some of these embodiments, the expression vector is pET28a (+).
In some of these embodiments, the E.coli competent cells are E.coli TOP10 or E.coli BL21 (DE 3) competent cells.
In a sixth aspect, the invention provides the use of an alcohol oxidase mutant as described above for catalyzing a redox reaction with cyclohexanol, an aromatic alcohol, as a substrate.
In some of these embodiments, the aromatic alcohol is a naphthalene alcohol, preferably, the naphthalene alcohol is naphthalene methanol.
In some of these embodimentsIn which the aromatic alcohol is benzyl alcohol, e.g.Wherein R is 1 Selected from: hydroxy-substituted C 1 -C 4 An alkyl group; r is R 2 Selected from: H. c (C) 1 -C 3 Alkyl, amino, hydroxy, C 1 -C 3 An alkoxy group.
In some more preferred embodiments, the aromatic alcohol is benzyl alcohol, phenethyl alcohol, p-methoxybenzyl alcohol, phenylpropanol.
In order to change the substrate selectivity of alcohol oxidase PcAOX, the invention improves the activity of the alcohol oxidase PcAOX on annular substrates such as cyclohexanol, aromatic alcohol and the like, improves the industrial application value and application range of the enzyme, and can be used for catalyzing alkane to be converted into corresponding aldehyde, ketone and acid by a one-pot method through the UPO cascade of non-specific peroxygenase. The invention creatively carries out proper site-directed mutagenesis by taking PcAOX as a target protein to obtain PcAOX mutant with obvious aromatic alcohol substrate activity, such as PcAOX-EFMH. The technology provides a new possibility for the biological enzyme synthesis of important food additives, especially benzaldehyde, phenylacetaldehyde and the like, and also provides a research foundation for further researching the structural and functional relationship of the enzyme.
Drawings
Structural analysis of pcaox: (a) PcAOx and MtAAOx and PeAAOx structures are compared, pcAOx (PDB ID 6H 3G) is shown as gray, mtAAOx (PD B ID 6O 9N) is shown as light brown, and PeAAOx (PDB ID 5OC 1) is shown as light blue. (b) key positions of PcAOx catalytic centers. yellow-FAD: rose bengal-active center bottom: blue-catalytic cavity wall: brown-top of the catalytic chamber.
FIG. 2 shows the results of agarose nucleic acid gel electrophoresis detection of PcAOX mutant.
FIG. 3 SDS-PAGE detection of PcAOX purification process, wherein lane M: protein Marker, lane 1: protein sample after total bacteria disruption, lane 2: after disruption, supernatant protein sample, lane 3: protein samples precipitated after disruption, lane 4: pcAOX crude enzyme solution, lane 5:50mM imidazole eluent sample, lane 6:300mM imidazole eluent sample (PcAOX pure enzyme solution), lane 7:500mM imidazole eluent sample.
FIG. 4 shows an analysis of purified SDS-PAGE of PcAOX mutants, wherein lane M: protein markers; lanes 1-13: R61G, R61E, R61D, R61S, R61N, R61E/Y407F, R61E/Y407L, R61E/Y407S, R61E/Y407F/W560F, R61E/Y407F/W560M, R61E/Y407F/W560L, R61E/Y407F/W560M/N604F, R61E/Y407F/W560M/N604H.
Fig. 5 is a hydrogen peroxide standard graph.
FIG. 6 shows results of alanine scans for PcAOX wild type and its mutants.
FIG. 7 is a graph showing the time profile and conversion results for mutant PcAOX-EFMH catalyzing different substrates, wherein (A) benzyl alcohol; (B) 2-phenylethanol; (C) S-1-phenethyl alcohol; (D) 3-phenylpropanol; (E) 4-methoxybenzyl alcohol; (F) conversion of different substrates.
FIG. 8 is a construction map of plasmid pET28a (+) -PcAOX.
Detailed Description
The present invention will be described more fully hereinafter in order to facilitate an understanding of the present invention. This invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Experimental methods, in which specific conditions are not noted in the examples below, are generally carried out according to conventional conditions, for example, green and Sambrook-s.A.fourth edition, molecular cloning, A.laboratory Manual (Molecular Cloning: A Laboratory Manual), published in 2013, or according to the conditions recommended by the manufacturer. The various chemicals commonly used in the examples are commercially available.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
According to the invention, pcAOX is taken as a target protein, a total gene synthesis technology is applied to obtain a PcAOX gene (Genbank ID: HG 425201) from Phanerochaete chrysosporium, and through two aspects of the catalytic active center and the catalytic substrate channel of the enzyme, proper rational design mutation is carried out, so that an amino acid site with stronger hydrophobicity and larger amino acid residue is mutated into an amino acid with weak hydrophobicity even hydrophilicity and only lower steric hindrance, thereby improving the catalytic conversion capability of the enzyme to aromatic alcohol benzyl alcohol and the like. The invention also takes pET28a (+) as an expression vector and E.coli BL21 (DE 3) as an expression host to obtain mutant PcAOX protein, and finally obtains mutant PcAOX-EFMH with obvious benzyl alcohol substrate activity. The mutant PcAOX improves the activity of alcohol oxidase PcAOX on aromatic alcohol substrates such as benzyl alcohol, so that the mutant PcAOX and the peroxygenase are cascaded to catalyze and synthesize a plurality of raw materials (such as benzaldehyde) with important application values. The method provides a new idea and a new technical basis for green and environment-friendly synthesis of chemical raw materials.
The amino acid composition of PcAOX is as follows:
MGHPEEVDVIVCGGGPAGCVVAGRLAYADPTLKVMLIEGGANNRDDPWVYRPGIYVRNMQRNGINDKATFYTDTMASSYLRGRRSIVPCANILGGGSSINFQMYTRASASDWDDFKTEGWTCKDLLPLMKRLENYQKPCNNDTHGYDGPIAISNGGQIMPVAQDFLRAAHAIGVPYSDDIQDLTTAHGAEIWAKYINRHTGRRSDAATAYVHSVMDVQDNLFLRCNARVSRVLFDDNNKAVGVAYVPSRNRTHGGKLHETIVKARKMVVLSSGTLGTPQILERSGVGNGELLRQLGIKIVSDLPGVGEQYQDHYTTLSIYRVSNESITTDDFLRGVKDVQRELFTEWEVSPEKARLSSNAIDAGFKIRPTEEELKEMGPEFNELWNRYFKDKPDKPVMFGSIVAGAYADHTLLPPGKYITMFQYLEYPASRGKIHIKSQNPYVEPFFDSGFMNNKADFAPIRWSYKKTREVARRMDAFRGELTSHHPRFHPASPAACKDIDIETAKQIYPDGLTVGIHMGSWHQPSEPYKHDKVIEDIPYTEEDDKAIDDWVADHVETTWHSLGTCAMKPREQGGVVDKRLNVYGTQNLKCVDLSICPDNLGTNTYSSALLVGEKGADLIAEELGLKIKTPHAPVPHAPVPTGRPATQQVRLE*(SEQ ID NO.1)。
materials and methods: the plasmid pET28a (+) -PcAOX is constructed by a conventional method. Competent cells of E.coli TOP10 and BL21 (DE 3), biotechnology Co., ltd; seamless cloning kit, medium maytai and biotechnology (beijing) limited; plasmid extraction kit, division of bioengineering (Shanghai); benzyl alcohol, phenethyl alcohol, cyclohexanol, benzoic acid, cyclohexanone, phenylacetic acid, etc., available from ala Ding Shiji, inc.
The present invention will be described in further detail with reference to specific examples.
Example 1: analysis of PcAOX three-dimensional Structure and design of mutants
The analysis result of the PcAOX three-dimensional structure shows that the PcAOX three-dimensional structure has a series of amino acid residues with strong hydrophobicity and large steric hindrance at the catalytic active center, so that the PcAOX active center is difficult to accept macromolecular substrates, the alcohol hydrophilic substrates have poor binding capacity due to the strong hydrophobicity, and the PcAOX is difficult to catalyze macromolecular annular alcohols. The technical personnel screens out a plurality of key amino acid sites such as F101, W560, M103, F422, Y407 and the like in the active center according to the past experience, and further designs series mutants such as F101S, W560F, W560M, M103A, F422A, F422G, Y A and the like; furthermore, according to the catalytic application and analysis of the oxidation reduction of heterocyclic alcohols, more amino acid residues with larger steric hindrance are found in the substrate channel, which makes the enzyme substrate enter the catalytic active center and release of the product more difficult, a plurality of key sites including M59, Q60 and R61 are screened in the research, and series of mutants such as M59A, Q60A, R A and the like are further designed.
(1) Primer design: site-directed mutagenesis was performed by amplifying the PcR product, and a fixed amino acid substitution was performed at the fixed position of the PcAOX mature peptide. Primer design was performed using snapge software, and the main primers involved in the present invention are shown in the following table:
the main primer design table of the invention:
TABLE 1.1 primer Table for alanine scanning
TABLE 1-2 design primer Table for mutants
Example 2: pcAOX wild site-directed mutagenesis and engineering bacterium construction thereof
(1) PcAOX wild-type site-directed mutagenesis: according to the designed primer, site-directed mutagenesis is performed on the PcAOX wild type by PcR reaction, and the mutated PcR product is digested with restriction endonuclease Dpn I. The procedures and systems for the site-directed mutagenesis PcR reaction, the digestion procedure and the systems are shown in table 2:
TABLE 2 PcR and digestion procedure and System used in the present invention
The result of the agarose gel electrophoresis detection of the PcAOX mutant is shown in FIG. 2.
Constructing PcAOX mutant engineering bacteria: the PCR product after template digestion is transformed into competent cells of escherichia coli BL21 (DE 3) by a heat shock transformation method.
The method comprises the following specific steps: the competent cells were removed from the-80℃refrigerator and placed on ice for 5-10min, after which 2-3. Mu.L of PCR product was added after thawing, and left on ice for 30min. Taking out the standing competent cells, placing in a water bath preheated to 42 ℃, immediately placing on ice after heat shock for 90s, adding 750 mu L of LB culture medium after 2-3min, and culturing for 40min at 37 ℃ and 220 rpm. After the completion of the culture, a certain proportion of the supernatant was removed by centrifugation at 1000rpm for 3min, and competent cells were plated on LB solid plates containing 50ng/mL kanamycin and cultured for 12-16h. Monoclonal clones were picked for colony PCR reactions to verify correct transformation, and positive clones were sequenced to further verify that the mutation was correct. And finally, storing the constructed engineering bacteria in 25% glycerol at the temperature of-20 ℃ for later use.
Example 3: fermentation and purification of PcAOX mutants
(1) Fermentation of PcAOX mutant: the preserved PcAOX mutant engineering bacteria are streaked and activated, single clone is selected and cultured in LB culture medium containing 50ng/mL kanamycin, when the temperature is 37 ℃, the culture speed is 220rpm, the OD600 is about 0.8-0.9, the single clone is inoculated in 100mL LB culture medium with the inoculation amount of 2 percent, when the culture speed is about 0.9-1.0, the single clone is inoculated in 500mL TB culture medium containing 1% (w/v) glucose with the inoculation amount of 5 percent, when the culture speed is 0.9-1.0, the IPTG with the final concentration of 1mM is added, and the single clone is induced for 20 hours under the conditions of 24 ℃ and 220 rpm.
(2) Purification of PcAOX: the culture completed by fermentation was centrifuged at 4000rpm at 4℃for 30min to remove the supernatant, and the supernatant was used as a culture medium at 1:10 (w/v) buffer A (50 mM phosphate buffer, pH 7.5, 400mM NaCl,100mM KCl) was added and suspended, and the cell suspension was sonicated on ice for 20min. The crushed solution was centrifuged at 12000rpm at 4℃for 25min, and the precipitate was discarded. Uploading the supernatant after centrifugation onto a Ni nucleophilic chromatographic column fully balanced by using buffer A, eluting the impurity protein by using 40mM imidazole, eluting the target protein by using 500mM imidazole, collecting the eluted components, performing SDS-PAGE detection and activity detection, combining active protein eluates with correct molecular weight (about 73 kDa), and performing desalting column to buffer C (pH 7.5) for standby at 4 ℃.
The result of SDS-PAGE detection in the PcAOX purification process is shown in FIG. 3, and the result of SDS-PAGE analysis of the PcAOX mutant is shown in FIG. 4.
Example 4: characterization of PcAOX mutants
(1) Drawing a hydrogen peroxide concentration standard curve: 30% (w/v) hydrogen peroxide standard solution was prepared by placing hydrogen peroxide with concentration gradients of 0.1, 0.2, 0.5, 0.8, 1.0, 1.5, 3,5 and 10mM in a 1.5mL EP tube, and adding buffer solution and color development solution to the system to determine the absorbance at 515nm at each concentration. The hydrogen peroxide standard curve was plotted with the hydrogen peroxide concentration gradient on the x-axis and the absorbance at 515nm on the y-axis (see FIG. 5). The measurement system in the above method is shown in the following table:
TABLE 3 determination of hydrogen peroxide Standard Curve reaction System
| Component (A) | Component addition amount (μL) | Final concentration of the components |
| PcAOX | 0 | -- |
| Hydrogen peroxide standard solution | 50 | -- |
| HRP | 40 | 12U/mL |
| 4-aminoantipyrine | 2 | 1.5mM |
| 3, 5-dichloro-2-hydroxybenzenesulfonic acid sodium salt | 4 | 3mM |
| Buffer(pH 7.5) | 104 | |
| Totals to | 200 |
(2) Measurement of enzyme activity of PcAOX mutant: the enzyme activity is measured according to the established detection method, the enzyme adding amount is firstly optimized, the substrate consumption amount in the detection time is controlled to be not more than 5% of the total substrate amount, the reaction detection time is 5min, and the change of the light absorption value at 515nm is detected every 10s for 30 times. The reaction system is shown in the following table:
TABLE 4 AOX enzyme Activity determination reaction System
The recorded data were calculated according to the enzyme activity definition.
FIG. 6 shows results of alanine scans for mutants that promote the activity (U/mg) of a circular substrate (benzyl alcohol is an example of the invention).
Example 5
This example designed a multi-site mutant, purified multi-site mutants were obtained according to the methods of examples 1 to 3, with 4 site mutant R61E/Y407F/W560M/N604H having the best PcAOX-EFMH activity. In the following examples, the results of the efficiency of catalyzing aromatic alcohols by wild type, R61E mutant, two-site R61E/Y407F mutant, three-site R61E/Y407F/W560M mutant, 4-site R61E/Y407F/W560M/N604H mutant are illustrated.
Wherein, the amino acid composition of the mutant PcAOX-EFMH is as follows:
MGHPEEVDVIVCGGGPAGCVVAGRLAYADPTLKVMLIEGGANNRDDPWVYRPGIYVRN
MQENGINDKATFYTDTMASSYLRGRRSIVPCANILGGGSSINFQMYTRASASDWDDFKTEG
WTCKDLLPLMKRLENYQKPCNNDTHGYDGPIAISNGGQIMPVAQDFLRAAHAIGVPYSDD
IQDLTTAHGAEIWAKYINRHTGRRSDAATAYVHSVMDVQDNLFLRCNARVSRVLFDDNN
KAVGVAYVPSRNRTHGGKLHETIVKARKMVVLSSGTLGTPQILERSGVGNGELLRQLGIKI
VSDLPGVGEQYQDHYTTLSIYRVSNESITTDDFLRGVKDVQRELFTEWEVSPEKARLSSNAI
DAGFKIRPTEEELKEMGPEFNELWNRYFKDKPDKPVMFGSIVAGAFADHTLLPPGKYITMF
QYLEYPASRGKIHIKSQNPYVEPFFDSGFMNNKADFAPIRWSYKKTREVARRMDAFRGELT
SHHPRFHPASPAACKDIDIETAKQIYPDGLTVGIHMGSWHQPSEPYKHDKVIEDIPYTEEDD
KAIDDWVADHVETTMHSLGTCAMKPREQGGVVDKRLNVYGTQNLKCVDLSICPDNLGTHTYSSALLVGEKGADLIAEELGLKIKTPHAPVPHAPVPTGRPATQQVRLE(SEQ ID NO.20)。
the coding gene of the mutant PcAOX-EFMH is as follows:
ATGGGTCATCCGGAAGAAGTTGATGTTATCGTGTGCGGTGGTGGCCCGGCTGGCTGTG
TCGTTGCTGGTCGCCTGGCTTACGCAGATCCGACCCTGAAAGTTATGCTGATTGAAGGC
GGTGCCAACAATCGTGATGACCCGTGGGTTTATCGCCCGGGTATTTACGTCCGTAACAT
GCAGGAAAACGGCATCAATGATAAAGCCACCTTTTATACCGACACGATGGCAAGCTCT
TACCTGCGTGGTCGTCGCAGCATTGTTCCGTGTGCCAACATCCTGGGCGGTGGCAGTTC
CATTAATTTTCAGATGTATACCCGCGCATCAGCTTCGGATTGGGATGACTTCAAAACCG
AAGGCTGGACGTGCAAAGACCTGCTGCCGCTGATGAAACGTCTGGAAAACTACCAAAA
ACCGTGTAACAACGATACCCACGGCTACGACGGTCCGATTGCGATCTCGAATGGTGGC
CAGATTATGCCGGTCGCTCAAGATTTTCTGCGTGCCGCACATGCCATTGGTGTGCCGTA
TAGCGATGACATCCAGGATCTGACCACGGCTCATGGTGCGGAAATTTGGGCTAAATAT
ATCAATCGTCATACCGGTCGTCGCAGCGATGCAGCTACCGCATACGTGCATTCTGTTAT
GGATGTCCAGGACAACCTGTTTCTGCGTTGTAATGCGCGTGTGTCCCGCGTTCTGTTCG
ATGACAACAATAAAGCCGTTGGCGTCGCGTATGTGCCGTCACGTAACCGTACCCACGG
CGGCAAACTGCACGAAACGATTGTCAAAGCCCGCAAAATGGTGGTTCTGTCAAGCGGT
ACCCTGGGTACGCCGCAGATCCTGGAACGTAGCGGTGTTGGCAATGGTGAACTGCTGC
GCCAACTGGGTATTAAAATCGTGTCTGATCTGCCGGGCGTTGGTGAACAGTATCAAGA
CCATTACACCACGCTGAGTATTTATCGTGTCAGCAACGAATCTATCACCACGGATGACT
TTCTGCGTGGCGTCAAAGATGTGCAGCGCGAACTGTTCACCGAATGGGAAGTGTCTCC
GGAAAAAGCCCGTCTGAGCTCTAATGCCATTGATGCAGGTTTTAAAATCCGCCCGACC
GAAGAAGAACTGAAAGAAATGGGCCCGGAATTTAACGAACTGTGGAATCGCTACTTCA
AAGATAAACCGGACAAACCGGTCATGTTTGGTAGCATTGTGGCGGGCGCCTTTGCAGA
TCATACCCTGCTGCCGCCGGGTAAATACATCACGATGTTCCAGTATCTGGAATACCCGG
CGTCACGTGGCAAAATTCACATCAAATCGCAAAACCCGTATGTTGAACCGTTTTTCGAT
TCAGGTTTCATGAACAACAAAGCTGACTTCGCGCCGATTCGTTGGTCGTACAAGAAAA
CCCGCGAAGTGGCCCGTCGCATGGATGCATTTCGTGGCGAACTGACGAGTCATCACCC
GCGCTTCCATCCGGCATCCCCGGCGGCATGCAAAGATATTGACATCGAAACCGCAAAA
CAGATTTATCCGGATGGCCTGACGGTGGGCATTCACATGGGCAGTTGGCACCAACCGT
CCGAACCGTACAAACACGATAAAGTTATCGAAGACATCCCGTACACCGAAGAAGATG
ACAAAGCTATTGATGACTGGGTTGCGGATCATGTCGAAACCACGATGCACTCTCTGGG
TACCTGTGCAATGAAACCGCGTGAACAGGGTGGCGTCGTGGATAAACGCCTGAACGTG
TATGGTACGCAAAATCTGAAATGCGTTGATCTGAGTATCTGTCCGGACAACCTGGGCA
CCCATACGTACAGTTCCGCGCTGCTGGTTGGCGAAAAAGGTGCTGATCTGATTGCGGA
AGAACTGGGCCTGAAAATCAAAACCCCGCACGCCCCGGTTCCGCACGCCCCGGTCCCGACGGGTCGCCCGGCTACGCAACAAGTCCGCCTCGAG(SEQ ID NO.21)。
(1) Drawing a benzaldehyde gas phase detection standard curve:
accurately weighing and accurately preparing 1M benzaldehyde ethyl acetate solution by using a 10mL volumetric flask according to the concentration gradient (mM): gradients of 0.5, 1, 3,5, 7, 10, 15, 20, 25, 30, 50, 100 were formulated to 1mL and placed in a 2mL brown gas detection chromatographic vial for detection.
(2) PcAOX and detection of efficiency of aromatic alcohol catalysis by mutants thereof:
PcAOX and its mutant were used to catalyze the aromatic alcohol reaction according to the following reaction system, and the detection was performed using a gas phase, and chromatographic separation was performed using a KB-FFAP gas chromatography column (30 m long by 0.25mm inner diameter by 0.25 μm thick).
The method comprises the following steps: injector temperature: 250 ℃; split ratio: 30:1; detector temperature: 280 ℃; the temperature program of the column box is as follows: the initial temperature is 50 ℃, 50-200 ℃ is increased at the rate of 10 ℃ min-1, and the temperature is kept for 3min, from 200-230 ℃ for 30min -1 Is then maintained for 1min. The conversion in this study was calculated by plotting the standard curves for the corresponding substrates and products.
TABLE 6AOX catalytic series annular substrate reaction System
(one) reaction process: in a 5mL reaction flask, 25. Mu.M enzyme, 25mM substrate (5% (v/v) DMSO-assist solution, reaction temperature 30 ℃, reaction time 24h, total system 1mL, and the remainder supplemented with 50mM phosphate buffer pH 8.0. Three experiments were performed in parallel and the average was taken. The results are shown in Table 7.
TABLE 7PcAOX wild-type and mutant catalytic series substrate conversion
As can be seen from Table 7, during the course of a reasonably selected mutant of this multiple site mutation, the activity of the mutant on the series benzyl alcohol substrates increased continuously, with benzyl alcohol as an example, the conversion increased gradually from 11% to 88% of the wild type, with an increase of about 8-fold. In addition, during the engineering process, the mutant has a gradually increasing conversion capacity for S-type substrates and hardly reacts for R-type substrates, which corresponds to the wild type. The best mutant PcAOX-EFMH has equivalent conversion capability on the series of benzyl alcohol substrates, and even has 20 times (2% -39%) improvement on the conversion capability on naphthalene alcohol compared with the wild type.
(II) reaction process: 25. Mu.M enzyme, 25mM substrate (final concentration 5% (v/v) DMSO to aid in solubilization) was added to a 5mL reaction flask, the reaction temperature was 30 ℃, the total system was 1mL, the remainder was supplemented with 50mM pH 8.0 phosphate buffer, samples were taken at different reaction times for detection, and time curves were plotted for the different substrates with time on the x-axis and conversion on the y-axis. Three experiments were performed in parallel and the average was taken.
As can be seen from fig. 7: mutant PcAOX-EFMH shows better activity on substrates benzyl alcohol, 2-phenethyl alcohol, S-1-phenethyl alcohol, p-methoxybenzyl alcohol and 3-phenylpropanol (red is a product in the figure, blue is a substrate), the conversion speed is higher 4 hours before the reaction, the enzyme molecules are inactivated due to accumulation of hydrogen peroxide in the system along with the progress of the reaction time, and O 2 In combination with the consumption of (a) to gradually decrease the reaction rate. The substrate spectrum differences between the wild type and mutant PcAOX-EFMH are shown in FIG. 7F, and the preference of the mutant for benzyl alcohol substrate is evident, indicating a shift in substrate selectivity.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (10)
1. An alcohol oxidase mutant having an amino acid sequence in which a site mutation of R61E, Y407F, W560M and N604H occurs in the amino acid composition of alcohol oxidase PcAOX.
2. The alcohol oxidase mutant according to claim 1, wherein the amino acid sequence of the alcohol oxidase mutant is shown in SEQ ID NO. 20.
3. A gene encoding the alcohol oxidase mutant according to claim 1 or 2.
4. The coding gene of claim 3, wherein the nucleotide sequence is shown in SEQ ID NO. 21.
5. A recombinant expression vector into which a gene encoding the alcohol oxidase mutant according to claim 3 or 4 has been inserted.
6. A recombinant genetically engineered bacterium comprising a gene encoding the alcohol oxidase mutant of claim 3 or 4.
7. A method for constructing the recombinant genetically engineered bacterium of claim 6, which is characterized in that the encoding gene of the alcohol oxidase mutant of claim 3 or 4 is cloned on an expression vector, and competent cells of escherichia coli are transformed to obtain the recombinant genetically engineered bacterium.
8. The method for constructing recombinant genetically engineered bacteria according to claim 7, wherein the expression vector is pET28a (+), and the escherichia coli competent cell is escherichia coli BL21 (DE 3) competent cell.
9. Use of the alcohol oxidase mutant according to claim 1 or 2 for catalyzing a redox reaction of cyclohexanol, or an aromatic alcohol, as a substrate.
10. The use according to claim 9, wherein the aromatic alcohol is naphthalene methanol; or the aromatic alcohol isWherein the method comprises the steps ofR 1 Selected from: hydroxy-substituted C 1 -C 4 An alkyl group; r is R 2 Selected from: H. c (C) 1 -C 3 Alkyl, amino, hydroxy, C 1 -C 3 An alkoxy group.
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