Ispetase-8×chimera pet hydrolase, and encoding gene, recombinant plasmid, engineering bacterium and use thereof

Abstract

The present invention belongs to the technical field of enzyme engineering, and particularly provided are an IsPETase-8×Chimera recombinase, and an encoding gene, a recombinant plasmid, an engineering bacterium and the use thereof. The ISpeTase-8×Chimera recombinase provided in the present application is obtained by means of replacing at least one of the amino acid sequences as shown in SEQ ID NOs. 1-8 on the basis of IsPETase, and has a high activity and thermal stability compared with a natural PET hydrolase. Further provided in the present application are an encoding gene for encoding the recombinase, a recombinant plasmid containing the encoding gene and an engineering bacterium containing the recombinant plasmid.

Description

IsPETase-8×Chimera recombinase and its encoding gene, recombinant plasmid, engineered bacteria and applications

[0001] This patent application claims priority to Chinese patent application No. CN 202311275023.9 ​​filed on September 28, 2023. The disclosure of the prior application is incorporated herein by reference in its entirety. Technical Field

[0002] The present application relates to the field of enzyme engineering technology, and specifically to an IsPETase-8×Chimera recombinase and its encoding gene, recombinant plasmid, engineered bacteria and application. Background Art

[0003] With the increasing consumption of plastics, the environmental impact of plastic waste has attracted widespread global attention. Polyethylene terephthalate (PET) is currently the most in-demand polyester plastic. Generally speaking, the main methods for disposing of PET include landfill, incineration, pyrolysis, and chemical degradation, all of which consume large amounts of energy and may cause secondary pollution. However, because PET is composed of terephthalic acid (TPA) and ethylene glycol (EG) connected by ester bonds, it can be degraded by the hydrolysis of various enzymes, such as lipase and cutinase. Degrading PET and recovering TPA not only reduces PET's environmental impact, but also provides additional social and economic benefits. Therefore, completely depolymerizing PET into monomers is a necessary step to reduce the environmental pollution caused by PET and to obtain the high-value-added product TPA.

[0004] Biological treatment technology has been gradually applied to the treatment of waste plastics due to its high efficiency, low cost and environmental friendliness. In the past few years, enzyme-based technology has made significant progress in the treatment of PET waste, providing a new environmentally friendly and cost-effective option. A variety of enzymes that can hydrolyze PET have been discovered, such as esterases, lipases and cutinases. However, the activity of these PET hydrolases at room temperature is very low, so they cannot degrade the remaining PET plastics in situ in the natural environment. PETase from the bacterium Ideonella sakaiensis, also known as IsPETase, is an enzyme that degrades PET. IsPETase can effectively hydrolyze PET into TPA and other products at room temperature, but its thermal stability and activity at high temperatures are poor, which still limits its practical application. Technical issues

[0005] In response to the above technical problems, the present application provides an IsPETase chimera (IsPETase-8×Chimera recombinase) and its encoding gene, recombinant plasmid, engineered bacteria and application. The recombinant enzyme has high activity and thermal stability, which makes up for the shortcomings of existing natural PET hydrolases. Technical Solutions

[0006] In order to achieve the above application objectives, this application adopts the following technical solutions:

[0007] In a first aspect, the present application provides an IsPETase-8×Chimera recombinase, wherein the IsPETase-8×Chimera recombinase has an amino acid sequence as follows: IsPETase (amino acid sequence as shown in SEQ ID No. 21) is replaced with at least one of the amino acid sequences shown in SEQ ID Nos. 1 to 8, wherein:

[0008] The amino acid sequence shown in SEQ ID No. 1 is replaced with the amino acid sequence shown in SEQ ID No. 9 or SEQ ID No. 10;

[0009] The amino acid sequence shown in SEQ ID No. 2 is replaced with the amino acid sequence shown in SEQ ID No. 11 or SEQ ID No. 12;

[0010] The amino acid sequence shown in SEQ ID No. 3 is replaced with the amino acid sequence shown in SEQ ID No. 13 or SEQ ID No. 14;

[0011] The amino acid sequence shown in SEQ ID No. 4 is replaced with the amino acid sequence shown in SEQ ID No. 15;

[0012] The amino acid sequence shown in SEQ ID No. 5 is replaced with the amino acid sequence shown in SEQ ID No. 16 or SEQ ID No. 17;

[0013] The amino acid sequence shown in SEQ ID No. 6 is replaced with the amino acid sequence shown in SEQ ID No. 18;

[0014] The amino acid sequence shown in SEQ ID No. 7 is replaced with the amino acid sequence shown in SEQ ID No. 19;

[0015] The amino acid sequence shown in SEQ ID No. 8 is replaced with the amino acid sequence shown in SEQ ID No. 20.

[0016] It should be noted that the amino acid sequences shown in SEQ ID Nos. 1 to 8 are partial sequences of IsPETase (amino acid sequence shown in SEQ ID No. 21). The peptide consisting of the first 26 amino acid residues in the IsPETase (amino acid sequence shown in SEQ ID No. 21) sequence is a signal peptide. To prevent it from affecting the correct folding of the protein, this signal peptide needs to be removed in advance. Therefore, this signal peptide is not included in the IsPETase-8×Chimera recombinant enzyme.

[0017] The IsPETase-8×Chimera recombinant enzyme constructed in this application has high activity and thermal stability, which makes up for the limitations of the natural IsPETase enzyme in practical applications, such as poor thermal stability and low reaction activity, and can be applied to PET hydrolysis catalysis reactions that are of great value to the environment and resources.

[0018] In combination with the first aspect, in some embodiments, the IsPETase-8×Chimera recombinase has an amino acid sequence: the amino acid sequences shown in SEQ ID No. 2, 3, 4, 5, and 7 are replaced.

[0019] In combination with the first aspect, as a more preferred technical solution, the IsPETase-8×Chimera recombinase has an amino acid sequence: the amino acid sequence shown in SEQ ID No.3 is replaced by the amino acid sequence shown in SEQ ID No.14.

[0020] Exemplarily, as a more preferred technical solution, the IsPETase-8×Chimera recombinase has an amino acid sequence as follows: on the basis of IsPETase, the amino acid sequence shown in SEQ ID No.1 is replaced with the amino acid sequence shown in SEQ ID No.9, the amino acid sequence shown in SEQ ID No.2 is replaced with the amino acid sequence shown in SEQ ID No.12, the amino acid sequence shown in SEQ ID No.3 is replaced with the amino acid sequence shown in SEQ ID No.14, the amino acid sequence shown in SEQ ID No.4 is replaced with the amino acid sequence shown in SEQ ID No.15, the amino acid sequence shown in SEQ ID No.5 is replaced with the amino acid sequence shown in SEQ ID No.17, the amino acid sequence shown in SEQ ID No.6 is replaced with the amino acid sequence shown in SEQ ID No.18, the amino acid sequence shown in SEQ ID No.7 is replaced with the amino acid sequence shown in SEQ ID No.19, and the amino acid sequence shown in SEQ ID No.8 is replaced with the amino acid sequence shown in SEQ ID No.20.

[0021] Exemplarily, as another more preferred technical solution, the IsPETase-8×Chimera recombinase has an amino acid sequence as follows: on the basis of IsPETase, the amino acid sequence shown in SEQ ID No.1 is replaced with the amino acid sequence shown in SEQ ID No.10, the amino acid sequence shown in SEQ ID No.2 is replaced with the amino acid sequence shown in SEQ ID No.12, the amino acid sequence shown in SEQ ID No.3 is replaced with the amino acid sequence shown in SEQ ID No.14, the amino acid sequence shown in SEQ ID No.4 is replaced with the amino acid sequence shown in SEQ ID No.15, the amino acid sequence shown in SEQ ID No.5 is replaced with the amino acid sequence shown in SEQ ID No.17, the amino acid sequence shown in SEQ ID No.6 is replaced with the amino acid sequence shown in SEQ ID No.18, the amino acid sequence shown in SEQ ID No.7 is replaced with the amino acid sequence shown in SEQ ID No.19, and the amino acid sequence shown in SEQ ID No.8 is replaced with the amino acid sequence shown in SEQ ID No.20.

[0022] Exemplarily, as another more preferred technical solution, the IsPETase-8×Chimera recombinase has an amino acid sequence as follows: on the basis of IsPETase, the amino acid sequence shown in SEQ ID No.1 is replaced with the amino acid sequence shown in SEQ ID No.9, the amino acid sequence shown in SEQ ID No.2 is replaced with the amino acid sequence shown in SEQ ID No.12, the amino acid sequence shown in SEQ ID No.3 is replaced with the amino acid sequence shown in SEQ ID No.14, the amino acid sequence shown in SEQ ID No.4 is replaced with the amino acid sequence shown in SEQ ID No.15, the amino acid sequence shown in SEQ ID No.5 is replaced with the amino acid sequence shown in SEQ ID No.16, the amino acid sequence shown in SEQ ID No.7 is replaced with the amino acid sequence shown in SEQ ID No.19, and the amino acid sequence shown in SEQ ID No.8 is replaced with the amino acid sequence shown in SEQ ID No.20.

[0023] Exemplarily, as another more preferred technical solution, the IsPETase-8×Chimera recombinase has an amino acid sequence as follows: on the basis of IsPETase, the amino acid sequence shown in SEQ ID No.2 is replaced with the amino acid sequence shown in SEQ ID No.12, the amino acid sequence shown in SEQ ID No.3 is replaced with the amino acid sequence shown in SEQ ID No.14, the amino acid sequence shown in SEQ ID No.4 is replaced with the amino acid sequence shown in SEQ ID No.15, the amino acid sequence shown in SEQ ID No.5 is replaced with the amino acid sequence shown in SEQ ID No.16, the amino acid sequence shown in SEQ ID No.6 is replaced with the amino acid sequence shown in SEQ ID No.18, the amino acid sequence shown in SEQ ID No.7 is replaced with the amino acid sequence shown in SEQ ID No.19, and the amino acid sequence shown in SEQ ID No.8 is replaced with the amino acid sequence shown in SEQ ID No.20.

[0024] Exemplarily, as another more preferred technical solution, the IsPETase-8×Chimera recombinase has an amino acid sequence as follows: on the basis of IsPETase, the amino acid sequence shown in SEQ ID No.1 is replaced with the amino acid sequence shown in SEQ ID No.9, the amino acid sequence shown in SEQ ID No.2 is replaced with the amino acid sequence shown in SEQ ID No.11, the amino acid sequence shown in SEQ ID No.3 is replaced with the amino acid sequence shown in SEQ ID No.14, the amino acid sequence shown in SEQ ID No.4 is replaced with the amino acid sequence shown in SEQ ID No.15, the amino acid sequence shown in SEQ ID No.5 is replaced with the amino acid sequence shown in SEQ ID No.16, the amino acid sequence shown in SEQ ID No.6 is replaced with the amino acid sequence shown in SEQ ID No.18, the amino acid sequence shown in SEQ ID No.7 is replaced with the amino acid sequence shown in SEQ ID No.19, and the amino acid sequence shown in SEQ ID No.8 is replaced with the amino acid sequence shown in SEQ ID No.20.

[0025] In the second aspect, the present application also provides the use of the above-mentioned IsPETase-8×Chimera recombinant enzyme in the hydrolysis of PET, including: adding PET to a glycine-NaOH buffer with a pH value of 8.0 to 10.0 and a concentration of 40mM (mmol / L) to 60mM to make a final concentration of 60mg / mL to 100mg / mL; then adding IsPETase-8×Chimera recombinant enzyme to the above solution to make the final concentration of IsPETase-8×Chimera recombinant enzyme 400nM (nmol / L) to 600nM; incubating for 1 to 3 days at a temperature of 30℃ to 50℃ and a rotation speed of 100rpm to 300rpm.

[0026] In a third aspect, the present application also provides a coding gene comprising a nucleotide sequence encoding the above-mentioned IsPETase-8×Chimera recombinase.

[0027] In a fourth aspect, the present application also provides a recombinant plasmid containing the nucleotide sequence of the above-mentioned encoding gene.

[0028] In a fifth aspect, the present application also provides a method for constructing the above-mentioned recombinant plasmid, comprising the following steps:

[0029] Plasmid pET-22b-IsPETase, plasmid pET-22b-TfCut2, and plasmid pET-22b-LCC were used as templates, and linear vector fragments corresponding to each template were obtained by PCR amplification; the linear vector fragments corresponding to each template were mixed with ligase to form circular recombinant plasmids; the circular recombinant plasmids were transformed into T1 competent cells, and the recombinant plasmid pET-22b-IsPETase-cSP, recombinant plasmid pET-22b-TfCut2-cSP, and recombinant plasmid pET-22b-LCC-cSP were obtained by ampicillin plate culture medium screening and plasmid extraction; the nucleotide sequence of the IsPETase-cSP gene in the recombinant plasmid pET-22b-IsPETase-cSP is shown in SEQ ID No. 51, and the amino acid sequence of the IsPETase-cSP enzyme is shown in SEQ ID No.52; the nucleotide sequence of the TfCut2-cSP gene in the recombinant plasmid pET-22b-TfCut2-cSP is shown in SEQ ID No.53, and the amino acid sequence of the TfCut2-cSP enzyme is shown in SEQ ID No.54; the nucleotide sequence of the LCC-cSP gene in the recombinant plasmid pET-22b-LCC-cSP is shown in SEQ ID No.55, and the amino acid sequence of the LCC-cSP enzyme is shown in SEQ ID No.56;

[0030] PCR amplification is performed using at least two of the recombinant plasmids pET-22b-IsPETase-cSP, pET-22b-TfCut2-cSP, and pET-22b-LCC-cSP as templates, the obtained amplified fragments are ligated, and transformed into T1 competent cells. The recombinant plasmid is obtained by screening with ampicillin plate culture medium and plasmid extraction.

[0031] In a sixth aspect, the present application also provides an engineered bacterium comprising the above-mentioned recombinant plasmid.

[0032] In a seventh aspect, the present application also provides a method for constructing the above-mentioned engineered bacteria, comprising: transforming the above-mentioned recombinant plasmid into BL21 (DE3) competent cells, spreading them on ampicillin plate culture medium, and obtaining positive recombinants, namely the engineered bacteria.

[0033] In an eighth aspect, the present application further provides the use of the above-mentioned engineered bacteria in degrading PET, comprising the following steps:

[0034] The engineered bacteria were cultured in LB liquid medium until OD600 was 0.7-0.9, 0.05%-0.2% (volume percentage v / v) of the inducer IPTG (isopropyl-β-D-thiogalactopyranoside) was added, the temperature was lowered to 14°C-20°C, expression was induced for 16h-24h, and the wet cells of the engineered bacteria were collected by centrifugation;

[0035] The engineered bacterial wet cells were subjected to a sterilization treatment, and cell debris was removed by centrifugation. The supernatant was eluted using a Ni-NTA packed column with an elution buffer as an elution solvent, the eluate was collected, and the eluate was concentrated to obtain a protein concentrate;

[0036] The protein concentrate is added to a glycine-sodium hydroxide buffer solution with a pH value of 8.0 to 10.0 and a concentration of 40 mM to 60 mM to make the protein concentration 400 nM to 600 nM, and PET degradation is performed at 30° C. to 60° C., wherein the concentration of glycine in the glycine-sodium hydroxide buffer solution is 50 mM to 200 mM. Beneficial effects

[0037] The beneficial effects of the present application are that the IsPETase-8×Chimera recombinant enzyme provided in the present application has high activity and thermal stability, and the thermal melting temperature Tm can be increased to 82.71°C, which is 36°C higher than the Tm of natural IsPETase, and the TPA production can reach up to 4856.49 μM, which is much higher than the TPA production of wild-type PET hydrolase IsPETase, TfCut2, and LCC at the same temperature. The amino acid fragments of the recombinant enzyme are derived from different natural enzyme parents, but compared with the natural enzyme, the recombinant enzyme has acquired new functions that it does not have, which is of great significance for achieving efficient degradation of PET. It can be applied to fields such as plastic waste conversion, resource utilization and environmental protection, and has a wide range of application value. Modes for Carrying Out the Invention

[0038] In order to make the purpose, technical solutions and advantages of this application more clear, the following further describes this application in detail with reference to specific embodiments. It should be understood that the specific embodiments described herein are only used to explain this application and are not intended to limit this application.

[0039] The degradation of PET can reduce its environmental pollution and produce the high-value-added product TPA. Enzymatic treatment technology can play a role in PET degradation. Currently, a variety of PET hydrolases have been discovered, such as esterases, lipases, and cutinases. However, these hydrolases have very low activity at room temperature. IsPETase can effectively hydrolyze PET at room temperature, but its hydrolysis mainly occurs in the amorphous portion. Natural IsPETase has poor stability and activity at high temperatures, with its activity largely lost after 24 hours of incubation at 37°C. This is insufficient to maintain efficient substrate degradation activity at higher reaction temperatures, severely limiting its application.

[0040] Based on the study of IsPETase, the present application combines rational design and directed evolution to modify its performance by replacing different structural domains with other amino acid sequences, thereby obtaining the highly active and thermostable IsPETase-8×Chimera recombinase. The recombinase is obtained by replacing at least one of the amino acid sequences shown in SEQ ID Nos. 1 to 8 on the basis of IsPETase (amino acid sequence shown in SEQ ID No. 21), wherein:

[0041] The amino acid sequence shown in SEQ ID No. 1 is replaced with the amino acid sequence shown in SEQ ID No. 9 or SEQ ID No. 10;

[0042] The amino acid sequence shown in SEQ ID No. 2 is replaced with the amino acid sequence shown in SEQ ID No. 11 or SEQ ID No. 12;

[0043] The amino acid sequence shown in SEQ ID No. 3 is replaced with the amino acid sequence shown in SEQ ID No. 13 or SEQ ID No. 14;

[0044] The amino acid sequence shown in SEQ ID No. 4 is replaced with the amino acid sequence shown in SEQ ID No. 15;

[0045] The amino acid sequence shown in SEQ ID No. 5 is replaced with the amino acid sequence shown in SEQ ID No. 16 or SEQ ID No. 17;

[0046] The amino acid sequence shown in SEQ ID No. 6 is replaced with the amino acid sequence shown in SEQ ID No. 18;

[0047] The amino acid sequence shown in SEQ ID No. 7 is replaced with the amino acid sequence shown in SEQ ID No. 19;

[0048] The amino acid sequence shown in SEQ ID No. 8 is replaced with the amino acid sequence shown in SEQ ID No. 20.

[0049] The examples of the present application also provide a method for constructing the above-mentioned IsPETase-8×Chimera recombinase and its application in hydrolyzing PET.

[0050] The present application also provides a gene encoding the recombinase.

[0051] The present application also provides a recombinant plasmid containing the above-mentioned nucleotide sequence, a method for constructing the same, and an engineered bacterium constructed using the recombinant plasmid.

[0052] The examples of this application also provide a method for constructing the engineered bacteria and its application in degrading PET.

[0053] The solution of this application is described below through specific embodiments.

[0054] Unless otherwise specified, the reagents in the following examples were commercially available or obtained according to methods known in the art.

[0055] Example 1

[0056] The present application embodiment provides an IsPETase-8×Chimera recombinase with high activity and thermal stability. The recombinase is based on IsPETase (amino acid sequence as shown in SEQ ID No. 21), wherein the amino acid sequence as shown in SEQ ID No. 1 is replaced with the amino acid sequence as shown in SEQ ID No. 9, the amino acid sequence as shown in SEQ ID No. 2 is replaced with the amino acid sequence as shown in SEQ ID No. 11, the amino acid sequence as shown in SEQ ID No. 3 is replaced with the amino acid sequence as shown in SEQ ID No. 13, the amino acid sequence as shown in SEQ ID No. 4 is replaced with the amino acid sequence as shown in SEQ ID No. 15, the amino acid sequence as shown in SEQ ID No. 5 is replaced with the amino acid sequence as shown in SEQ ID No. 16, the amino acid sequence as shown in SEQ ID No. 6 is replaced with the amino acid sequence as shown in SEQ ID No. 18, the amino acid sequence as shown in SEQ ID No. 7 is replaced with the amino acid sequence as shown in SEQ ID No. 19, and the amino acid sequence as shown in SEQ ID No. 8 is replaced with the amino acid sequence as shown in SEQ ID No. 20. The nucleotide sequence encoding the recombinase is shown in SEQ ID No. 22.

[0057] Example 2

[0058] The present application embodiment provides an IsPETase-8×Chimera recombinase with high activity and thermal stability. The recombinase is based on IsPETase (amino acid sequence as shown in SEQ ID No. 21), wherein the amino acid sequence shown in SEQ ID No. 1 is replaced with the amino acid sequence shown in SEQ ID No. 9, the amino acid sequence shown in SEQ ID No. 2 is replaced with the amino acid sequence shown in SEQ ID No. 11, the amino acid sequence shown in SEQ ID No. 3 is replaced with the amino acid sequence shown in SEQ ID No. 14, the amino acid sequence shown in SEQ ID No. 4 is replaced with the amino acid sequence shown in SEQ ID No. 15, the amino acid sequence shown in SEQ ID No. 5 is replaced with the amino acid sequence shown in SEQ ID No. 16, the amino acid sequence shown in SEQ ID No. 7 is replaced with the amino acid sequence shown in SEQ ID No. 19, and the amino acid sequence shown in SEQ ID No. 8 is replaced with the amino acid sequence shown in SEQ ID No. 20. The nucleotide sequence encoding the recombinase is shown in SEQ ID No. 23.

[0059] Example 3

[0060] The embodiment of the present application provides an IsPETase-8×Chimera recombinase with high activity and thermal stability. The recombinase is IsPETase (amino acid sequence is shown in SEQ ID No.21), wherein the amino acid sequence shown in SEQ ID No.1 is replaced with the amino acid sequence shown in SEQ ID No.10, the amino acid sequence shown in SEQ ID No.2 is replaced with the amino acid sequence shown in SEQ ID No.11, the amino acid sequence shown in SEQ ID No.3 is replaced with the amino acid sequence shown in SEQ ID No.14, the amino acid sequence shown in SEQ ID No.4 is replaced with the amino acid sequence shown in SEQ ID No.15, the amino acid sequence shown in SEQ ID No.5 is replaced with the amino acid sequence shown in SEQ ID No.17, the amino acid sequence shown in SEQ ID No.6 is replaced with the amino acid sequence shown in SEQ ID No.18, the amino acid sequence shown in SEQ ID No.7 is replaced with the amino acid sequence shown in SEQ ID No.19, and the amino acid sequence shown in SEQ ID No.8 is replaced with the amino acid sequence shown in SEQ ID No.20. The nucleotide sequence encoding the recombinase is shown in SEQ ID No. 24.

[0061] Example 4

[0062] The present application provides an IsPETase-8×Chimera recombinase with high activity and thermal stability. The recombinase is based on IsPETase (amino acid sequence as shown in SEQ ID No. 21), wherein the amino acid sequence shown in SEQ ID No. 1 is replaced with the amino acid sequence shown in SEQ ID No. 9, the amino acid sequence shown in SEQ ID No. 2 is replaced with the amino acid sequence shown in SEQ ID No. 12, the amino acid sequence shown in SEQ ID No. 3 is replaced with the amino acid sequence shown in SEQ ID No. 14, the amino acid sequence shown in SEQ ID No. 4 is replaced with the amino acid sequence shown in SEQ ID No. 15, the amino acid sequence shown in SEQ ID No. 5 is replaced with the amino acid sequence shown in SEQ ID No. 17, the amino acid sequence shown in SEQ ID No. 6 is replaced with the amino acid sequence shown in SEQ ID No. 18, and the amino acid sequence shown in SEQ ID No. 7 is replaced with the amino acid sequence shown in SEQ ID No. 19. The nucleotide sequence encoding the recombinase is shown in SEQ ID No. 25.

[0063] Example 5

[0064] The present application embodiment provides an IsPETase-8×Chimera recombinase with high activity and thermal stability. The recombinase is based on IsPETase (amino acid sequence as shown in SEQ ID No. 21), wherein the amino acid sequence as shown in SEQ ID No. 1 is replaced with the amino acid sequence as shown in SEQ ID No. 9, the amino acid sequence as shown in SEQ ID No. 2 is replaced with the amino acid sequence as shown in SEQ ID No. 12, the amino acid sequence as shown in SEQ ID No. 3 is replaced with the amino acid sequence as shown in SEQ ID No. 13, the amino acid sequence as shown in SEQ ID No. 4 is replaced with the amino acid sequence as shown in SEQ ID No. 15, the amino acid sequence as shown in SEQ ID No. 5 is replaced with the amino acid sequence as shown in SEQ ID No. 17, the amino acid sequence as shown in SEQ ID No. 6 is replaced with the amino acid sequence as shown in SEQ ID No. 18, the amino acid sequence as shown in SEQ ID No. 7 is replaced with the amino acid sequence as shown in SEQ ID No. 19, and the amino acid sequence as shown in SEQ ID No. 8 is replaced with the amino acid sequence as shown in SEQ ID No. 20. The nucleotide sequence encoding the recombinase is shown in SEQ ID No. 26.

[0065] Example 6

[0066] The present application provides an IsPETase-8×Chimera recombinase with high activity and thermal stability. The recombinase is based on IsPETase (amino acid sequence as shown in SEQ ID No. 21), wherein the amino acid sequence shown in SEQ ID No. 1 is replaced with the amino acid sequence shown in SEQ ID No. 10, the amino acid sequence shown in SEQ ID No. 2 is replaced with the amino acid sequence shown in SEQ ID No. 12, the amino acid sequence shown in SEQ ID No. 3 is replaced with the amino acid sequence shown in SEQ ID No. 14, the amino acid sequence shown in SEQ ID No. 4 is replaced with the amino acid sequence shown in SEQ ID No. 15, the amino acid sequence shown in SEQ ID No. 5 is replaced with the amino acid sequence shown in SEQ ID No. 16, the amino acid sequence shown in SEQ ID No. 7 is replaced with the amino acid sequence shown in SEQ ID No. 19, and the amino acid sequence shown in SEQ ID No. 8 is replaced with the amino acid sequence shown in SEQ ID No. 20. The nucleotide sequence encoding the recombinase is shown in SEQ ID No. 27.

[0067] Example 7

[0068] The present application embodiment provides an IsPETase-8×Chimera recombinase with high activity and thermal stability. The recombinase is based on IsPETase (amino acid sequence as shown in SEQ ID No. 21), wherein the amino acid sequence shown in SEQ ID No. 2 is replaced with the amino acid sequence shown in SEQ ID No. 11, the amino acid sequence shown in SEQ ID No. 3 is replaced with the amino acid sequence shown in SEQ ID No. 14, the amino acid sequence shown in SEQ ID No. 4 is replaced with the amino acid sequence shown in SEQ ID No. 15, the amino acid sequence shown in SEQ ID No. 5 is replaced with the amino acid sequence shown in SEQ ID No. 16, the amino acid sequence shown in SEQ ID No. 6 is replaced with the amino acid sequence shown in SEQ ID No. 18, the amino acid sequence shown in SEQ ID No. 7 is replaced with the amino acid sequence shown in SEQ ID No. 19, and the amino acid sequence shown in SEQ ID No. 8 is replaced with the amino acid sequence shown in SEQ ID No. 20. The nucleotide sequence encoding the recombinase is shown in SEQ ID No. 28.

[0069] Example 8

[0070] The present application provides an IsPETase-8×Chimera recombinase with high activity and thermal stability. The recombinase is based on IsPETase (amino acid sequence as shown in SEQ ID No. 21), wherein the amino acid sequence as shown in SEQ ID No. 2 is replaced with the amino acid sequence as shown in SEQ ID No. 12, the amino acid sequence as shown in SEQ ID No. 3 is replaced with the amino acid sequence as shown in SEQ ID No. 14, the amino acid sequence as shown in SEQ ID No. 4 is replaced with the amino acid sequence as shown in SEQ ID No. 15, the amino acid sequence as shown in SEQ ID No. 5 is replaced with the amino acid sequence as shown in SEQ ID No. 17, the amino acid sequence as shown in SEQ ID No. 6 is replaced with the amino acid sequence as shown in SEQ ID No. 18, the amino acid sequence as shown in SEQ ID No. 7 is replaced with the amino acid sequence as shown in SEQ ID No. 19, and the amino acid sequence as shown in SEQ ID No. 8 is replaced with the amino acid sequence as shown in SEQ ID No. 20. The nucleotide sequence encoding the recombinase is shown in SEQ ID No. 29.

[0071] Example 9

[0072] The present application provides an IsPETase-8×Chimera recombinase with high activity and thermal stability. The recombinase is based on IsPETase (amino acid sequence as shown in SEQ ID No. 21), wherein the amino acid sequence shown in SEQ ID No. 1 is replaced with the amino acid sequence shown in SEQ ID No. 9, the amino acid sequence shown in SEQ ID No. 2 is replaced with the amino acid sequence shown in SEQ ID No. 12, the amino acid sequence shown in SEQ ID No. 3 is replaced with the amino acid sequence shown in SEQ ID No. 14, the amino acid sequence shown in SEQ ID No. 4 is replaced with the amino acid sequence shown in SEQ ID No. 15, the amino acid sequence shown in SEQ ID No. 5 is replaced with the amino acid sequence shown in SEQ ID No. 17, the amino acid sequence shown in SEQ ID No. 7 is replaced with the amino acid sequence shown in SEQ ID No. 19, and the amino acid sequence shown in SEQ ID No. 8 is replaced with the amino acid sequence shown in SEQ ID No. 20. The nucleotide sequence encoding the recombinase is shown in SEQ ID No. 30.

[0073] Example 10

[0074] The present application embodiment provides an IsPETase-8×Chimera recombinase with high activity and thermal stability. The recombinase is based on IsPETase (amino acid sequence as shown in SEQ ID No. 21), wherein the amino acid sequence shown in SEQ ID No. 1 is replaced with the amino acid sequence shown in SEQ ID No. 9, the amino acid sequence shown in SEQ ID No. 2 is replaced with the amino acid sequence shown in SEQ ID No. 12, the amino acid sequence shown in SEQ ID No. 3 is replaced with the amino acid sequence shown in SEQ ID No. 14, the amino acid sequence shown in SEQ ID No. 4 is replaced with the amino acid sequence shown in SEQ ID No. 15, the amino acid sequence shown in SEQ ID No. 5 is replaced with the amino acid sequence shown in SEQ ID No. 16, and the amino acid sequence shown in SEQ ID No. 7 is replaced with the amino acid sequence shown in SEQ ID No. 19. The nucleotide sequence encoding the recombinase is shown in SEQ ID No. 31.

[0075] Example 11

[0076] The embodiment of the present application provides an IsPETase-8×Chimera recombinase with high activity and thermal stability. The recombinase is IsPETase (amino acid sequence is shown in SEQ ID No.21), wherein the amino acid sequence shown in SEQ ID No.1 is replaced with the amino acid sequence shown in SEQ ID No.10, the amino acid sequence shown in SEQ ID No.2 is replaced with the amino acid sequence shown in SEQ ID No.11, the amino acid sequence shown in SEQ ID No.3 is replaced with the amino acid sequence shown in SEQ ID No.14, the amino acid sequence shown in SEQ ID No.4 is replaced with the amino acid sequence shown in SEQ ID No.15, the amino acid sequence shown in SEQ ID No.5 is replaced with the amino acid sequence shown in SEQ ID No.16, the amino acid sequence shown in SEQ ID No.6 is replaced with the amino acid sequence shown in SEQ ID No.18, the amino acid sequence shown in SEQ ID No.7 is replaced with the amino acid sequence shown in SEQ ID No.19, and the amino acid sequence shown in SEQ ID No.8 is replaced with the amino acid sequence shown in SEQ ID No.20. The nucleotide sequence encoding the recombinase is shown in SEQ ID No.32.

[0077] Example 12

[0078] The present application embodiment provides an IsPETase-8×Chimera recombinase with high activity and thermal stability. The recombinase is based on IsPETase (amino acid sequence as shown in SEQ ID No. 21), wherein the amino acid sequence shown in SEQ ID No. 1 is replaced with the amino acid sequence shown in SEQ ID No. 9, the amino acid sequence shown in SEQ ID No. 2 is replaced with the amino acid sequence shown in SEQ ID No. 12, the amino acid sequence shown in SEQ ID No. 3 is replaced with the amino acid sequence shown in SEQ ID No. 13, the amino acid sequence shown in SEQ ID No. 4 is replaced with the amino acid sequence shown in SEQ ID No. 15, the amino acid sequence shown in SEQ ID No. 5 is replaced with the amino acid sequence shown in SEQ ID No. 16, the amino acid sequence shown in SEQ ID No. 6 is replaced with the amino acid sequence shown in SEQ ID No. 18, the amino acid sequence shown in SEQ ID No. 7 is replaced with the amino acid sequence shown in SEQ ID No. 19, and the amino acid sequence shown in SEQ ID No. 8 is replaced with the amino acid sequence shown in SEQ ID No. 20. The nucleotide sequence encoding the recombinase is shown in SEQ ID No. 33.

[0079] Example 13

[0080] The embodiment of the present application provides an IsPETase-8×Chimera recombinase with high activity and thermal stability. The recombinase is IsPETase (amino acid sequence is shown in SEQ ID No.21), wherein the amino acid sequence shown in SEQ ID No.1 is replaced with the amino acid sequence shown in SEQ ID No.10, the amino acid sequence shown in SEQ ID No.2 is replaced with the amino acid sequence shown in SEQ ID No.12, the amino acid sequence shown in SEQ ID No.3 is replaced with the amino acid sequence shown in SEQ ID No.14, the amino acid sequence shown in SEQ ID No.4 is replaced with the amino acid sequence shown in SEQ ID No.15, the amino acid sequence shown in SEQ ID No.5 is replaced with the amino acid sequence shown in SEQ ID No.17, the amino acid sequence shown in SEQ ID No.6 is replaced with the amino acid sequence shown in SEQ ID No.18, the amino acid sequence shown in SEQ ID No.7 is replaced with the amino acid sequence shown in SEQ ID No.19, and the amino acid sequence shown in SEQ ID No.8 is replaced with the amino acid sequence shown in SEQ ID No.20. The nucleotide sequence encoding the recombinase is shown in SEQ ID No.34.

[0081] Example 14

[0082] The present application embodiment provides an IsPETase-8×Chimera recombinase with high activity and thermal stability. The recombinase is based on IsPETase (amino acid sequence as shown in SEQ ID No. 21), wherein the amino acid sequence shown in SEQ ID No. 1 is replaced with the amino acid sequence shown in SEQ ID No. 9, the amino acid sequence shown in SEQ ID No. 2 is replaced with the amino acid sequence shown in SEQ ID No. 11, the amino acid sequence shown in SEQ ID No. 3 is replaced with the amino acid sequence shown in SEQ ID No. 14, the amino acid sequence shown in SEQ ID No. 4 is replaced with the amino acid sequence shown in SEQ ID No. 15, the amino acid sequence shown in SEQ ID No. 5 is replaced with the amino acid sequence shown in SEQ ID No. 17, the amino acid sequence shown in SEQ ID No. 6 is replaced with the amino acid sequence shown in SEQ ID No. 18, the amino acid sequence shown in SEQ ID No. 7 is replaced with the amino acid sequence shown in SEQ ID No. 19, and the amino acid sequence shown in SEQ ID No. 8 is replaced with the amino acid sequence shown in SEQ ID No. 20. The nucleotide sequence encoding the recombinase is shown in SEQ ID No. 35.

[0083] Example 15

[0084] The present application provides an IsPETase-8×Chimera recombinase with high activity and thermal stability. The recombinase is based on IsPETase (amino acid sequence as shown in SEQ ID No. 21), wherein the amino acid sequence shown in SEQ ID No. 1 is replaced with the amino acid sequence shown in SEQ ID No. 9, the amino acid sequence shown in SEQ ID No. 2 is replaced with the amino acid sequence shown in SEQ ID No. 12, the amino acid sequence shown in SEQ ID No. 3 is replaced with the amino acid sequence shown in SEQ ID No. 14, the amino acid sequence shown in SEQ ID No. 4 is replaced with the amino acid sequence shown in SEQ ID No. 15, the amino acid sequence shown in SEQ ID No. 5 is replaced with the amino acid sequence shown in SEQ ID No. 16, the amino acid sequence shown in SEQ ID No. 7 is replaced with the amino acid sequence shown in SEQ ID No. 19, and the amino acid sequence shown in SEQ ID No. 8 is replaced with the amino acid sequence shown in SEQ ID No. 20. The nucleotide sequence encoding the recombinase is shown in SEQ ID No. 36.

[0085] Example 16

[0086] The present application provides an IsPETase-8×Chimera recombinase with high activity and thermal stability. The recombinase is based on IsPETase (amino acid sequence as shown in SEQ ID No. 21), wherein the amino acid sequence as shown in SEQ ID No. 2 is replaced with the amino acid sequence as shown in SEQ ID No. 12, the amino acid sequence as shown in SEQ ID No. 3 is replaced with the amino acid sequence as shown in SEQ ID No. 14, the amino acid sequence as shown in SEQ ID No. 4 is replaced with the amino acid sequence as shown in SEQ ID No. 15, the amino acid sequence as shown in SEQ ID No. 5 is replaced with the amino acid sequence as shown in SEQ ID No. 16, the amino acid sequence as shown in SEQ ID No. 6 is replaced with the amino acid sequence as shown in SEQ ID No. 18, the amino acid sequence as shown in SEQ ID No. 7 is replaced with the amino acid sequence as shown in SEQ ID No. 19, and the amino acid sequence as shown in SEQ ID No. 8 is replaced with the amino acid sequence as shown in SEQ ID No. 20. The nucleotide sequence encoding the recombinase is shown in SEQ ID No. 37.

[0087] Example 17

[0088] The embodiment of the present application provides an IsPETase-8×Chimera recombinase with high activity and thermal stability. The recombinase is IsPETase (amino acid sequence is shown in SEQ ID No.21), wherein the amino acid sequence shown in SEQ ID No.1 is replaced with the amino acid sequence shown in SEQ ID No.10, the amino acid sequence shown in SEQ ID No.2 is replaced with the amino acid sequence shown in SEQ ID No.12, the amino acid sequence shown in SEQ ID No.3 is replaced with the amino acid sequence shown in SEQ ID No.14, the amino acid sequence shown in SEQ ID No.4 is replaced with the amino acid sequence shown in SEQ ID No.15, the amino acid sequence shown in SEQ ID No.5 is replaced with the amino acid sequence shown in SEQ ID No.16, the amino acid sequence shown in SEQ ID No.6 is replaced with the amino acid sequence shown in SEQ ID No.18, the amino acid sequence shown in SEQ ID No.7 is replaced with the amino acid sequence shown in SEQ ID No.19, and the amino acid sequence shown in SEQ ID No.8 is replaced with the amino acid sequence shown in SEQ ID No.20. The nucleotide sequence encoding the recombinase is shown in SEQ ID No.38.

[0089] Example 18

[0090] The present application embodiment provides an IsPETase-8×Chimera recombinase with high activity and thermal stability. The recombinase is based on IsPETase (amino acid sequence as shown in SEQ ID No. 21), wherein the amino acid sequence as shown in SEQ ID No. 1 is replaced with the amino acid sequence as shown in SEQ ID No. 9, the amino acid sequence as shown in SEQ ID No. 2 is replaced with the amino acid sequence as shown in SEQ ID No. 11, the amino acid sequence as shown in SEQ ID No. 3 is replaced with the amino acid sequence as shown in SEQ ID No. 14, the amino acid sequence as shown in SEQ ID No. 4 is replaced with the amino acid sequence as shown in SEQ ID No. 15, the amino acid sequence as shown in SEQ ID No. 5 is replaced with the amino acid sequence as shown in SEQ ID No. 16, the amino acid sequence as shown in SEQ ID No. 6 is replaced with the amino acid sequence as shown in SEQ ID No. 18, the amino acid sequence as shown in SEQ ID No. 7 is replaced with the amino acid sequence as shown in SEQ ID No. 19, and the amino acid sequence as shown in SEQ ID No. 8 is replaced with the amino acid sequence as shown in SEQ ID No. 20. The nucleotide sequence encoding the recombinase is shown in SEQ ID No. 39.

[0091] Example 19

[0092] The present application provides an IsPETase-8×Chimera recombinase with high activity and thermal stability. The recombinase is based on IsPETase (amino acid sequence as shown in SEQ ID No. 21), wherein the amino acid sequence as shown in SEQ ID No. 1 is replaced with the amino acid sequence as shown in SEQ ID No. 9, the amino acid sequence as shown in SEQ ID No. 2 is replaced with the amino acid sequence as shown in SEQ ID No. 12, the amino acid sequence as shown in SEQ ID No. 3 is replaced with the amino acid sequence as shown in SEQ ID No. 14, the amino acid sequence as shown in SEQ ID No. 4 is replaced with the amino acid sequence as shown in SEQ ID No. 15, the amino acid sequence as shown in SEQ ID No. 5 is replaced with the amino acid sequence as shown in SEQ ID No. 17, the amino acid sequence as shown in SEQ ID No. 6 is replaced with the amino acid sequence as shown in SEQ ID No. 18, the amino acid sequence as shown in SEQ ID No. 7 is replaced with the amino acid sequence as shown in SEQ ID No. 19, and the amino acid sequence as shown in SEQ ID No. 8 is replaced with the amino acid sequence as shown in SEQ ID No. 20. The nucleotide sequence encoding the recombinase is shown in SEQ ID No. 40.

[0093] Example 20

[0094] The present application embodiment provides an IsPETase-8×Chimera recombinase with high activity and thermal stability. The recombinase is based on IsPETase (amino acid sequence as shown in SEQ ID No. 21), wherein the amino acid sequence as shown in SEQ ID No. 1 is replaced with the amino acid sequence as shown in SEQ ID No. 9, the amino acid sequence as shown in SEQ ID No. 2 is replaced with the amino acid sequence as shown in SEQ ID No. 12, the amino acid sequence as shown in SEQ ID No. 3 is replaced with the amino acid sequence as shown in SEQ ID No. 14, the amino acid sequence as shown in SEQ ID No. 4 is replaced with the amino acid sequence as shown in SEQ ID No. 15, the amino acid sequence as shown in SEQ ID No. 5 is replaced with the amino acid sequence as shown in SEQ ID No. 16, the amino acid sequence as shown in SEQ ID No. 6 is replaced with the amino acid sequence as shown in SEQ ID No. 18, the amino acid sequence as shown in SEQ ID No. 7 is replaced with the amino acid sequence as shown in SEQ ID No. 19, and the amino acid sequence as shown in SEQ ID No. 8 is replaced with the amino acid sequence as shown in SEQ ID No. 20. The nucleotide sequence encoding the recombinase is shown in SEQ ID No. 41.

[0095] Example 21

[0096] The examples of this application provide the use of the IsPETase-8×Chimera recombinant enzyme in Examples 1 to 20 in hydrolyzing PET:

[0097] A PET film (diameter ); add IsPETase-8×Chimera recombinant enzyme to a final concentration of 400 nM to 600 nM; and incubate at 30° C. to 50° C. and 100 rpm to 300 rpm for 1 to 3 days.

[0098] Test Example 1

[0099] This test example investigated the Tm values ​​of the IsPETase-8×Chimera recombinant enzymes of Examples 1 to 20 and their effects on PET films. degradation effect.

[0100] The Tm value was measured using a Q-PCR instrument.

[0101] PET film The degradation effect of PET film was investigated by the following method: PET film was added to 50 mM glycine-NaOH buffer with a pH of 9.0. IsPETase-8×Chimera recombinant enzyme was added to a final concentration of 500 nM and incubated at 40°C–60°C, 250 rpm, for one day. The TPA yield in the reaction solution obtained after PET degradation was determined by high-performance liquid chromatography (HPLC). HPLC detection conditions included a UV detector with a characteristic absorption peak at 240 nm, a ZORBAX Eclipse Plus C18 reversed-phase column (5 μl, 250 mm × 4.6 mm), mobile phase A consisting of 0.1% formic acid in water, and mobile phase B consisting of acetonitrile, with the mobile phase B increasing from 5% to 70% acetonitrile over 20 minutes. The column temperature was 30°C, the injection volume was 10 μL, and the flow rate was 0.8 ml / min.

[0102] The Tm values ​​and TPA yields are shown in Table 1.

[0103] Table 1 Tm values ​​and TPA yields of IsPETase-8×Chimera recombinases of Examples 1 to 20

[0104] In this test example, the Tm and TPA production of wild-type PET hydrolase IsPETase, TfCut2, and LCC were also investigated using the same method, as shown in Table 2.

[0105] Table 2 Tm values ​​and TPA yields of wild-type PET hydrolase IsPETase, TfCut2, and LCC

[0106] Example 22

[0107] The present application provides a recombinant plasmid containing a gene encoding the PET hydrolase IsPETase-8×Chimera recombinase in Examples 1 to 20 and a construction method thereof:

[0108] 1. Construction of recombinant plasmids pET-22b-IsPETase-cSP, pET-22b-TfCut2-cSP and pET-22b-LCC-cSP

[0109] Construction of recombinant plasmid pET-22b-IsPETase: The wild-type PET hydrolase IsPETase gene (SEQ ID No. 42) was connected to the pET-22b plasmid.

[0110] Construction of recombinant plasmid pET-22b-TfCut2: The wild-type TfCut2 gene (SEQ ID NO.43) was connected to the pET-22b plasmid.

[0111] Construction of recombinant plasmid pET-22b-LCC: The wild-type LCC gene (SEQ ID NO.44) was connected to the pET-22b plasmid.

[0112] The wild-type PET hydrolase IsPETase gene is from Ideonella sakaiensis (GI: 1028065175), the wild-type TfCut2 gene is from Thermobifida fusca (GenBank: JN129500.1), and the wild-type LCC gene is from uncultured bacteria (GenBank: HQ704839.1).

[0113] The pET-22b plasmid is a commercially available product.

[0114] The preparation method of recombinant plasmids (recombinant plasmids pET-22b-IsPETase-cSP, pET-22b-TfCut2-cSP and pET-22b-LCC-cSP) is as follows:

[0115] According to Beijing Quanshijin Biotechnology Co., Ltd. According to the requirements of the FastPfuPCR SuperMix kit, the recombinant plasmids pET-22b-IsPETase, pET-22b-TfCut2, and pET-22b-LCC were used as templates, and the primers were designed as follows:

[0116] IsPETase-cSP-F:5'-GAGATATACATATGCAGACCAACCCCTACGCCCGCGGCCCGAACC (SEQ ID No. 45);

[0117] IsPETase-cSP-R:5'-GTAGGGGTTGGTCTGCATATGTATATCTCCTTCTTAAAGTTAAAC (SEQ ID No. 46);

[0118] TfCut2-cSP-F:5'-GGAGATATAcatatgGCCAACCCCTACGAGCGCGGCCCCAACCCG (SEQ ID No. 47);

[0119] TfCut2-cSP-R:5'-CTCGTAGGGGTTGGCcatatgTATATCTCCTTCTTAAAGTTAAAC (SEQ ID No. 48);

[0120] LCC-cSP-F:5'-GGAGATATAcatatgGACGGAGTTCTCTGGCGAGTGCGAACCGCG (SEQ ID No. 49);

[0121] LCC-cSP-R:5'-CCAGAGAACTCCGTCcatatgTATATCTCCTTCTTAAAGTTAAAC (SEQ ID No. 50);

[0122] PCR amplification was performed to obtain linear vector fragments of 6.2 kb, 6.2 kb, and 6.2 kb corresponding to each template, respectively. The linear vector fragments obtained by amplification of each template were mixed with ligase to form circular recombinant plasmids under the action of ligase (three circular recombinant plasmids were obtained). The circular recombinant plasmids were transformed into T1 competent cells, and the recombinant plasmids pET-22b-IsPETase-cSP, pET-22b-TfCut2-cSP, and pET-22b-LCC-cSP were obtained, respectively, through ampicillin plate culture medium screening, plasmid extraction, and sequencing verification. The nucleotide sequence of the IsPETase-cSP (cut off signal peptide, cSP) gene in the recombinant plasmid pET-22b-IsPETase-cSP is shown in SEQ ID No. 51, and the amino acid sequence of the IsPETase-cSP enzyme is shown in SEQ ID No. 52; the nucleotide sequence of the TfCut2-cSP gene in the recombinant plasmid pET-22b-TfCut2-cSP is shown in SEQ ID No. 53, and the amino acid sequence of the TfCut2-cSP enzyme is shown in SEQ ID No. 54; the nucleotide sequence of the LCC-cSP gene in the recombinant plasmid pET-22b-LCC-cSP is shown in SEQ ID No. 55, and the amino acid sequence of the LCC-cSP enzyme is shown in SEQ ID No. 56.

[0123] The formula of ampicillin plate medium is: yeast extract 5g / L, tryptone 10g / L, sodium chloride 10g / L, agar powder 15g / L, ampicillin 50mg / L.

[0124] PCR reaction system and PCR procedure were based on the data from Beijing Quanshijin Biotechnology Co., Ltd. The requirements for the FastPfu PCR SuperMix Kit are shown in Table 3:

[0125] Table 3 PCR reaction system

[0126] 2. Construction of a recombinant plasmid containing the IsPETase-8×Chimera recombinase gene (using Example 18 as an example)

[0127] Design and synthesize the primers required for recombination:

[0128] 8×chimera18-1-F:5'-GAAGCTGTGGGTGGATAACGACACCCGCTACACCCAGTTCCTCTG (SEQ ID No. 57)

[0129] 8×chimera18-1-R:5'-GCTCCACGTACACCTTGCTGATGGAGCTCGGCAGG (SEQ ID No. 58)

[0130] 8×chimera18-2-F:5'-CCGAGCTCCATCAGCAAGGTGTACGTGGAGCTCGAC (SEQ ID No. 59)

[0131] 8×chimera18-2-R:5'-CAGAGGAACTGGGTGTAGCGGGTGTCGTTATCCACCCACAGCTTC (SEQ ID No. 60)

[0132] The recombinant plasmid containing the IsPETase-8×Chimera recombinase gene encoding Example 18 was constructed by PCR technology (amplification system is the same as Table 3) and seamless connection according to the requirements of the seamless recombination kit. The steps are mainly divided into three steps: (1) using the recombinant plasmid pET-22b-TfCut2-cSP as a template, 8×chimera18-1-F and 8×chimera18-1-R as forward (upstream) and reverse (downstream) primers, respectively, to perform PCR amplification (95°C for 2 min; 95°C for 20 s, 76°C for 20 s, 72°C for 5 min, 30 cycles; 72°C for 5 min), and the PCR product was subjected to DMT enzyme digestion, nucleic acid electrophoresis and gel cutting recovery to obtain the purified gene fragment 1; (2) using the recombinant plasmid pET-22b-LCC-cSP as a template, 8×chimera18- 2-F and 8×chimera18-2-R were used as forward and reverse primers for PCR amplification (95°C for 2 min; 95°C for 20 s, 76°C for 20 s, 72°C for 5 min, 30 cycles; 72°C for 5 min), and the PCR products were subjected to DMT enzyme digestion, nucleic acid electrophoresis and gel excision recovery to obtain purified gene fragment 2; (3) under the action of seamless recombinant ligase, gene fragment 1 and gene fragment 2 were connected, and then transformed into T1 competent cells. After screening with ampicillin plate culture medium, plasmid extraction and sequencing verification, a recombinant plasmid pET-22b-IsPETase-8×Chimera18 containing the IsPETase-8×Chimera recombinase gene encoding Example 18 was obtained; wherein the nucleotide sequence of the IsPETase-8×Chimera18 recombinase gene is shown in SEQ ID No.39.

[0133] The formula of the ampicillin plate medium is: 5 g / L yeast extract, 10 g / L tryptone, 10 g / L sodium chloride, 15 g / L agar powder, and 50 mg / L ampicillin. The seamless recombinant ligase is a seamless cloning enzyme, and this application uses 2X MultiF Seamless Assembly Mix produced by ABclonal.

[0134] When constructing the IsPETase-8×Chimera recombinase in other embodiments, the primers required for recombination were designed and synthesized corresponding to each recombinase (see Table 4), and the recombinant plasmids pET-22b-IsPETase-cSP, recombinant plasmid pET-22b-TfCut2-cSP and recombinant plasmid pET-22b-LCC-cSP in step 1 were used to construct recombinant plasmids containing genes encoding each recombinase using seamless ligation and PCR technology according to the requirements of the seamless recombination kit.

[0135] Table 4 Primers and templates corresponding to IsPETase-8×Chimera recombinase of Examples 1 to 20

[0136] Example 23

[0137] The present application provides an engineering bacterium containing the recombinant plasmid of Example 22 and a method for constructing the same:

[0138] The recombinant plasmid of Example 22 was transformed into BL21 (DE3) competent cells and plated on ampicillin plate culture medium to obtain positive recombinants, which were engineered bacteria containing the recombinant plasmid of Example 22.

[0139] Example 24

[0140] The present application provides the method for inducing expression of the engineered bacteria and purifying the target protein of Example 23:

[0141] The engineered bacteria of Example 23 were inoculated into LB liquid medium and cultured overnight at 37°C, 220 rpm. A 1% inoculum of the overnight culture was inoculated into fresh LB liquid medium and cultured at 37°C, 220 rpm until the OD600 reached approximately 0.8. Then, 0.1% (v / v) of the inducer IPTG was added, and the temperature was lowered to 16°C to induce expression for 20 hours to obtain a fermentation culture. The fermentation culture was centrifuged at 4000 rpm for 15 minutes, and the wet cells of the engineered bacteria were collected.

[0142] Resuspend the engineered bacterial wet cells in lysis buffer and disrupt them using a high-pressure cell disruptor. Centrifuge the lysis solution at 10,000 rpm for 60 minutes to remove cell debris. The supernatant is then passed through a Ni-NTA column to adsorb the target protein. Nonspecific adsorbed proteins are then removed using wash buffer. The target protein is then eluted using elution buffer, and the eluate is concentrated using a protein concentrator.

[0143] The formula of LB liquid medium is: yeast extract 5g / L, tryptone 10g / L, sodium chloride 10g / L;

[0144] The formula of the lysis buffer is: 50 mM Tris-HCl, 150 mM NaCl, 10 mM Imidazole, pH = 7.5;

[0145] The formula of the washing buffer is: 50mM Tris-HCl, 150mM NaCl, 20mM Imidazole, pH=7.5;

[0146] The formula of the elution buffer is: 50 mM Tris-HCl, 300 mM NaCl, 300 mM Imidazole, pH = 7.5.

[0147] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent replacements or improvements made within the spirit and principles of the present application should be included in the scope of protection of the present application.

Claims

1. An IsPETase-8×Chimera recombinase, It is characterized in that The amino acid sequence of the IsPETase-8×Chimera recombinase is as follows: IsPETase is replaced with at least one of the amino acid sequences shown in SEQ ID No. 1 to 8, wherein: The amino acid sequence shown in SEQ ID No.1 is replaced with the amino acid sequence shown in SEQ ID No.9 or SEQ ID No.10; The amino acid sequence shown in SEQ ID No.2 is replaced with the amino acid sequence shown in SEQ ID No.11 or SEQ ID No.12; The amino acid sequence shown in SEQ ID No.3 is replaced with the amino acid sequence shown in SEQ ID No.13 or SEQ ID No.14; The amino acid sequence shown in SEQ ID No.4 is replaced with the amino acid sequence shown in SEQ ID No.15; The amino acid sequence shown in SEQ ID No.5 is replaced with the amino acid sequence shown in SEQ ID No.16 or SEQ ID No.17; The amino acid sequence shown in SEQ ID No.6 is replaced with the amino acid sequence shown in SEQ ID No.18; The amino acid sequence shown in SEQ ID No.7 is replaced with the amino acid sequence shown in SEQ ID No.19; The amino acid sequence shown in SEQ ID No.8 is replaced with the amino acid sequence shown in SEQ ID No.

20.

2. The IsPETase-8×Chimera recombinant enzyme according to claim 1, It is characterized in that The amino acid sequence of the IsPETase-8×Chimera recombinase is as follows: the amino acid sequences shown in SEQ ID No. 2, 3, 4, 5, and 7 are replaced.

3. IsPETase-8×Chimera recombinant enzyme according to claim 1 or 2, It is characterized in that The amino acid sequence of the IsPETase-8×Chimera recombinase is as follows: based on IsPETase, the amino acid sequence shown in SEQ ID No.3 is replaced with the amino acid sequence shown in SEQ ID No.

14.

4. IsPETase-8×Chimera recombinant enzyme according to claim 1 or 2, It is characterized in that The IsPETase-8×Chimera recombinase has an amino acid sequence as follows: on the basis of IsPETase, the amino acid sequence shown in SEQ ID No.1 is replaced with the amino acid sequence shown in SEQ ID No.9, the amino acid sequence shown in SEQ ID No.2 is replaced with the amino acid sequence shown in SEQ ID No.12, the amino acid sequence shown in SEQ ID No.3 is replaced with the amino acid sequence shown in SEQ ID No.14, the amino acid sequence shown in SEQ ID No.4 is replaced with the amino acid sequence shown in SEQ ID No.15, the amino acid sequence shown in SEQ ID No.5 is replaced with the amino acid sequence shown in SEQ ID No.17, the amino acid sequence shown in SEQ ID No.6 is replaced with the amino acid sequence shown in SEQ ID No.18, the amino acid sequence shown in SEQ ID No.7 is replaced with the amino acid sequence shown in SEQ ID No.19, and the amino acid sequence shown in SEQ ID No.8 is replaced with the amino acid sequence shown in SEQ ID No.20; or On the basis of IsPETase, the amino acid sequence shown in SEQ ID No.1 is replaced with the amino acid sequence shown in SEQ ID No.10, the amino acid sequence shown in SEQ ID No.2 is replaced with the amino acid sequence shown in SEQ ID No.12, the amino acid sequence shown in SEQ ID No.3 is replaced with the amino acid sequence shown in SEQ ID No.14, the amino acid sequence shown in SEQ ID No.4 is replaced with the amino acid sequence shown in SEQ ID No.15, the amino acid sequence shown in SEQ ID No.5 is replaced with the amino acid sequence shown in SEQ ID No.17, the amino acid sequence shown in SEQ ID No.6 is replaced with the amino acid sequence shown in SEQ ID No.18, the amino acid sequence shown in SEQ ID No.7 is replaced with the amino acid sequence shown in SEQ ID No.19, and the amino acid sequence shown in SEQ ID No.8 is replaced with the amino acid sequence shown in SEQ ID No.20; or On the basis of IsPETase, the amino acid sequence shown in SEQ ID No.1 is replaced with the amino acid sequence shown in SEQ ID No.9, the amino acid sequence shown in SEQ ID No.2 is replaced with the amino acid sequence shown in SEQ ID No.12, the amino acid sequence shown in SEQ ID No.3 is replaced with the amino acid sequence shown in SEQ ID No.14, the amino acid sequence shown in SEQ ID No.4 is replaced with the amino acid sequence shown in SEQ ID No.15, the amino acid sequence shown in SEQ ID No.5 is replaced with the amino acid sequence shown in SEQ ID No.16, the amino acid sequence shown in SEQ ID No.7 is replaced with the amino acid sequence shown in SEQ ID No.19, and the amino acid sequence shown in SEQ ID No.8 is replaced with the amino acid sequence shown in SEQ ID No.20; or On the basis of IsPETase, the amino acid sequence shown in SEQ ID No.2 is replaced by the amino acid sequence shown in SEQ ID No.12, the amino acid sequence shown in SEQ ID No.3 is replaced by the amino acid sequence shown in SEQ ID No.14, the amino acid sequence shown in SEQ ID No.4 is replaced by the amino acid sequence shown in SEQ ID No.15, the amino acid sequence shown in SEQ ID No.5 is replaced by the amino acid sequence shown in SEQ ID No.16, the amino acid sequence shown in SEQ ID No.6 is replaced by the amino acid sequence shown in SEQ ID No.18, the amino acid sequence shown in SEQ ID No.7 is replaced by the amino acid sequence shown in SEQ ID No.19, and the amino acid sequence shown in SEQ ID No.8 is replaced by the amino acid sequence shown in SEQ ID No.20; or On the basis of IsPETase, the amino acid sequence shown in SEQ ID No.1 is replaced with the amino acid sequence shown in SEQ ID No.9, the amino acid sequence shown in SEQ ID No.2 is replaced with the amino acid sequence shown in SEQ ID No.11, the amino acid sequence shown in SEQ ID No.3 is replaced with the amino acid sequence shown in SEQ ID No.14, the amino acid sequence shown in SEQ ID No.4 is replaced with the amino acid sequence shown in SEQ ID No.15, the amino acid sequence shown in SEQ ID No.5 is replaced with the amino acid sequence shown in SEQ ID No.16, the amino acid sequence shown in SEQ ID No.6 is replaced with the amino acid sequence shown in SEQ ID No.18, the amino acid sequence shown in SEQ ID No.7 is replaced with the amino acid sequence shown in SEQ ID No.19, and the amino acid sequence shown in SEQ ID No.8 is replaced with the amino acid sequence shown in SEQ ID No.

20.

5. Use of the IsPETase-8×Chimera recombinant enzyme according to any one of claims 1 to 4 in hydrolyzing PET, It is characterized in that include: Add PET to a glycine-NaOH buffer solution with a pH value of 8.0 to 10.0 and a concentration of 40 mM to 60 mM to make a final concentration of 60 mg / mL to 100 mg / mL; then add the IsPETase-8×Chimera recombinant enzyme to the above solution to make a final concentration of 400 nM to 600 nM; incubate at a temperature of 30°C to 50°C and a rotation speed of 100 rpm to 300 rpm for 1 to 3 days.

6. A gene encoding It is characterized in that It comprises a nucleotide sequence encoding the IsPETase-8×Chimera recombinase according to any one of claims 1 to 4.

7. A recombinant plasmid, It is characterized in that Containing the nucleotide sequence encoding the gene according to claim 6.

8. The method for constructing the recombinant plasmid according to claim 7, It is characterized in that The method comprises the following steps: using plasmid pET-22b-IsPETase, plasmid pET-22b-TfCut2 and plasmid pET-22b-LCC as templates respectively, obtaining linear vector fragments corresponding to each template by PCR amplification; mixing the linear vector fragments corresponding to each template with ligase respectively to form circular recombinant plasmids; transforming the circular recombinant plasmids into T1 competent cells respectively, screening by ampicillin plate culture medium and extracting plasmids to obtain recombinant plasmid pET-22b-IsPETase-cSP, recombinant plasmid pET-22b-TfCut2-cSP and recombinant plasmid pET-22b-LCC-cSP; the nucleotide sequence of the IsPETase-cSP gene in the recombinant plasmid pET-22b-IsPETase-cSP is shown in SEQ ID No.51, and the amino acid sequence of the IsPETase-cSP enzyme is shown in SEQ ID No.52; the nucleotide sequence of the TfCut2-cSP gene in the recombinant plasmid pET-22b-TfCut2-cSP is shown in SEQ ID No.53, and the amino acid sequence of the TfCut2-cSP enzyme is shown in SEQ ID No.54; the nucleotide sequence of the LCC-cSP gene in the recombinant plasmid pET-22b-LCC-cSP is shown in SEQ ID No.55, and the amino acid sequence of the LCC-cSP enzyme is shown in SEQ ID No.56; At least two of the recombinant plasmids pET-22b-IsPETase-cSP, pET-22b-TfCut2-cSP and pET-22b-LCC-cSP are used as templates for PCR amplification, the obtained amplified fragments are connected, transformed into T1 competent cells, and the recombinant plasmid is obtained by screening with ampicillin plate culture medium and extracting the plasmid.

9. An engineered bacterium, It is characterized in that Comprising the recombinant plasmid described in claim 7.

10. The method for constructing the engineered bacteria according to claim 9, It is characterized in that include: The recombinant plasmid described in claim 8 is transformed into BL21 (DE3) competent cells, and spread on ampicillin plate culture medium to obtain positive recombinants, which are the engineered bacteria.

11. Use of the engineered bacteria according to claim 9 in degrading PET, It is characterized in that The following steps are involved: The engineered bacteria were cultured in LB liquid medium until OD600 was 0.7-0.9, 0.05%-0.2% of inducer IPTG was added, the temperature was lowered to 14°C-20°C to induce expression for 16h-24h, and the wet cells of the engineered bacteria were collected by centrifugation; After the wet cells of the engineered bacteria are subjected to a sterilization treatment, the cell debris are removed by centrifugation, the supernatant is eluted using a Ni-NTA packed column with an elution buffer as an elution solvent, the eluate is collected, and the eluate is concentrated to obtain a protein concentrate; The concentrated solution is added to a glycine-sodium hydroxide buffer solution with a pH value of 8.0 to 10.0 and a concentration of 40 mM to 60 mM to make the protein concentration 400 nM to 600 nM, and PET is degraded at 30° C. to 60° C. The concentration of glycine in the sodium buffer is 50 mM to 200 mM.