Application of anchoring protein INP-N in surface-displayed PET degrading enzyme system

Abstract

The invention belongs to the technical field of genetic engineering, and discloses application of an anchoring protein INP-N in a PET degrading enzyme system displayed on the surface. According to the invention, after efficient anchoring protein INP-N is screened, FAST-PETase is mediated by the efficient anchoring protein INP-N to construct recombinant escherichia coli of which the surface displays PET degrading enzyme. Furthermore, hydrophobin HFBII is introduced into the surface display system, so that the relative enzyme activity of the PET degrading enzyme is improved by 1.1 times or more. Meanwhile, FAST-PETase and carboxylesterase Est30KL are synchronously expressed, so that the proportion of TPA in a degradation product can reach 95% or above. In addition, the PET degrading enzyme surface display system provided by the invention is excellent in cycle performance, more than 65% of activity can still be retained when PET is cyclically degraded for the third time, and the application cost can be greatly reduced. The method can be applied to the fields of degradation of PET products, preparation of PET degradation agents or BHET degradation agents and the like.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology and relates to the application of an anchoring protein INP-N in a PET degradation enzyme system displayed on a surface. Background Technology

[0002] The massive accumulation of plastic waste, especially polyethylene terephthalate (PET), has become a major challenge for global environmental governance, and enzymatic degradation has become a research hotspot due to its green and environmentally friendly nature. In existing PET enzymatic degradation systems, extracellular secreted enzymes not only suffer from low secretion efficiency and poor catalytic effect, but also face the cumbersome process of requiring bacterial disruption treatment of the crude enzyme solution; furthermore, cell debris easily covers the substrate surface, further hindering enzyme-substrate binding. Meanwhile, purifying PET enzymes is costly and cannot be reused. While surface display technology can solve the reuse problem, some existing anchoring proteins (such as PgsA, AIDA-I, and Lpp-OmpA) have low catalytic efficiency, making it difficult to meet practical needs for PET degradation efficiency. Therefore, developing a PET degradation system based on highly efficient anchoring proteins is of great significance.

[0003] Furthermore, in existing PET biodegradation systems, the contact efficiency between the enzyme and the hydrophobic substrate PET is one of the key bottlenecks limiting degradation efficiency. Even when using highly efficient anchoring proteins to construct surface display systems, insufficient enzyme-substrate affinity still limits catalytic activity. Simultaneously, existing PET degradation products often contain residual intermediate MHET, resulting in a low proportion of terephthalic acid (TPA), which affects the subsequent recycling of degradation products. Summary of the Invention

[0004] In view of the aforementioned problems in the prior art, this invention provides an application of the anchoring protein INP-N in a surface-displayed PET degrading enzyme system by screening a combination of a highly efficient anchoring protein and a PET degrading enzyme. Among the four display systems constructed in this invention, FAST-PETase anchored by INP-N exhibited the highest release of degradation products and enzyme activity. Furthermore, this invention creatively introduces the hydrophobic protein HHFBII into the surface-displayed PET degrading enzyme system, increasing the relative enzyme activity by approximately 1.1 times. In addition, the synergistic effect of Est30KL and FAST-PETase can efficiently degrade PET products, not only significantly increasing the proportion of TPA in the degradation products but also enhancing the recyclability of the system, providing a new and efficient strategy for PET biodegradation.

[0005] To achieve the above-mentioned objectives, the embodiments of the present invention employ the following technical solutions: In a first aspect, the present invention provides the application of the anchoring protein INP-N in a surface-displayed PET degrading enzyme system.

[0006] In existing PET biodegradation systems, extracellularly secreted PET hydrolases suffer from low secretion efficiency and weak catalytic activity. Crude enzyme solutions are hindered by substrate coverage by cell debris, and enzyme purification is costly and cannot be reused. The recombinant E. coli with surface-displayed PET-degrading enzymes provided by this invention eliminates the need for lysis and enzyme purification steps when used to degrade PET, simplifying the operation process. Furthermore, the whole-cell catalyst can be recycled, significantly reducing application costs.

[0007] This invention experimentally demonstrates that, compared with preferred anchoring proteins such as Lpp-OmpA, AIDA-I, or PgsA in surface-displayed PET degradation systems, the system constructed using INP-N as the anchoring protein exhibits significantly superior degradation activity for BHET.

[0008] Secondly, the present invention provides a recombinant Escherichia coli with a surface display of PET-degrading enzyme, which heterologously expresses the encoding gene of PET hydrolase FAST-PETase and the anchoring protein INP-N.

[0009] This invention demonstrates that in a PET degradation system based on surface display of E. coli, using INP-N as the anchoring protein, FAST-PETase mediates the degradation of 10 mM BHET at 30°C for 1 hour, with a degradation product amount of 8.9 mM, which is significantly higher than that of anchoring proteins such as Lpp-OmpA, AIDA-I, and PgsA.

[0010] Preferably, the recombinant Escherichia coli can express the hydrophobic protein HFBII.

[0011] This invention creatively introduces the hydrophobic protein HHFBII into the surface-displayed PET degrading enzyme system, effectively breaking through the bottleneck of enzyme-hydrophobic substrate contact efficiency, and increasing the relative enzyme activity of PET degrading enzyme by more than 1.1 times.

[0012] More preferably, the recombinant Escherichia coli may also express the anchoring protein AIDA-I or the anchoring protein PgsA.

[0013] Preferably, the recombinant Escherichia coli can express carboxylesterase Est30KL.

[0014] In this invention, FAST-PETase and carboxylesterase Est30KL are expressed simultaneously, which can make the TPA content in the degradation products reach more than 95%, solving the problem of low TPA content in existing PET degradation products due to the accumulation of intermediate product MHET, and laying the foundation for the efficient recycling of PET.

[0015] Thirdly, the present invention provides a method for constructing recombinant Escherichia coli with surface display of PET degrading enzyme, the method comprising the following steps: cloning the encoding gene of the PET hydrolase FAST-PETase and the encoding gene of the anchoring protein INP-N into the pETDuet-1 plasmid vector, and then introducing them into competent Escherichia coli cells to obtain recombinant Escherichia coli with surface display of PET degrading enzyme.

[0016] Preferably, the construction method specifically includes the following steps: cloning the coding gene of the PET hydrolase FAST-PETase and the coding gene of the anchoring protein INP-N into the pETDuet-1 plasmid vector to obtain recombinant pETDuet-1 plasmid 1; cloning the coding gene of the anchoring protein AIDA-I and the coding gene of the hydrophobic protein HFBII into the pBAD33 plasmid vector to obtain recombinant pBAD33 plasmid 1; and then introducing the recombinant pETDuet-1 plasmid 1 and the recombinant pBAD33 plasmid 1 into competent Escherichia coli cells to obtain the recombinant Escherichia coli.

[0017] Preferably, the construction method specifically includes the following steps: cloning the encoding gene of the PET hydrolase FAST-PETase and the encoding gene of the anchoring protein INP-N into the pETDuet-1 plasmid vector to obtain recombinant pETDuet-1 plasmid 1; cloning the encoding gene of the anchoring protein PgsA and the encoding gene of the hydrophobic protein HFBII into the pBAD33 plasmid vector to obtain recombinant pBAD33 plasmid 2; and then introducing the recombinant pETDuet-1 plasmid 1 and the recombinant pBAD33 plasmid 2 into competent Escherichia coli cells to obtain the recombinant Escherichia coli.

[0018] Preferably, the construction method specifically includes the following steps: cloning the encoding gene of the PET hydrolase FAST-PETase and the encoding gene of the anchoring protein INP-N into the pETDuet-1 plasmid vector to obtain recombinant pETDuet-1 plasmid 1; cloning the encoding gene of the anchoring protein INP-N and the encoding gene of the carboxylesterase Est30KL into the pETDuet-1 plasmid vector to obtain recombinant pETDuet-1 plasmid 2; and then introducing recombinant pETDuet-1 plasmid 1 and recombinant pETDuet-1 plasmid 2 into competent Escherichia coli cells to obtain recombinant Escherichia coli.

[0019] The method for constructing recombinant Escherichia coli with surface-displaying PET-degrading enzymes provided by this invention has the advantages of strong compatibility, flexible and diverse combinations that can be adapted to different degradation needs, targeted performance enhancement, strong functional specificity, and simple operation and easy promotion and application.

[0020] Fourthly, the present invention provides the application of the above-mentioned recombinant Escherichia coli with PET-degrading enzymes on its surface in the degradation of PET products or the preparation of PET-degrading agents.

[0021] The recombinant Escherichia coli with surface-displayed PET-degrading enzyme provided by this invention can be used as a whole-cell catalyst. It can be recycled—it can retain more than 65% of its activity in the third cycle of PET degradation, which can significantly reduce the application cost. Under the same conditions, its activity can be up to 3.5 times higher than that of the free enzyme.

[0022] Fifthly, the present invention also provides a method for degrading PET products, specifically comprising: culturing the above-mentioned recombinant Escherichia coli with PET-degrading enzymes on its surface in LB medium until the bacterial concentration reaches OD0.05. 600 When the concentration reaches 0.8-1.0, add at least one of isopropyl-β-D-thiogalactoside or L-arabinose for induction culture for 18-24 hours, and separate the bacterial cells from the solid and liquid. After resuspending the bacterial cells, mix them with the PET product and carry out the PET degradation reaction at 40℃-50℃ to obtain the PET degradation product.

[0023] The surface-display PET degradation system provided by this invention has a wide range of applications and is recyclable. It can cover several practical scenarios, including: in daily waste disposal, it can efficiently degrade waste PET beverage bottles, food packaging films, cosmetic bottles, etc., reducing urban plastic waste accumulation; in industrial recycling, it provides a green degradation process for PET recycled plastic production lines, converting recycled PET products into high-purity TPA raw materials, contributing to the construction of a circular economy industrial chain; in environmental governance, it is suitable for the remediation of PET microplastics in soil and water, and can also remove agricultural residue film fragments, alleviating ecological pollution problems; in scientific research and environmental protection industries, it can provide an efficient model for the development of new biodegradation technologies and can be applied to the purification of industrial wastewater containing PET degradation intermediates, reducing wastewater treatment costs; furthermore, it can promote the green transformation of industries such as textiles and packaging, realizing the resource utilization of industry waste through the degradation of waste PET fiber products and packaging materials. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 The results of surface fluorescence intensity measurement of recombinant Escherichia coli containing a single anchoring protein in the effect example of this invention; Figure 2The results of BHET enzyme activity assays for recombinant Escherichia coli LF, recombinant Escherichia coli AF, and recombinant Escherichia coli PF in the efficacy examples of this invention; Figure 3 This is a comparison chart of the degradation PET results of an intermediate amount of free enzyme and an IF surface display system in the example of the effectiveness of this invention; Figure 4 The results of surface fluorescence intensity measurement of recombinant Escherichia coli containing multiple anchoring proteins in the effect examples of this invention; Figure 5 The results of relative enzyme activity assays for surface display systems containing hydrophobic anchoring proteins and those without hydrophobic proteins are shown in the effect examples of this invention. Figure 6 The results of relative enzyme activity assays for recombinant Escherichia coli IF, recombinant Escherichia coli I-F+PH, recombinant Escherichia coli I-F+IE, and recombinant Escherichia coli P-H+IE in the efficacy examples of this invention; Figure 7 The results show the cycling performance of the P-H+IE surface display system for PET degradation enzymes in the example of this invention. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. The naming of mutants described in this invention follows the conventional naming methods used by those skilled in the art.

[0027] Example 1 This embodiment provides a recombinant Escherichia coli with PET-degrading enzymes displayed on its surface and its construction method, as detailed below: This invention obtains the target gene and expression vector through polymerase chain reaction (PCR), prepares recombinant plasmids using molecular biology methods such as DMT enzyme and seamless cloning, and transforms them into Escherichia coli BL21(DE3) (TransGen, CD601) competent cells. After culturing, recombinant E. coli expressing the target protein heterologously are obtained.

[0028] This invention commissioned Genewiz to synthesize recombinant plasmids and recombinant bacterial strains. Specifically, the recombinant plasmids INP-N-FAST (IF), Lpp-OmpA-FAST (LF), AIDA-I-FAST (AF), PgsA-FAST (PF), INP-N-Est30KL (IE), Lpp-OmpA-Est30KL (LE), AIDA-I-Est30KL (AE), and PgsA-Est30KL (PE) were all constructed on the pETDuet-1 plasmid, while the recombinant plasmids PgsA-HFBII (PH) and AIDA-I-HFBII (AH) were both constructed on the pBAD33 plasmid.

[0029] In constructing the recombinant plasmid AIDA-I-FAST, a signal peptide SP was linked between the gene encoding the anchoring protein AIDA-I and the gene encoding the PET hydrolase FAST-PETase. The gene encoding the functional fragment GFP11 of green fluorescent protein was linked to the end of the gene encoding the relevant protein in the recombinant plasmid. After heterologous expression of the GFP11 fragment, it bound to the GFP1-10 fragment added to the assay system, generating fluorescence to determine the relative expression level of the target protein in the system. Furthermore, the primer pair for amplifying the encoding genes of "FAST-Linker" and "Linker-FAST" was FAST-F / R, and the primer pair for PCR amplification of the encoding genes of "Linker-HFBII" and "HFBII-Linker" was HFBII-F / R.

[0030] The recombinant plasmids obtained above were introduced into Escherichia coli BL21 competent cells via heat shock. After resuscitation, screening, and single-clone amplification culture, a recombinant Escherichia coli strain containing the corresponding recombinant plasmid was finally constructed.

[0031] 1. The amino acid sequence of the target protein used to synthesize the recombinant plasmid and the coding gene of the codon-optimized protein are as follows.

[0032] (1) The amino acid sequence of the anchoring protein INP-N is shown in SEQ ID No.1, and the coding gene after codon optimization is shown in SEQ ID No.2.

[0033] (2) The amino acid sequence of PET hydrolase FAST-PETase is shown in SEQ ID No.3, and the coding gene after codon optimization is shown in SEQ ID No.4.

[0034] (3) The amino acid sequence of the hydrophobic protein HFBII is shown in SEQ ID No. 5, and the coding gene after codon optimization is shown in SEQ ID No. 6.

[0035] (4) The amino acid sequence of carboxylesterase Est30KL is shown in SEQ ID No.7, and the coding gene after codon optimization is shown in SEQ ID No.8.

[0036] (5) The amino acid sequence of the anchoring protein Lpp-OmpA is shown in SEQ ID No. 9, and the coding gene after codon optimization is shown in SEQ ID No. 10.

[0037] (6) The amino acid sequence of the anchoring protein AIDA-I is shown in SEQ ID No.11, and the coding gene after codon optimization is shown in SEQ ID No.12.

[0038] (7) The amino acid sequence of the anchoring protein PgsA is shown in SEQ ID No.13, and the coding gene after codon optimization is shown in SEQ ID No.14.

[0039] (8) The amino acid sequence of the functional fragment GFP11 of green fluorescent protein is shown in SEQ ID No.15, and the coding gene after codon optimization is shown in SEQ ID No.16.

[0040] (9) The amino acid sequence of the functional fragment GFP1-10 of green fluorescent protein is shown in SEQ ID No.17.

[0041] (10) The amino acid sequence of the signal peptide SP used in constructing the recombinant plasmid AIDA-I-FAST is shown in SEQ ID No.18, and the coding gene after codon optimization is shown in SEQ ID No.19.

[0042] (11) Construct recombinant plasmids IF, PF or LF, the amino acid sequence of the Linker-FAST used is shown in SEQ ID No. 20, and the codon-optimized Linker-FAST encoding gene is shown in SEQ ID No. 21.

[0043] (12) The amino acid sequence of Linker-Est30KL used to construct the recombinant plasmid IE is shown in SEQ ID No.22, and the codon-optimized Linker-Est30KL encoding gene is shown in SEQ ID No.23.

[0044] (13) The amino acid sequence of FAST-Linker used to construct recombinant plasmid AF is shown in SEQ ID No. 24, and the FAST-Linker encoding gene after codon optimization is shown in SEQ ID No. 25.

[0045] (14) The amino acid sequence of Linker-HEBII used to construct the recombinant plasmid PH is shown in SEQ ID No. 26, and the codon-optimized Linker-HFBII encoding gene is shown in SEQ ID No. 27.

[0046] (15) The amino acid sequence of the HFBII-Linker used to construct the recombinant plasmid AH is shown in SEQ ID No. 28, and the HFBII-Linker encoding gene after codon optimization is shown in SEQ ID No. 29.

[0047] 2. Construction of recombinant plasmids and recombinant Escherichia coli, and acquisition of recombinant proteins. The primers used in the construction of the recombinant plasmid are shown in Table 1 below.

[0048] Table 1

[0049] (1) This invention uses the construction method of recombinant plasmid INP-N-FAST (IF) as an example for illustration. The construction methods of recombinant plasmids Lpp-OmpA-FAST (LF), AIDA-I-FAST (AF), and PgsA-FAST (PF) are basically the same, except that the target gene and its corresponding primers are different, as follows: Using the coding genes of INP-N, Linker-FAST, and GFP11, as well as the pETDuet plasmid, as templates, PCR amplification was performed using the corresponding primer pairs listed in Table 1 to obtain the corresponding linearized plasmid fragments. Then, the templates were digested using DMT enzyme (TransGen, GD111), and the coding genes of INP-N, FAST-PETase, and GFP11 were cloned into the pETDuet-1 plasmid using molecular biology methods such as seamless cloning. Sequencing verification yielded the recombinant plasmid IF.

[0050] I) Using the INP-N encoding gene as a template and INP-NF and INP-NR as primers, PCR amplification was performed to obtain the nucleotide sequence shown in SEQ ID No. 2. INP-N Gene fragments; II) Using the Linker-FAST encoding gene as a template and FAST-F and FAST-R as primers, PCR amplification was performed to obtain the nucleotide sequence shown in SEQ ID No. 21. Linker-FAST Gene fragments; III) Using the GFP11 encoding gene as a template and GFP11-F and GFP11-R as primers, PCR amplification was performed to obtain the nucleotide sequence shown in SEQ ID No. 16. GFP11 Gene fragments; (IV) Using pETDuet-1 plasmid as a template and pETDuet-F and pETDuet-R as primers, PCR amplification was performed to obtain the linearized fragment sequence of the pETDuet plasmid vector. V) will the INP-N Gene fragments, Linker-FAST Gene fragments and GFP11 The gene fragment and the linearized fragment of the pETDuet plasmid vector were amplified by PCR, verified by 1% agarose gel electrophoresis, and the PCR products were recovered by gel extraction. The resulting fragments were then ligated using seamless cloning technology. INP-N Gene fragments, Linker-FAST Gene fragments and GFP11 The gene fragment and the linearized fragment sequence of the pETDuet plasmid vector were ligated by circularization at 50°C for 30 min using 2×MultiF Seamless. The resulting fragment was then heat-shocked and transformed into E. coli T1 competent cells and sent for verification. The plasmid that was successfully sequenced was the recombinant plasmid IF. For recombinant plasmid construction, the PCR reaction volume was 20 μL, including 1 μL template (plasmid), 1 μL forward primer (F), 1 μL reverse primer (R), 10 μL high-fidelity amplification reagent, and the remainder being enzyme-free water. The PCR reaction conditions were as follows: pre-denaturation at 98℃ for 3 min; followed by 30 cycles, each cycle consisting of: denaturation at 98℃ for 15 s, annealing at 60℃ for 15 s, extension at 72℃ for 4 min; and final extension at 72℃ for 5 min.

[0051] (2) Construction of recombinant Escherichia coli IF and obtaining recombinant protein IF The recombinant plasmid IF (5 μL) was heat-shocked into 50 μL of BL21 competent cells (TransGen, CD601). 500 μL of sterile LB medium (antibiotic-free) was added, mixed, and incubated at 37°C and 200 rpm for 1 hour to allow bacterial recovery. After recovery, the cells were centrifuged at 6000 rpm for 90 seconds, and 450 μL of supernatant was discarded. The remainder was added to LB agar, and the cells were spread evenly until the liquid was absorbed. The plates were inverted and incubated at 37°C for 12 hours. Then, the cultured recombinant strain was placed in 5 mL of LB medium (containing 100 mg / L ampicillin) and cultured in a shaker at 37°C and 220 rpm for 12 hours to obtain recombinant Escherichia coli IF.

[0052] Recombinant Escherichia coli IF was transferred to a 200 mL LB medium shake flask for fermentation. It was cultured at 37 °C and 220 rpm for 3 h. When the bacterial concentration OD600 reached between 0.8 and 1.0, isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 1 mM, and expression was induced at 16 °C and 160 rpm for 20 h to obtain a bacterial culture rich in PET degrading enzyme.

[0053] (3) Construction of recombinant plasmid PgsA-HFBII (PH for short) In this invention, the construction of recombinant plasmid PH is used as an example to illustrate the construction method of recombinant plasmid containing the HFBII gene encoding hydrophobic protein. The construction method of recombinant plasmid AIDA-I-HFBII (abbreviated as AH) is basically the same.

[0054] I) Using the PgsA encoding gene as a template and PgsA-F and PgsA-R as primers, PCR amplification was performed to obtain the nucleotide sequence shown in SEQ ID No. 14. PgsA Gene fragments; II) Using the Linker-HFBII encoding gene as a template and HFBII-F and HFBII-R as primers, PCR amplification was performed to obtain the nucleotide sequence shown in SEQ ID No. 29. Linker-HFBII Gene fragments; III) Using the GFP11 encoding gene as a template and GFP11-F and GFP11-R as primers, PCR amplification was performed to obtain the nucleotide sequence shown in SEQ ID No. 16. GFP11 Gene fragments; IV) Using pBAD33 plasmid as a template and pBAD33-F and pBAD33-R as primers, reverse PCR was performed to amplify the linearized fragment sequence of the pBAD33 plasmid vector. V) PgsA Gene fragments, Linker-HFBII Gene fragments, GFP11 The gene fragment and the linearized fragment of the pBAD33 plasmid vector were amplified by PCR, verified by 1% agarose gel electrophoresis, and the PCR products were recovered by gel extraction. Seamless cloning ligation technology was then used to... PgsA Gene fragments, Linker-HFBII Gene fragments, GFP11 The gene fragment and the linearized fragment sequence of the pBAD33 plasmid vector were ligated by circularization at 50℃ for 30 min using 2×MultiF Seamless, and then introduced into E. coli T1 competent cells and sent for verification. The plasmid that was successfully sequenced is the recombinant plasmid PH. The method for constructing recombinant Escherichia coli PH and the corresponding recombinant protein PH is basically the same as the method described in "(2) Construction of recombinant Escherichia coli IF and acquisition of recombinant protein IF" in this embodiment. The only difference is that during induction expression, in addition to adding IPTG with a final concentration of 1mM, L-arabinose with a final concentration of 0.3%w / v is also added. The other construction parameters are the same as those described above.

[0055] (4) Obtaining recombinant proteins INP-N-FAST+ PgsA-HFBII (abbreviated as I-F+PH) and INP-N-FAST+ AIDA-I-HFBII (abbreviated as I-F+AH) After constructing recombinant plasmids IF and PH according to the method described above, 3 μL of recombinant plasmid IF and 3 μL of recombinant plasmid PH were introduced into 50 μL of BL21 competent cells (TransGen, CD601) using a heat shock method. 500 μL of sterile LB medium (antibiotic-free) was added, and the mixture was incubated at 37°C and 200 rpm for 1 hour to allow bacterial recovery. After recovery, the cells were centrifuged at 6000 rpm for 90 seconds, and 450 μL of supernatant was discarded. The remainder was added to LB agar medium, and the cells were spread evenly until the liquid was absorbed. The plates were inverted and incubated at 37°C for 12 hours. Then, the cultured recombinant strain was placed in 5 mL of LB medium (containing 100 mg / L ampicillin) and cultured in a shaker at 37°C and 220 rpm for 12 hours to obtain recombinant Escherichia coli I-F+AH.

[0056] Recombinant Escherichia coli I-F+AH was transferred to a 200 mL LB medium shake flask for fermentation. The culture was carried out at 37°C and 220 rpm in a shaker for 3 h 10 min. The bacterial concentration (OD) was determined by the following steps: 600 When the concentration reaches between 0.8 and 1.0, IPTG (final concentration 1 mM) and L-arabinose (final concentration 0.3% w / v) are added, and expression is induced at 16℃ and 160 rpm for 20 h to obtain a bacterial culture rich in PET degrading enzyme and hydrophobic protein.

[0057] After constructing the recombinant plasmid AH according to the corresponding method, 3 μL of recombinant plasmid IF and 3 μL of recombinant plasmid AH were introduced into 50 μL of BL21 competent cells (TransGen, CD601) by heat shock. Recombinant Escherichia coli I-F+AH and the corresponding recombinant protein I-F+AH were constructed according to the method described above in this section.

[0058] (5) Construction of recombinant plasmid IE and corresponding recombinant Escherichia coli The construction method of the recombinant plasmid INP-N-Est30KL (IE) of this invention is basically the same as the construction method of the recombinant plasmid IF, except that in step II), the Est30KL-Linker encoding gene is used as a template, and Est30KL-F and Est30KL-R are used as primers to amplify the nucleotide sequence as shown in SEQ ID No. 27. Est30KL-Linker Gene fragments; In step V), during amplification and ligation, Linker-FAST The gene fragments were replaced accordingly. Est30KL-Linker Gene fragment. The remaining construction steps are the same, and the recombinant plasmid IE is obtained after sequencing verification.

[0059] Recombinant plasmid IE was used to construct recombinant Escherichia coli IE according to the construction method of recombinant Escherichia coli IF.

[0060] (6) Construction of recombinant Escherichia coli P-H+IE and I-F+IE and the acquisition of corresponding recombinant proteins The construction methods for recombinant *E. coli* PgsA-HFBII+INP-N-Est30KL (abbreviated as P-H+IE) and recombinant *E. coli* INP-N-FAST+INP-N-Est30KL (abbreviated as I-F+IE) are basically the same as those for recombinant *E. coli* I-F+AH, the only difference being the replacement of the corresponding recombinant plasmids. Following the method for obtaining recombinant protein IF, the corresponding recombinant *E. coli* were fermented to obtain the corresponding recombinant protein.

[0061] Example of effect This invention measures the relative enzyme activity and surface fluorescence intensity of different recombinant Escherichia coli strains displaying PET-degrading enzymes on their surfaces, as detailed below: 1. Measurement methods for relevant indicators The induced bacterial cells were collected using a high-speed refrigerated centrifuge (4000 rpm, 15 min), and washed once with 10 mL of PBS buffer (pH=7.4) to remove the influence of the culture medium. The bacterial concentration was adjusted to OD using PBS buffer. 600 =20, used to determine the amount of surface-displaying enzymes, the OD of the bacterial culture was adjusted using 50mM glycine-NaOH (pH=9.0) reaction solution. 600 =2.5, used for subsequent determination of relative enzyme activity.

[0062] (1) Surface fluorescence measurement method 100μL OD 600Add 100 μL of purified GFP1-10 to a bacterial culture of 20 μL and incubate in a black 96-well plate at 30 °C and 220 rpm until saturation. Measure the saturation fluorescence value using a fluorescence microplate reader.

[0063] (2) Relative enzyme activity assay method The total amount of PET degradation products released corresponding to a unit mass of enzyme solution added.

[0064] (3) Determination methods for PET and BHET degradation activity The PET substrate of this invention is an amorphous model substrate (with a crystallinity of about 8%) purchased from GoodFellow. The model substrate was washed sequentially with 1% SDS, anhydrous ethanol and double-distilled water, and then punched into discs with a diameter of 6 mm (weighing about 8 mg, accurately recorded). One disc was taken for each enzymatic hydrolysis reaction.

[0065] The substrate for BHET (CAS No. 959-26-2) was purchased from McLean Company and was 95% pure.

[0066] BHET enzyme activity assay method: The above-resuspended OD 600 Add 300 μL of bacterial culture with pH 2.5 (containing 50 mM glycine-NaOH buffer, pH=9.0) to BHET, ensuring a final BHET concentration of 10 mM, and react in a 30°C water bath for 1 h.

[0067] PET enzyme activity assay method: The above-resuspended OD 600 300 μL of a bacterial culture with a pH of 2.5 (containing 50 mM glycine-NaOH buffer, pH=9.0) was added to a 6 mm diameter PET disc, and the mixture was reacted at 45 °C in a metal bath at 900 rpm for 24 h. After the reaction was completed, acetonitrile was added to terminate the reaction, and the TPA, MHET, and BHET produced in the reaction were analyzed by high performance liquid chromatography (HPLC). The sum of the concentrations of TPA, MHET, and BHET was used to characterize the PET degradation activity.

[0068] (4) Cyclic performance testing method Following the method described above for PET enzyme activity assay, after one PET enzyme activity assay, the PET disc was removed from the system, and the mixture was centrifuged at 10,000 rpm for 10 min. The supernatant was collected and inactivated with acetonitrile. The precipitate (i.e., bacterial cells) was washed once with 300 μL of 50 mM glycine-NaOH buffer (pH=9.0), and then resuspended in 300 μL of 50 mM glycine-NaOH buffer. A 6 mm diameter PET disc was added again, and the mixture was reacted at 45°C in a metal bath at 900 rpm for 24 h. This method was repeated to examine the relative enzyme activity at different cycles.

[0069] 2. Measurement results of relevant indicators (1) Surface fluorescence intensity of recombinant Escherichia coli containing a single anchoring protein Recombinant Escherichia coli IF, Recombinant Escherichia coli LF, Recombinant Escherichia coli AF, and Recombinant Escherichia coli PF OD 600 The surface fluorescence intensity of a bacterial culture with a concentration of 20 g / L was measured after adding excess GFP1-10 and incubating under the conditions described in the surface fluorescence intensity measurement method until saturation. The results are as follows: Figure 1 As shown.

[0070] Depend on Figure 1 It can be seen that the recombinant proteins IF, LF, AF and PF were successfully anchored on the surface of recombinant Escherichia coli.

[0071] (2) BHET enzyme activity The BHET degradation activities of recombinant Escherichia coli IF, recombinant Escherichia coli LF, recombinant Escherichia coli AF, and recombinant Escherichia coli PF were determined according to the BHET enzyme activity assay method. The results are as follows: Figure 2 As shown.

[0072] Depend on Figure 2 It can be seen that, compared with recombinant Escherichia coli LF, recombinant Escherichia coli AF and recombinant Escherichia coli PF, recombinant Escherichia coli IF has a significant advantage in degrading BHET.

[0073] (3) Comparison of free enzymes and recombinant Escherichia coli with PET-degrading enzymes on the surface This invention includes a comparative example, in which recombinant Escherichia coli IF with equal viable bacterial concentrations are divided into two groups. One group is inoculated into LB medium and fermented at 37°C and 220 rpm until OD reaches 100%. 600 =0.8-1.0, after adding IPTG to induce culture for 20h, FAST-PETase was obtained by purification and separation using Ni column; another group was cultured under the same conditions to obtain the corresponding bacterial culture.

[0074] Method for obtaining free enzyme: Bacterial cells were collected using a high-speed refrigerated centrifuge (8000xg, 5 min). The cells were resuspended in 10 mL of lysis buffer (containing 50 mM Tris-HCl, 150 mM NaCl, and 10 mM imidazole per liter, pH=7.5). The collected cells were then lysed using a high-pressure homogenizer. After lysis, the cells were centrifuged at 10000 rpm for 1 h to remove cell debris. The supernatant was the total protein solution containing FAST-PETase. The total protein solution was passed through a 0.45 μm membrane to remove impurities, followed by purification using a Ni-NTA packed column with gradient elution to obtain the target protein. The specific steps are as follows: First, equilibrate with the lysis buffer for 2 minutes. Then, repeat the column loading process three times with the whole protein solution after membrane transfer. Wash three times with the washing buffer (containing 50 mM Tris-HCl, 150 mM NaCl, and 80 mM imidazole per liter, pH=7.5) to remove impurities. Finally, elute with the elution buffer (containing 50 mM Tris-HCl, 300 mM NaCl, and 300 mM imidazole per liter, pH=7.5) to further concentrate the protein and remove the high concentration of imidazole, thus obtaining the concentrated protein solution.

[0075] This invention compares equal amounts of free enzyme FAST-PETase with recombinant Escherichia coli IF bacterial suspension (i.e., IF surface display system, the bacterial suspension obtained after induced expression was resuspended in reaction solution to OD). 600 =2.5), both were degraded at 45℃ / pH 9.0 for 24 h for 8% crystallinity PET film, and the PET degradation products were measured. A comparison of the degradation results of PET by equal amounts of free enzyme and IF surface display system is shown in the figure below. Figure 3 As shown.

[0076] Depend on Figure 3 It was found that, under the same conditions, the total release of PET degradation products in the IF surface display system reached 3.4 mM, which was 3.5 times higher than the total release of PET degradation products in the same amount of free enzyme FAST-PETase (758 μM). At the same time, the IF surface display system degraded PET more thoroughly, with TPA accounting for up to 56% of the degradation products, which was significantly higher than the 35% TPA content in the free enzyme degradation products.

[0077] (4) Surface fluorescence intensity of recombinant Escherichia coli containing multiple anchoring proteins Recombinant Escherichia coli I-F+PH and Recombinant Escherichia coli I-F+AH OD 600 The fluorescence intensity measurement results after incubating a bacterial culture of 20 μL with excess GFP1-10 until saturation under the conditions described in the surface fluorescence intensity measurement method are as follows: Figure 4 As shown. Among them Figure 4 a) is the fluorescence intensity of FAST; Figure 4 b) is the fluorescence intensity of HFBII.

[0078] Depend on Figure 4 It can be seen that in surface display systems I-F+P+H and I-F+AH, FAST-PETase and hydrophobic protein HFBII are simultaneously anchored on the surface of Escherichia coli.

[0079] (5) Comparison of relative enzyme activity between surface display systems containing hydrophobic anchoring proteins and those without hydrophobic proteins This invention, following the PET enzyme activity assay method, determined the amounts of degradation products of recombinant Escherichia coli IF, recombinant Escherichia coli I-F+PH, and recombinant Escherichia coli I-F+AH after degrading PET, and calculated the relative enzyme activity. The results are as follows: Figure 5 As shown.

[0080] Depend on Figure 5 It can be seen that the introduction of hydrophobic protein HFBII into the PET degrading enzyme surface display system can increase the PET enzyme activity by 18%-112%. Among them, compared with the surface display system IF, the PET degrading enzyme activity of the surface display system I-F+PH can be increased by 1.12 times.

[0081] (6) Comparison of the effects of dual-enzyme synergy on PET degradation This invention, following the PET enzyme activity assay method, determined the amounts of degradation products of recombinant *E. coli* IF, recombinant *E. coli* I-F+PH, recombinant *E. coli* I-F+IE, and recombinant *E. coli* P-H+IE after degrading PET, and calculated the relative enzyme activities. The results are as follows: Figure 6 As shown.

[0082] Depend on Figure 6 It is known that after co-expressing carboxylesterase Est30KL, carboxylesterase can synergize with FAST-PETase, and TPA accounts for more than 95% of PET degradation products.

[0083] (7) Cyclic performance of the PET degradation enzyme surface display system This invention investigated the recycling performance of recombinant Escherichia coli P-H+IE, and the results are as follows: Figure 7 As shown.

[0084] Depend on Figure 7 It can be seen that the PET degrading enzyme surface display system P-H+IE retains 92.4% of the PET degrading enzyme activity after the second cycle and 65.6% of the PET degrading enzyme activity after the third cycle.

[0085] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Use of ankyrin INP-N in a surface-displayed PET-degrading enzyme system.

2. A recombinant Escherichia coli displaying a PET-degrading enzyme on its surface, characterized in that: The recombinant E. coli can express a hydrophobin HFBII.

3. The recombinant E. coli surface displaying PET-degrading enzymes of claim 2, wherein: the PET-degrading enzyme is a PETase; and the PETase is a polypeptide having at least 80% sequence identity to SEQ ID NO:

1. The recombinant E. coli can express a carboxylesterase Est30KL.

4. The recombinant E. coli bacterium of claim 3, wherein the PET-degrading enzyme is a surface-displayed PETase. 5 The recombinant E. coli can express ankyrin AIDA-I or ankyrin PgsA.

5. The recombinant E. coli surface displaying PET-degrading enzyme of claim 2, wherein: the PET-degrading enzyme is a PETase; and the PETase is a polypeptide having at least 80% sequence identity to SEQ ID NO:

1. The recombinant E. coli can express a carboxylesterase Est30KL.

6. The method for constructing a recombinant E. coli displaying a PET-degrading enzyme on the cell surface according to claim 2, wherein the recombinant E. coli is constructed by introducing the expression vector into the E. coli. The gene coding for the PET-hydrolytic enzyme FAST-PETase and the gene coding for the ankyrin INP-N are cloned into a pETDuet-1 plasmid vector to obtain a recombinant pETDuet-1 plasmid 1, and the gene coding for the ankyrin AIDA-I and the gene coding for the hydrophobin HFBII are cloned into a pBAD33 plasmid vector to obtain a recombinant pBAD33 plasmid 1, and then the recombinant pETDuet-1 plasmid 1 and the recombinant pBAD33 plasmid 1 are introduced into E. coli competent cells to obtain the recombinant E. coli for surface-displaying PET-degrading enzymes; and / or 7. The method for constructing a recombinant E. coli displaying a PET-degrading enzyme on the cell surface according to claim 4, wherein the PET-degrading enzyme is selected from the group consisting of a PETase, a cutinase, and a lipase. 8 The gene coding for the PET-hydrolytic enzyme FAST-PETase and the gene coding for the ankyrin INP-N are cloned into a pETDuet-1 plasmid vector to obtain a recombinant pETDuet-1 plasmid 1, and the gene coding for the ankyrin PgsA and the gene coding for the hydrophobin HFBII are cloned into a pBAD33 plasmid vector to obtain a recombinant pBAD33 plasmid 2, and then the recombinant pETDuet-1 plasmid 1 and the recombinant pBAD33 plasmid 2 are introduced into E. coli competent cells to obtain the recombinant E. coli for surface-displaying PET-degrading enzymes. The gene coding for the PET-hydrolytic enzyme FAST-PETase and the gene coding for the ankyrin INP-N are cloned into a pETDuet-1 plasmid vector to obtain a recombinant pETDuet-1 plasmid 1, and the gene coding for the ankyrin INP-N and the gene coding for the carboxylesterase Est30KL are cloned into a pETDuet-1 plasmid vector to obtain a recombinant pETDuet-1 plasmid 2, and then the recombinant pETDuet-1 plasmid 1 and the recombinant pETDuet-1 plasmid 2 are introduced into E. coli competent cells to obtain the recombinant E. coli for surface-displaying PET-degrading enzymes.

8. The method for constructing a recombinant E. coli displaying a PET-degrading enzyme on the cell surface according to claim 5, wherein the PET-degrading enzyme is selected from the group consisting of a PETase, a cutinase, and a lipase. 8 9. Use of the recombinant E. coli for surface-displaying PET-degrading enzymes according to any one of claims 2-5 in degrading PET products or preparing a PET-degrading agent or a BHET-degrading agent. ​ 10. A method of degrading a PET article, characterized by: The recombinant E. coli displaying PET-degrading enzyme on the surface according to any one of claims 2-5 is cultured in LB medium until the bacterial concentration OD 600 reaches 0.8-1.0, at least one of isopropyl-β-D-thiogalactoside or L-arabinose is added as an inducer to induce the culture for 18-24 hours, and the bacterial cells are separated from the liquid; the bacterial cells are resuspended and mixed with the PET product, and a PET degradation reaction is carried out at 40-50°C to obtain a PET degradation product.

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