PET plastic degrading enzyme LCC mutant as well as protein crystal and application thereof

Abstract

The invention discloses a PET plastic degrading enzyme LCC mutant as well as a protein crystal and application thereof, and relates to the technical field of biology, the LCC mutant is obtained by modification on the basis of a wild PET plastic degrading enzyme LCC, and the amino acid sequence of the LCC mutant is shown as SEQ ID NO.1. Compared with a wild type LCC, the LCC mutant has higher thermal stability, the Tm value of the LCC mutant is 91.30 DEG C, the protein Tm value of the wild type LCC is 86.76 DEG C and is increased by 4.54 DEG C compared with that of the wild type LCC, good activity is still kept, and the yield of the mutant LCC-M4 is 2 times higher than that of the wild type LCC. In addition, the invention further provides a protein crystal and a structure of the LCC mutant, and a research basis is provided for further modifying the PET plastic degrading enzyme LCC with high activity and high stability.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a PET plastic degrading enzyme LCC mutant and a protein crystal and application thereof. Background Art

[0002] PET (polyethylene terephthalate) is a widely used plastic, mainly used to make beverage bottles, food packaging and fibers. With the increase in plastic consumption, the amount of PET waste is also increasing. However, the degradation rate of PET is very slow, leading to increasingly serious environmental pollution problems. A large amount of PET waste enters the natural environment, causing soil and water pollution; at the same time, the production of PET consumes a lot of petroleum resources, and if the discarded PET cannot be effectively recycled, it will cause a waste of resources.

[0003] In order to solve this problem, scientists began to study enzymes that can degrade PET. The application of PET plastic degrading enzymes not only provides an effective means to solve the problem of plastic pollution, but also opens up a new path for the recycling and reuse of resources. With the continuous advancement of technology, PET degrading enzymes will play an increasingly important role in environmental protection and sustainable development.

[0004] Leaf-branch compost cutinase (LCC) is an enzyme found in nature. It is a classic PET plastic degrading enzyme that can specifically degrade PET plastic. It can cut PET molecular chains into smaller molecules, thereby achieving plastic degradation. LCC has broad application prospects in the field of plastic degradation. It can be used for PET plastic degradation in industrial production, and can also be used for plastic waste treatment in the environment, which helps to reduce plastic pollution. With the global emphasis on sustainable development and environmental protection, the use of PET enzymes for biodegradation and recycling of plastic waste will become an important direction in the future. The search for PET plastic degrading enzymes with higher thermal stability not only provides new ideas for solving the problem of plastic pollution, but also promotes the development of biotechnology and environmental science. In the future, technological innovation and commercial applications in this field will be more abundant. Summary of the invention

[0005] The purpose of the present invention is to provide a PET plastic degrading enzyme LCC mutant and its protein crystal and application.

[0006] The present invention achieves the above-mentioned purpose through the following technical solutions:

[0007] As a first aspect of the present invention, a PET plastic degrading enzyme LCC mutant is provided, and the amino acid sequence of the PET plastic degrading enzyme LCC mutant is shown in SEQ ID NO.1.

[0008] As a second aspect of the present invention, a polynucleotide encoding the PET plastic degrading enzyme LCC mutant as claimed in claim 1 is also provided, and the polynucleotide sequence is shown as SEQ ID NO.2.

[0009] As a third aspect of the present invention, a recombinant plasmid is also provided, wherein the recombinant plasmid contains the polynucleotide sequence as claimed in claim 2 and can correspondingly translate and express the PET plastic degrading enzyme LCC mutant as claimed in claim 1.

[0010] A further improvement is that the vector for expressing the PET plastic degrading enzyme LCC mutant is pET-28a, and the strain for expressing the PET plastic degrading enzyme LCC mutant is Escherichia coli BL21 (DE3).

[0011] As a fourth aspect of the present invention, there is also provided a use of the above-mentioned PET plastic degrading enzyme LCC mutant in degrading PET plastic.

[0012] As a fifth aspect of the present invention, a protein crystal of a PET plastic degrading enzyme LCC mutant is also provided. The protein crystal is obtained by crystallizing the purified PET plastic degrading enzyme LCC mutant as described above using a sitting drop method.

[0013] A further improvement is that the crystallization conditions of the protein crystals are 0.2 M ammonium sulfate, 0.1 M 2-(N-morpholino)ethanesulfonic acid, pH 6.5 and 30% w / v PEG 5000MME.

[0014] A further improvement is that the unit cell parameters of the protein crystal are 39.61, 74.69, 157.45, 90.00, 90.00, 90.000; the space group is P212121; the resolution is

[0015] As a sixth aspect of the present invention, there is also provided an application of the protein crystal as described above in the transformation of a highly stable and highly active PET plastic degrading enzyme LCC.

[0016] As a seventh aspect of the present invention, a protein structure of a PET plastic degrading enzyme LCC mutant is also provided, wherein the protein structure is obtained by using X-ray diffraction of any of the above-mentioned protein crystals.

[0017] The present invention has the following beneficial effects:

[0018] The present invention provides a PET plastic degrading enzyme mutant, which has higher thermal stability than wild-type LCC, and its Tm value is 91.30°C, while the Tm value of wild-type LCC protein is 86.76°C, which is 4.54°C higher than that of the wild-type, and the output of the mutant is also 2 times higher than that of the wild-type, so it has wider application conditions and stronger practical application value, and is more suitable for large-scale production and industrial use. In addition, the present invention also provides protein crystals of the modified PET plastic degrading enzyme mutant, which provides a research basis for further modifying the high-activity and high-stability PET plastic degrading enzyme LCC. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The results of small-scale expression test of PET plastic degradation enzyme LCC and four mutant proteins;

[0020] Figure 2 The results of thermal stability test of PET plastic degrading enzyme LCC and four mutant proteins;

[0021] Figure 3 The activity test results of PET plastic degrading enzyme LCC and mutants;

[0022] Figure 4 The results of affinity purification of PET plastic degrading enzyme LCC and mutant LCC-M4;

[0023] Figure 5 This is a crystal photo of the PET plastic degrading enzyme mutant LCC-M4;

[0024] Figure 6 This is a three-dimensional structure comparison of the PET plastic degrading enzyme mutant LCC-M4 and PDB 7VVE. DETAILED DESCRIPTION

[0025] The present application is further described in detail below in conjunction with the accompanying drawings. It is necessary to point out here that the following specific implementation methods are only used to further illustrate the present application and cannot be understood as limiting the scope of protection of the present application. Technical personnel in this field can make some non-essential improvements and adjustments to the present application based on the above application content.

[0026] 1. Materials and reagents

[0027] Unless otherwise specified, the methods used in the present invention are conventional methods known to those skilled in the art. If no specific conditions are specified, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0028] 2. Methods

[0029] 2.1. Gene synthesis of recombinant PET plastic degrading enzyme LCC and its mutants

[0030] The gene sequences of the recombinant PET plastic degrading enzyme provided by the present invention are all obtained through gene synthesis.

[0031] The amino acid sequence of the wild-type PET plastic degrading enzyme LCC was synthesized with reference to the PDB 7VVE model, and its amino acid sequence is shown in SEQ ID NO.3, which is the sequence from the 36th position to the C-terminus of the amino acid sequence of the wild-type PET plastic degrading enzyme LCC, excluding the signal peptide sequence of the first 35 positions.

[0032] The sequences of the four mutant proteins are all mutated based on the amino acid sequence of the wild-type PET plastic degrading enzyme LCC. The four modified PET plastic degrading enzymes are LCC-M1 (amino acid sequence see SEQ ID NO.4), LCC-M2 (amino acid sequence see SEQ ID NO.5), LCC-M3 (amino acid sequence see SEQ ID NO.6), and LCC-M4 (amino acid sequence see SEQ ID NO.1, nucleotide sequence see SEQ ID NO.2).

[0033] In order to improve the solubility of LCC, the N-termini of all LCC and its mutants are tagged with Strep II tag and SUMO tag. The amino acid sequence of Strep II-SUMO is shown in SEQ ID NO.7. All synthesized genes are verified to be correct by sequencing companies.

[0034] 2.2. Small-scale expression test of recombinant PET plastic degrading enzyme LCC and its mutants

[0035] Using conventional molecular biological methods, the recombinant PET plastic degrading enzyme LCC and its mutant plasmids constructed above were transformed into BL21 (DE3) Escherichia coli competent cells in a clean bench and cultured at 37°C overnight. The monoclonal colonies cultured overnight were picked and placed in 5 ml LB liquid culture medium and cultured at 37°C until the OD of the bacterial solution reached 0. 600 When the p-value is 0.6-0.8, take a small amount of bacterial solution and fix it with loading buffer, and take a small amount of bacterial solution and add glycerol to freeze to -80℃, add 0.5mM IPTG to the remaining bacterial solution, induce at 15℃ for 16 hours, collect the bacteria and take the induced bacterial solution for SDS-PAGE detection.

[0036] In order to further detect the expression level of the protein, all expressed samples were subjected to a small amount of purification test. The specific purification scheme is to collect the bacteria after centrifugation of 5mL sample at 5000rpm for 10 minutes using a centrifuge. Add 1ml lysis buffer 50mM Tris-HCl pH 7.5, 500mM NaCl, 5% glycerol to the collected bacteria, and use an ultrasonic crusher to crush the broken samples. After centrifugation at 12000rpm for 30 minutes, collect the supernatant, add 50μL of Strep-tactin filler pre-treated with lysis buffer, and then wash with 1ml lysis buffer and lysis buffer containing 75mM biotin, respectively, and collect the eluted samples at each step for SDS-PAGE detection.

[0037] The experimental results are shown in Figure 1 Compared with the wild-type LCC, the sample bands of the 75 mM biotin system in the modified LCC-M1, LCC-M2, LCC-M3 and LCC-M4 were thicker, indicating that the yield of the mutants was higher than that of the wild-type.

[0038] 2.3 Thermal stability test of recombinant PET plastic degrading enzyme LCC and its mutants

[0039] After obtaining LCC mutant samples with good purity, nanoDifferential Scanning Fluorimetry (nanoDSF) was used to detect the Tm values ​​of wild-type LCC and its mutants LCC-M1, LCC-M2, LCC-M3 and LCC-M4.

[0040] Specifically, 20 μL of 0.5 mg / ml wild-type LCC and four mutant LCC-M1, LCC-M2, LCC-M3 and LCC-M4 proteins were added to a 384-well test plate. After centrifugation (to avoid uneven samples or bubbles during aspiration), the test plate was placed on a sampling rack and the sample was aspirated using a Nano DSF capillary to ensure that the sample filled the entire capillary. The capillary was placed in the nanoDSF instrument, the initial temperature was set to 20°C, and the temperature was raised to 90°C at a rate of 2.0°C per minute. The instrument will perform heating and real-time detection according to the set parameters.

[0041] The experimental results are shown in Figure 2The Tm value of LCC is 86.76℃, the Tm value of LCC-M1 is 82.04℃, the Tm value of LCC-M2 is 85.89℃, the Tm value of LCC-M3 is 80.10℃, and the Tm value of LCC-M4 is 91.30℃. Judging from the Tm value detection results, the Tm value of LCC-M4 increased by 4.54℃ compared with LCC, and its thermal stability has been significantly improved.

[0042] 2.4 Activity determination of recombinant PET plastic degrading enzyme LCC and its mutants

[0043] The specific operation of the activity determination method is as follows:

[0044] (1) Prepare buffer: 75 mM PBS, pH 8.0, substrate 2.5 mM BHET-OH (dissolved in DMSO, then added with 10 mM sodium carbonate solution), reaction temperature 30°C and 60°C. Dilute the LCC mutants in a 2-fold gradient from 1 μM with the buffer, for a total of 12 dilutions.

[0045] (2) 20 μL of substrate was transferred to a 384-well plate and two replicate wells were set up. 40 μL of the LCC mutant to be tested was transferred to the corresponding well plate, and the mixture was immediately centrifuged and oscillated to mix. The fluorescence signal value generated by the reaction was collected using a TECAN F200 microplate reader. GraphPad Prism9 analysis software was used for data analysis, and the enzyme activity parameters of the protease to be tested were finally obtained. The results are shown in the figure. Figure 3 shown.

[0046] Results: According to the enzyme activity data, the wild-type PET plastic degradation enzyme LCC and the LCC mutants were active at reaction temperatures of 30°C and 60°C. The activity of the LCC mutants decreased to a certain extent compared with the wild type, among which the activity of LCC-M1 decreased the most, with a decrease of 20% at 30°C and 60°C, and the activity of LCC-M4 decreased less, with a decrease of 12% at 30°C and only 7.5% at 60°C. This shows that the modified LCC-M4 has not significantly reduced its activity while improving the thermal stability of the LCC wild type.

[0047] 2.5. Large-scale expression and purification of PET plastic degrading enzyme LCC and its mutant LCC-M4

[0048] In order to further compare the yield of LCC-M4 and LCC, LCC-M4 and wild-type LCC were expressed and purified in large quantities. The method is as follows: the above two strains were inoculated into 50ml LB liquid medium and cultured at 37℃ overnight. The bacteria cultured overnight were inoculated into 1L LB liquid medium at a ratio of 1:100 and cultured at 37℃ until the bacterial solution OD 600When the pH value was 0.6-0.8, 0.5 mM IPTG was added, cultured overnight at 15°C, and the cells were collected by centrifugation at 5000 rpm for purification.

[0049] The specific purification steps are as follows: weigh the collected bacteria, add the corresponding volume of buffer (50mM Tris-HCl pH 7.5, 500mM NaCl, 5% glycerol) at a ratio of 1:10, use a high-pressure homogenizer to break the bacteria, and collect the supernatant by high-speed centrifugation at 16000rpm. First, use the buffer to wash 10 column volumes of 1mL Strep-Tactin XT pre-loaded affinity column, then load the lysis supernatant onto the column, and wash the column with buffer for 5 column volumes to remove proteins that are not bound to the column, then add a buffer containing 0.1mg / ml SUMO enzyme, and the LCC and LCC-M4 proteins that have been removed from the label will not bind to the column because they do not have Strep tags, and will penetrate out. Collect the penetrating proteins, perform SDS-PAGE detection, and use Nanodrop to determine the protein concentration and calculate the protein yield.

[0050] According to the SDS-PAGE results (see Figure 4 ), the yield of the wild-type PET plastic degrading enzyme LCC after purification was 12.2 mg / L, while the yield of the mutant LCC-M4 was 24.6 mg / L. After affinity purification, the yield of LCC-M4 was twice that of LCC. In addition, the purity of LCC-M4 was also significantly improved compared with the wild-type.

[0051] 2.6. Crystallization and structural analysis of modified PET plastic degrading enzyme LCC-M4 protein

[0052] The purified mutant LCC-M4 protein was crystallized by the sitting drop method. PEG Kit, Index Kit produced by Hampton Research and MorpheusⅡ, MorpheusⅢKit, Proplex produced by QIAGEN were selected for crystallization screening. 15μL of crystallization reagent was taken as buffer in a 96-well crystallization plate, and the Mosquito LCP protein crystallization screening instrument was used for spotting. 200nL protein + 200nL crystallization solution was taken. The 96-well plate sealed with MicroAmp Optical Adhesive film was placed in a constant temperature incubator at 20℃ for cultivation, and the crystal growth was observed regularly.

[0053] Through crystallization screening of LCC-M4, mutant LCC-M4 crystals were obtained in about 11 days at a protein concentration of 18.99 mg / ml. The crystallization conditions were 0.2 M ammonium sulfate, 0.1 M 2-(N-morpholino)ethanesulfonic acid, pH 6.5, and 30% w / v PEG5000MME (Polyethylene glycol Monomethyl ether). The crystal photos are shown in Figure 5 The unit cell parameters of the protein crystals are 39.61, 74.69, 157.45, 90.00, 90.00, 90.000; the space group is P212121. After obtaining a crystal with a good shape, the crystal was sent to the Shanghai Synchrotron Radiation Facility (SSRF) for X-ray data collection. The data was processed using HKL2000 software and a resolution of The high-resolution structure of LCC-M4 was obtained by using CCP4 software and PDB 7VVE as a model. The structure of LCC-M4 was compared with that of wild-type LCC. The experimental results are shown in Figure 6 , the difference between the two is very small, and the RMSD value is This indicates that the LCC-M4 mutant has no structural changes after the yield and Tm value are improved compared with the wild type. The structural information of the mutant LCC-M4 provided by the present invention provides a molecular basis for designing LCC with higher activity and higher stability based on structure-directed modification of LCC.

[0054] 3. Conclusion

[0055] The above shows that the modified PET plastic degrading enzyme LCC-M4 provided by the present invention has a higher protein yield and better stability than the wild-type PET plastic degrading enzyme LCC, with a Tm value of 91.3°C and still maintains good activity, has broader application conditions and stronger practical application value, and is more suitable for large-scale production and industrial use.

[0056] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A PET plastic degrading enzyme LCC mutant, characterized in that: The amino acid sequence of the PET plastic degrading enzyme LCC mutant is shown in SEQ ID NO.

1.

2. A polynucleotide encoding the PET plastic degrading enzyme LCC mutant according to claim 1, characterized in that: The polynucleotide sequence is shown as SEQ ID NO.

2.

3. A recombinant plasmid, characterized in that: The recombinant plasmid contains the polynucleotide sequence as claimed in claim 2, and can correspondingly translate and express the PET plastic degrading enzyme LCC mutant as claimed in claim 1.

4. The recombinant plasmid according to claim 3, characterized in that The vector for expressing the PET plastic degrading enzyme LCC mutant is pET-28a, and the strain for expressing the PET plastic degrading enzyme LCC mutant is Escherichia coli BL21 (DE3).

5. Use of the PET plastic degrading enzyme LCC mutant as claimed in claim 1 in degrading PET plastic.

6. A protein crystal of a PET plastic degrading enzyme LCC mutant, characterized in that: The protein crystals are obtained by crystallizing the purified PET plastic degrading enzyme LCC mutant as claimed in claim 1 using a sitting drop method.

7. The protein crystal of a PET plastic degrading enzyme LCC mutant according to claim 6, characterized in that: The crystallization conditions of the protein crystals were 0.2 M ammonium sulfate, 0.1 M 2-(N-morpholino)ethanesulfonic acid, pH 6.5 and 30% w / v PEG 5000MME.

8. The protein crystal of a PET plastic degrading enzyme LCC mutant according to claim 6, characterized in that: The unit cell parameters of the protein crystals are 39.61, 74.69, 157.45, 90.00, 90.00, 90.000; the space group is P212121; the resolution is 9. Use of the protein crystal as described in any one of claims 6 to 8 in transforming a highly stable and highly active PET plastic degrading enzyme LCC.

10. A protein structure of a PET plastic degrading enzyme LCC mutant, characterized in that: The protein structure is obtained by using X-ray diffraction on the protein crystal as described in any one of claims 6 to 8.

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