A kl-ccf-mhetase mutant and related biological materials and applications thereof

Abstract

The application belongs to the technical field of enzyme engineering, and specifically discloses a KL-CCF-MHETase mutant and related biological materials and applications thereof. The amino acid sequence of the KL-CCF-MHETase mutant provided by the application is shown in SEQ ID No. 3. Compared with the KL-MHETase mutant, the BHET degradation activity of the KL-CCF-MHETase mutant is significantly improved, and the KL-CCF-MHETase mutant can rapidly convert BHET into a target product TPA. Meanwhile, the application provides a PET degradation method. After an intermediate is prepared by chemical depolymerization of PET, the intermediate is combined with the KL-MHETase mutant for efficient conversion, and under the strategy of a crude substrate-crude enzyme, efficient degradation of high-crystallinity PET and green recovery of TPA are achieved. The application can be applied to the fields of degrading PET or BHET, preparing a degrading agent for PET or BHET, and recycling TPA.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering technology and relates to a PET degrading enzyme, specifically a KL-CCF-MHETase mutant and its related biomaterials and applications. Background Technology

[0002] The increasing amount of waste polyethylene terephthalate (PET) poses a serious threat to the ecological environment, and efficient recycling and high-value utilization of waste PET has become a current research focus. At present, the main recycling methods for PET include mechanical methods, chemical methods and bio-enzymatic methods. Among them, the bio-enzymatic method has received widespread attention due to its advantages such as mild reaction conditions and high product purity. However, this method still faces problems such as low enzymatic hydrolysis efficiency and limited ability to degrade highly crystalline PET. Moreover, the formate-containing wastewater generated during the product purification process also increases the treatment cost. Specifically: (1) Waste PET usually has a high degree of crystallinity, which makes it difficult for bio-enzymes to effectively contact and act, resulting in low degradation efficiency; (2) Most commonly used PET degradation enzymes are intracellular enzymes, which require multiple steps such as sterile fermentation, cell disruption, and protein purification. The overall process has high energy consumption, long cycle, and low equipment utilization; (3) Terephthalic acid (TPA) in the enzymatic hydrolysis product is recovered after acid precipitation, while the by-product ethylene glycol (EG) has a low concentration in the reaction system, making separation and purification difficult. Existing methods often use electrocatalytic oxidation to convert it into formic acid or formate, but this still produces saline wastewater, increasing the burden of subsequent treatment and environmental risks. These problems have limited the industrial application of biological recycling processes. Summary of the Invention

[0003] This invention provides a KL-CCF-MHETase mutant with significantly enhanced BHET degradation activity, along with related biomaterials and applications, through modification of the KL-MHETase mutant. The KL-CCF-MHETase mutant provided by this invention exhibits a significantly higher yield of the target product TPA when degrading BHET compared to the KL-MHETase mutant. Furthermore, this invention provides its encoding gene, recombinant plasmid, and recombinant bacterial strain, among other biomaterials, to support industrial applications. In constructing the recombinant bacterial strain, [the following is mentioned:]... V. natriegens As a host cell, it enables the treatment and resource utilization of formate wastewater. This invention also provides a PET degradation method, which achieves efficient degradation of high-crystallinity PET and green recycling of TPA through a synergistic process of chemical depolymerization and enzymatic hydrolysis. At the same time, it adopts a "crude substrate-crude enzyme" strategy in the enzymatic hydrolysis stage and a non-sterile culture mode in the fermentation stage, which greatly reduces costs, energy consumption and operation difficulty, and improves the economics and scalability of the process.

[0004] To achieve the above-mentioned objectives, the embodiments of the present invention employ the following technical solutions:

[0005] In a first aspect, the present invention provides a KL-CCF-MHETase mutant, which is obtained by site-directed mutation of valine at position 62 to cysteine, glutamic acid at position 78 to phenylalanine, and methionine at position 161 to cysteine ​​in the amino acid sequence of the KL-MHETase mutant, as shown in SEQ ID No. 3.

[0006] Compared with the KL-MHETase mutant, the KL-CCF-MHETase mutant provided by this invention has significantly enhanced BHET degradation activity, which can rapidly convert BHET into the target product TPA.

[0007] Secondly, the present invention provides the encoding gene of the above-mentioned KL-CCF-MHETase mutant, the nucleotide sequence of which is shown in SEQ ID No. 4.

[0008] Thirdly, the present invention provides a recombinant plasmid comprising the coding gene provided in the second aspect.

[0009] Fourthly, the present invention provides a recombinant strain comprising the recombinant plasmid provided in the third aspect.

[0010] Preferably, the host cell of the recombinant strain is a sodium-dependent Vibrio. Vibrio natriegens .

[0011] This invention utilizes formate-containing wastewater generated during PET degradation as a carbon source in the fermentation process of sodium-dependent Vibrio bacteria to produce PET-degrading enzymes. This not only avoids the end-of-pipe treatment costs of formate-containing wastewater but also achieves resource utilization of waste within the process system.

[0012] The coding gene, recombinant plasmid, and recombinant strain of the KL-CCF-MHETase mutant provided by this invention can lay the foundation for the efficient preparation, large-scale production, and industrial application of the KL-CCF-MHETase mutant.

[0013] Fifthly, the present invention provides the application of the above-mentioned KL-CCF-MHETase mutant and its encoding gene, recombinant plasmid and recombinant strain in the degradation of PET and its degradation products, the preparation of degradation agents for PET and its degradation products, or the recovery of terephthalic acid.

[0014] The degradation products include MHET or BHET.

[0015] Sixthly, the present invention provides a method for degrading PET, the method comprising the following steps:

[0016] S1. BHET crude product is obtained by glycolysis of PET under bicarbonate catalysis;

[0017] S2. The lysate of the recombinant strain from the fourth aspect is mixed with the crude BHET product and hydrolyzed at 40℃-50℃ to obtain PET degradation products.

[0018] To address the challenge of directly biodegrading highly crystalline PET, this invention first employs a rapid glycolysis reaction to pretreat PET, depolymerizing it into intermediates BHET and MHET, significantly improving substrate accessibility to the enzyme. Subsequently, a specific hydrolytic enzyme is used to further convert the intermediates into TPA monomers. This two-step synergistic process effectively overcomes the limitations of single biological methods, achieving a significant improvement in degradation efficiency.

[0019] In the enzymatic hydrolysis stage, this invention directly uses the crude BHET product obtained from chemical depolymerization as the substrate, and uses unpurified lysis buffer for the reaction. This "crude substrate-crude enzyme" strategy avoids cumbersome and expensive purification steps, greatly simplifies the operation process, and reduces the overall cost.

[0020] For example, the present invention uses the biocompatible catalyst KHCO3 to catalyze the glycolysis reaction of PET using PET and ethylene glycol as substrates, and PET can be completely depolymerized in just 1.5 hours.

[0021] Preferably, the method for preparing the lysate of the recombinant strain includes the following steps:

[0022] Step 1: Inoculate the above recombinant strain into LB3 medium for large-scale culture to obtain recombinant strain seed culture;

[0023] Step 2: Inoculate the above-mentioned recombinant strain seed culture into LBv2-sodium formate medium and culture until OD200. 600 When the concentration reaches 0.8-1.0, add isopropyl-β-D-thiogalactoside for induction culture;

[0024] Step 3: After the induction culture is completed, collect the bacterial cells, resuspend them, and break them up to obtain the lysate of the recombinant strain.

[0025] In preparing the lysate, the core fermentation stage of this invention adopts a non-sterile culture mode, eliminating the need for sterilization of the LBv2-formate sodium culture medium used for fermentation. This not only significantly reduces energy consumption in equipment operation and steam sterilization, but also greatly reduces production costs, effectively improving the economic efficiency and large-scale application potential of the process.

[0026] More preferably, in step one, the conditions for the expanded culture are: culture temperature 35℃-39℃, rotation speed 180rpm-240rpm, and culture time 10h-14h.

[0027] For example, the present invention is illustrated by culturing at 37°C and 220 rpm for 12 hours.

[0028] More preferably, in step two, the inoculation amount of the recombinant strain seed liquid is 1%-5%;

[0029] The LBv2 sodium formate medium is LBv2 medium supplemented with 20g / L-35g / L sodium formate.

[0030] In step two, the LBv2-sodium formate culture medium includes at least one of unsterilized LBv2-sodium formate culture medium without antibiotics or unsterilized LBv2-sodium formate culture medium containing antibiotics.

[0031] In step two, the conditions for induction culture are: culture temperature 14℃-18℃, rotation speed 150pm-170rpm, and culture time 26h-32h.

[0032] Preferably, in step S2, when mixing the lysate of the recombinant strain with the crude BHET product, FAST-PETase is also added.

[0033] When used in combination with FAST-PETase, the KL-CCF-MHETase mutant showed an 85.95% higher degradation efficiency of a 50 g / L BHET mixture within 1 hour compared to the KL-MHETase mutant.

[0034] In summary, compared to the KL-MHETase mutant, the KL-CCF-MHETase mutant provided by this invention can rapidly and efficiently degrade BHET to TPA. Simultaneously, this invention provides biological materials such as the coding gene of the KL-MHETase mutant, recombinant plasmids, and recombinant strains, ensuring the industrial application of the KL-MHETase mutant. Based on this, this invention achieves efficient degradation of highly crystalline PET and green recycling of TPA through a process combining "rapid chemical depolymerization of PET to prepare intermediates" and "efficient conversion of KL-MHETase mutant intermediates." It is worth emphasizing that this invention utilizes... V . natriegens Based on the formate metabolism characteristics, recombinant strains expressing KL-MHETase mutants were constructed using these strains as host cells to achieve the treatment and resource utilization of formate wastewater.

[0035] Furthermore, in the enzymatic hydrolysis stage, this invention directly uses the crude BHET product obtained from chemical depolymerization as the substrate, and uses unpurified lysis buffer for the reaction. This "crude substrate-crude enzyme" strategy eliminates complex and costly purification steps, and also reduces the overall cost.

[0036] The fermentation stage for preparing lysate uses a non-sterile culture mode, eliminating the need for sterilization of the fermentation medium. This significantly reduces energy consumption in equipment operation and steam sterilization, lowers production costs, and effectively enhances the economic efficiency and scalability of the process. Attached Figure Description

[0037] 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.

[0038] Figure 1 This is a schematic diagram of the plasmid map of the recombinant plasmid pET-22b-KL-MHETase in Example 1 of the present invention;

[0039] Figure 2 This refers to the effect of different sodium formate concentrations on the growth and formic acid consumption of the recombinant strain after 20 hours of induced expression, as described in Example 1 of this invention. Figure 2 'a' represents the recombinant strain FAST-PETase. Figure 2 b represents the recombinant strain KL-MHETase;

[0040] Figure 3 This invention, in Example 1, investigates the effects of different induction times on the growth and formic acid consumption of the recombinant strain in LBv2-sodium formate medium containing 441 mM sodium formate. Figure 3 'a' represents the recombinant strain FAST-PETase. Figure 3 b represents the recombinant strain KL-MHETase;

[0041] Figure 4 The results of this study show the effect of different pH values ​​on the BHET degradation activity of the mixed lysate of recombinant strain FAST-PETase and recombinant strain KL-MHETase under the conditions of a final BHET mixture concentration of 50 g / L, a reaction temperature of 45 °C, and a KH2PO4 concentration of 100 mM in the buffer solution in Example 2 of this invention.

[0042] Figure 5The results show the effect of different reaction temperatures on the BHET degradation activity of the mixed lysate of recombinant strain FAST-PETase and recombinant strain KL-MHETase under the conditions of a final BHET mixture concentration of 50 g / L, a KH2PO4 concentration of 100 mM in the buffer, and a reaction system pH of 8.0 in Example 2 of this invention.

[0043] Figure 6 The results show the effect of different KH2PO4 concentrations in the buffer solution on the BHET degradation activity of the mixed lysate of recombinant strain FAST-PETase and recombinant strain KL-MHETase under the conditions of a final BHET mixture concentration of 50 g / L, a reaction system pH of 8.0, and a reaction temperature of 40 °C in Example 2 of this invention.

[0044] Figure 7 This study investigated the effect of different final concentrations of BHET mixtures on the BHET degradation activity of a mixed lysate of recombinant strains FAST-PETase and KL-MHETase under the conditions of 150 mM KH2PO4 concentration in the buffer solution, pH of the reaction system, and reaction temperature of 40 °C in Example 2 of the present invention.

[0045] Figure 8 This refers to the results of investigating the effects of different fermentation media on the growth and formic acid consumption of recombinant strain KL-MHETase or recombinant strain FAST-PETase after 28 hours of induction expression in Example 2 of the present invention; wherein, Figure 8 'a' represents the recombinant strain FAST-PETase. Figure 8 b represents the recombinant strain KL-MHETase;

[0046] Figure 9 This illustrates the effect of different fermentation media on the BHET degradation activity of the mixed lysate of recombinant strain FAST-PETase and recombinant strain KL-MHETase in Example 2 of the present invention.

[0047] Figure 10 This is a comparison chart of the BHET degradation activities of the two mixed lysis solutions in Example 2 of the present invention. Detailed Implementation

[0048] 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.

[0049] The mutants described in this invention are named according to the conventional naming methods used by those skilled in the art. In this invention, when examining the formate tolerance of recombinant strains and the BHET degradation activity of lysates from different recombinant strains, three replicate experiments were conducted.

[0050] Example 1

[0051] This embodiment provides a KL-CCF-MHETase mutant and its preparation method, as detailed below:

[0052] The KL-CCF-MHETase mutant provided by this invention is formed by site-directed mutation of valine at position 62 to cysteine, glutamic acid at position 78 to phenylalanine, and methionine at position 161 to cysteine ​​in the amino acid sequence of the KL-MHETase mutant, while the amino acids at other positions remain unchanged.

[0053] The amino acid sequence of the KL-MHETase mutant is shown in SEQ ID No. 1, and its codon-optimized coding gene is shown in SEQ ID No. 2; the amino acid sequence of the KL-CCF-MHETase mutant is shown in SEQ ID No. 3.

[0054] 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 (TransGen GD111) and seamless cloning, and transforms them into sodium-dependent Vibrio natriureticis Vmax competent cells. After culturing, recombinant sodium-dependent Vibrio natriureticis expressing the target protein heterologously is obtained. The specific process is as follows:

[0055] 1. Construction of recombinant plasmids

[0056] This invention commissioned Genewiz to synthesize recombinant plasmids. The gene encoding the KL-MHETase mutant (SEQ ID No. 2) was cloned into the *E. coli* pET-22b plasmid, denoted as the recombinant plasmid pET-22b-KL-MHETase. A schematic diagram of the plasmid map is shown below. Figure 1 As shown.

[0057] Using site-directed mutagenesis, the recombinant plasmid pET was used. 22b Using KL-MHETase as a template, PCR was performed using the primer pairs shown in Table 1. The PCR reaction volume was 20 μL, including 1 μL of template (plasmid), 1 μL of forward primer (F), 1 μL of reverse primer (R), 10 μL of high-fidelity amplification reagent, and the remainder being enzyme-free water. The PCR product was digested with DMT enzyme (TransGen, GD111) to obtain the recombinant plasmid pET using molecular biology methods such as seamless cloning. 22b-KL-CCF-MHETase. PCR reaction conditions: pre-denaturation 98℃, 3 min; then 30 cycles, each cycle consisting of: denaturation 98℃, 15 s; annealing 55℃, 15 s; extension 72℃, 3 min 30 s; final extension 72℃, 5 min. Based on recombinant plasmid pET... 22b Table 1 shows the statistics of site-directed mutagenesis primers used in constructing the recombinant plasmid pET-22b-KL-CCF-MHETase using KL-MHETase.

[0058] Table 1

[0059]

[0060] The FAST-PETase encoding gene was cloned into the E. coli pET-22b plasmid, denoted as recombinant plasmid pET-22b-FAST-PETase. The amino acid sequence of FAST-PETase is shown in SEQ ID No. 5, and its encoding gene is shown in SEQ ID No. 6.

[0061] 2. Construction of recombinant strains

[0062] (1) Preparation of competent cells. 10 μL of empty vector *Vibrio natans* (NaH) was used to prepare the cells. Vibrio natriegens Vmax glycerol bacteria were inoculated into 5 mL of sterile LB3 medium and cultured at 37°C and 220 rpm for 12 h. 1% of the inoculum was then transferred to 40 mL of sterile LB3 medium and cultured at 37°C and 220 rpm for 1 h until OD reached [value missing]. 600 When the concentration is around 0.7, immediately incubate the bacterial culture on ice for at least 15 minutes, then centrifuge at 4°C and 6500 rpm for 10 minutes. Discard the supernatant, resuspend the precipitate in 10 mL of 1 mol / L sorbitol solution, repeat twice, discard the supernatant, gently resuspend the bacterial cells using the residual liquid in the tube, aliquot 50 μL into 1.5 mL centrifuge tubes and store on ice.

[0063] (2) Electroporation. Take the 1mm electroporation cup out of anhydrous ethanol, sterilize it in a clean bench with ultraviolet light, dry it and place it on ice for later use; at the same time, preheat the LB3 medium in a 42℃ water bath.

[0064] Place the plasmid to be transformed (recombinant plasmid pET-22b-KL-MHETase, pET-22b-KL-CCF-MHETase, or pET-22b-FAST-PETase) on ice to pre-cool. Add 15 μL of the above recombinant plasmid to each tube of competent cells and mix gently with a pipette tip (do not blow or aspirate forcefully to prevent rupture of competent cells).

[0065] The mixture of competent cells and plasmids was rapidly added to a pre-chilled electroporation vessel and transformed using an electroporator at 0.9 kV. After electroporation, the vessel was placed at room temperature, and then 500 μL of preheated LB3 medium was quickly added to resuspend the cells. The resuspended culture was transferred to a 1.5 mL centrifuge tube and cultured at 37°C and 200 rpm with shaking for 3 h.

[0066] After the culture was completed, the bacterial cells were collected by centrifugation at 6000 rpm for 5 min. After discarding the supernatant, 70 μL-100 μL of bacterial suspension was retained and gently resuspended. The resuspended bacterial suspension was evenly spread on LB3 solid medium plates containing the corresponding antibiotics. After spreading, the plates were incubated overnight at 37°C. Single colonies were picked and inoculated into LB3 liquid medium containing the corresponding antibiotics to obtain the recombinant strains KL-MHETase, KL-CCF-MHETase and FAST-PETase corresponding to each recombinant plasmid.

[0067] 3. Investigation of formate tolerance of recombinant strains

[0068] This example uses recombinant strains KL-MHETase and FAST-PETase as examples to illustrate the formate resistance of different recombinant strains. Recombinant strain KL-CCF-MHETase can also achieve formate resistance comparable to that of recombinant strain KL-MHETase. Details are as follows:

[0069] Take 10 μL of recombinant strain KL-MHETase or recombinant strain FAST-PETase and inoculate them into 5 mL of LB3 medium. Incubate at 37℃ and 220 rpm for 12 h to obtain seed culture.

[0070] Take 1 mL of seed culture and transfer it to 100 mL of sterile LBv2 medium containing 100 μg / mL ampicillin and 0-441 mM sodium formate (referred to as LBv2-sodium formate medium containing antibiotics). Continue to culture at 37°C and 220 rpm with shaking until OD. 600 When the concentration reached 0.9, isopropyl-β-D-thiogalactoside (IPTG) was added to the culture system to a final concentration of 1 mM. Subsequently, expression was induced at 16℃ and 160 rpm for 20-28 h.

[0071] The effects of different sodium formate concentrations on the growth and formic acid consumption of the recombinant strain after 20 hours of induction expression are as follows: Figure 2 As shown; where Figure 2 'a' represents the recombinant strain FAST-PETase. Figure 2 b represents the recombinant strain KL-MHETase;

[0072] The effects of different induction times on the growth and formic acid consumption of recombinant strains in LBv2-sodium formate medium containing 441 mM sodium formate were investigated. Figure 3 As shown; where, Figure 3 'a' represents the recombinant strain FAST-PETase. Figure 3 b represents the recombinant strain KL-MHETase.

[0073] Depend on Figure 2-3 It was found that recombinant strains KL-MHETase and FAST-PETase exhibited good growth adaptability and efficient formic acid assimilation in a high-concentration sodium formate environment (30 g / L, 441 mM). Extending the induction time of recombinant strain FAST-PETase from 20 h to 28 h increased the formate utilization rate from 28.6% (20 h, sodium formate consumption 124 mM) to 39.2% (28 h, sodium formate consumption 171 mM). Recombinant strain KL-MHETase showed similar growth characteristics to recombinant strain FAST-PETase; therefore, extending its induction time from 24 h to 28 h increased the formate utilization rate from 26.5% (20 h, sodium formate consumption 117 mM) to 40.4% (28 h, sodium formate consumption 178 mM).

[0074] 4. Preparation of recombinant strain lysate

[0075] 10 μL of each of the above recombinant strains was inoculated into 5 mL of LB3 medium and cultured at 37°C and 220 rpm for 12 h to obtain seed culture. 1 mL of seed culture was transferred to 100 mL of LBv2-formate sodium medium containing 100 μg / mL ampicillin and cultured with shaking at 37°C and 220 rpm until OD... 600When the concentration reached 0.8–1.0, isopropyl-β-D-thiogalactoside (IPTG) was added to the culture system to a final concentration of 1 mM. Expression was then induced for 28 h at 16°C and 160 rpm. The resulting fermentation broth was then centrifuged for 10 min at 4°C and 10,000 rpm, the supernatant was discarded, and the cell pellet was collected. The cell pellet was resuspended in 10 mL of pre-cooled buffer (150 mM KH₂PO₄, pH adjusted to 8.0 with KOH), and the cell suspension was lysed three times at 1000 bar using a low-temperature ultra-high pressure cell disruptor to obtain the lysis buffer. This lysis buffer is the crude enzyme solution containing the target enzyme and is used for later use.

[0076] The LBv2 sodium formate medium was prepared by adding 30 g / L of sodium formate to the LBv2 medium.

[0077] The lysate of the recombinant strain KL-CCF-MHETase prepared by the above method is a crude enzyme solution containing KL-CCF-MHETase.

[0078] Example 2

[0079] This example investigated the performance of different lysis buffers prepared in Example 1 in the PET degradation product BHET. The specific details are as follows:

[0080] 1. Preparation of BHET mixture

[0081] This invention produces a BHET mixture via KHCO3-catalyzed PET glycolysis. To determine the optimal process conditions, the effects of reaction temperature (180℃-200℃), catalyst dosage (1wt%-5wt%), and reaction time (1-3 h) on PET degradation rate and monomer yield were investigated. The results show that, with a PET:EG mass ratio of 1:10, using 4wt% KHCO3 based on PET mass, and reacting at 195℃ for 1.5 h, complete PET degradation can be achieved, with a total monomer yield of 96.49% (of which BHET accounts for 90.34% and MHET accounts for 6.15%). After recycling ethylene glycol as a solvent three times, the PET degradation rate, total monomer yield, and BHET selectivity did not show a significant decrease, indicating that ethylene glycol has good recycling performance in this catalytic system. This method can effectively reduce the reaction cost of the PET glycolysis process.

[0082] The optimized process for preparing the BHET mixture is as follows: Add 20g of ethylene glycol, 2g of PET fragments (approximately 0.5cm x 0.5cm in size, sourced from Zaoqiang County Hongrunda Plastic Products Factory, item number 005#), and 0.08g of KHCO3 to a 100mL three-necked round-bottom flask. Mix thoroughly and heat to 195℃ for 1.5h under magnetic stirring at 400rpm to induce PET glycolysis. After the reaction, vacuum filter while hot to separate unreacted PET solids and any impurities, and collect the filtrate. Allow the filtrate to crystallize at 0-4℃ for 24h, then separate the crystals from the mother liquor under vacuum. Dry the resulting crystals in an electric heating oven at 50℃ to obtain crude BHET product.

[0083] Meanwhile, the ethylene glycol obtained from the separation can be collected and recycled as a solvent for a new round of glycolysis reaction.

[0084] 2. BHET degradation activity of different lysis buffers

[0085] In Example 1, 10 mL of lysis buffer was prepared from every 100 mL of fermentation broth (if less than 10 mL, it was made up to 10 mL with buffer). This lysis buffer was used directly and entirely for the enzymatic hydrolysis reaction. In this invention, the total volume of the enzymatic hydrolysis reaction system for both single and dual enzymes was set to 20 mL. The specific enzymatic hydrolysis reaction system and hydrolysis conditions for single enzymes were as follows: an appropriate amount of BHET mixture was added to 10 mL of crude enzyme solution to make the final concentration of BHET mixture in the reaction system 10-200 g / L. 50-150 mM KH2PO4 buffer was added to make up to 20 mL, and the pH of the reaction system was adjusted to 6.0-8.0. The reaction system was then subjected to isothermal shaking at 40℃-50℃ and 160 rpm for 48 h, and the TPA concentration in the reaction system was determined by HPLC. Unless otherwise specified, the concentration of KH2PO4 in the buffer used for resuspending the bacterial cells when preparing the lysis buffer is 100 mM, and the pH of the buffer is adjusted to 8.0 using KOH. If the effect of the concentration of KH2PO4 in the buffer or the pH of the system on the degradation activity of BHET is investigated, the concentration of KH2PO4 in the buffer for resuspending the bacterial cells or the pH of the buffer should be replaced accordingly.

[0086] The specific enzymatic hydrolysis reaction system and hydrolysis conditions are as follows: The lysates of the two recombinant strains were mixed at a volume ratio of 1:1 to prepare 20 mL of mixed lysate. An appropriate amount of BHET mixture was added to the 20 mL mixed lysate to bring the final concentration of the BHET mixture in the reaction system to 10-200 g / L. Unless otherwise specified, the KH₂PO₄ concentration in the buffer used for resuspending the bacterial cells during the preparation of the lysate was 100 mM, and the pH of the buffer was adjusted to 8.0 using KOH. If the effect of adjusting the KH₂PO₄ concentration in the buffer or the pH of the system on the BHET degradation activity was investigated, the KH₂PO₄ concentration in the buffer for resuspending the bacterial cells was adjusted to 50-150 mM, or the pH of the buffer was adjusted to 6.0-8.0. The reaction system containing the mixed lysate and the BHET mixture was enzymatically hydrolyzed at 40-50℃ and 160 rpm for 48 h under constant temperature shaking. The TPA concentration in the reaction system was determined by HPLC.

[0087] For example, the preparation method of the mixed lysis buffer of recombinant strain KL-CCF-MHETase and recombinant strain FAST-PETase is as follows: 10 mL of recombinant strain KL-CCF-MHETase lysis buffer and 10 mL of recombinant strain FAST-PETase lysis buffer are mixed evenly. The 10 mL of recombinant strain KL-CCF-MHETase lysis buffer or the 10 mL of recombinant strain FAST-PETase lysis buffer are obtained by crushing 100 mL of fermentation broth three times according to "4. Preparation of recombinant strain lysis buffer" in Example 1. However, the phosphate concentration and pH of the buffer solution in the preparation process of the lysis buffer can be adjusted according to the system optimization conditions.

[0088] In this invention, the concentration of TPA generated in the reaction system is used to characterize the BHET degradation activity of different crude enzyme solutions.

[0089] The effects of different pH values ​​on the BHET degradation activity of a mixed lysate of recombinant strains FAST-PETase and KL-MHETase were investigated under the following conditions: a final BHET mixture concentration of 50 g / L, a reaction temperature of 45 °C, and a KH2PO4 concentration of 100 mM in the buffer solution. Figure 4 As shown.

[0090] The effects of different reaction temperatures on the BHET degradation activity of a mixed lysate of recombinant strains FAST-PETase and KL-MHETase were investigated under the following conditions: a final BHET mixture concentration of 50 g / L, a KH2PO4 concentration of 100 mM in the buffer, and a reaction system pH of 8.0. The results are as follows: Figure 5 As shown.

[0091] The effects of different KH2PO4 concentrations in the buffer solution on the BHET degradation activity of the mixed lysate of recombinant strains FAST-PETase and KL-MHETase were investigated under the following conditions: a final BHET mixture concentration of 50 g / L, a reaction system pH of 8.0, and a reaction temperature of 40 °C. Figure 6 As shown.

[0092] The effects of different final concentrations of BHET mixtures on the BHET degradation activity of mixed lysates of recombinant strains FAST-PETase and KL-MHETase were investigated under the conditions of 150 mM KH2PO4 in the buffer solution, pH 8.0 of the reaction system, and reaction temperature of 40 °C. The results are as follows: Figure 7 As shown.

[0093] Depend on Figure 4-7 Experimental results determined the optimal pH of the reaction system to be 8.0 and the optimal reaction temperature to be 40℃. As shown in the figure, increasing the phosphate concentration in the buffer to 150 mM resulted in better synergistic effects of the FAST-PETase and KL-MHETase enzymes, enabling complete degradation of a 50 g / L BHET mixture within 4 hours. During the 48-hour reaction period, when the BHET mixture concentration was 100-200 g / L, the lysis buffer provided by this invention effectively catalyzed the degradation of BHET into TPA; the highest TPA yield (81.38 g / L) was observed when the BHET mixture concentration was 150 g / L.

[0094] 3. Effect of non-sterile fermentation medium on the yield of target enzyme expressed by recombinant strains

[0095] Compared to sterilization of the inoculation medium, this embodiment further investigated the effect of non-sterilized fermentation medium on the growth of recombinant strains and the expression level of the target enzyme. This invention uses recombinant strains KL-MHETase and FAST-PETase as examples, and comparable effects can be achieved with recombinant strain KL-CCF-MHETase. The specific details are as follows:

[0096] In this invention, the fermentation medium used in the sterilized + Amp group was LBv2-formate sodium medium sterilized by high temperature and autoclave and supplemented with 100 μg / mL ampicillin; the fermentation medium used in the non-sterile group was LBv2-formate sodium medium that was not sterilized by high temperature and autoclave and did not contain antibiotics; and the fermentation medium used in the non-sterile + Amp group was unsterilized LBv2-formate sodium medium supplemented with 100 μg / mL ampicillin. The concentration of sodium formate in the LBv2-formate sodium medium was 30 g / L.

[0097] Seed cultures of recombinant strain KL-MHETase or recombinant strain FAST-PETase were prepared according to the method in Example 1. 1 mL of seed culture was transferred to 100 mL of fermentation medium from each group, and cultured with shaking at 37°C and 220 rpm until OD reached. 600 When the concentration reached 0.9, IPTG was added to the culture system to a final concentration of 1 mM. Expression was then induced for 28 h at 16 °C and 160 rpm.

[0098] After 28 hours of induction, the effects of different fermentation media on the growth and formic acid consumption of recombinant strain KL-MHETase or recombinant strain FAST-PETase were investigated. The results are as follows: Figure 8 As shown. Figure 8 middle, Figure 8 'a' represents the recombinant strain FAST-PETase. Figure 8 b represents the recombinant strain KL-MHETase.

[0099] Depend on Figure 8 It can be seen that, compared with the sterile + Amp group, there was no significant difference in the growth of recombinant strain KL-MHETase or recombinant strain FAST-PETase and the consumption of formic acid in the non-sterile group and the non-sterile + Amp group.

[0100] The fermentation broths of recombinant strains KL-MHETase or FAST-PETase, from the same fermentation medium group described above, were used to prepare corresponding lysates according to the method of this invention. Following the method described in "2. BHET degradation activity of different lysates" of this embodiment, the reaction was carried out for 48 h under the following conditions: KH2PO4 concentration in buffer was 150 mM, pH of the reaction system was 8.0, reaction temperature was 40 °C, and the final concentration of the BHET mixture was 150 g / L. The effect of different fermentation media on the BHET degradation activity of the mixed lysate of recombinant strains FAST-PETase and KL-MHETase was investigated. The results are as follows: Figure 9 As shown.

[0101] Depend on Figure 9 It can be seen that after 48 hours of reaction, the TPA yield of each system ranged from 79.5 g / L to 82.6 g / L, indicating that there was no significant difference in the degradation activity of BHET by the three fermentation media.

[0102] Based on the above experimental results, an open formate wastewater culture system can be constructed to achieve sustainable, efficient and stable production of PETase and MHETase, providing a low-energy, low-cost and easy-to-operate technical path for industrial-scale enzyme production and PET biorecycling.

[0103] 4. Comparison of BHET degradation activities of two mixed lysis buffers prepared from recombinant strain FAST-PETase lysis buffer and recombinant strain KL-MHETase lysis buffer or recombinant strain KL-CCF-MHETase lysis buffer.

[0104] In this invention, 1 mL of seed culture of recombinant strain FAST-PETase, recombinant strain KL-MHETase, or recombinant strain KL-CCF-MHETase was inoculated into 100 mL of LBv2-formate sodium agar medium that had not been autoclaved and contained no antibiotics. The medium was then cultured with shaking at 37°C and 220 rpm until OD reached [value missing]. 600 When the concentration reached 0.9, IPTG was added to the culture system to a final concentration of 1 mM. Expression was then induced for 28 h at 16 °C and 160 rpm. The corresponding lysis buffer was prepared according to the method described in Example 1. Following the method described in "2. BHET degradation activity of different lysis buffers" of this example, the reaction was carried out for 4 h at a buffer concentration of 150 mM, a reaction system pH of 8.0, a reaction temperature of 40 °C, and a final BHET mixture concentration of 50 g / L. The TPA concentration at different reaction times was investigated in the reaction system containing a mixed lysis buffer of recombinant strain FAST-PETase and recombinant strain KL-MHETase (referred to as mixed lysis buffer 1) or a mixed lysis buffer of recombinant strain FAST-PETase and recombinant strain KL-CCF-MHETase (referred to as mixed lysis buffer 2).

[0105] The comparison of the BHET degradation activities of the two mixed lysis buffers is shown in the figure below. Figure 10 As shown. By Figure 10 It is evident that, within the experimental scope of this invention, both mixed lysis buffers can completely hydrolyze a 50 g / L BHET mixture within 4 hours. However, there is a significant difference in their degradation rates. Mixed lysis buffer 2 shows significantly better hydrolysis performance on the BHET mixture than mixed lysis buffer 1 within the first 3 hours; specifically, after 1 hour of reaction, the BHET degradation efficiency of mixed lysis buffer 2 is 85.95% higher than that of mixed lysis buffer 1. In other words, under the same conditions, the degradation rate of BHET by the dual-enzyme system composed of FAST-PETase and KL-CCF-MHETase is significantly better than that of the dual-enzyme system composed of FAST-PETase and KL-MHETase.

[0106] 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. A KL-CCF-MHETase mutant, characterized in that: Its amino acid sequence is shown in SEQ ID No.

3.

2. The encoding gene of the KL-CCF-MHETase mutant as described in claim 1, characterized in that: Its nucleotide sequence is shown in SEQ ID No.

4.

3. A recombinant plasmid, characterized in that: The recombinant plasmid contains the encoding gene as described in claim 2.

4. A recombinant bacterial strain, characterized in that: The recombinant strain comprises the recombinant plasmid as described in claim 3.

5. The recombinant strain according to claim 4, characterized in that: The host cell of the recombinant strain is a sodium-dependent Vibrio. Vibrio natriegens .

6. The application of the KL-CCF-MHETase mutant of claim 1, the encoding gene of claim 2, the recombinant plasmid of claim 3, or the recombinant strain of claim 4 or 5 in the degradation of PET degradation products, the preparation of degradation agents for PET degradation products, or the recovery of terephthalic acid; wherein the PET degradation product is MHET.

7. A method for degrading PET, characterized in that: The degradation method includes the following steps: S1. BHET crude product is obtained by glycolysis of PET under bicarbonate catalysis; S2. The lysate of the recombinant strain described in claim 4 or 5, FAST-PETase, and the crude BHET product are mixed and hydrolyzed at 40°C-50°C to obtain PET degradation products.

8. The method for degrading PET as described in claim 7, characterized in that: The method for preparing the lysate of the recombinant strain includes the following steps: Step 1: Inoculate the recombinant strain described in claim 4 or 5 into LB3 medium for expansion culture to obtain recombinant strain seed liquid; Step 2: Inoculate the recombinant strain seed culture into LBv2 sodium formate medium and culture until OD200. 600 When the concentration reaches 0.8-1.0, add isopropyl-β-D-thiogalactoside for induction culture; Step 3: After the induction culture is completed, collect the bacterial cells, resuspend the bacterial cells, break the cells, and obtain the lysate of the recombinant strain.

9. The method for degrading PET as described in claim 8, characterized in that: In step one, the conditions for the scale-up culture are: culture temperature 35℃-39℃, rotation speed 180rpm-240rpm, and culture time 10h-14h; and / or In step two, the inoculation amount of the recombinant strain seed solution is 1%-5%; and / or In step two, the LBv2 sodium formate medium is LBv2 medium supplemented with 20 g / L-35 g / L sodium formate; and / or In step two, the LBv2-sodium formate medium includes at least one of unsterilized LBv2-sodium formate medium without antibiotics or unsterilized LBv2-sodium formate medium containing antibiotics; and / or In step two, the conditions for induction culture are: culture temperature 14℃-18℃, rotation speed 150pm-170rpm, and culture time 26h-32h.

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