Acyltransferase for synthesizing polyester intermediate and application of acyltransferase

Abstract

The invention discloses acyltransferase for synthesizing a polyester intermediate and application of the acyltransferase. The acyltransferase is specifically a wild-type enzyme with an amino acid sequence as shown in SEQ ID NO: 1 or an acyltransferase variant which is obtained by mutation of the wild-type enzyme and has acyl transfer reaction catalysis activity, and the acyltransferase variant is specifically obtained by replacing at least one amino acid residue in the 209th site, the 166th site and the 119th site in the amino acid sequence of the wild-type enzyme. And the protein with improved acyltransferase activity is obtained. The wild type enzyme and the acyltransferase variant provided by the invention both have acyltransferase activity. The method has an important application value.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme engineering, and specifically relates to an acyltransferase for synthesizing polyester intermediates and application thereof. Background Art

[0002] Studies have found that some hydrolases (such as MsAcT from Mycobacterium smegmatis, EstCE1 from the eighth family of carboxylesterases, and Est8 from the metagenomics) can catalyze the synthesis of esters and amides in water in addition to their hydrolysis function. These enzymes are called hybrid hydrolases / acyltransferases. Compared with traditional chemical synthesis and lipase synthesis, the synthesis reaction catalyzed by hybrid hydrolases / acyltransferases does not require the participation of expensive and toxic organic solvents, and can be used for multi-enzyme driven biocatalytic cascade reactions, which has broad application prospects. However, only a small number of hydrolases have been explored for their acyltransferase function, and the ester synthesis reaction catalyzed by them in water is usually carried out under kinetic control, and there are generally problems such as low yield of synthetic products and serious hydrolysis side reactions. It is urgent to solve the above problems through enzyme molecular modification strategies, that is, to obtain hydrolases with high acyltransferase activity.

[0003] PETase, derived from Ideonella sakaiensis 201-F6, can hydrolyze the difficult-to-degrade plastic polyethylene glycol terephthalate (PET) into monomers of terephthalic acid (TPA) and ethylene glycol (EG) at 20-40°C. PETase is generally used as a plastic hydrolase and is widely studied and used. Summary of the invention

[0004] The purpose of the present invention is to provide an acyltransferase for synthesizing polyester intermediates. The acyltransferase is specifically a hydrolase PETase (wild type) with an amino acid sequence as shown in SEQ ID NO: 1 or a mutant thereof having catalytic acyl transfer reaction activity.

[0005] The present invention first protects a protein, which may be any one of C1)-C4):

[0006] C1) replacing at least one of the amino acid residues at positions 209, 166 and 119 in the amino acid sequence of the hydrolase PETase to obtain a protein having acyltransferase activity;

[0007] C2) A fusion protein obtained by connecting a tag to the N-terminus or / and the C-terminus of the protein shown in C1);

[0008] C3) A protein obtained by substituting and / or deleting and / or adding one or more amino acid residues of the protein shown in C1) or C2) and having acyltransferase activity;

[0009] C4) a protein having an amino acid sequence homology of 80% or more to the protein shown in C1) or C2) and having acyltransferase activity;

[0010] The amino acid sequence of the hydrolase PETase is shown in SEQ ID NO:1.

[0011] The protein is an acyltransferase variant obtained by mutation of the hydrolase PETase.

[0012] In the above protein, the A residue at position 209 is substituted with a T residue, a V residue, a N residue, a G residue, an I residue, a C residue, an F residue, a Q residue, an R residue, an M residue, a D residue, a P residue, an L residue, a K residue, an H residue, a W residue or a Y residue. The S residue at position 166 is substituted with a T residue, a V residue, a C residue, an E residue, an M residue or a G residue. The Q residue at position 119 is substituted with an R residue, a C residue, a W residue, a P residue, an F residue, an I residue or a G residue.

[0013] In the above, the 209th, 166th and 119th positions in the amino acid sequence of the hydrolase PETase refer to the 209th, 166th and 119th positions from the N-terminus of SEQ ID NO: 1, respectively.

[0014] The acyltransferase activity of any of the above proteins is higher than that of the hydrolase PETase.

[0015] Any of the above-mentioned proteins can specifically be any of the 37 acyltransferase mutants in Table 1 of the Examples (ie, acyltransferase mutants other than acyltransferase mutant Q119L and acyltransferase mutant Q119S).

[0016] Nucleic acid molecules encoding any of the above-mentioned proteins also fall within the protection scope of the present invention.

[0017] An expression cassette, recombinant vector or recombinant microorganism containing any of the above-mentioned nucleic acid molecules also falls within the protection scope of the present invention.

[0018] The recombinant vector may be a recombinant plasmid obtained by inserting the nucleic acid molecule into an expression vector or a cloning vector. The expression vector may specifically be the pET-22b(+) vector mentioned in the examples.

[0019] The recombinant microorganism is a recombinant bacterium obtained by introducing the recombinant vector into the starting microorganism.

[0020] The starting microorganism may be Escherichia coli.

[0021] The Escherichia coli may specifically be Escherichia coli BL21 Gold (DE3).

[0022] The use of any of the above-mentioned proteins, the above-mentioned hydrolase PETase, any of the above-mentioned nucleic acid molecules, or an expression cassette, recombinant vector or recombinant microorganism containing any of the above-mentioned nucleic acid molecules in the preparation of acyltransferase also falls within the protection scope of the present invention.

[0023] The use of any of the above-mentioned proteins, the hydrolase PETase, any of the above-mentioned nucleic acid molecules, or an expression cassette, recombinant vector or recombinant microorganism containing any of the above-mentioned nucleic acid molecules as acyltransferase also falls within the protection scope of the present invention.

[0024] In the above application, the acyltransferase activity of any of the above proteins is higher than that of the hydrolase PETase; the amino acid sequence of the hydrolase PETase is shown in SEQ ID NO:1.

[0025] The use of any of the above-mentioned proteins, any of the above-mentioned nucleic acid molecules, or an expression cassette, a recombinant vector or a recombinant microorganism containing any of the above-mentioned nucleic acid molecules in PET hydrolysis also falls within the protection scope of the present invention.

[0026] The use of any of the above-mentioned proteins, the hydrolase PETase, any of the above-mentioned nucleic acid molecules, or an expression cassette, recombinant vector or recombinant microorganism containing any of the above-mentioned nucleic acid molecules in catalyzing the acyl transfer reaction of BHET also falls within the protection scope of the present invention.

[0027] The use of any of the above-mentioned proteins, the hydrolase PETase, any of the above-mentioned nucleic acid molecules, or an expression cassette, recombinant vector or recombinant microorganism containing any of the above-mentioned nucleic acid molecules in catalyzing acyl transfer reactions of aromatic, furanic or aliphatic polymer precursors also falls within the scope of protection of the present invention.

[0028] The aromatic polymer precursor may be bis(4-hydroxybutyl)terephthalate, 2-hydroxyethyl benzoate, 4-hydroxybutyl benzoate or 6-hydroxyhexyl benzoate.

[0029] The furan-based polymer precursor may be bis(2-hydroxyethyl)furan-2,5-dicarboxylate or bis(4-hydroxybutyl)furan-2,5-dicarboxylate.

[0030] The aliphatic polymer precursor may be 2-hydroxyethyl 3-phenylpropanoate, 4-hydroxybutyl3-phenylpropanoate or 6-hydroxyhexyl 3-phenylpropanoate.

[0031] bis(4-hydroxybutyl)terephthalate, bis(2-hydroxyethyl)furan-2,5-dicarboxylate, bis(4-hydroxybutyl)furan-2,5-dicarboxylate, 2-hydroxyethyl 3-phenylpropanoate, 4-hydroxybutyl 3-phenylpropanoate, 6-hydroxyhexyl 3-phenylpropanoate, 2-hydroxyethyl The structural formulas of benzoate, 4-hydroxybutyl benzoate and 6-hydroxyhexyl benzoate are detailed in Table 5 in the Examples.

[0032] The use of any of the above-mentioned proteins, the above-mentioned hydrolase PETase, any of the above-mentioned nucleic acid molecules, or an expression cassette, recombinant vector or recombinant microorganism containing any of the above-mentioned nucleic acid molecules in catalyzing the synthesis of esters and amides also falls within the protection scope of the present invention.

[0033] In view of the fact that the hydrolase PETase with an amino acid sequence as shown in SEQ ID NO: 1 has weak acyltransferase activity, the inventors of the present invention have conducted a large number of experiments to explore and transform the hydrolase PETase, that is, point mutations were performed on the existing amino acid sequence as shown in SEQ ID NO: 1, and a series of PETase mutants with significantly improved acyltransferase activity were prepared, thereby achieving efficient synthesis of esters in an aqueous phase. The present invention has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The results are for the purification of pure enzymes of the hydrolase PETase (ie, PETase WT), the acyltransferase mutant A209T / S166V (ie, A209T / S166V), the acyltransferase mutant A209T (ie, A209T), and the acyltransferase mutant S166V (ie, S166V).

[0035] Figure 2 For BHET-MHET 1H-NMR spectrum.

[0036] Figure 3 For BHET-MHET 13 C-NMR spectrum.

[0037] Figure 4 is the substrate 1 in Example 8 1 H-NMR spectrum.

[0038] Figure 5 is the product 1p in Example 8 1 H-NMR spectrum.

[0039] Figure 6 is the substrate 2 in Example 8 1 H-NMR spectrum.

[0040] Figure 7 is the product 2p in Example 8 1 H-NMR spectrum.

[0041] Figure 8 is the substrate 3 in Example 8 1 H-NMR spectrum.

[0042] Fig. 9 is the product 3p in Example 8 1 H-NMR spectrum.

[0043] Fig.10 is the substrate 4 in Example 8 1 H-NMR spectrum.

[0044] Fig.11 is the product 4p in Example 8 1 H-NMR spectrum.

[0045] Fig.12 is the substrate 5 in Example 8 1 H-NMR spectrum.

[0046] Fig.13 is the product 5p in Example 8 1 H-NMR spectrum.

[0047] Fig.14 is the substrate 6 in Example 8 1 H-NMR spectrum.

[0048] Fig.15 is the product 6p in Example 8 1 H-NMR spectrum.

[0049] Fig.16 is the substrate 7 in Example 8 1 H-NMR spectrum.

[0050] Fig.17 is the product 7p in Example 8 1 H-NMR spectrum.

[0051] Fig.18 is the substrate 8 in Example 8 1 H-NMR spectrum.

[0052] Fig.19 is the product 8p in Example 8 1 H-NMR spectrum.

[0053] Fig. 20 is the substrate 9 in Example 8 1 H-NMR spectrum.

[0054] Fig.21 is the product 9p in Example 8 1 H-NMR spectrum. DETAILED DESCRIPTION

[0055] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0056] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0057] Example 1. Preparation of hydrolase PETase (referred to as PETase or PETase WT) and PETase mutants (also known as acyltransferase variants or acyltransferase mutants)

[0058] 1. Construction of recombinant plasmid

[0059] 1. Replace the small DNA fragment between the restriction endonuclease EcoRI and XhoI recognition sites of the pET-22b(+) vector with the hydrolase PETase gene (Genebank No. GAP38373.1) from the Ideonella sakaiensis 201-F6 strain to obtain the recombinant plasmid pET22b-PETase.

[0060] The recombinant plasmid pET22b-PETase expresses the PETase shown in SEQ ID No: 1. PETase has weak acyltransferase activity.

[0061] 2. Primer F: 5'-GGCCATGGATATCGGAATTAATTCGGATCCGAATTCG-3' and primer R: 5'-TCAGTGGTGGTGGTGGTGGTGCTCGAG-3' were used to randomly mutate the nucleotide sequence of the hydrolase PETase gene from Ideonella sakaiensis 201-F6 strain by error-prone PCR to obtain several mutant nucleotide sequences.

[0062] 3. Replace the small DNA fragments between the restriction endonuclease EcoRI and XhoI recognition sites of the pET-22b(+) vector with several mutant nucleotide sequences obtained in step 2 to obtain the corresponding recombinant plasmid pET22b-PETase mutant.

[0063] 2. Acyltransferase mutant acquisition

[0064] Each recombinant plasmid constructed in step 1 was subjected to the following experiment:

[0065] 1. Transform the recombinant plasmid into Escherichia coli BL21 Gold (DE3) to obtain recombinant bacteria.

[0066] 2. Pick a single clone of the recombinant bacteria and inoculate it into 150 μL LB liquid culture medium (containing ampicillin), and culture it overnight in a 96-well plate shaking incubator at 37° C. and 800 rpm to obtain a culture solution.

[0067] 3. Inoculate the culture obtained in step 2 into 150 μL LB liquid medium (containing ampicillin), culture at 37°C for 3 h, add IPTG to a final concentration of 0.1 mM, culture at 20°C, 800 rpm, and shake for 24 h. Centrifuge at 4°C, 4000 rpm, and collect the supernatant.

[0068] 4. Preparation of screening system. The screening was carried out in a 96-well plate, and the screening system was 200 μL, consisting of pH 8.0, 100 mM phosphate buffer, the supernatant collected in step 3, and 15 mM bis(hydroxyethyl) terephthalate (BHET) solution.

[0069] 5. Take the screening system prepared in step 4 and react at 30℃ for 24h; use an ELISA reader to detect the OD at different time points 600nm .

[0070] Compared with the recombinant bacteria transformed with the recombinant plasmid pET22b-PETase, OD 600nm The acyltransferase activity of the mutant expressed by the recombinant bacteria transformed with the recombinant plasmid pET22b-PETase mutant was significantly increased. 600nmThe significantly increased recombinant bacteria were subjected to gene sequencing, and the mutation sites were determined based on the sequencing results to obtain the amino acid sequence of the PETase mutant with improved acyltransferase activity.

[0071] The names of the acyltransferase mutants obtained by screening are shown in the first column of Table 1. Compared with the amino acid sequence of PETase (the mutation site corresponds to the sequence shown in SEQ ID No: 1), the different amino acid residue substitutions of the acyltransferase mutants are shown in the second column of Table 1.

[0072] Table 1

[0073]

[0074]

[0075] Example 2. Preparation of crude enzyme of acyltransferase mutant screened in Example 1

[0076] The inoculation needle picked up the recombinant bacteria transformed by the recombinant plasmid pET22b-PETase and the recombinant bacteria transformed by the recombinant plasmid pET22b-PETase mutant obtained in Example 1 and inoculated them into 3 mL LB liquid culture medium, cultured overnight at 37°C and 220 rpm, and then inoculated into 15 mL LB liquid culture medium at a 1% (V / V) inoculation amount, and cultured at 37°C and 220 rpm until OD 600nm When the concentration of IPTG reached about 0.8, IPTG with a final concentration of 0.1 mM was added to induce expression at 20°C and 220 rpm for 24 h. The cells were centrifuged at 4°C and 4000 rpm for 30 min, and the supernatant was collected.

[0077] The supernatant is the corresponding crude enzyme.

[0078] Example 3. Preparation of pure enzyme of acyltransferase mutant screened in Example 1

[0079] The supernatant collected in Example 2 was purified according to the operating procedures of the nickel column (GE Healthcare product, catalog number 17-5248-02) instructions, and the final eluted target protein was stored in 100 mM pH 8.0 phosphate buffer after removing imidazole.

[0080] The target protein obtained from the recombinant bacteria transformed with the recombinant plasmid pET22b-PETase through the above purification steps is the pure enzyme of PETase.

[0081] The target protein obtained from the recombinant bacteria transformed with the recombinant plasmid pET22b-PETase mutant obtained in Example 1 through the above purification step is the pure enzyme of the acyltransferase mutant.

[0082] The partial purification results are shown in Figure 1(1 is the pure enzyme of PETase, 2 is the pure enzyme of the acyltransferase mutant A209T / S166V, 3 is the acyltransferase mutant A209T, and 4 is the acyltransferase mutant S166V). The results showed that the pure enzyme of PETase and the pure enzyme of the acyltransferase mutant had basically no mixed bands, and the protein purification effect was good.

[0083] Example 4. Enzymatic Preparation and Identification of Acyl Transfer Product O,O'-(Ethane-1,2-diacyl)bis(2-hydroxyethyl)terephthalate (BHET-MHET)

[0084] 1. Add 50 mL of 100 mM, pH 8.0 phosphate buffer and 0.5 g of BHET powder to a 250 mL conical flask, then add 50 μL of pure enzyme of acyltransferase mutant A209T / S166V, and shake at 30°C and 220 rpm. After the reaction, centrifuge the reaction solution (4°C, 12000 rpm, 10 min), collect the precipitate, wash it with ddH2O for 3 to 5 times, use acetonitrile to completely dissolve the precipitate, then add ddH2O, centrifuge and collect the precipitated solid, and obtain the purified product BHET-MHET.

[0085] 2. Dissolve 10-20 mg of the purified product BHET-MHET in deuterated DMSO and collect a one-dimensional NMR spectrum using a 400 MHz NMR spectrometer.

[0086] BHET-MHET 1 H-NMR spectrum is shown in Figure 2 .

[0087] BHET-MHET 13 C-NMR spectrum is shown in Figure 3 .

[0088] The structural formula of BHET-MHET is finally determined as shown in formula (I):

[0089]

[0090] According to the above steps, the pure enzyme of acyltransferase mutant A209T / S166V is replaced with acyltransferase mutant A209T, acyltransferase mutant S166V, acyltransferase mutant A209D, acyltransferase mutant A209V, acyltransferase mutant S166T, acyltransferase mutant A209T / S166T, acyltransferase mutant A209T / S166T / Q119R or acyltransferase mutant A209T / S166V / Q119I, and the other steps remain unchanged. The results show that the purified product BHET-MHET can be prepared.

[0091] Example 5. Quantitative Analysis of Acyl Transfer Product BHET-MHET

[0092] 1. Prepare a 5 mM BHET-MHET stock solution (solvent: acetonitrile).

[0093] 2. Prepare BHET-MHET standards of different concentrations according to Table 2 (1 mL of each standard), filter with a 0.22 μm filter membrane, and perform high performance liquid chromatography (HPLC) analysis.

[0094] HPLC analysis conditions are as follows: chromatographic column: C18 reverse phase column; temperature: 25°C; detection wavelength: 260nm; flow rate: 0.8mL / min; injection volume: 10μL; mobile phase: 0.1% formic acid, acetonitrile; elution conditions: 0-20min, 5% acetonitrile to 70% acetonitrile gradient elution.

[0095] The results showed that the standard curve of BHET-MHET was y=0.1265x-0.013, R 2 =0.9994.

[0096] Table 2

[0097]

[0098] Example 6: Acyltransferase activity detection of crude enzyme of acyltransferase mutant prepared in Example 2

[0099] 1. Prepare the reaction system. The reaction system is 200 μL, consisting of 50 μL of pH 8.0, 100 mM phosphate buffer, 50 μL of the supernatant collected in Example 2 (i.e., crude enzyme of PETase or crude enzyme of acyltransferase mutant), and 100 μL of bis(hydroxyethyl)terephthalate (BHET) solution with a final concentration of 15 mM (solvent is pH 8.0, 100 mM phosphate buffer).

[0100] 2. Use high performance liquid chromatography (HPLC) to analyze the yield of BHET-MHET in the reaction product. The HPLC analysis conditions are the same as those in Example 5.

[0101] The test results are shown in Table 3. The results showed that compared with the crude enzyme of PETase, the maximum BHET-MHET yield of the crude enzyme of most acyltransferase mutants was significantly increased, about 1.2-16.7 times.

[0102] Table 3

[0103]

[0104]

[0105]

[0106] Example 7: Use of the crude enzyme of the best acyltransferase mutant (i.e., acyltransferase mutant A209T / S166V) prepared in Example 2 in the preparation of polyester prepolymers (e.g., product 1p, product 2p, product 3p, product 4p, product 5p, product 6p, product 7p, product 8p, product 9p)

[0107] 1. Obtain the standard of each substrate and each product. The structural formula of each substrate and each product standard is shown in Table 4.

[0108] Table 4

[0109]

[0110]

[0111] 10-20 mg of substrate 1-substrate 9, product 1p, product 3p, product 4p, product 5p, product 6p, product 7p, product 8p and product 9p were fully dissolved in 500 μL CDCl3, and a one-dimensional NMR spectrum was collected using a 400 MHz NMR instrument. 10-20 mg of product 2p was fully dissolved in 500 μL deuterated DMSO, and a one-dimensional NMR spectrum was collected using a 400 MHz NMR instrument.

[0112] Substrate 1 1 H-NMR spectrum is shown in Figure 4 .

[0113] Product 1p 1 H-NMR spectrum is shown in Figure 5 .

[0114] Substrate 2 1 H-NMR spectrum is shown in Figure 6 .

[0115] Product 2p 1 H-NMR spectrum is shown in Figure 7 .

[0116] Substrate 3 1 H-NMR spectrum is shown in Figure 8 .

[0117] Product 3p 1 H-NMR spectrum is shown in Fig. 9 .

[0118] Substrate 4 1 H-NMR spectrum is shown in Fig.10 .

[0119] Product 4p 1 H-NMR spectrum is shown in Fig.11 .

[0120] Substrate 5 1 H-NMR spectrum is shown in Fig.12 .

[0121] Product 5p 1 H-NMR spectrum is shown in Fig.13 .

[0122] Substrate 6 1 H-NMR spectrum is shown in Fig.14 .

[0123] Product 6p 1 H-NMR spectrum is shown in Fig.15 .

[0124] Substrate 7 1 H-NMR spectrum is shown in Fig.16 .

[0125] Product 7p 1 H-NMR spectrum is shown in Fig.17 .

[0126] Substrate 8 1 H-NMR spectrum is shown in Fig.18 .

[0127] Product 8p 1 H-NMR spectrum is shown in Fig.19 .

[0128] Substrate 9 1 H-NMR spectrum is shown in Fig. 20 .

[0129] Product 9p 1 H-NMR spectrum is shown in Fig.21 .

[0130] 2. Quantitative analysis of substrate 1-substrate 9 and product 1p-product 9p.

[0131] (1) Prepare mother solutions of substrate 1-substrate 9 and product 1p-product 9p at concentrations of 5 mM, 10 mM or 20 mM (the solvent is acetonitrile).

[0132] (2) The mother solutions of substrate 1-substrate 3 and product 1p-product 3p were diluted with 25% acetonitrile (the solvent consisted of 1 volume part acetonitrile and 3 volume parts pH 7.0, 20mM phosphate buffer) to prepare standard solutions of substrate 1, substrate 2, substrate 3, product 2p, and product 3p with concentrations of 50, 100, 250, 500, 750, and 1000 μM, respectively, and standard solutions of product 1p with concentrations of 25, 50, 125, 250, 375, and 500 μM, respectively. 1 mL of each standard solution was then taken, filtered with a 0.22 μm filter membrane, and analyzed by high performance liquid chromatography (HPLC). The HPLC analysis conditions were the same as those in Example 5.

[0133] The results showed that the standard curve of substrate 1 was y = 0.0669x + 0.4967, R 2 =0.9997; the standard curve of product 1p is y=0.0961x-1.3883, R 2 =0.9955; the standard curve of substrate 2 is y=0.2422x-0.2205, R 2 =1; the standard curve of product 2p is y=0.443x-9.5306, R 2 =0.9949; the standard curve of substrate 3 is y=0.185x+0.0209, R 2 =0.9999; the standard curve of product 3p is y=0.4084x-6.8961, R 2 =0.995.

[0134] (3) The mother solutions of substrate 4-substrate 9 and product 4p-product 9p were diluted with 80% acetonitrile (the solvent consisted of 4 parts by volume of acetonitrile and 1 part by volume of pH 7.0, 20 mM phosphate buffer) to prepare standard solutions of substrate 4, product 4p, substrate 7, product 7p, substrate 8, and product 8p with concentrations of 50, 100, 250, 500, 750, and 1000 μM, respectively; standard solutions of substrate 9 and product 9p with concentrations of 50, 100, 200, 250, 400, and 500 μM, respectively; and standard solutions of substrate 5, substrate 6, product 5p, and product 6p with concentrations of 1, 2, 3, 4, and 5 mM, respectively. Then, 1 mL of each standard solution was taken, filtered with a 0.22 μm filter membrane, and then analyzed by high performance liquid chromatography (HPLC). The HPLC analysis conditions were as follows: chromatographic column: C18 reverse phase column; temperature: room temperature; detection wavelength: 260 nm; flow rate: 1.0 mL / min; injection volume: 10 μL; mobile phase: H2O, acetonitrile; elution conditions: 83% acetonitrile / 17% H2O.

[0135] The results showed that the standard curve of substrate 4 was y = 0.0119x + 0.103, R2 =0.9999; the standard curve of product 4p is y=0.0565x-1.0896, R 2 =0.9976; the standard curve of substrate 5 is y=16.7x+0.6656, R 2 =0.9994; the standard curve of product 5p is y=7.4979x-0.6792, R 2 =0.9978; the standard curve of substrate 6 is y=18.044x+1.0662, R 2 =0.9949; the standard curve of product 6p is y=4.1178x+0.1525, R 2 =0.9998; the standard curve of substrate 7 is y=0.0099x+0.1301, R 2 =0.9996; the standard curve of product 7p is y=0.0124x-0.0425, R 2 =1; the standard curve of substrate 8 is y=0.0071x+0.0426, R 2 =0.9996; the standard curve of product 8p is y=0.0121x-0.2214, R 2 =0.9991; the standard curve of substrate 9 is y=0.0042x+0.0179, R 2 =0.9987; the standard curve of product 9p is y=0.0075x-0.1287, R 2 =0.983.

[0136] 3. Prepare a reaction system. The reaction system is 2.5 mL and consists of 1 mL of pH 6.5, 100 mM phosphate buffer, 1.5 mL of the crude enzyme freeze-dried powder of the acyltransferase mutant A209T / S166V prepared in Example 2, and 0.1 g of substrate (BHET, substrate 1, substrate 2, substrate 3, substrate 4, substrate 5, substrate 6, substrate 7, substrate 8 or substrate 9).

[0137] 4. Take the reaction system prepared in step 3, react at room temperature for 29h to obtain a reaction solution. Add 25% acetonitrile (the solvent is composed of 1 volume part of acetonitrile and 3 volume parts of pH7.0, 20mM phosphate buffer) to the reaction solution obtained by 20-30 μL substrate BHET, substrate 1, substrate 2 or substrate 3 to dilute so that the solid is completely dissolved, and the dilution factor is 200-400 times. Add 80% acetonitrile (the solvent is composed of 4 volume parts of acetonitrile and 1 volume part of pH 7.0, 20mM phosphate buffer) to the reaction solution obtained by 20-30 μL substrate 4, substrate 5, substrate 6, substrate 7, substrate 8 or substrate 9 to dilute so that the solid is completely dissolved, and the dilution factor is 50-300 times.

[0138] 5. Use high performance liquid chromatography (HPLC) to determine the yields of the esterification products BHET-MHET, product 1p, product 2p and product 3p corresponding to BHET, substrate 1, substrate 2 and substrate 3. The HPLC analysis conditions are the same as the HPLC analysis conditions in Example 5.

[0139] HPLC was used to determine the yields of the esterification products 4p, 5p, 6p, 7p, 8p and 9p corresponding to substrate 4, substrate 5, substrate 6, substrate 7, substrate 8 and substrate 9, and the HPLC analysis conditions were as follows: chromatographic column: C18 reverse phase column; temperature: room temperature; detection wavelength: 260 nm; flow rate: 1.0 mL / min; injection volume: 10 μL; mobile phase: H2O, acetonitrile; elution conditions: 83% acetonitrile / 17% H2O.

[0140] The test results are shown in Table 5. The results show that the acyltransferase mutant A209T / S166V can catalyze acyl transfer reactions of various substrates and exhibits high catalytic efficiency for furan ring substrates (such as substrate 2 and substrate 3). Among all the tested substrates, the conversion effect of substrate 3 is the best, with a product yield of more than 90%, and the catalytic efficiency of benzoate substrates (such as substrate 7, substrate 8, and substrate 9) is relatively low, within 10%.

[0141] Table 5

[0142]

[0143]

[0144] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. Protein, any one of C1)-C4): C1) replacing at least one of the amino acid residues at positions 209, 166 and 119 in the amino acid sequence of the hydrolase PETase to obtain a protein having acyltransferase activity; C2) A fusion protein obtained by connecting a tag to the N-terminus or / and the C-terminus of the protein shown in C1); C3) A protein obtained by substituting and / or deleting and / or adding one or more amino acid residues of the protein shown in C1) or C2) and having acyltransferase activity; C4) a protein having an amino acid sequence homology of 80% or more to the protein shown in C1) or C2) and having acyltransferase activity; The amino acid sequence of the hydrolase PETase is shown in SEQ ID NO:

1.

2. The protein according to claim 1, characterized in that: The A residue at position 209 is replaced by a T residue, a V residue, an N residue, a G residue, an I residue, a C residue, an F residue, a Q residue, an R residue, an M residue, a D residue, a P residue, an L residue, a K residue, an H residue, a W residue or a Y residue; The S residue at position 166 is replaced by a T residue, a V residue, a C residue, an E residue, an M residue or a G residue; The Q residue at position 119 is substituted with an R residue, a C residue, a W residue, a P residue, an F residue, an I residue or a G residue.

3. The protein according to claim 1 or 2, characterized in that: The acyltransferase activity of the protein is higher than that of the hydrolase PETase.

4. A nucleic acid molecule encoding the protein according to any one of claims 1 to 3.

5. An expression cassette, recombinant vector or recombinant microorganism containing the nucleic acid molecule according to claim 4.

6. Use of the protein according to any one of claims 1 to 3, the hydrolase PETase according to claim 1, the nucleic acid molecule according to claim 4, or an expression cassette, a recombinant vector or a recombinant microorganism containing the nucleic acid molecule according to claim 4 in the preparation of an acyltransferase.

7. Use of the protein according to any one of claims 1 to 3, the hydrolase PETase according to claim 1, the nucleic acid molecule according to claim 4, or an expression cassette, a recombinant vector or a recombinant microorganism containing the nucleic acid molecule according to claim 4 as an acyltransferase.

8. The use according to claim 6 or 7, characterized in that: The acyltransferase activity of the protein according to any one of claims 1 to 3 is higher than that of the hydrolase PETase according to claim 1.

9. Use of the protein according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, or an expression cassette, a recombinant vector or a recombinant microorganism containing the nucleic acid molecule according to claim 4 in PET hydrolysis.

10. Use of the protein according to any one of claims 1 to 3, the hydrolase PETase according to claim 1, the nucleic acid molecule according to claim 4, or an expression cassette, a recombinant vector or a recombinant microorganism containing the nucleic acid molecule according to claim 4, wherein: K1) or K2) or K3): K1) catalyzes the acyl transfer reaction of BHET; K2) catalyzing acyl transfer reactions of aromatic, furanic or aliphatic polymer precursors; K3) Catalyzes the synthesis of esters and amides.

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