Novel polyester intermediate product as well as preparation method and application thereof

Abstract

The invention discloses a novel polyester intermediate product as well as a preparation method and application thereof, and the novel polyester intermediate product is O, O '-(ethane-1, 2-diacyl) bis (2-hydroxyethyl) diterephthalate (BHET-MHET). Experiments prove that BHET-MHET is taken as a substrate and can be widely applied to activity detection and screening of polyester depolymerases by detecting OD600nm. The polyester degrading enzyme can be a polyester depolymerases LCC, a polyester depolymerases TfCut2 or a polyester depolymerases PETase. The method for screening the polyester depolymerases by using BHET-MHET has the characteristics of high accuracy and high reliability. The method has an important application value.

Description

Technical Field

[0001] The invention belongs to the technical field of chemistry and enzyme engineering, and specifically relates to a novel polyester intermediate product and a preparation method and application thereof. The novel polyester intermediate product is O,O'-(ethane-1,2-diacyl)bis(2-hydroxyethyl)terephthalate (BHET-MHET). Background Art

[0002] Polyester depolymerase is a type of hydrolase that can hydrolyze high molecular weight polyesters such as PET. Polyester depolymerases include PETase, LCC, TfCut2, etc. At present, the analysis of polyester depolymerase activity is mainly achieved by detecting the degradation products MHET and TPA of PET or PET oligomers by high performance liquid chromatography (HPLC). HPLC detection has high sensitivity, good separation effect, and relatively accurate and reliable results, but its analysis is limited in number and time-consuming and is not suitable for screening polyester depolymerase mutant libraries containing thousands of clones. Therefore, it is very important to establish a simple and easy-to-operate method for high-throughput screening of polyester depolymerases with high hydrolytic activity, which can effectively promote the development of enzymatic depolymerization of polyesters. Summary of the invention

[0003] The object of the present invention is to provide a method for screening polyester depolymerase.

[0004] The present invention first provides a compound that can be used to screen polyester depolymerases, namely, O,O'-(ethane-1,2-diacyl)bis(2-hydroxyethyl)terephthalate, namely, BHET-MHET, whose structural formula is shown in formula (I):

[0005]

[0006] The present invention also protects a method for preparing the BHET-MHET, which can be obtained by catalyzing BHET with a protein having acyltransferase activity.

[0007] In the above preparation method, the step of "catalyzing BHET with a protein having acyltransferase activity to obtain BHET-MHET" specifically includes steps (1) and (2):

[0008] (1) reacting BHET with a protein having acyltransferase activity to obtain a reaction solution;

[0009] (2) After completing step (1), the reaction solution is centrifuged and the precipitate is collected; the precipitate contains BHET-MHET.

[0010] The step (1) may be: adding a buffer solution and a protein having acyltransferase activity to BHET, reacting to obtain a reaction solution.

[0011] Any of the above-mentioned proteins having acyltransferase activity may be any one of C1)-C4):

[0012] 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;

[0013] C2) A fusion protein obtained by connecting a tag to the N-terminus or / and the C-terminus of the protein shown in C1);

[0014] 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;

[0015] 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;

[0016] The amino acid sequence of the hydrolase PETase is shown in SEQ ID NO:1.

[0017] Any of the above-mentioned proteins having acyltransferase activity is an acyltransferase variant obtained by mutation of the hydrolase PETase.

[0018] In any of the above proteins having acyltransferase activity, 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.

[0019] 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.

[0020] The acyltransferase activity of any of the above-mentioned proteins having acyltransferase activity is higher than that of the hydrolase PETase.

[0021] Any of the above-mentioned proteins having acyltransferase activity 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).

[0022] In the step (2), the centrifugation may be performed at 4° C. and 12,000 rpm for 10 min.

[0023] The preparation method may further include step (3): after completing step (2), separating BHET-MHET from the precipitate. The "separating BHET-MHET from the precipitate" may specifically be dissolving the precipitate in acetonitrile, then slowly adding ddH2O in an amount not less than the volume of acetonitrile, and collecting the precipitated solid, which is BHET-MHET.

[0024] The application of BHET-MHET in detecting the activity of polyester depolymerase also falls within the protection scope of the present invention.

[0025] The application of BHET-MHET in screening polyester depolymerase also belongs to the protection scope of the present invention.

[0026] The present invention also protects a method for identifying or assisting in identifying whether a polyester depolymerase to be tested has polyester depolymerase activity, comprising the following steps:

[0027] (a1) Mixing the polyester depolymerase to be tested, buffer and BHET-MHET to obtain a mixed solution 1; then detecting the OD of the mixed solution 1 600nm As the reaction time changes, the OD of the mixture 1 is obtained. 600nm Curves of changes with reaction time;

[0028] (a2) mixing the buffer solution and BHET-MHET to obtain a mixed solution 2; and then detecting the OD of the mixed solution 2. 600nm As the reaction time changes, the OD of the mixed solution 2 is obtained. 600nm Curves of changes with reaction time;

[0029] (a3) Make the following judgment: If the OD of mixture 1 increases with the increase of reaction time compared with mixture 2, 600nm If the rate of decrease of the slope of the curve changing with the reaction time is faster, the polyester depolymerase to be tested has or is suspected to have polyester depolymerase activity; otherwise, the polyester depolymerase to be tested does not have or is suspected to not have polyester depolymerase activity.

[0030] The present invention also protects a method for screening polyester depolymerase, comprising the following steps:

[0031] (a1) Mixing polyester degrading enzyme, buffer and BHET-MHET to obtain mixed solution A; then detecting the OD of the mixed solution A. 600nm As the reaction time changes, the OD of the mixed solution A is obtained. 600nm Curves of changes with reaction time;

[0032] (a2) preparing a polyester depolymerase mutant to be screened; then mixing the polyester depolymerase to be screened, a buffer solution and BHET-MHET to obtain a mixed solution B; then detecting the OD of the mixed solution B. 600nm As the reaction time changes, the OD of the mixed solution B is obtained. 600nm Curve of change with reaction time;

[0033] (a3) Compare the OD of mixture A and mixture B 600nm The curve of the reaction time is as follows: If the OD of the mixed solution B 600nm If the rate of decrease of the slope of the curve varying with reaction time is faster than that of the mixed solution A, the polyester depolymerase to be screened has a higher or suspected higher polyester depolymerase activity than the polyester degrading enzyme.

[0034] In any of the above methods, the polyester degrading enzyme may be polyester depolymerase LCC, polyester depolymerase TfCut2 or polyester depolymerase PETase. The amino acid sequence of polyester depolymerase LCC may be as shown in SEQ ID No: 2. The amino acid sequence of polyester depolymerase TfCut2 may be as shown in SEQ ID No: 3. The amino acid sequence of polyester depolymerase PETase may be as shown in SEQ ID No: 1.

[0035] The present invention also protects a BHET-MHET derivative, characterized in that the BHET-MHET derivative can be obtained by one or more steps of esterification reaction of BHET-MHET.

[0036] Experiments have shown that using BHET-MHET as a substrate, by detecting OD 600nm The method can be widely used in the activity detection and screening of polyester depolymerase. The polyester degrading enzyme can be polyester depolymerase LCC, polyester depolymerase TfCut2 or polyester depolymerase PETase. The screening of polyester depolymerase by BHET-MHET has the characteristics of high accuracy and strong reliability. The invention has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Schematic diagram of the BHET reaction catalyzed by the acyltransferase mutant to produce BHET-MHET.

[0038] Figure 2 For BHET-MHET 1 H-NMR spectrum.

[0039] Figure 3 For BHET-MHET 13 C-NMR spectrum.

[0040] Figure 4The standard curves of BHET, MHET and BHET-MHET obtained by HPLC analysis are shown in Figure 1. A is BHET-MHET; B is BHET; C is MHET.

[0041] Figure 5 This is the HPLC analysis spectrum of the acyltransferase mutant A209T / S166V catalyzing 20mM BHET for 1h.

[0042] Figure 6 This is the HPLC analysis spectrum of 2g BHET catalyzed by the acyltransferase mutant A209T / S166V.

[0043] Figure 7 The method for determining the hydrolysis activity of polyester depolymerase using BHET-MHET as substrate was established. A is OD 600nm Standard curve of the method; B is OD 600nm The method is used to determine the hydrolytic activity of polyester depolymerase PETase.

[0044] Figure 8 BHET-MHET was used as a substrate for the screening of polyester depolymerase mutants and the determination of their activity. A is OD 600nm A is the standard deviation determination for 96-well plate screening; B is the activity determination of PETase and its mutants; C is the activity determination of polyester depolymerase LCC and TfCut2. DETAILED DESCRIPTION

[0045] 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.

[0046] 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.

[0047] Example 1. Preparation of polyester depolymerase PETase (PETase for short, also known as PETase wild type or PETaseWT) and PETase mutants

[0048] 1. Construction of recombinant plasmid

[0049] 1. Replace the small DNA fragment between the restriction endonuclease EcoRI and XhoI recognition sites of the pET-22b(+) vector with the polyester depolymerase PETase gene (Genebank No. GAP38373.1) from the Ideonella sakaiensis 201-F6 strain to obtain the recombinant plasmid pET22b-PETase.

[0050] The recombinant plasmid pET22b-PETase expresses the PETase shown in SEQ ID No: 1. PETase has weak acyltransferase activity.

[0051] 2. Primer F: 5'-GGCCATGGATATCGGAATTAATTCGGATCCGAATTCG-3' and primer R: 5'-TCAGTGGTGGTGGTGGTGGTGCTCGAG-3' were used to randomly mutate the nucleotide sequence of the polyester depolymerase PETase gene from Ideonella sakaiensis 201-F6 strain by error-prone PCR to obtain several mutant nucleotide sequences.

[0052] 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.

[0053] 2. Obtaining PETase mutants with improved acyltransferase activity (referred to as acyltransferase mutants)

[0054] Each recombinant plasmid constructed in step 1 was subjected to the following experiment:

[0055] 1. Transform the recombinant plasmid into Escherichia coli BL21 Gold (DE3) to obtain recombinant bacteria.

[0056] 2. Inoculate the recombinant bacterial monoclone into 150 μL LB medium (containing 100 μg / mL ampicillin), and culture in a 96-well plate shaking incubator at 37°C and 800 rpm for 16 h to obtain a culture solution.

[0057] 3. Inoculate the culture solution obtained in step 2 into 150 μL LB liquid medium (containing 100 μg / mL ampicillin), culture at 37°C for 3 h, add 50 μL 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 for 30 min, and collect the supernatant.

[0058] 4. Preparation of screening system. The screening was carried out in a 96-well plate, and the screening system was 200 μL, consisting of 50 μL of pH 8.0, 100 mM phosphate buffer, 50 μL of the supernatant collected in step 3, and 100 μL of bis(hydroxyethyl)terephthalate (BHET) solution with a final concentration of 15 mM.

[0059] 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 , test every 10 minutes in the first hour, and then test every 1 hour.

[0060] 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. 600nm The 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 a PETase mutant with improved acyltransferase activity (referred to as an acyltransferase mutant).

[0061] 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 (taking A209T as an example, A209T means that the A at position 209 from the N-terminus of SEQ ID No: 1 is replaced by T).

[0062] Table 1

[0063]

[0064]

[0065] Example 2. Preparation of crude enzyme of acyltransferase mutant screened in Example 1

[0066] The recombinant bacteria (recombinant bacteria transformed with the pET-22b(+) vector (i.e., the pET-22b(+) vector was transformed into Escherichia coli BL21 Gold(DE3) to obtain the recombinant bacteria), the recombinant bacteria transformed with the recombinant plasmid pET22b-PETase, or the recombinant bacteria transformed with the recombinant plasmid pET22b-PETase mutant) were inoculated into 3 mL of LB medium with an inoculation needle, and cultured at 37°C and 220 rpm for 16 h. Then, the inoculation amount was inoculated into 15 mL of LB medium at 1% (V / V), and cultured at 37°C and 220 rpm until the OD 600nmWhen the p-value reached about 0.8, IPTG was added at a final concentration of 0.1 mM, and the expression was induced at 20°C and 220 rpm for 24 h. The supernatant was collected after centrifugation.

[0067] The supernatant is the corresponding crude enzyme.

[0068] Example 3. Enzymatic preparation and identification of acyl transfer product O,O'-(ethane-1,2-diacyl)bis(2-hydroxyethyl)terephthalate (BHET-MHET)

[0069] At 30°C, the PETase mutant (i.e., acyltransferase mutant) screened in Example 1 was used to catalyze the transesterification of bis(hydroxyethyl) terephthalate (BHET) as a single substrate to obtain a novel polyester intermediate, namely, O,O'-(ethane-1,2-diacyl)bis(2-hydroxyethyl) diterephthalate (BHET-MHET). The schematic diagram of the acyltransferase mutant catalyzing the BHET reaction to produce BHET-MHET is shown in Figure 1 The specific steps are as follows:

[0070] 1. Add 50 mL of pH 8.0, 100 mM phosphate buffer and 0.5 g of BHET powder to a 250 mL conical flask, then add 50 μL of crude enzyme of acyltransferase mutant A209T / S166V, and shake the reaction 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, completely dissolve the precipitate with acetonitrile, then add ddH2O with a volume of about 2 times that of acetonitrile, and centrifuge to collect the precipitated solid to obtain the purified product BHET-MHET.

[0071] 2. Dissolve 10-20 mg of the purified product BHET-MHET in 500 μL of deuterated DMSO and collect a one-dimensional NMR spectrum using a 400 MHz NMR spectrometer.

[0072] BHET-MHET 1 H-NMR spectrum is shown in Figure 2 .

[0073] BHET-MHET 13 C-NMR spectrum is shown in Figure 3 .

[0074] The structural formula of BHET-MHET is finally determined as shown in formula (I):

[0075]

[0076] According to the above steps, the crude enzyme of acyltransferase mutant A209T / S166V is replaced with the crude enzyme of 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.

[0077] Example 4, quantitative analysis of BHET-MHET

[0078] 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: 20μL; mobile phase: 0.1% formic acid, acetonitrile; elution conditions: 0-20min, 5% acetonitrile to 70% acetonitrile gradient elution.

[0079] 1. Preparation of standard curves for BHET, MHET, and BHET-MHET

[0080] 1. Prepare a 5 mM BHET-MHET mother solution (the solvent is acetonitrile). Prepare BHET-MHET standards of different concentrations according to Table 2 (1 mL for each standard), filter with a 0.22 μm filter membrane, and perform high performance liquid chromatography (HPLC) analysis.

[0081] Table 2

[0082]

[0083] The results showed that the standard curve of BHET-MHET was y = 0.1265x-0.013, R 2 =0.9994 (see Figure 4 (A).

[0084] 2. Follow the method of step 1, replacing BHET-MHET with BHET, and keep other steps unchanged.

[0085] The results show that the standard curve of BHET is y=0.0775x+2.4513, R 2 =0.9944 (see Figure 4 (middle B).

[0086] 3. Follow the method in step 1, replacing BHET-MHET with MHET, and keep other steps unchanged.

[0087] The results showed that the standard curve of MHET was y = 0.0813x + 0.1874, R 2 =0.9998 (see Figure 4 Middle C).

[0088] 2. Quantitative Analysis

[0089] 1. Prepare the reaction system. The reaction system is 200 μL, consisting of 50 μL of pH 8.0, 100 mM phosphate buffer, 100 μL of 40 mM BHET solution, and 50 μL of 0.05 mg / mL PETase acyltransferase active mutant A209T / S166V.

[0090] 2. Take the reaction system prepared in step 1, react at 30°C and 900 rpm for 1 hour, then dilute the reaction system with 25% acetonitrile and terminate the reaction to obtain a dilution. Filter the dilution with a 0.22 μm filter membrane and perform high performance liquid chromatography (HPLC) analysis.

[0091] The HPLC analysis of the diluent is shown in Figure 5 .

[0092] The results showed that BHET-MHET peaked at around 10 min. According to the standard curve of BHET, MHET and BHET-MHET in step 1, the yield of BHET-MHET after acyltransferase mutant A209T / S166V catalyzed 20 mM BHET for 1 h was 5 mM, and the conversion rate was 50%.

[0093] Example 5, preparation-scale reaction of BHET-MHET

[0094] A total of about 50 g of BHET-MHET was prepared. The specific preparation method is as follows:

[0095] The reaction was carried out in batches in a 250 mL conical flask at room temperature and 220 rpm. 2 g of BHET powder and 20 mL of pH 6.5, 100 mM sodium phosphate buffer were added to each bottle, and 30 mL of crude enzyme of the acyltransferase mutant A209T / S166V was added in batches. The final reaction volume was 50 mL. The reaction was oscillated for 24 h and the reaction was stopped. The precipitate was filtered and washed 3 to 5 times with ddH2O and dried to obtain a sample containing BHET-MHET.

[0096] HPLC was used to detect the yield of BHET-MHET in the sample under the above reaction conditions. The specific steps are as follows: 7 volumes of acetonitrile and 1 volume of pH 6.5, 100 mM sodium phosphate buffer were added to the sample until the solid was basically completely dissolved, centrifuged at 12000 rpm for 5 minutes, and the supernatant was collected. The supernatant was filtered with a 0.22 μm filter membrane and analyzed by HPLC.

[0097] 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: 20μL; mobile phase: 0.1% formic acid, acetonitrile; elution conditions: 0-20min, 5% acetonitrile to 70% acetonitrile gradient elution.

[0098] The analysis results are shown in Figure 6 The results showed that the yield of BHET-MHET was 81.31 ± 0.38%.

[0099] Example 6: Application of BHET-MHET in screening and detecting polyester depolymerase and its mutants

[0100] 1. BHET-MHET prepared in Example 5 was dried to obtain a powder, which was then completely dissolved in dimethyl sulfoxide (DMSO). pH 8.0, 100 mM phosphate buffer was slowly added until the final DMSO concentration was 10%. BHET-MHET gradually precipitated, and a stable white emulsion was finally obtained. As the concentration of BHET-MHET decreased, the turbidity of the emulsion decreased, which could be measured by OD 600nm To reflect the change of turbidity.

[0101] 2. Prepare a 5mM BHET-MHET stock solution, dilute it to 1mM, 2mM, 3mM and 4mM respectively using pH 8.0, 100mM phosphate buffer containing 10% DMSO to obtain the corresponding BHET-MHET solutions. Use MD microplate reader to detect the OD values ​​of BHET-MHET solutions (0-5mM) of different concentrations. 600nm value.

[0102] The concentration of BHET-MHET is taken as the horizontal axis, OD 600 The value is the vertical axis, and the standard curve is drawn. The result is as follows Figure 7 As shown in A. The results show that the concentration of BHET-MHET is related to OD 600nm There is a good linear relationship, so it can be measured by OD 600nm To characterize the consumption of BHET-MHET.

[0103] 3. Prepare the reaction system. The reaction system is 200 μL, consisting of 80 μL pH 8.0, 100 mM phosphate buffer, 20 μL supernatant (the supernatant collected from the recombinant bacteria transformed with the pET-22b(+) vector or the supernatant collected from the recombinant bacteria transformed with the recombinant plasmid pET22b-PETase in Example 2) and 100 μL of 10 mM BHET-MHET solution. Use an ELISA reader to detect the OD of the reaction system at different times. 600nm The test lasted for 230 minutes. The test results are shown in Figure 7 The results showed that the OD of BHET-MHET hydrolyzed by the supernatant collected from the recombinant bacteria transformed with the recombinant plasmid pET22b-PETase increased with time. 600nm The value decreased continuously, while the supernatant collected by the recombinant bacteria transformed with pET-22b(+) vector hydrolyzed the OD of BHET-MHET. 600nm The value was basically unchanged. It can be seen that BHET-MHET can be hydrolyzed by PETase and the concentration decreases. The higher the hydrolysis activity of PETase, the higher the OD 600nm The faster the rate of descent.

[0104] 4. Establishment of a method for screening PETase mutants using BHET-MHET as a substrate

[0105] The inventors of the present invention have established a method for screening PETase mutants using BHET-MHET as a substrate, comprising the following steps:

[0106] (1) Take reaction system 1 and react at 30°C and 900 rpm. During the reaction, measure OD every 5 minutes. 600nm , O.D. 600nm After no more changes, the OD of reaction system 1 is obtained. 600nm A graph of values ​​changing over time;

[0107] Reaction system 1 was 200 μL, consisting of 80 μL of pH 8.0, 100 mM phosphate buffer, 20 μL of the PETase hydrolysis mutant to be screened, and 100 μL of 10 mM BHET-MHET solution;

[0108] (2) Take reaction system 2 and react at 30°C and 900 rpm. During the reaction, measure OD every 5 minutes. 600nm , O.D. 600nm After no more changes, the OD of reaction system 2 is obtained. 600nm A graph of values ​​changing over time;

[0109] Reaction system 2 was 200 μL, consisting of 80 μL of 100 mM phosphate buffer, pH 8.0, 20 μL of PETase, and 100 μL of 10 mM BHET-MHET solution;

[0110] (3) Compare the OD of reaction system 1 and reaction system 2 600nm The curve graph of reaction time changes. If the OD of reaction system 1 600nm If the slope of the curve changing with the reaction time decreases faster than that of reaction system 2, the PETase hydrolysis mutant to be screened has a higher or suspected higher hydrolase activity than the PETase.

[0111] 5. Application of BHET-MHET as substrate for screening PETase mutants

[0112] The method established in step 4 was applied to the screening of a mutant library of PETase from Ideonella sakaiensis (amino acid sequence as shown in SEQ ID No: 1), and the specific steps were as follows:

[0113] (1) Prepare a 96-well plate and prepare a reaction system in each well. The reaction system is 200 μL, consisting of 80 μL of pH 8.0, 100 mM phosphate buffer, 20 μL of PETase mutant crude enzyme solution, and 100 μL of 10 mM BHET-MHET solution.

[0114] (2) After completing step (1), react at 30°C and 900 rpm, and take the OD value before and after the reaction for 30 minutes. 600nm The difference is used to calculate the standard deviation.

[0115] Results Figure 8 A. The results show that the standard deviation of the method established by the present invention is less than 10%.

[0116] (3) Using the recombinant plasmid pET-22b-PETase as a template, error-prone PCR was performed with primers F: 5′-GGCCATGGATATCGGAATTAATTCGGATCCGAATTCG-3′ and R: 5′-TCAGTGGTGGTGGTGGTGGTGCTCGAG-3′ to randomly mutate the target gene and obtain a random mutation library.

[0117] (4) Using a 96-well plate to express and screen mutants, specifically as follows: Inoculate a single clone from the random mutation library into 150 μL of LB liquid culture medium containing 100 μg / mL ampicillin, and culture in a shaking incubator at 37°C and 800 rpm for 16 h to obtain a culture solution. Inoculate the culture solution into 150 μL of LB liquid culture medium containing 100 μg / mL ampicillin, add 50 μL of IPTG to a final concentration of 0.1 mM, shake culture at 20°C and 800 rpm for 24 h, and collect the supernatant by centrifugation, which is the crude enzyme solution of the PETase mutant.

[0118] (5) Preparation of reaction system: The reaction system is 200 μL, consisting of 80 μL of pH 8.0, 100 mM phosphate buffer, 20 μL of crude enzyme solution of PETase mutant, and 100 μL of 10 mM BHET-MHET solution.

[0119] (6) Take the reaction system prepared in step (6) and start the reaction at 30°C and 900 rpm. Measure the OD of the reaction system every 10 min for 1 h before the reaction. 600nm , until OD 600nm The measurement can be terminated when there is no more change.

[0120] The crude enzyme solution of the PETase mutant was replaced with pH 8.0, 100 mM phosphate buffer, and other steps remained unchanged as a control.

[0121] After 96-well plate screening, multiple PETase mutants with improved hydrolysis activity were obtained, among which the activity data of mutant N246D (the mutation site corresponds to the amino acid sequence of SEQ ID No: 1) are as follows: Figure 8 As shown in B. The hydrolysis activity of the PETase mutant N246D was significantly increased compared with the PETase from Ideonella sakaiensis, as shown by the OD 600nm The value decreased faster than that of PETase from Ideonella sakaiensis.

[0122] 6. Application of BHET-MHET in detecting the activity of thermostable polyester depolymerase LCC and TfCut2

[0123] (1) Construction of recombinant plasmid

[0124] The small DNA fragment between the restriction endonuclease BamHI and XhoI recognition sites of the pET-22b(+) vector was replaced with the polyester depolymerase gene LCC derived from the Leaf-branch compost to obtain the recombinant plasmid pET22b-LCC. The recombinant plasmid pET22b-LCC expressed the LCC shown in SEQ ID No:2.

[0125] The small DNA fragment between the restriction endonuclease EcoRI and XhoI recognition sites of the pET-22b(+) vector was replaced with the polyester depolymerase gene TfCut2 from the Thermobifidafusca KW3 strain to obtain the recombinant plasmid pET22b-TfCut2. The recombinant plasmid pET22b-TfCut2 expressed TfCut2 shown in SEQ ID No:3.

[0126] (2) Preparation of LCC crude enzyme solution and TfCut2 crude enzyme solution

[0127] The recombinant plasmid pET22b-LCC was transformed into Escherichia coli BL21 Gold (DE3) to obtain recombinant bacteria 1.

[0128] The recombinant plasmid pET22b-TfCut2 was transformed into Escherichia coli BL21 Gold (DE3) to obtain recombinant bacteria 2.

[0129] According to the method of Example 2, the recombinant bacteria were replaced by recombinant bacteria 1 or recombinant bacteria 2, and the other steps remained unchanged, and the supernatant was collected.

[0130] The supernatant collected from recombinant bacteria 1 is the crude LCC enzyme solution, and the supernatant collected from recombinant bacteria 2 is the crude TfCut2 enzyme solution.

[0131] (3) Preparation of reaction system: The reaction system was 200 μL and consisted of 50 μL of pH 8.0, 100 mM phosphate buffer, 50 μL of crude enzyme solution of polyester depolymerase (LCC crude enzyme solution or TfCut2 crude enzyme solution), and 100 μL of 10 mM BHET-MHET solution.

[0132] (4) Take the reaction system prepared in step (3) and start the reaction at 60°C and 900 rpm. Measure the OD of the reaction system every 10 min for 1 h before the reaction. 600nm , until OD 600nm The measurement can be terminated when there is no more change.

[0133] The crude enzyme solution of polyester depolymerase was replaced with pH 8.0, 100 mM phosphate buffer, while other steps remained unchanged, as a control.

[0134] Test results see Figure 8 C. The results showed that at 60°C, the OD values ​​of polyester depolymerase LCC and polyester depolymerase TfCut2 600nm The values ​​decreased with the extension of reaction time, while the OD 600nm Basically stable.

[0135] The above results show that using BHET-MHET as substrate, the OD600nm The method can be widely used in the activity detection and screening of polyester depolymerase.

[0136] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. O,O'-(ethane-1,2-diacyl)bis(2-hydroxyethyl)terephthalate, namely BHET-MHET, has a structural formula as shown in formula (I):

2. The method for preparing BHET-MHET according to claim 1, wherein BHET-MHET is obtained by catalyzing BHET with a protein having acyltransferase activity.

3. The preparation method according to claim 2, characterized in that: The "catalyzing BHET with a protein having acyltransferase activity to obtain BHET-MHET" comprises the following steps: (1) reacting BHET with a protein having acyltransferase activity to obtain a reaction solution; (2) After completing step (1), the reaction solution is centrifuged to collect the precipitate; (3) After completing step (2), BHET-MHET is separated from the precipitate.

4. Use of the BHET-MHET according to claim 1 in detecting the activity of polyester depolymerase.

5. Use of the BHET-MHET described in claim 1 in screening polyester depolymerase.

6. A method for identifying or assisting in identifying whether a polyester depolymerase to be tested has polyester depolymerase activity, comprising the following steps: (a1) mixing the polyester depolymerase to be tested, a buffer and the BHET-MHET described in claim 1 to obtain a mixed solution 1; then detecting the OD of the mixed solution 1 600nm As the reaction time changes, the OD of the mixture 1 is obtained. 600nm Curves of changes with reaction time; (a2) mixing the buffer solution and the BHET-MHET according to claim 1 to obtain a mixed solution 2; and then detecting the OD of the mixed solution 2. 600nm As the reaction time changes, the OD of the mixed solution 2 is obtained. 600nm Curves of changes with reaction time; (a3) Make the following judgment: If the OD of mixture 1 increases with the increase of reaction time compared with mixture 2, 600nm If the rate of decrease of the slope of the curve changing with the reaction time is faster, the polyester depolymerase to be tested has or is suspected to have polyester depolymerase activity; otherwise, the polyester depolymerase to be tested does not have or is suspected to not have polyester depolymerase activity.

7. A method for screening polyester depolymerase, comprising the following steps: (a1) mixing a polyester degrading enzyme, a buffer and the BHET-MHET described in claim 1 to obtain a mixed solution A; and then detecting the OD of the mixed solution A. 600nm As the reaction time changes, the OD of the mixed solution A is obtained. 600nm Curves of changes with reaction time; (a2) preparing a polyester depolymerase mutant to be screened; then mixing the polyester depolymerase to be screened, a buffer and the BHET-MHET according to claim 1 to obtain a mixed solution B; then detecting the OD of the mixed solution B. 600nm As the reaction time changes, the OD of the mixed solution B is obtained. 600nm Curves of changes with reaction time; (a3) Compare the OD of mixture A and mixture B 600nm The curve of the reaction time is as follows: If the OD of the mixed solution B 600nm If the rate of decrease of the slope of the curve changing with reaction time is faster than that of the mixed solution A, the polyester depolymerase to be screened has a higher or suspected higher polyester depolymerase activity than the polyester degrading enzyme.

8. The method according to claim 7, characterized in that: The polyester degrading enzyme is polyester depolymerase LCC, polyester depolymerase TfCut2 or polyester depolymerase PETase.

9. The method according to claim 8, characterized in that: The amino acid sequence of polyester depolymerase LCC is shown in SEQ ID No: 2; The amino acid sequence of polyester depolymerase TfCut2 is shown in SEQ ID No: 3; The amino acid sequence of polyester depolymerase PETase is shown in SEQ ID No:

1.

10. A BHET-MHET derivative, characterized in that: The BHET-MHET derivative can be obtained by subjecting the BHET-MHET described in claim 1 to one or more esterification reactions.

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