Mutants of alcohol oxidase PcAOX and their applications

Abstract

The present invention discloses a mutant of alcohol oxidase PcAOX and its application. The amino acid sequence of the mutant of alcohol oxidase PcAOX is as shown in SEQ ID No. 3. Through multi-faceted comparative analysis of the three-dimensional structure of alcohol oxidase PcAOX, the present invention analyzes key amino acid sites and designs a series of mutants, and obtains a mutant of alcohol oxidase PcAOX that not only has alcohol oxidase activity but also has transacylase activity. By using this mutant, it becomes possible to synthesize certain special products, providing new ideas and technical basis for the green and environmentally friendly synthesis of chemical raw materials.

Description

Technical Field

[0001] The present invention belongs to the technical field of enzyme engineering. Specifically, the present invention relates to a mutant of alcohol oxidase PcAOX and its application. Background Art

[0002] Alcohol oxidases (AOX, EC 1.1.3.13) are a class of oxidoreductases containing flavin adenine dinucleotide (FAD) cofactors and belong to the GMC (glucose-methanol-choline) oxidoreductase superfamily. They can use oxygen to catalytically oxidize alcohols to generate the corresponding aldehydes or ketones and produce hydrogen peroxide. Their products are of great significance in the fields of biochemistry, food processing, etc.

[0003] Acyl transfer reaction is a reaction in which an acyl group on an acyl donor is transferred to an acyl acceptor to form a new ester or amide. This reaction widely exists in the life metabolism activities of organisms and also has important applications in the fields of organic synthesis and biosynthesis. For example, methyl 4-chlorobenzoate is used as an acyl acceptor, and 4-(2-aminoethyl)morpholine is used to synthesize the important antidepressant drug moclobemide.

[0004] PcAOX is an alcohol oxidase derived from Phanerochaete chrysosporium. It was reported in 2014 for its ability to oxidize glycerol to produce glyceraldehyde. Subsequently, Marco W. Fraaije et al. rationally modified this enzyme to enhance its catalytic ability to oxidize glycerol. At the same time, it was also found that this enzyme has catalytic functional promiscuity, not only can catalyze the oxidation of methanol, ethanol, etc. to produce hydrogen peroxide, but also can catalyze the multi-step oxidation of other linear polyols to produce the corresponding aldehydic acids or dibasic acids.

[0005] Currently, no transacylation activity of PcAOX has been found. Summary of the Invention

[0006] Based on this, one of the purposes of the present invention is to provide a mutant of alcohol oxidase PcAOX, which has transacylation activity while possessing alcohol oxidase activity.

[0007] The specific technical solutions for achieving the above invention purposes include the following:

[0008] A mutant of alcohol oxidase PcAOX, the amino acid sequence of the mutant is as shown in SEQ ID No. 3.

[0009] The present invention also provides a coding gene for a mutant of the above-mentioned alcohol oxidase PcAOX.

[0010] In some embodiments, the nucleotide sequence of the coding gene is as shown in SEQ ID No.4.

[0011] The present invention also provides the use of the above-mentioned mutant of alcohol oxidase PcAOX and / or the coding gene in catalyzing acyl transfer reactions.

[0012] The present invention also provides a recombinant expression vector inserted with the above-mentioned coding gene.

[0013] The present invention also provides a recombinant engineering strain transfected with the above-mentioned recombinant expression vector.

[0014] The present invention also provides the use of the above-mentioned recombinant expression vector or the above-mentioned recombinant engineering strain in catalyzing acyl transfer reactions.

[0015] The present invention also provides a method for catalyzing acyl transfer reactions, using the above-mentioned mutant of alcohol oxidase PcAOX for the catalytic reaction.

[0016] In some embodiments, the catalytic reaction is: catalyzing benzyl alcohol and vinyl acetate to form benzyl acetate.

[0017] In some embodiments, the catalytic reaction is: catalyzing phenethyl alcohol and vinyl acetate to form phenethyl acetate.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] Through multi-faceted comparative analysis of the three-dimensional structure of alcohol oxidase PcAOX, the present invention analyzes key amino acid sites and designs a series of mutants, obtaining a mutant of alcohol oxidase PcAOX that not only has alcohol oxidase activity but also has transacylase activity. Using this mutant makes it possible to synthesize certain special products, providing new ideas and a technical basis for the green and environmentally friendly synthesis of chemical raw materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is the SDS-PAGE result of each component during the purification process of wild-type alcohol oxidase PcAOX in Example 2 of the present invention. Among them, lane M: marker; lane 1: total bacterial lysate; lane 2: supernatant after disruption; lane 3: precipitate after disruption; lane 4: purified flow-through; lanes 5-7: pure PcAOX enzyme solutions with different concentrations.

[0021] Figure 2This is the SDS-PAGE result of each component in the purification process of the alcohol oxidase PcAOX mutant in Example 2 of the present invention. Among them, lane M: marker; lane 1: crude enzyme solution of PcAOX-VPN; lane 2: precipitate of PcAOX-VPN; lane 3: permeate of PcAOX-VPN; lane 4: pure enzyme solution of PcAOX-VPN; lane 5: crude enzyme solution of PcAOX-VPN; lane 6: precipitate of PcAOX-VPN; lane 7: permeate of PcAOX-VPN; lane 8: pure enzyme solution of PcAOX-VPN.

[0022] Figure 3 This is the standard curve of hydrogen peroxide in Example 3 of the present invention.

[0023] Figure 4 This is the reaction result diagram with benzyl alcohol as the acyl acceptor in Example 4 of the present invention.

[0024] Figure 5 This is the reaction result diagram with phenethyl alcohol as the acyl acceptor in Example 4 of the present invention.

[0025] Figure 6 This is the molecular sieve chromatogram of the PcAOX mutant (PcAOX-VPN) in Example 5 of the present invention.

[0026] Figure 7 This is the SDS-PAGE detection result diagram of the molecular sieve sample of the PcAOX mutant (PcAOX-VPN) in Example 5 of the present invention, where M is the marker, and lanes 1-11 are the samples eluted at different times.

[0027] Figure 8 This is the preliminary crystal screening result of the PcAOX mutant (PcAOX-VPN) in Example 5 of the present invention.

[0028] Figure 9 This is the schematic diagram of crystal optimization of the PcAOX mutant (PcAOX-VPN) in Example 5 of the present invention.

[0029] Figure 10 This is the shape diagram of the PcAOX mutant (PcAOX-VPN) crystal after step-by-step optimization in Example 5 of the present invention.

[0030] Figure 11 This is the structural analysis diagram of the PcAOX mutant (PcAOX-VPN) crystal in Example 5 of the present invention. Detailed implementation manners

[0031] For the convenience of understanding the present invention, the present invention will be described more comprehensively below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0032] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the description of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0033] In the present invention, the inventors used PcAOX enzyme as the target protein, obtained the PcAOX gene (Genbank ID: HG425201) from Phanerochaete chrysosporium by applying the total gene synthesis technology, constructed the pET28a(+)-PcAOX expression vector, and obtained the PcAOX protein using E. coli BL21(DE3) as the expression host. By analyzing the three-dimensional structure of the wild-type alcohol oxidase PcAOX (the nucleotide sequence is shown in SEQ ID No. 2 and the amino acid sequence is shown in SEQ ID No. 1), through structural comparison and analysis in the database, and comparing the structure and function from the catalytic active center of the enzyme, it was found that its catalytic center might have a second activity. After multi-scale sequence comparison, it was considered that the narrow catalytic channel might prevent the manifestation of the transacylase activity. Therefore, the key amino acid sites were analyzed and a series of mutants were designed to expand the original substrate catalytic channel, expose the catalytic center, and enable the transacylase activity of the enzyme to be demonstrated. Subsequently, experimental verification was carried out to characterize the properties of the mutants, and finally a mutant with dual-functional activity of alcohol oxidase activity and catalytic acyl transfer activity (hereinafter named PcAOX-VPN, the nucleotide sequence is shown in SEQ ID No. 4 and the amino acid sequence is shown in SEQ ID No. 3) was obtained, and the three-dimensional structure of the mutant was further analyzed. Existing studies have not found that the alcohol oxidase PcAOX has the activity of acyl transferase. Therefore, this is the first discovery that the oxidase of this family has the activity of acyl transferase. This provides a new idea for the discovery, exploration, design, and research of multifunctional enzymes, and also provides a research basis for further studying the structure-function relationship of such enzymes.

[0034] Amino acid sequence of wild-type alcohol oxidase PcAOX SEQ ID No.1 MGHPEEVDVIVCGGGPAGCVVAGRLAYADPTLKVMLIEGGANNRDDPWVYRPGIYVRNMQRNGINDKATFYTDTMASSYLRGRRSIVPCANILGGGSSINFQMYTRASASDWDDFKTEGWTCKDLLPLMKRLENYQKPCNNDTHGYDGPIAISNGGQIMPVAQDFLRAAHAIGVPYSDDIQDLTTAHGAEIWAKYINRHTGRRSDAATAYVHSVMDVQDNLFLRCNARVSRVLFDDNNKAVGVAYVPSRNRTHGGKLHETIVKARKMVVLSSGTLGTPQILERSGVGNGELLRQLGIKIVSDLPGVGEQYQDHYTTLSIYRVSNESITTDDFLRGVKDVQRELFTEWEVSPEKARLSSNAIDAGFKIRPTEEELKEMGPEFNELWNRYFKDKPDKPVMFGSIVAGAYADHTLLPPGKYITMFQYLEYPASRGKIHIKSQNPYVEPFFDSGFMNNKADFAPIRWSYKKTREVARRMDAFRGELTSHHPRFHPASPAACKDIDIETAKQIYPDGLTVGIHMGSWHQPSEPYKHDKVIEDIPYTEEDDKAIDDWVADHVETTWHSLGTCAMKPREQGGVVDKRLNVYGTQNLKCVDLSICPDNLGTNTYSSALLVGEKGADLIAEELGLKIKTPHAPVPHAPVPTGRPATQQVR

[0035]

[0036] Amino acid sequence of the alcohol oxidase PcAOX mutant, SEQ ID No. 3

[0037] MGHPEEVDVIVCGGGPAGCVVAGRLAYADPTLKVMLIEGGANNRDDPWVY

[0038] RPGIYVRNVPNNGINDKATFYTDTMASSYLRGRRSIVPCANILGGGSSINFQM

[0039] YTRASASDWDDFKTEGWTCKDLLPLMKRLENYQKPCNNDTHGYDGPIAISN

[0040] GGQIMPVAQDFLRAAHAIGVPYSDDIQDLTTAHGAEIWAKYINRHTGRRSDA

[0041] ATAYVHSVMDVQDNLFLRCNARVSRVLFDDNNKAVGVAYVPSRNRTHGGKL

[0042] HETIVKARKMVVLSSGTLGTPQILERSGVGNGELLRQLGIKIVSDLPGVGEQY

[0043] QDHYTTLSIYRVSNESITTDDFLRGVKDVQRELFTEWEVSPEKARLSSNAIDA

[0044] GFKIRPTEEELKEMGPEFNELWNRYFKDKPDKPVMFGSIVAGAYADHTLLPPG

[0045] KYITMFQYLEYPASRGKIHIKSQNPYVEPFFDSGFMNNKADFAPIRWSYKKTR

[0046] EVARRMDAFRGELTSHHPRFHPASPAACKDIDIETAKQIYPDGLTVGIHMGSW

[0047] HQPSEPYKHDKVIEDIPYTEEDDKAIDDWVADHVETTWHSLGTCAMKPREQ

[0048] GGVVDKRLNVYGTQNLKCVDLSICPDNLGTNTYSSALLVGEKGADLIAEELG

[0049] LKIKTPHAPVPHAPVPTGRPATQQVRLE

[0050] Nucleotide sequence of alcohol oxidase PcAOX mutant, SEQ ID No. 4

[0051] ATGGGTCATCCGGAAGAAGTTGATGTTATCGTGTGCGGTGGTGGCCCGGCT

[0052] GGCTGTGTCGTTGCTGGTCGCCTGGCTTACGCAGATCCGACCCTGAAAGTT

[0053] ATGCTGATTGAAGGCGGTGCCAACAATCGTGATGACCCGTGGGTTTATCGC

[0054] CCGGGTATTTACGTCCGTAACGTTCCGAATAACGGCATCAATGATAAAGCC

[0055] ACCTTTTATACCGACACGATGGCAAGCTCTTACCTGCGTGGTCGTCGCAGC

[0056] ATTGTTCCGTGTGCCAACATCCTGGGCGGTGGCAGTTCCATTAATTTTCAGA

[0057] TGTATACCCGCGCATCAGCTTCGGATTGGGATGACTTCAAAACCGAAGGCT

[0058] GGACGTGCAAAGACCTGCTGCCGCTGATGAAACGTCTGGAAAACTACCAA

[0059] AAACCGTGTAACAACGATACCCACGGCTACGACGGTCCGATTGCGATCTCG

[0060] AATGGTGGCCAGATTATGCCGGTCGCTCAAGATTTTCTGCGTGCCGCACAT

[0061] GCCATTGGTGTGCCGTATAGCGATGACATCCAGGATCTGACCACGGCTCAT

[0062] GGTGCGGAAATTTGGGCTAAATATATCAATCGTCATACCGGTCGTCGCAGC

[0063] GATGCAGCTACCGCATACGTGCATTCTGTTATGGATGTCCAGGACAACCTG

[0064] TTTCTGCGTTGTAATGCGCGTGTGTCCCGCGTTCTGTTCGATGACAACAATA

[0065] AAGCCGTTGGCGTCGCGTATGTGCCGTCACGTAACCGTACCCACGGCGGC

[0066] AAACTGCACGAAACGATTGTCAAAGCCCGCAAAATGGTGGTTCTGTCAAG

[0067] CGGTACCCTGGGTACGCCGCAGATCCTGGAACGTAGCGGTGTTGGCAATG

[0068] GTGAACTGCTGCGCCAACTGGGTATTAAAATCGTGTCTGATCTGCCGGGCG

[0069] TTGGTGAACAGTATCAAGACCATTACACCACGCTGAGTATTTATCGTGTCA

[0070] GCAACGAATCTATCACCACGGATGACTTTCTGCGTGGCGTCAAAGATGTGC

[0071] AGCGCGAACTGTTCACCGAATGGGAAGTGTCTCCGGAAAAAGCCCGTCTG

[0072] AGCTCTAATGCCATTGATGCAGGTTTTAAAATCCGCCCGACCGAAGAAGAA

[0073] CTGAAAGAAATGGGCCCGGAATTTAACGAACTGTGGAATCGCTACTTCAA

[0074] AGATAAACCGGACAAACCGGTCATGTTTGGTAGCATTGTGGCGGGCGCCTA

[0075] TGCAGATCATACCCTGCTGCCGCCGGGTAAATACATCACGATGTTCCAGTAT

[0076] CTGGAATACCCGGCGTCACGTGGCAAAATTCACATCAAATCGCAAAACCC

[0077] GTATGTTGAACCGTTTTTCGATTCAGGTTTCATGAACAACAAAGCTGACTT

[0078] CGCGCCGATTCGTTGGTCGTACAAGAAAACCCGCGAAGTGGCCCGTCGCA

[0079] TGGATGCATTTCGTGGCGAACTGACGAGTCATCACCCGCGCTTCCATCCGG

[0080] CATCCCCGGCGGCATGCAAAGATATTGACATCGAAACCGCAAAACAGATTT

[0081] ATCCGGATGGCCTGACGGTGGGCATTCACATGGGCAGTTGGCACCAACCG

[0082] TCCGAACCGTACAAACACGATAAAGTTATCGAAGACATCCCGTACACCGA

[0083] AGAAGATGACAAAGCTATTGATGACTGGGTTGCGGATCATGTCGAAACCA

[0084] CGTGGCACTCTCTGGGTACCTGTGCAATGAAACCGCGTGAACAGGGTGGC

[0085] GTCGTGGATAAACGCCTGAACGTGTATGGTACGCAAAATCTGAAATGCGTT

[0086] GATCTGAGTATCTGTCCGGACAACCTGGGCACCAATACGTACAGTTCCGCG

[0087] CTGCTGGTTGGCGAAAAAGGTGCTGATCTGATTGCGGAAGAACTGGGCCT

[0088] GAAAATCAAAACCCCGCACGCCCCGGTTCCGCACGCCCCGGTCCCGACGG

[0089] GTCGCCCGGCTACGCAACAAGTCCGCCTCGAG

[0090] In the following examples, the materials used include: plasmid pET28a(+)-PcAOX, which is preserved in the applicant's laboratory; Escherichia coli TOP10 and BL21(DE3) competent cells, purchased from Weidi Biotechnology Co., Ltd.; seamless cloning kit, purchased from Zhongmei Taihe Biotechnology (Beijing) Co., Ltd.; plasmid extraction kit, purchased from Sangon Biotech (Shanghai) Co., Ltd.; pre-crystallization kit, purchased from Hampton Research, USA; reagents such as benzyl alcohol, phenethyl alcohol, vinyl acetate, ethyl acetate, etc., purchased from Aladdin Reagent Co., Ltd.

[0091] The present invention will be described in detail below in conjunction with the accompanying drawings and specific examples.

[0092] Example 1 Analysis of the three-dimensional structure of PcAOX and construction of mutant engineering bacteria

[0093] The results of the three-dimensional structure analysis of PcAOX show that there are multiple amino acid residues above the catalytic channel side that block the catalytic center, which makes it impossible for macromolecular substrates to enter the PcAOX active center and the products after the transacylation reaction cannot be released. It is speculated that this original structural characteristic may be an important reason for its inability to exhibit transacylation activity.

[0094] After multi-scale comparative analysis, the inventor locked three consecutive amino acid sites M59, Q60, and R61, and designed 5 mutants M59A, Q60A, R61A, F101S, and M59V-Q60P-R61N without changing their hydrophilicity-hydrophobicity and charge.

[0095] Specifically, it includes the following steps:

[0096] (1) Primer design

[0097] Site-directed mutagenesis was carried out by PCR product amplification method to replace fixed amino acids at fixed positions of the PcAOX mature peptide. Primer design was carried out using SnapGene software, and the main primers involved are shown in Table 1.

[0098] Table 1 Main primer design table

[0099]

[0100]

[0101] (2), Site-directed mutagenesis of PcAOX wild type

[0102] Using the primers designed according to Table 1, site-directed mutagenesis was performed on the PcAOX wild type by PCR reaction. The PCR product after mutagenesis was digested with the restriction endonuclease Dpn I.

[0103] The reaction system and procedure of site-directed mutagenesis PCR and the digestion system and procedure are shown in Table 2 respectively.

[0104] Table 2 PCR and digestion procedures and systems

[0105]

[0106] (3), Construction of engineering bacteria of PcAOX mutants (M59A, Q60A, R61A, F101S and M59V-Q60P-R61N)

[0107] The above-mentioned PCR product after template digestion was transformed into Escherichia coli BL21(DE3) competent cells by heat shock transformation. The specific steps are as follows: Take the competent cells out of the -80°C refrigerator and place them on ice for 5 - 10 min. After melting, add 2 - 3 μL of the PCR product and let it stand on ice for 30 min. Take out the standing competent cells and place them in a water bath preheated to 42°C. After heat shock for 90 s, immediately place them on ice. After 2 - 3 min, add 750 μL of LB medium and culture at 37°C and 220 rpm for 40 min. After the culture is completed, centrifuge at 1000 rpm for 3 min to remove a certain proportion of the supernatant, and spread the competent cells on an LB solid plate containing 50 ng / mL kanamycin and culture for 12 - 16 h. Pick monoclonal colonies for colony PCR reaction to verify whether the transformation is correct, and sequence the positive clones to further verify whether the mutation is correct. Finally, store the constructed engineering bacteria at -20°C in 25% glycerol for later use.

[0108] Example 2 Fermentation and purification of PcAOX mutants

[0109] 1. Fermentation of PcAOX mutants

[0110] The engineering bacteria of PcAOX mutants (M59A, Q60A, R61A, F101S and M59V-Q60P-R61N) stored in Example 1 (using the PcAOX wild-type engineering bacteria as a control) were streaked and activated. Pick monoclonal colonies into an LB medium containing 50 ng / mL kanamycin and culture at 37°C and 220 rpm until the OD 600 reaches about 0.8 - 0.9, and then inoculate into 100 mL of LB medium at an inoculation amount of 2%, and culture until the OD 600When it is about 0.9 - 1.0, inoculate it into 500 mL of TB medium containing 1% (w / v) glucose at an inoculation amount of 5%, and culture until OD 600 When it is 0.9 - 1.0, add IPTG with a final concentration of 1 mM, and induce for 20 h at 24 °C and 220 rpm.

[0111] 2. Purification of wild-type PcAOX and its 5 mutants

[0112] Centrifuge the completed fermentation culture at 4000 rpm and 4 °C for 30 min to remove the supernatant. Suspend it in buffer A (50 mM phosphate buffer, pH 7.5, 400 mM NaCl, 100 mM KCl) at a ratio of 1:10 (w / v). Ultrasonically disrupt the cell suspension on ice for 20 min. Centrifuge the disrupted solution at 12000 rpm and 4 °C for 25 min, and discard the precipitate. Load the supernatant after centrifugation onto a Ni affinity chromatography column that has been fully equilibrated with buffer A. First, elute the miscellaneous proteins with 40 mM imidazole, and then elute the target protein with 500 mM imidazole and collect it. Detect the eluted components by SDS-PAGE and activity detection. Finally, combine the protein eluates with the correct molecular weight and activity, and perform desalting column salt exchange to buffer C (50 mM phosphate buffer, pH 7.5), and store it at 4 °C for later use.

[0113] Example 3 Alcohol oxidase activity of wild-type PcAOX and its 5 mutants

[0114] 1. The reaction system for alcohol oxidase activity determination is shown in Table 3.

[0115] Table 3 Reaction system for AOX activity determination

[0116]

[0117] The formula for alcohol oxidase activity determination is as follows:

[0118]

[0119] Where:

[0120] ΔAbs 515 -----------------Difference in absorbance at 515 nm

[0121] 12.236 / 0.0474----------a / b value of the standard curve

[0122] V 体 --------------------Total volume of the reaction system (μL)

[0123] nx --------------------Dilution factor of enzyme solution

[0124] Δt--------------------Time corresponding to the difference in absorbance value (min)

[0125] N--------------------Protein concentration of enzyme solution (mg / mL)

[0126] 2. The standard curve is as Figure 3 shown.

[0127] 3. The alcohol oxidase activity results of PcAOX wild type and its 5 mutants are shown in Table 4.

[0128] Table 4 Determination results of alcohol oxidase activity

[0129] enzyme substrate enzyme activity (U / mg) PcAOX wild type ethanol 2.4 PcAOX mutant M59A ethanol 2.1 PcAOX mutant Q60A ethanol 2.2 PcAOX mutant R61A ethanol 1.4 PcAOX mutant F101S ethanol 1.8 PcAOX mutant M59V-Q60P-R61N ethanol 1.9

[0130] As can be seen from the results in Table 4, both the PcAOX wild type and its 5 mutants have alcohol oxidase activity.

[0131] Example 4 Determination of the transacylation activity of PcAOX wild type and its 5 mutants

[0132] 1. Establishment of the catalytic transacylation reaction system

[0133] Using benzyl alcohol and phenethyl alcohol as acyl acceptors, vinyl acetate as acyl donor, and PcAOX wild type and its 5 mutants as enzymes, the catalytic transacylation reaction systems were constructed respectively according to the reaction system table in Table 5.

[0134] Table 5 Reaction system table

[0135] component final concentration enzyme 5 μM acyl acceptor 20 mM, 40 mM, 100 mM, 200 mM acyl donor 20 mM buffer make up to 1 mL

[0136] 2. Establishment of the gas chromatography detection method

[0137] Using an HP-5 chromatographic column, the initial temperature of the column oven was 80 °C, heated to 150 °C at 8 °C / min and held for 3 min, then heated to 300 °C at 15 °C / min and held for 10 min. The detector temperature was 280 °C and the injection port temperature was 250 °C.

[0138] 3. Detection results of the transacylation activity of PcAOX mutants

[0139] (1). The reaction formula with benzyl alcohol as acyl acceptor is as follows:

[0140]

[0141] The reaction formula with phenethyl alcohol as acyl acceptor is as follows:

[0142]

[0143] (2) In the transacylation verification reaction of wild-type PcAOX and its five mutants, only the PcAOX mutant M59V-Q60P-R61N (PcAOX-VPN) has transacylation activity.

[0144] The reaction results with benzyl alcohol as the acyl acceptor and the PcAOX mutant (M59V-Q60P-R61N) as the enzyme are as Figure 4 shown. In the reaction catalyzed in the aqueous phase to produce benzyl acetate with benzyl alcohol as the acyl acceptor and vinyl acetate as the acyl donor, the optimal condition is that the molar ratio of the acyl acceptor to the acyl donor is 1:5, and the final highest conversion rate is 35%.

[0145] The reaction results with phenethyl alcohol as the acyl acceptor and the PcAOX mutant (M59V-Q60P-R61N) as the enzyme are as Figure 5 shown. In the reaction to produce phenethyl acetate with phenethyl alcohol as the acyl acceptor and vinyl acetate as the acyl donor, the optimal condition is that the molar ratio of the acyl acceptor to the acyl donor is 1:5, and the final highest conversion rate is 51%.

[0146] The results of this example show that the PcAOX mutant M59V-Q60P-R61N (abbreviated as PcAOX-VPN) has transacylation activity while having alcohol oxidase activity.

[0147] Example 5 Crystal Preparation and Structure Analysis of PcAOX Mutant (PcAOX-VPN)

[0148] 1. Initial Screening of Crystal Growth and Preparation of High-Purity Protein

[0149] The PcAOX mutant (PcAOX-VPN) purified by nickel column was desalted and concentrated using a 10 kDa cut-off ultrafiltration concentrator until it was within 5 mL. The concentrated protein was loaded onto a molecular sieve (200 pg) equilibrated with a 10 mM Tris-HCl pH 8.0 buffer containing 150 mM NaCl. The molecular sieve chromatogram of the PcAOX mutant is as Figure 6 shown. The results show that there is only one peak, and the peak shape is symmetric, indicating high protein homogeneity, providing a basis for the initial screening of crystallization. When the UV absorption value began to rise, the samples were collected at a rate of 1 mL / min, and the collected samples were labeled and subjected to SDS-PAGE detection. The SDS-PAGE detection results are as Figure 7 shown. The results show that the purity reaches over 90%, and the samples meet the requirements for the initial screening of protein crystallization.

[0150] 2. Precrystallization Detection of PcAOX Mutant (PcAOX-VPN)

[0151] Use a pre-crystallization kit to detect whether the current protein concentration is suitable for initial crystal screening. Accurately pipette 400 μL of solutions A1 and B1, and A2 and B2 of the kit into a 24-well hanging-drop plate, and perform crystal seeding at a ratio of protein:reservoir solution = 1:1 (v / v). After 30 min, observe the droplet precipitation using an optical crystal microscope and judge whether initial crystal screening can be carried out according to the kit instructions.

[0152] Pipette the reagents in the initial crystal screening kit into a 96-well sitting-drop plate (a total of 8 * 48 conditions), 60 μL for each condition. Place the PcAOX mutant (PcAOX-VPN) protein solution in the crystal spotting robot tray and perform spotting according to the set program. The results are as Figure 8 and Table 6 show ( Figure 8 in the left figure, the crystallization condition is Classic 1-12; in the middle figure, the crystallization condition is HR2112-23; in the right figure, the crystallization condition is Classic 4-8).

[0153] Table 6 Initial crystal screening results

[0154]

[0155] From Figure 8 and the initial crystal screening results of the PcAOX mutant (PcAOX-VPN) in Table 6, it can be seen that crystals can grow, but the crystal quality is poor and fails to meet the diffraction standard. Compared with the other two crystallization conditions, the crystal form of the crystallization condition HR2112-23 is better. Therefore, the crystallization condition HR2112-23 is used for crystal optimization.

[0156] 3. Crystal optimization of PcAOX mutant (PcAOX-VPN)

[0157] Under the crystallized conditions, further optimize the crystal form and size. There are two optimization conditions: one is the concentration of the precipitant, and the other is the pH value of the reservoir solution. After preparing the optimized reagents under each condition, accurately pipette 150 μL of the reservoir solution into a 24-well hanging-drop plate and perform spotting on a silanized glass slide with a spotting specification of 1 μL:1 μL. The spotted hanging-drop plate is left to stand at 18 °C to wait for crystal growth.

[0158] The crystal optimization results are as Figure 9 and Figure 10 shown. From Figure 9 and Figure 10 it can be seen that the crystals change from the original small particles with irregular shapes to crystals with clear boundaries, regular shapes and appropriate sizes (1.6 M ammonium sulfate, 0.1 M MES pH 7.5, 8% (v / v) 1,4-Dioxane), meeting the requirements for X-ray diffraction.

[0159] 4. Resolution of the crystal structure of the PcAOX mutant (PcAOX-VPN)

[0160] The diffraction data of the crystal of the PcAOX mutant (PcAOX-VPN) are shown in Table 7.

[0161] Table 7 Crystal diffraction data

[0162]

[0163] The structure of the crystal of the PcAOX mutant (PcAOX-VPN) is as Figure 11 shown. It can be seen from Figure 11 that except for the mutation sites, the structural information of the other positions of the PcAOX mutant (PcAOX-VPN) is the same, which indicates that the mutations at these three sites (M59V-Q60P-R61N) do not change the original structure of the enzyme. After the mutations at the three sites of M59V-Q60P-R61N, the result shows that the internal active center FAD active group head can be exposed to a certain extent.

[0164] Regarding the generation of the transacylation activity, the catalytic mechanism of this reaction is not yet clear. It is speculated that because the enzyme itself has a bifunctional catalytic center, the channel is narrow and the active center is not exposed before mutation, so it cannot catalyze the transacylation activity. However, after the mutations at the three sites of M59V-Q60P-R61N, the exposure of the active center makes this mutant show transacylation activity.

[0165] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope recorded in this specification.

[0166] The above-described embodiments only represent several implementation manners of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several deformations and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the invention patent should be subject to the appended claims.

Claims

1. An alcohol oxidase Pc A mutant of AOX, characterized in that The alcohol oxidase Pc The amino acid sequence of the mutant of AOX is shown in SEQ ID No.

3.

2. The alcohol oxidase according to claim 1 Pc A coding gene for a mutant of AOX.

3. The coding gene according to claim 2, wherein The nucleotide sequence of the coding gene is shown in SEQ ID No.

4.

4. The alcohol oxidase according to claim 1 Pc Use of the mutant of AOX and the coding gene according to claim 2 or 3 in catalyzing an acyl transfer reaction, wherein the acyl acceptor of the acyl transfer reaction is benzyl alcohol or phenethyl alcohol, and the acyl donor of the acyl transfer reaction is vinyl acetate.

5. A recombinant expression vector inserted with the coding gene according to claim 2 or 3.

6. A recombinant engineering strain transfected with the recombinant expression vector according to claim 5.

7. Use of the recombinant expression vector according to claim 5 or the recombinant engineering strain according to claim 6 in catalyzing an acyl transfer reaction, wherein the acyl acceptor of the acyl transfer reaction is benzyl alcohol or phenethyl alcohol, and the acyl donor of the acyl transfer reaction is vinyl acetate.

8. A method for catalyzing an acyl transfer reaction, characterized in that, Use the alcohol oxidase according to claim 1 Pc The mutant of AOX catalyzes benzyl alcohol and vinyl acetate to produce benzyl acetate or catalyzes phenethyl alcohol and vinyl acetate to produce phenethyl acetate.

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