High-expression high-efficiency PET hydrolase mutant and application thereof

Abstract

The invention belongs to the technical field of protein engineering and biological catalysis application, and discloses a high-expression efficient PET hydrolase mutant and application thereof. The PET hydrolase mutant with high activity, high thermal stability and high expression level obtained through calculation design and site-specific mutagenesis shows great potential to be applied to industrial application of PET recovery. Experiments prove that the fusion temperature of the modified mutant protein reaches 92.4 DEG C; under the industrial-grade PET substrate concentration, the maximum reaction speed of catalyzing PET hydrolysis is 1.76 times that of the template enzyme; and the expression level of the gene in a shake flask is 3.3 times that of a template enzyme, and the protein yield in a fermentation tank reaches 1.49 g / L.

Description

Technical Field

[0001] The present invention belongs to the field of biocatalysis and relates to a highly expressed and efficient PET hydrolase mutant and its application. Background Art

[0002] Plastics are widely used in various fields due to their excellent heat resistance, good mechanical properties, and stable chemical properties. However, only 9% of plastics globally are effectively recycled. Polyethylene terephthalate (PET), one of the most produced synthetic polyesters globally with an annual output of over 82 million tons, is widely used in products such as beverage bottles, textiles, and food packaging. Due to the strong chemical stability of PET, a large amount of waste PET is difficult to degrade in the natural environment, ultimately entering the ocean and soil and potentially affecting the ecosystem through the food chain, causing serious long-term harm to the environment. Traditional chemical degradation methods (such as methanolysis, hydrolysis, aminolysis, and glycolysis, etc.) usually require high-temperature and high-pressure environments and rely on hazardous chemicals as catalysts, thus having many limitations in actual industrial applications. In contrast, enzymatic degradation of PET has relatively mild reaction conditions, the degradation products are purer, and it helps in the efficient recycling of PET, thus showing greater potential in environmental protection and industrial applications.

[0003] In the past two decades, researchers have used protein engineering methods to discover and modify multiple PET hydrolases, significantly improving the thermal stability and catalytic efficiency of the enzymes. Among them, the mutant LCC-ICCG of the thermophilic cutinase leaf-branch compost cutinase (LCC) designed by Tournier et al. in 2020 achieved more than 90% PET degradation within 10 hours under industrial reaction conditions (PET substrate concentration of 200 g / L), demonstrating excellent production efficiency (Tournier, V., etal. "An engineered PET depolymerase to break down and recycle plastic bottles." Nature 580.7802 (2020): 216-219.). By introducing disulfide bonds, the melting temperature ( T m ) of the mutant LCC-ICCG was increased by 9.5 °C compared to the wild type, but its insufficient activity forced the addition of an enzyme amount of three-thousandths of the PET mass to achieve a 90% degradation rate.

[0004] The properties of PET hydrolase, such as its activity, thermal stability, and expression level, limit its economic feasibility in industrial applications. Through process simulation to analyze the techno-economic performance of the enzymatic PET recycling process, it was found that the cost of the enzyme is higher than the energy cost required to maintain the reaction temperature, and the high usage cost of PET hydrolase is mainly attributed to its insufficient expression level. In published studies, the protein production of PET hydrolase with high catalytic activity in shake flasks usually does not exceed 30 mg / L; while the maximum target protein production in fed-batch fermentation in a fermenter is only 1.2 g / L (Soong, Ya‐Hue Valerie, et al. "Enzyme selection, optimization, and production toward biodegradation of post‐consumer poly (ethylene terephthalate) at scale." Biotechnology Journal 18.12 (2023): 2300119.), which makes it difficult to achieve economic feasibility in industrial applications in terms of its production cost. Therefore, improving the expression level of PET hydrolase is one of the key challenges for its industrial application.

[0005] In summary, in order to make the enzymatic PET depolymerization technology economically feasible in industrial applications, the PET hydrolase involved must possess excellent thermal stability, high catalytic activity, and good expression level. However, currently engineered PET hydrolases have not been able to meet these key performance indicators simultaneously. Therefore, on the premise of maintaining the thermal stability and catalytic activity of the enzyme, developing a PET hydrolase that can achieve high-level expression in Escherichia coli has important practical significance for addressing the increasingly severe plastic pollution problem. This research can not only promote the sustainable recycling of waste PET but also has long-term strategic value for reducing environmental pollution. Summary of the Invention

[0006] The technical problem solved by the present invention is to provide a PET hydrolase with improved catalytic activity and expression level while maintaining the thermal stability of the enzyme, thereby effectively reducing the usage cost of the enzyme, enhancing its competitiveness in industrial production, and solving the economic feasibility problem of PET hydrolase in the industrial application of PET recycling.

[0007] To solve the above technical problem, in the first aspect, the present invention provides a mutant protein of PET hydrolase, which is any one of the following: The mutant protein shown in A1) is obtained by mutating the amino acid residues at positions 184 and 188 of SEQ ID NO.2 to obtain a protein with the same function; A2) A protein with more than 99%, more than 95%, more than 90%, more than 85% or more than 80% homology of other amino acid residues except the mutated amino acid residues in the mutant protein shown in A1), and having the same function; A3) A protein obtained by linking a tag to the N-terminus and / or C-terminus of the mutant protein shown in A1) or A2).

[0008] In the mutant protein described above, the mutation mode of each amino acid residue is as follows: The 184th amino acid residue His is mutated to Asn; The 188th amino acid residue Phe is mutated to Met.

[0009] In the mutant protein described above, the amino acid sequence of the mutant protein shown in A1) is SEQ ID NO.4, named LCC-ICCG-NM.

[0010] Compared with the template PET hydrolase LCC-ICCG, the mutant protein has high activity and high thermal stability for PET hydrolysis, specifically manifested as being stable and efficiently catalyzing PET hydrolysis above 68 °C, that is, the degradation rate of PET in 3 hours is 176% of that of LCC-ICCG.

[0011] In the second aspect, the present invention provides a biological material related to the mutant protein described in the first aspect, which is any one of the following B1) to B4): B1) A nucleic acid molecule encoding the mutant protein described in the first aspect; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) A recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3).

[0012] The recombinant vector described above is a recombinant expression vector obtained by inserting the encoding nucleic acid described in the second aspect of the present invention into the multiple cloning sites Nde I and Xho I of the pET-22b(+) vector, and can be named pET-22b(+)-LCC-ICCG-NM.

[0013] The above recombinant microorganism is a recombinant Escherichia coli containing the above recombinant vector.

[0014] In the biological material described above, the nucleic acid molecule described in B1) is any one of the following b1)-b6): b1) The DNA molecule shown in SEQ ID NO.3; b2) A DNA molecule having 75% or more identity with the nucleotide sequence defined in b1) and encoding the mutant protein described in the first aspect; b3) A DNA molecule that hybridizes with the nucleotide sequence defined in b1) under stringent conditions and encodes the mutant protein described in the first aspect.

[0015] In the third aspect, the present invention provides the use of the mutant protein described in the first aspect in any of the following; C1) Degrading PET; C2) Accelerating the degradation rate of PET; C4) Preparing a product for degrading PET; C5) Preparing a product for accelerating the degradation rate of PET.

[0016] In the fourth aspect, the present invention provides the use of the biological material described in the second aspect in any of the following: C1) Degrading PET; C2) Accelerating the degradation rate of PET; C3) Increasing the yield of PET hydrolase; C4) Preparing a product for degrading PET; C5) Preparing a product for accelerating the degradation rate of PET; C6) Preparing a product for increasing the yield of PET hydrolase.

[0017] In the fifth aspect, the present invention provides a product comprising the mutant protein described in the first aspect or the biological material described in the second aspect.

[0018] In the sixth aspect, the present invention provides a method for degrading PET, comprising the following steps: degrading PET with the mutant protein described in the first aspect to achieve degradation.

[0019] In the above, the degradation temperature is 68°C.

[0020] In the above, the mass ratio of the mutant protein to PET is 1:1000.

[0021] In the above, the concentration of PET as a substrate can be greater than or equal to 1.6 g / L or greater than or equal to 165 g / L.

[0022] In the seventh aspect, the present invention provides a method for preparing the mutant protein described in the first aspect, comprising the following steps: inducing and culturing the recombinant microorganism described in the second aspect with IPTG, collecting the culture product, and obtaining the mutant protein described in the first aspect.

[0023] The induced culture can be shake flask culture or fermenter fermentation culture.

[0024] The fermentation culture in the fermenter is to perform fed-batch fermentation of recombinant Escherichia coli in the fermenter to obtain the mutant protein described in the first aspect.

[0025] Through rational design and site-directed mutagenesis, the present invention is designed with the highly efficient PET hydrolase LCC-ICCG reported in the literature as a template. The maximum reaction rate of the modified mutant has increased by 76%, and its thermal stability is comparable to that of the template LCC-ICCG, and it can maintain activity for a long time at the optimal reaction temperature for PET depolymerization (68 - 70 °C). Under industrial-grade PET substrate concentration, with only 0.1% (by mass) of the enzyme added to the PET, the mutant enzyme can almost completely degrade PET within 24 hours. In terms of expression level, the protein yield of the mutant enzyme in the shake flask is 3.3 times that of the template enzyme, and the recombinant Escherichia coli containing the mutant can produce 1.49 g / L of the target protein through fed-batch high-density fermentation in the fermenter. Therefore, the mutant enzyme has shown great value in the industrial application of enzymatic catalysis for PET depolymerization and will promote the further development of PET recycling.

[0026] Compared with the prior art, the present invention has the following advantages: (1) The mutant LCC-ICCG-NM in the present invention can maintain activity for a long time at the optimal reaction temperature for PET depolymerization (68 - 70 °C), and its protein melting temperature reaches 92.4 °C.

[0027] (2) The mutant LCC-ICCG-NM in the present invention significantly improves the catalytic efficiency. Under the conditions of a PET substrate concentration of 1.6 g / L and an enzyme addition amount of 0.1% (by mass) of the PET (enzyme concentration of 0.057 μM), after a 12-hour reaction, the degradation rates at 50 °C, 60 °C, 68 °C, 72 °C, and 75 °C reach 16.6%, 47.8%, 66.5%, 69.4%, and 71.1% respectively, which are 1.60, 1.38, 1.20, 1.10, and 1.07 times that of the PET hydrolase LCC-ICCG under the same conditions.

[0028] (3) The mutant LCC-ICCG-NM in the present invention shows good catalytic activity and application value in the PET depolymerization experiment at industrial-grade substrate concentration. Under the conditions of 68 °C, a PET substrate concentration of 165 g / L, and an enzyme addition amount of 0.1% (by mass) of the PET (enzyme concentration of 4.46 μM), the degradation rate reaches 53.7% in 3 hours and 89.4% in 18 hours, which are 1.76 times and 1.05 times faster than the PET hydrolase LCC-ICCG under the same reaction conditions respectively.

[0029] (4)In the present invention, the recombinant bacterium containing the pET-22b(+)-LCC-ICCG-NM gene was subjected to fed-batch fermentation, and the amount of the LCC-ICCG-NM mutant protein produced reached 1.49 g / L, effectively reducing the production cost of the highly efficient PET hydrolase. Description of the Drawings

[0030] Figure 1 It is a reaction route diagram for the enzymatic catalysis of PET degradation into ethylene terephthalate (BHET), mono-hydroxyethyl terephthalate (MHET), terephthalic acid (TPA), and ethylene glycol (EG).

[0031] Figure 2 It is a map of the recombinant plasmid pET-22b(+)-LCC-ICCG-NM.

[0032] Figure 3 It is a high-performance liquid chromatography diagram of the PET hydrolase LCC-ICCG and the mutant LCC-ICCG-NM reacting for 3 hours at a high PET substrate concentration.

[0033] Figure 4 It is a product time curve diagram of the PET hydrolase LCC-ICCG and the mutant LCC-ICCG-NM catalyzing the depolymerization of PET at a high PET substrate concentration. Detailed Embodiments

[0034] The present invention will be further described in detail below in conjunction with the specific embodiments. The provided embodiments are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not constitute any limitation to the present invention in any way.

[0035] In the experimental methods in the following embodiments, unless otherwise specified, they are all conventional methods, carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following embodiments, unless otherwise specified, can all be obtained from commercial channels.

[0036] Unless otherwise specified, in the following embodiments, all quantitative tests are set with three repeated experiments, and the results are averaged.

[0037] The medium formulations involved in the following embodiments are as follows: LB liquid medium: 10 g / L of peptone, 5 g / L of NaCl, 5 g / L of yeast powder, the balance is water, pH = 7.0.

[0038] LB solid medium: peptone 10 g / L, NaCl 5 g / L, yeast powder 5 g / L, agar powder 15 g / L, the balance is water, pH = 7.0.

[0039] Fermentation medium: citric acid 3 g / L, KH2PO4 3.1 g / L, (NH4)2SO4 1 g / L, (NH4)2HPO4 4 g / L, Na2HPO4 2.55 g / L, MgSO4 0.55 g / L, NaH2PO4 2.15 g / L, NH4Cl 0.1 g / L, glucose 10 g / L, yeast 10 g / L, peptone 16 g / L, antifoaming agent 1 mL / L, the balance is water, pH = 7.0.

[0040] Feed medium: glucose 750 g / L, MgSO4 9.8 g / L, citric acid 3 g / L, KH2PO4 4 g / L, Na2HPO4 3 g / L, the balance is water, pH = 7.0.

[0041] Example 1: Preparation and purification of mutants of template PET hydrolase LCC-ICCG I. Obtaining the gene encoding the mutant of template PET hydrolase LCC-ICCG The nucleotide sequence of the template PET hydrolase LCC-ICCG was obtained from the published literature (Tournier, V., et al. "An engineered PET depolymerase to break down and recycle plastic bottles." Nature 580.7802 (2020): 216-219.) as SEQ ID NO.1; the amino acid sequence of the encoded LCC-ICCG was SEQ ID NO.2.

[0042] First, the gene of the template PET hydrolase LCC-ICCG was obtained by total gene synthesis, and then site-directed mutagenesis was performed on the encoding gene of LCC-ICCG to obtain the gene of the LCC-ICCG mutant. The above LCC-ICCG mutant is the LCC-ICCG-NM mutant protein.

[0043] The LCC-ICCG-NM mutant protein is a mutant obtained by mutating His at position 184 of the template PET hydrolase LCC-ICCG shown in SEQ ID NO.2 to Asn, and mutating Phe at position 188 to Met, with other amino acid residues unchanged; the amino acid sequence of the LCC-ICCG-NM mutant protein is SEQ ID NO.4.

[0044] II. Construction of the recombinant expression vector First, the LCC-ICCG gene (the nucleotide sequence is shown in SEQ ID NO.1, containing 783 bases) was obtained by total gene synthesis, and Nde I and Xho I restriction enzymes were used to construct the gene into the pET-22b(+) vector (containing the ampicillin resistance gene). Then, the recombinant plasmid was transformed into the competent cell BL21(DE3) to obtain the recombinant expression plasmid pET-22b(+)-LCC-ICCG. The plasmid pET-22b(+) was purchased from BGI. The pET-22b(+) plasmid carries a carboxyl-terminal histidine tag His6 coding sequence at the XhoI digestion end, facilitating the purification of the target protein. After sequencing, the sequence of pET-22b(+)-LCC-ICCG was correct. This plasmid is the recombinant plasmid pET-22b(+)-LCC-ICCG formed by replacing the LCC-ICCG gene shown in positions 1 - 783 of SEQ ID NO.1 in the sequence listing with the fragment between the Nde I and Xho I sites of pET-22b(+), and the LCC-ICCG gene is fused with the His6 tag on the vector for expression.

[0045] According to the above method, the coding gene (SEQ ID NO.3) of the mutant LCC-ICCG-NM was inserted between the Nde I and Xho I digestion sites of pET-22b(+) to obtain the recombinant expression vector pET-22b(+)-LCC-ICCG-NM. The map of its recombinant plasmid is as shown in Figure 2 .

[0046] III. Construction method of engineering bacteria The recombinant expression vectors pET-22b(+)-LCC-ICCG and pET-22b(+)-LCC-ICCG-NM obtained in step II were respectively transformed into the Escherichia coli competent cell Escherichia coli BL21(DE3) (the competent cell was purchased from Beijing Solarbio Science & Technology Co., Ltd., product number C1400), to obtain the recombinant engineering bacteria E. coli BL21(DE3) / pET-22b(+)-LCC-ICCG and E.coli BL21(DE3) / pET-22b(+)-LCC-ICCG-NM. The plasmids were extracted and verified correct by sequencing.

[0047] IV. The PET hydrolase LCC-ICCG and its mutants were obtained by shake flask fermentation 1. Induced expression of engineering bacteria The constructed engineering strains in step three were streaked on LB solid medium containing 100 μg / mL ampicillin and then cultured at 37 °C for 14 hours. Then, well-grown single colonies were selected from these media and inoculated into 5 mL LB liquid medium (also containing 100 μg / mL ampicillin), and continued to be cultured at 37 °C and 200 rpm for 7 hours until the OD 600 value reached 1.2 - 2.0. Subsequently, these engineering bacteria were transferred to two bottles of 200 mL LB liquid medium (containing 100 μg / mL ampicillin) according to a volume ratio of 1%, and continued to be cultured at 37 °C and 200 rpm until the OD 600 value reached 0.6 - 0.8. To induce the expression of PET hydrolase LCC-ICCG or its mutant, then isopropyl-β-D-thiogalactopyranoside (IPTG) with a final concentration of 1 mM was added, and cultured at 20 °C and 180 rpm for 16 hours and 30 minutes.

[0048] After the cultivation was completed, first, the bacterial liquid was centrifuged at 4 °C and 6000 rpm for 20 minutes to collect the cell precipitate. Subsequently, the precipitate was resuspended by shaking with 50 mL of protein lysis buffer (containing 100 mM potassium dihydrogen phosphate and 300 mM sodium chloride, the balance being water, pH 8.0). Next, the resuspended liquid was subjected to cell disruption using an ultrasonic cell disruptor in an ice-water bath, adopting a mode of working for 4 seconds and resting for 6 seconds, and disrupting the cells at 60% of the maximum power for a total of 50 minutes. The disrupted liquid was centrifuged at 4 °C and 7000 rpm for 15 minutes, and the supernatant was collected by further centrifugation at 4 °C and 12,000 rpm for 10 minutes. The finally obtained supernatant was the crude enzyme solution containing PET hydrolase LCC-ICCG or its mutant LCC-ICCG-NM.

[0049] 2. Purification of PET hydrolase LCC-ICCG and its mutant LCC-ICCG-NM proteins The crude enzyme solution containing PET hydrolase LCC-ICCG or its mutant LCC-ICCG-NM was loaded onto a Ni-NTA affinity column, and nickel ions were used as the affinity medium. The protein was gradually eluted with imidazole solutions of different concentrations to obtain the purified target protein. Since proteins have a specific absorption peak at a wavelength of 280 nm, the absorbance of the eluates with different imidazole concentrations can be measured at this wavelength to identify and remove the non-target proteins. Specifically as follows: The crude enzyme solution was purified using a 20 mL nickel agarose gel column (Beijing Weishi Bohui Chromatography Technology Co., Ltd., model FF) and a nucleic acid and protein detector (Shanghai Huxi Analytical Instrument Factory, model HD-21-1). First, the column bed was pre-equilibrated with binding buffer A. Then, the impurity proteins were gradually eluted using binding buffer B containing imidazole until the absorbance of the eluate at 280 nm no longer changed. Subsequently, the target protein was collected using elution buffer. To improve the purity of the target protein, the collected protein solution was heated in a water bath at 60 °C for 2 hours, and then centrifuged at 4 °C and 12,000 rpm for 30 minutes to remove the residual impurity proteins. Next, the supernatant after centrifugation was transferred to an ultrafiltration centrifugal tube with a molecular weight cut-off of 10 kD and centrifuged at 4 °C and 6,000 rpm for 20 minutes, and this process was repeated 3 times to remove the imidazole in the solution. The purified target protein was finally stored in phosphate buffer and refrigerated in a 4 °C refrigerator to obtain the target protein solutions (PET hydrolase LCC-ICCG solution and mutant LCC-ICCG-NM solution) respectively.

[0050] The presence and purity of the target protein were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A 5% stacking gel and a 10% separating gel were used, and electrophoresis was carried out at a constant voltage of 120 V for 90 minutes. According to the analysis results of SDS-PAGE, the purity of the collected protein exceeded 90%.

[0051] To determine the concentration of the target protein in the purified target protein solution, the Coomassie brilliant blue method was used. Using an ultraviolet spectrophotometer (Shanghai Muxi Instrument Co., Ltd., model UV-1200), with bovine serum albumin as the standard, the absorbance was measured at a wavelength of 595 nm. The results showed that in each target protein solution, the purified protein concentrations of the corresponding PET hydrolase LCC-ICCG and mutant LCC-ICCG-NM were between 2 - 10 mg / mL.

[0052] The formula of the above-mentioned binding buffer A is: 100 mM potassium dihydrogen phosphate, 300 mM sodium chloride, the balance is water, pH 8.0.

[0053] The formula of the above-mentioned binding buffer B is: 100 mM potassium dihydrogen phosphate, 300 mM NaCl, 50 mM imidazole, the balance is water, pH 8.0.

[0054] The formula of the above-mentioned elution buffer is: 100 mM potassium dihydrogen phosphate, 300 mM sodium chloride, 200 mM imidazole, the balance is water, pH 8.0.

[0055] The formula of the above phosphate buffer is: 100 mM potassium dihydrogen phosphate, 300 mM sodium chloride, the balance is water, pH 8.0.

[0056] Example 2. Determination of the ability of PET hydrolase LCC-ICCG mutant to catalyze the depolymerization of post-consumer bottle-grade PET I. Thermal stability test of PET hydrolase LCC-ICCG mutant Many enzyme modifications are carried out by increasing the structural flexibility to promote the binding of the enzyme to the substrate, which may cause the loss of structural stability of the enzyme. However, PET hydrolase needs to have sufficient thermal stability to maintain its activity at the optimal reaction temperature for PET depolymerization. By using nano differential scanning fluorimetry (NanoDSF) and a protein stability analyzer (Prometheus NT. 48, Nanotemper Technologies, Germany), the melting temperature ( T m ) of PET hydrolase LCC-ICCG and its mutant LCC-ICCG-NM in a protein buffer (100 mM potassium dihydrogen phosphate, 300 mM sodium chloride, the balance is water, pH 8.0) was measured. During the measurement, the sample was heated from 25 °C to 110 °C at a rate of 2 °C per minute. Each enzyme was analyzed in triplicate, and its T m was obtained from the melting curve of the protein, and the average value and standard deviation of the measured T m were calculated. The T m of the mutant LCC-ICCG-NM was 92.4 ± 1.1 °C, which was comparable to that of its template PET hydrolase LCC-ICCG ( T m = 95.6 ± 0.2 °C), exceeding the glass transition temperature of PET (70 °C) by more than 15 °C, ensuring that the enzyme would not lose its activity under long-term industrial reaction conditions.

[0057] II. Activity test of PET hydrolase LCC-ICCG and mutant LCC-ICCG-NM in catalyzing the depolymerization of PET at low substrate concentrations Post-consumer bottle-grade PET is sourced from the beverage bottles produced by The Coca-Cola Company. First, the bottle bodies are cut into small pieces of 1 cm × 1 cm. Then, they are successively washed with water, surfactant, and ethanol, and dried. The treated PET bottle chips are put into a high-speed rotating grinder (AM500S, Ant Source Scientific Instruments Beijing Co., Ltd.) equipped with a 0.5 mm trapezoidal hole sieve ring and pulverized at a rotational speed of 8000 rpm. The ground powder is filtered through sieves with pore sizes of 200 μm and 355 μm respectively, and finally, post-consumer PET bottle powder with a particle size range of 200 - 355 μm is obtained.

[0058] The post-consumer PET bottle powder described below refers to the pre-treated post-consumer bottle-grade PET powder with a particle size of 200 - 355 μm.

[0059] Buffer 1 is a solution with a pH of 8.0 composed of 100 mM potassium dihydrogen phosphate, 300 mM sodium chloride, and water.

[0060] 1. Activity test of template PET hydrolase LCC-ICCG and mutant LCC-ICCG-NM in catalyzing PET depolymerization at different temperatures Template PET hydrolase LCC-ICCG experimental group (3 mL): Weigh approximately 5 mg of post-consumer PET bottle powder into a 5 mL glass reaction flask, and then add 3 mL of Buffer 1 with a pH of 8.0 containing 0.057 μM PET hydrolase LCC-ICCG (obtained by dissolving the PET hydrolase LCC-ICCG solution (protein content 5 μg) in Buffer 1, where the concentration of PET hydrolase LCC-ICCG is 0.057 μM).

[0061] Mutant LCC-ICCG-NM experimental group (3 mL): Weigh approximately 5 mg of post-consumer PET bottle powder into a 5 mL glass reaction flask, and then add 3 mL of Buffer 1 with a pH of 8.0 containing 0.057 μM mutant LCC-ICCG-NM (obtained by dissolving the mutant LCC-ICCG-NM solution (protein content 5 μg) in Buffer 1, where the concentration of mutant LCC-ICCG-NM is 0.057 μM).

[0062] The above-mentioned substances in each group were placed in a constant temperature shaker mixer (DH300, Hangzhou Ruicheng Instrument Co., Ltd.). Under the condition of a rotation speed of 400 rpm, reactions were carried out at 50 °C, 60 °C, 68 °C, 72 °C and 75 °C for 12 hours respectively. At the end of the reaction, an equal volume of 100% methanol was added to terminate the reaction, and samples were obtained. The samples were diluted 10 times by adding buffer solution 1, centrifuged (12000 rpm, 2 minutes) and the supernatant was collected. Then, the supernatant was taken with a syringe and passed through a 0.22 μm filter membrane to remove the inactivated enzymes in the solution, and various hydrolysis product samples were obtained.

[0063] The process of PET depolymerization is as Figure 1 shown, in which only terephthalic acid (TPA), mono(2-hydroxyethyl) terephthalate (MHET) and bis(2-hydroxyethyl) terephthalate (BHET) in the products can be detected by liquid chromatography.

[0064] Standard products TPA (Shanghai Macklin Biochemical Co., Ltd.), MHET (Biode Pharmaceutical Technology Co., Ltd.) and BHET (Biode Pharmaceutical Technology Co., Ltd.) with different concentrations were respectively determined for peak areas by high performance liquid chromatography HPLC. With the peak area (X) as the abscissa and the concentration (Y) as the ordinate, standard curves were plotted. The standard curve functions corresponding to the three products are as follows.

[0065] Concentration (TPA) = TPA peak area × 0.13602 × 10 -6 Concentration (MHET) = MHET peak area × 0.11342 × 10 -6 Concentration (BHET) = BHET peak area × 0.99696 × 10 -7 The samples to be measured were detected by high performance liquid chromatography HPLC. According to the corresponding peak emergence time and peak area, substituting them into the corresponding standard curves can obtain the contents of TPA, MHET and BHET in the samples.

[0066] The above high performance liquid chromatography (HPLC) (Shimadzu Corporation, LC-20AD) analysis used a SunFire™ C18 reverse phase column (GL Sciences, 5 μm, 250×4.6 mm). The sample injection volume for each sample was 10 μL. The flow rate was 0.8 mL / minute, the detection wavelength was 254 nm, and the column oven was maintained at a constant temperature of 40 °C. Mobile phase A was distilled water containing 0.1% formic acid (v / v), and mobile phase B was chromatographically pure acetonitrile. From 0 - 5 minutes, the concentration of mobile phase B remained constant at 5%; from 5 - 13 minutes, the concentration of mobile phase B increased from 5% to 44%; from 13 - 18 minutes, the concentration of mobile phase B increased from 44% to 70%; from 18 - 23 minutes, the concentration of mobile phase B remained constant at 70%; from 23 - 24 minutes, the concentration of mobile phase B decreased from 70% to 5%; from 24 - 28 minutes, the concentration of mobile phase B remained constant at 5%.

[0067] After HPLC analysis, the chromatographic peaks of the PET degradation product TPA appeared near the retention time t R = 14.88 minutes, the chromatographic peaks of the PET degradation product MHET appeared near the retention time t R = 15.46 minutes, and the chromatographic peaks of the PET degradation product BHET appeared near the retention time t R = 15.76 minutes.

[0068] The concentrations of TPA, MHET, and BHET in the hydrolysis products after the template PET hydrolase LCC-ICCG and the mutant LCC-ICCG-NM degraded PET at 50 - 75 °C for 12 hours, as well as the degradation rate of PET, are shown in Table 1.

[0069] The calculation formula for the degradation rate in Table 1 is as follows, where the total product concentration is the sum of the concentrations of TPA, MHET, and BHET detected by HPLC. The reaction volume in this example is 3 mL, and the dilution factor in this example is 10; the PET mass is 5 mg, and the molecular weight of the PET monomer is 182.14 Da.

[0070] Degradation rate = total product concentration × dilution factor × reaction volume / (1000×PET mass / PET monomer molecular weight) × 100% It can be seen that the degradation rates of the mutant LCC-ICCG-NM catalyzing PET hydrolysis at 50 °C, 60 °C, 68 °C, 72 °C, and 75 °C for 12 hours are 1.60, 1.38, 1.20, 1.10, and 1.07 times that of the template PET hydrolase LCC-ICCG, respectively, proving that the catalytic activity of the mutant is higher than that of the template PET hydrolase at different temperatures.

[0071] Table 1 shows the products of PET hydrolysis catalyzed by PET hydrolase LCC-ICCG and mutant LCC-ICCG-NM at 50 - 75 °C

[0072] III. Activity test of template PET hydrolase LCC-ICCG and mutant LCC-ICCG-NM in catalyzing PET depolymerization at high substrate concentration In order to compare the ability of template PET hydrolase LCC-ICCG and mutant LCC-ICCG-NM to catalyze PET depolymerization under conditions that meet the requirements of industrial applications. PET depolymerization tests with a PET substrate concentration of 16.5% (w / v) were carried out on the two enzymes respectively in a three-necked flask.

[0073] The PET substrate for the reaction was from the body of post-consumer colorless transparent PET bottles. First, the PET bottle body was placed in an aluminum foil paper box and then put into a muffle furnace (Hefei Kejing Materials Technology Co., Ltd., KSL-1200X), heated at 290 °C for 15 minutes until completely melted, and then the aluminum foil paper box was placed in an ice-water bath to cool and solidify. The solidified PET was washed with water and then dried and put into a high-speed rotary grinder (Antiyuan Scientific Instruments Beijing Co., Ltd., AM500S) equipped with a 0.5 mm trapezoidal hole sieve ring to crush the PET. The rotation speed of the grinder was 8000 rpm. The crushed powder was filtered through sieves with pore sizes of 200 μm and 355 μm respectively to obtain post-consumer PET bottle powder with a particle size of 200 - 355 μm.

[0074] Template PET hydrolase LCC-ICCG experimental group (50 ml): Weigh about 8.25 g of the treated PET powder into a 100 mL three-necked flask, and then add 44 mL of buffer 1 with pH 8.0 containing 2.23 μM template PET hydrolase LCC-ICCG (obtained by dissolving the PET hydrolase LCC-ICCG solution (protein content is 4.125 mg) in buffer 1, and the concentration of PET hydrolase LCC-ICCG is 2.23 μM).

[0075] Mutant LCC-ICCG-NM experimental group (50 ml): Weigh about 8.25 g of the treated PET powder into a 100 mL three-necked flask, and then add 44 mL of buffer 1 with pH 8.0 containing 2.23 μM purified mutant LCC-ICCG-NM (obtained by dissolving the mutant LCC-ICCG-NM solution (protein content is 4.125 mg) in buffer 1, and the concentration of mutant LCC-ICCG-NM is 2.23 μM).

[0076] Place the three-necked flask containing the above-mentioned substances in a heating water bath constant temperature magnetic stirrer (Shanghai Yuhua Instrument Co., Ltd., ZNCL-G), stir the reaction solution, and keep the reaction temperature at 70 °C. At the same time, use a pH meter FE28 (Mettler Toledo, FE28) to measure the pH value of the reaction solution in real time. By adding 5 M sodium hydroxide solution, maintain the pH value of the reaction solution between 7.8 - 8.2, and record the added volume of the sodium hydroxide solution. Take 1000 μL of samples at 3, 6, 9, 12, 18, and 24 hours respectively, dilute the samples by adding buffer 1 by 400, 400, 400, 400, 800, and 800 times respectively, centrifuge (12000 rpm, 2 minutes) and collect the supernatant. Then use a syringe to take the supernatant through a 0.22 μm filter membrane to remove the inactivated enzymes in the solution. Obtain the hydrolyzate samples.

[0077] According to the HPLC detection method described above, quantitatively analyze the products of PET depolymerization, and calculate the degradation rate of PET based on the production amount of the products. The HPLC chromatograms of the reaction products of the mutant LCC-ICCG-NM and PET hydrolase LCC-ICCG at 3 hours are presented in Figure 3 Figure (a) and Figure 3 Figure (b). It can be seen from Figure 3 that chromatographic peaks of the hydrolysis product TPA appear near the retention time t R = 14.9 min (consistent with the peak time of the TPA standard), chromatographic peaks of the substrate MHET appear near the retention time t R = 15.5 min (consistent with the peak time of the MHET standard), and chromatographic peaks of the substrate BHET appear near the retention time t R = 15.8 min (consistent with the peak time of the BHET standard). Figure 3 In Figure (a), the chromatographic peak of MHET is significantly larger than that in Figure 3 Figure (b) in terms of both peak height and peak area.

[0078] The graphs showing the changes in the degradation rates of the mutant LCC-ICCG-NM and PET hydrolase LCC-ICCG at different times are presented in Figure 4 Figure. It can be seen that the degradation rate of the mutant LCC-ICCG-NM after 3 hours of reaction is 1.08 times that of PET hydrolase LCC-ICCG, and the degradation rate at 9 hours has exceeded 80%, which is 10% more than the degradation rate of LCC-ICCG at the same reaction time. After 24 hours of reaction, the degradation rate of the mutant LCC-ICCG-NM reached 90%, meeting the minimum standard for industrial applications, while the final degradation rate of the template PET hydrolase LCC-ICCG stopped at 85%.

[0079] These results indicate that the mutant LCC-ICCG-NM catalyzes the depolymerization of PET much faster than its template LCC-ICCG in the early stage of the reaction, and its catalytic efficiency is not affected at high substrate concentrations, showing good application value in the industrialization of PET recycling.

[0080] Example 3. Evaluation of the Fermentation Production of the Target Protein by Recombinant Escherichia coli BL21(DE3) / pET-22b(+)-LCC-ICCG-NM I. Comparison of the Yields of the Target Protein Produced by Recombinant Escherichia coli BL21(DE3) / pET-22b(+)-LCC-ICCG-NM and Recombinant Escherichia coli BL21(DE3) / pET-22b(+)-LCC-ICCG in Shake Flasks According to the method in item 4 of Example 1, the preparation of the seed solution, the scale-up culture of the bacteria, the induction expression of the protein, the disruption of the bacteria, and the purification of the target protein were carried out for recombinant Escherichia coli BL21(DE3) / pET-22b(+)-LCC-ICCG-NM and recombinant Escherichia coli BL21(DE3) / pET-22b(+)-LCC-ICCG respectively. The target protein solutions (PET hydrolase LCC-ICCG solution and mutant LCC-ICCG-NM solution) were obtained.

[0081] For the purified protein, the protein concentration was determined by the Coomassie brilliant blue method using a UV spectrophotometer, and the yields of PET hydrolase LCC-ICCG and mutant LCC-ICCG-NM under shake flask culture conditions were calculated. The formula for calculating the protein yield is as follows, where the unit of the protein concentration in the purified solution is mg / mL, the unit of the volume of the purified solution is mL, and the volume of the fermentation broth is 0.4 L. Protein yield = (Protein concentration in the purified solution × Volume of the purified solution) ÷ Volume of the fermentation broth The yield of PET hydrolase LCC-ICCG was 28.1 ± 1.75 mg / L, and the yield of mutant LCC-ICCG-NM was 93.0 ± 2.63 mg / L, which was 3.3 times that of the former.

[0082] II. Cultivation of Recombinant Escherichia coli BL21(DE3) / pET-22b(+)-LCC-ICCG-NM in a 5L Fermenter to Produce the Target Protein The constructed recombinant Escherichia coli BL21(DE3) / pET-22b(+)-LCC-ICCG-NM was streaked on an LB agar plate containing 100 μg / mL ampicillin and then cultured at 37 °C for 14 hours. Well-grown single colonies were selected and inoculated into 5 mL of LB liquid medium (containing 100 μg / mL ampicillin), and further cultured at 37 °C and 200 rpm for 12 hours until the OD 600 reached 3.0 - 4.0. Then, 3 mL of the bacterial solution was taken, and each 1 mL of the bacterial solution was inoculated into 35 mL of LB medium and cultured at 30 °C and 200 rpm for 8 hours until the OD 600 reached 1.5 - 2.5, and the obtained 100 mL of bacterial solution was used as the seed solution.

[0083] First, the 5 L automatic fermenter (Shanghai Baoxing Biological Equipment Engineering Co., Ltd., 5JGZ) was sterilized empty, and then 2000 mL of fermentation medium was added to the fermenter for sterilization. The sterilized fermenter was connected to the main unit, and the pH electrode and dissolved oxygen electrode were calibrated. 100 mL of the seed solution was added to the fermenter by the flame inoculation method, and at the same time, ampicillin with a final concentration of 100 μg / mL was added to start fermentation. The stirring speed was controlled in the range of 300 - 700 rpm by cascade control, the gas flow rate was 0.3 - 0.6 vvm, and the pure oxygen enrichment was 0 - 50%, so that the dissolved oxygen level of the fermentation broth was always greater than 5% of the air saturation. The fermentation adopted a two-stage temperature control. The 0 - 14 hours was the bacterial growth stage (counting from the time of inoculation into the fermenter as the 0 hour), and the temperature was 37 °C; the 14 - 38 hours was the stage of induced expression of the target protein, and the temperature was 25 °C. During the fermentation process, the pH value of the fermentation broth was controlled at 7.0 by adding ammonia water solution with a volume fraction of 25% (25% ammonia water + 75% water) and phosphoric acid water solution with a volume fraction of 20% (20% phosphoric acid + 80% water). The glucose content in the fermentation broth was measured by a blood glucose meter (Roche, ACCU-CHEK). When the glucose in the fermentation broth was exhausted, the feeding medium was added to the fermenter according to the pre-set feeding curve to maintain the glucose concentration at 0 - 0.1 g / L. At 14 hours of fermentation (counting from the time of inoculation into the fermenter as the 0 hour), IPTG with a final concentration of 1 mM was added, and then fermentation was continued at 25 °C for another 24 hours.

[0084] At 22, 28, 33, and 38 hours of fermentation respectively, 50 mL of the fermentation broth was taken for the analysis of the target protein yield. The fermentation broth was centrifuged at 4 °C and 6000 rpm for 20 minutes. The obtained cell pellet was resuspended by shaking with 50 mL of protein lysis buffer (100 mM potassium dihydrogen phosphate, 300 mM sodium chloride, the balance being water, pH 8.0). Then, an ultrasonic cell disruptor was used to disrupt the cells in an ice-water bath. At 60% of the maximum power, in the mode of working for 4 seconds and resting for 6 seconds, the cells were disrupted for a total of 50 minutes. The obtained liquid was centrifuged at 4 °C and 7000 rpm for 15 minutes, and then the supernatant was taken and centrifuged at 4 °C and 12000 rpm for 10 minutes. The finally obtained supernatant was the crude enzyme solution containing the mutant LCC-ICCG-NM.

[0085] Different from the method of purification by nickel column in shake flask fermentation, only the method of heat purification was used to remove the impurity proteins in fermentor fermentation. This purification method is easy to operate and more suitable for industrial applications. The crude enzyme solution containing the mutant LCC-ICCG-NM was placed in a water bath at 60 °C and heated for 2.5 hours, and then centrifuged at 4 °C and 12000 rpm for 30 minutes. The obtained supernatant was the pure solution of the mutant LCC-ICCG-NM.

[0086] The protein concentration in the pure solution of the mutant LCC-ICCG-NM obtained at different fermentation times was determined by the Coomassie brilliant blue method using an ultraviolet spectrophotometer. The protein concentrations in the mutant LCC-ICCG-NM solutions generated at 22, 28, 33, and 38 hours of fermentation were 1.49, 1.46, 1.29, and 1.28 g / L respectively.

[0087] Compared with shake flask fermentation, fed-batch fermentation of recombinant Escherichia coli BL21(DE3) / pET-22b(+)-LCC-ICCG-NM using a fermentor greatly increased the yield of the mutant LCC-ICCG-NM.

[0088] III. Activity test of the mutant LCC-ICCG-NM produced by 5 L fermentor culture for PET depolymerization Samples of the fermentation broth in the fermentor were taken at 22 hours, 28 hours, 33 hours, and 38 hours of fermentation in the above II respectively. Pure solutions of the mutant LCC-ICCG-NM were obtained according to the methods of crude enzyme solution preparation and heat purification in the above II.

[0089] Mutant LCC-ICCG-NM experimental group obtained by shake flask culture: Weigh approximately 5 mg of post-consumer PET bottle powder into a 5 mL glass reaction flask, and then add 3 mL of buffer 1 with a pH of 8.0 containing 0.057 μM mutant LCC-ICCG-NM (the enzyme was purified by the method of nickel column purification from the shake flask culture, and the mutant LCC-ICCG-NM solution (protein content is 5 μg) was dissolved in buffer 1 to obtain a concentration of 0.057 μM of mutant LCC-ICCG-NM).

[0090] Mutant LCC-ICCG-NM experimental group obtained by fermenter culture: Weigh approximately 5 mg of post-consumer PET bottle powder into a 5 mL glass reaction flask, and then add 3 mL of buffer 1 with a pH of 8.0 containing 0.057 μM mutant LCC-ICCG-NM (the enzyme was purified from the fermentation broth at 22 hours, 28 hours, 33 hours, and 38 hours in the fermenter according to the above-mentioned thermal purification method, and the mutant LCC-ICCG-NM solution (protein content is 5 μg) was dissolved in buffer 1 to obtain a concentration of 0.057 μM of mutant LCC-ICCG-NM).

[0091] The above-mentioned substances were placed in a thermostatic shaker mixer (Hangzhou Ruicheng Instrument Co., Ltd., DH300), and reacted for 12 hours under the conditions of a rotation speed of 400 rpm and a temperature of 68 °C. At the end of the reaction, an equal volume of 100% methanol was added to terminate the reaction. The sample was diluted 10 times by adding buffer 1, centrifuged (12000 rpm, 2 minutes), and the supernatant was collected. Then, the supernatant was taken with a syringe and passed through a 0.22 μm filter membrane to remove the inactivated enzyme in the solution, obtaining a hydrolysis product sample.

[0092] According to the HPLC detection method described above, quantitative analysis was performed on the products of PET depolymerization. The degradation rate of the mutant LCC-ICCG-NM obtained by shake flask fermentation after 12 hours was 66.5%, while the degradation rates of the mutant LCC-ICCG-NM obtained by fermenter fermentation for 22 hours, 28 hours, 33 hours, and 38 hours after 12 hours were 59.1%, 60.1%, 57.5%, and 59.2% respectively.

[0093] The results showed that the activity of the mutant LCC-ICCG-NM obtained by fed-batch culture in the fermenter and then thermal purification was basically the same as that of the enzyme obtained by shake flask production and then nickel column purification in the laboratory, proving the feasibility of the fermenter production of mutant LCC-ICCG-NM in industrial applications.

[0094] The above has described the present invention in detail. For those skilled in the art, without departing from the gist and scope of the present invention and without the need for unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that further improvements can be made to the present invention. In short, according to the principle of the present invention, this application intends to cover any modifications, uses, or improvements to the present invention, including those that depart from the scope disclosed in this application but are made using conventional techniques known in the art. Applications of some basic features can be made within the scope of the appended claims below.

Claims

1. A mutant protein of a PET hydrolase, which is any one of the following: A1) The mutant protein shown in A1) is a protein obtained by mutating the amino acid residues at positions 184 and 188 of SEQ ID NO.2 to obtain a protein with the same function; A2) In the mutant protein shown in A1), except for the mutated amino acid residues, the homology of other amino acid residues is greater than 99%, 95%, 90%, 85% or 80% homology, and it is a protein with the same function; A3) A protein obtained by connecting a tag to the N-terminus and / or C-terminus of the mutant protein shown in A1) or A2).

2. The mutant protein according to claim 1, wherein The mutation mode of each amino acid residue is as follows: The amino acid residue His at position 184 is mutated to Asn; The amino acid residue Phe at position 188 is mutated to Met.

3. The mutant protein according to claim 1 or 2, wherein: The amino acid sequence of the mutant protein shown in A1) is SEQ ID NO.

4.

4. A biological material related to the mutant protein according to any one of claims 1-3, which is any one of the following B1) to B4): B1) A nucleic acid molecule encoding the mutant protein according to any one of claims 1-3; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) A recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3).

5. The biomaterial according to claim 4, wherein: The nucleic acid molecule described in B1) is any one of the following b1)-b6): b1) The DNA molecule shown in SEQ ID NO.3; b2) A DNA molecule having 75% or more identity with the nucleotide sequence defined in b1) and encoding the mutant protein according to any one of claims 1-3; b3) A DNA molecule that hybridizes with the nucleotide sequence defined in b1) under stringent conditions and encodes the mutant protein according to any one of claims 1-3.

6. The application of the mutant protein according to any one of claims 1-3 in any of the following; C1) Degrading PET; C2) Accelerating the degradation rate of PET; C4) Preparing a product for degrading PET; C5) Preparing a product for accelerating the degradation rate of PET.

7. The application of the biological material according to claim 4 or 5 in any of the following: C1) Degrading PET; C2) Accelerating the degradation rate of PET; C3) Increasing the yield of PET hydrolase; C4) Preparing a product for degrading PET; C5) Preparing a product for accelerating the degradation rate of PET; C6) Preparing a product for increasing the yield of PET hydrolase.

8. A product, which comprises the mutant protein according to any one of claims 1-3 or the biological material according to claim 4 or 5.

9. A method for degrading PET, comprising the following steps: degrading PET with the mutant protein according to any one of claims 1-3 to achieve degradation.

10. A method for preparing the mutant protein according to any one of claims 1-3, comprising the following steps: inducing and culturing the recombinant microorganism according to claim 4 with IPTG, collecting the culture product, and obtaining the mutant protein according to any one of claims 1-3.

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