A PTT hydrolase, its encoding gene and its application

Abstract

The present invention relates to the field of bioengineering technology, and in particular to a PTT hydrolase, a coding gene thereof and an application thereof, wherein the PTT hydrolase comprises any of the following amino acid sequences: (1) the amino acid sequence is as shown in SEQ ID NO:1; (2) the amino acid sequence has more than 80% homology with that shown in SEQ ID NO:1 and has PTT hydrolase activity. The nucleotide sequence of the PTT hydrolase coding gene is as shown in SEQ ID NO:2. The present invention also provides the application of the PTT hydrolase or its recombinant expression vector or its recombinant expression transformant for degrading PTT plastics. The PPT hydrolase of the present invention exhibits efficient PTT hydrolysis ability, generates mono(3-hydroxypropyl)terephthalate (MHPT), terephthalic acid (TPA) and 1,3-propylene glycol (1,3-PDO), thereby achieving enzymatic degradation of PTT.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and in particular to a PTT hydrolase, a coding gene thereof and an application thereof. Background Art

[0002] PTT (polytrimethylene terephthalate) is a non-degradable polyester plastic with excellent performance, and its glass transition temperature is close to room temperature. It is soft, anti-fouling, fluffy and highly elastic, making it widely used in textile and decoration fields.

[0003] At present, there is no research on the degradation of pure PTT. The common treatment methods for PTT plastic waste are incineration or landfill, which have a series of problems and limitations, including high energy consumption, pollution and waste of resources. As time goes by, the demand for the degradation of its waste will increase. Therefore, it is becoming more and more meaningful to develop an enzyme with the ability to degrade PTT.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The purpose of the present invention is to provide a PTT hydrolase, a coding gene thereof and an application thereof. The PTT hydrolase has a highly efficient PTT hydrolysis ability.

[0006] The first aspect of the present invention provides a PTT hydrolase comprising any of the following amino acid sequences:

[0007] (1) The amino acid sequence is as shown in SEQ ID NO: 1;

[0008] (2) The amino acid sequence has 80% or more homology with that of SEQ ID NO: 1, more preferably 90% or more homology, and has PTT hydrolase activity.

[0009] Preferably, the PTT hydrolase is derived from a Brevundimonas sp. strain, which has been deposited in the China Center for Type Culture Collection (address: Wuhan University, Wuhan, China), with a deposit date of June 28, 2021, and a deposit number of: CCTCCNO:M2021791.

[0010] The second aspect of the present invention provides a PTT hydrolase encoding gene, which encodes the PTT hydrolase described in claim 1.

[0011] Preferably, the nucleotide sequence of the PTT hydrolase encoding gene is shown in SEQ ID NO:2.

[0012] The third aspect of the present invention provides a recombinant expression vector comprising the above-mentioned PTT hydrolase encoding gene.

[0013] Preferably, the recombinant expression vector has resistance to kanamycin and tetracycline.

[0014] The PTT hydrolase encoding gene of the present invention is connected to various suitable vectors by conventional methods in the art to construct. The vector can be various conventional vectors in the art, and the expression vector is selected from plasmids, and the plasmid is selected from pET series expression plasmids, preferably pET28a. The PTT hydrolase encoding gene can be operably connected to the downstream of the regulatory sequence suitable for expression in the selected vector to achieve constitutive or inducible expression of the PTT hydrolase.

[0015] In a fourth aspect, the present invention provides a method for constructing a recombinant expression vector, comprising the following steps: removing all the sequences from the ATG start codon to the second His·Tag at the Nco I site after the pET28aT7 promoter and then inserting the PTT hydrolase encoding gene.

[0016] The fifth aspect of the present invention provides a recombinant expression transformant comprising the above-mentioned PTT hydrolase encoding gene or the above-mentioned recombinant expression vector.

[0017] The recombinant expression transformant is prepared by transforming the recombinant expression vector of the present invention into a host cell. The host cell can be any conventional host cell in the art, provided that the recombinant expression vector can be used to stably replicate itself and the PTT hydrolase encoding gene carried by the recombinant expression vector can be effectively expressed.

[0018] Preferably, the host cell is Escherichia coli; more preferably, the host cell is Escherichia coli Rosetta-gami 2 (DE3).

[0019] The sixth aspect of the present invention provides the application of the above-mentioned PTT hydrolase, the above-mentioned recombinant expression vector or the above-mentioned recombinant expression transformant, and uses the above-mentioned PTT hydrolase, the above-mentioned recombinant expression vector or the above-mentioned recombinant expression transformant to degrade PTT plastic.

[0020] Preferably, the reaction conditions for PTT hydrolase to degrade PTT plastic are: reaction at a temperature of 25-50°C and a pH of 8-10; more preferably, reaction at a temperature of 30°C and a pH of 9.

[0021] Beneficial effects of the present invention:

[0022] The invention provides a PPT hydrolase from the genome of Brevundimonas sp. and a coding gene thereof, a recombinant expression vector containing the coding gene and a recombinant expression transformant. The PPT hydrolase exhibits efficient PTT hydrolysis ability to generate mono(3-hydroxypropyl)terephthalate (MHPT), terephthalic acid (TPA) and 1,3-propylene glycol (1,3-PDO), thereby achieving enzymatic degradation of PTT. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 This is a diagram showing the experimental results of PPT hydrolase degrading PTT membrane provided in Example 1 of the present invention.

[0025] Figure 2 Schematic diagram of the recombinant expression vector pET28a-XXII-G655 of Example 4 of the present invention.

[0026] Figure 3 The electrophoresis gel shows a phase diagram of the linearized vector pET-28a(+) amplification of Example 4 of the present invention; wherein A is a DNA molecular weight standard Marker (250-10000 bp); B is the amplification result under 56°C annealing conditions; C is the amplification result under 57°C annealing conditions; D is the amplification result under 58°C annealing conditions; and E is the amplification result under 59°C annealing conditions.

[0027] Figure 4 This is the electrophoresis gel phase of the gene amplification of Example 4 of the present invention; wherein A is D2000 Plus DNA Ladder; and B is gene XXII-G655.

[0028] Figure 5 This is a phase diagram of the electrophoresis gel for PCR verification of Example 4 of the present invention; wherein A is a DNA molecular weight standard Marker (250-10000 bp); and B is the T7 expression region of the gene XXII-G655 transformant. DETAILED DESCRIPTION

[0029] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0030] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form also includes the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0031] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] Example 1

[0033] The present embodiment provides a PTT hydrolase (PTTaseXXII-G655), and the amino acid sequence is as follows: MQDGVTTELVTFDSRSRAVRTAVFRPGSNTNGAGVVFLHGSGGMGE NQLAFARRFAEGGWLVLAPTYLDAAADTVIRAAPVMDAWRKCAVDAVDWMIGQGVDRRRTAAIGYSLGSHIAVDGALGAGRAAAAVSICGGWDVYIPRRPRRRIPVLMVRTANDRVVSAESTQRWRRFLQEAGTPVRERVIPDAGHLMNAAQWDAVYTVTSEFLRESQDLA (SEQ ID NO: 1).

[0034] In this embodiment, the PTT hydrolase is derived from the Brevundimonas sp. strain, which has been deposited in the China Center for Type Culture Collection (address: Wuhan University, Wuhan, China), the preservation date is June 28, 2021, and the biological preservation number is: CCTCCNO:M2021791.

[0035] In the specific implementation process, the waste PTT material can be treated by dissolving and then evaporating the solvent or adding a powdering process to obtain a low crystallinity / high specific surface area PTT film waste to improve the hydrolysis efficiency of PTT.

[0036] In the degradation experiment of PTT membrane made by evaporation (PTT plastic was first dissolved into hexafluoroisopropanol to become a 40 mg / mL solution, and then 4 mL of the solution was evenly spread on a round glass plate with a diameter of 10 cm and evaporated naturally, and a 6 mm round membrane was punched out with a hole puncher), 3 parallel groups were set up under the same reaction conditions (500 μL reaction system, PTT hydrolase concentration of 100 nM, 50 mM glycine-sodium hydroxide buffer at 30°C and pH = 9.0, and reaction for 24 hours). The results are as follows Figure 1 As shown: PTT hydrolase exhibited efficient PTT hydrolysis ability, producing degradation products with a concentration of 1.43 μM, among which 1.22 μM mono(3-hydroxypropyl)terephthalate (MHPT), 0.21 μM terephthalic acid (TPA) and 1,3-propylene glycol (1,3-PDO) were generated, thereby achieving enzymatic degradation of PTT.

[0037] Example 2

[0038] This embodiment provides a PTT hydrolase, the amino acid sequence of which has more than 80% homology with SEQ ID NO: 1 and has PTT hydrolase activity.

[0039] Example 3

[0040] This example provides a PTT hydrolase encoding gene (XXII-G655), which encodes the PTT hydrolase of Example 1.

[0041] In this example, the nucleotide sequence of the gene encoding PTT hydrolase is as follows: 5'-ATGCAGGA -3' (SEQ ID NO: 2).

[0042] Example 4

[0043] like Figure 2 As shown, this embodiment provides a recombinant expression vector (pET28a-XXII-G655) of the PTT hydrolase encoding gene of Example 3.

[0044] This embodiment also provides a method for constructing a recombinant expression vector, comprising the following steps: removing all the sequences from the ATG start codon to the second His Tag at the Nco I site after the pET28a T7 promoter, and inserting the PTT hydrolase encoding gene (XXII-G655), and the connected recombinant expression vector has the ability to express the PTT hydrolase PTTaseXXII-G655 and resistance to kanamycin and tetracycline. The specific implementation steps are as follows:

[0045] (1) PCR amplification

[0046] Corresponding primers are required for amplifying the XXII-G655 gene series. The primers used in this example are synthesized by Shanghai Shenggong Biotechnology Co., Ltd. The specific primer information is shown in Table 1, the PCR gene amplification system is shown in Table 2, and the PCR pET-28a (+) linearized vector amplification system is shown in Table 3.

[0047] Table 1 Primer information

[0048] name Primer nucleotide sequence (5' to 3') Vector upstream primer F CACCACCACCACCACCACTG(SEQ ID NO:3) Vector downstream primer R CATGGTATATCTCCTTCTTAAAGTTAAACA(SEQ ID NO:4) Gene upstream primer F taagaaggagatataccatgCAGGATGGGGTCACGACAGA(SEQ ID NO:5) Gene downstream primer R cagtggtggtggtggtggtgGGCGAGGTCCTGCGACTC(SEQ ID NO:6) T7 TAATACGACTCACTATAGG(SEQ ID NO:7) T7-TER GCTAGTTATTGCTCAGCGG(SEQ ID NO:8)

[0049] Note: The lowercase sequence is the sequence that pairs with the template DNA.

[0050] The gene amplification procedure was as follows: ① pre-denaturation: 98°C, 10 min; ② denaturation: 98°C, 10 s; ③ annealing: 60°C, 30 s; ④ extension: 72°C, 15 s (②~④ were repeated 30 times); ⑤ final extension: 72°C, 5 min; and finally kept at 4°C.

[0051] The linear amplification program was as follows: ① pre-denaturation: 98°C, 10 min; ② denaturation: 98°C, 10 s; ③ annealing: six gradients of 55°C, 56°C, 57°C, 58°C, 59°C, and 60°C, 30 s; ④ extension: 72°C, 1.5 min (② to ④ were repeated 30 times); ⑤ final extension: 72°C, 5 min; and finally kept at 4°C.

[0052] Table 2 PCR gene amplification system

[0053] name Dosage Gloria Nova HS2X Master Mix 25μL Gene upstream primer F 1μL Gene downstream primer R 1μL Deionized water 23μL Brevendimonas sp. strain single colony Trace

[0054] Table 3 PCR linearization vector amplification system

[0055] name Dosage Gloria Nova HS2X Master Mix 25μL Vector upstream primer F 1μL Vector downstream primer R 1μL Deionized water 22.5μL pET-28a(+) 0.5μL

[0056] (2) Agarose gel electrophoresis. First, dissolve 0.6g of agarose in 50mL of diluted normal concentration TAE buffer and dissolve it by microwave heating. Then cool it to 50℃ in a water bath and add GenGreen nucleic acid gel stain to mix and stain. Put it in the tank and insert the hole to solidify the gel. Then put it in the electrophoresis tank and let the normal concentration TAE solution cover the gel. Add DNA marker to the small hole, use D2000 Plus DNA Ladder to compare the gene, and use DNA molecular weight standard Marker (250~10000bp) to compare the linearized vector. Mix the DNA fragment with DNA gel loading dye (6X) and add it to the large hole, and run the gel at 180V for 20 minutes. The linearized pET-28a(+) amplicon is visible on the electrophoresis gel. Figure 3 , gene amplification electrophoresis gel Figure 4 .

[0057] (3) Gel extraction. Use a blue light gel exciter to cut the DNA fragments under ultraviolet light. Use the Omega Gel Extraction Kit D2500 to extract the DNA according to its instructions. The solution product is purified DNA.

[0058] (4) Recombination reaction. Reaction system: 2 μL linearized pET-28a(+) solution, 1 μL gene fragment solution, 2 μL 5xCE II Buffer, 1 μL Exnase II. Mix gently, use a PCR instrument to react at 37°C for 30 min, and then reduce the temperature to 4°C. Obtain the ligation product solution.

[0059] (5) Transformation. Perform aseptic operation (except for the ligation product solution). ① Take 50 μL of competent cells melted on ice, add the ligation product solution, mix gently, and let stand on ice for 30 minutes. ② Heat shock in a 42°C water bath for 45 seconds, quickly transfer to an ice bath, and let stand for 2 minutes. ③ Add 500 μL of resistance-free LB culture medium to a centrifuge tube and resuscitate at 37°C and 120 rpm for 60 minutes. ④ Take an appropriate volume of the resuscitation solution and evenly spread it on the LB solid culture medium containing 50 μg / mL kanamycin, and invert and culture it in a 37°C incubator overnight.

[0060] (6) PCR verification. Table 4 is the PCR verification system. Add a small amount of colonies to the PCR verification system, and plant the remaining part of the same colony on another LB solid culture medium containing 50μg / mL kanamycin. After marking the position, invert and culture it in a 37℃ incubator overnight (sterile operation). PCR verification is performed on a maximum of 5 colonies for strains containing the same genes. The PCR program is set as: ① Pre-denaturation: 98℃, 10min; ② Denaturation: 98℃, 10s; ③ Annealing: 60℃, 30s; ④ Extension: 72℃, 15s (②~④ repeated 30 times); ⑤ Final extension: 72℃, 5min; finally keep at 4℃. Perform small-hole agarose gel electrophoresis and use DNA molecular weight standard Marker (250~10000bp) as a control. The results of PCR verification are shown in Figure 5 A small portion of the transformants verified by PCR were sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing verification (primers T7 and T7-TER were selected), and bidirectional sequencing obtained a positive result.

[0061] Table 4 PCR verification system

[0062] name Dosage Gloria Nova HS2X Master Mix 10μL T7 Primer 1μL T7-TER Primer 1μL Deionized water 8μL Colony Trace

[0063] Example 5

[0064] This embodiment provides a recombinant expression transformant comprising the PTT hydrolase encoding gene of Example 3 or the recombinant expression vector of Example 4.

[0065] In this example, the host cell is Escherichia coli Rosetta-gami2 (DE3).

[0066] Specifically, the recombinant expression vector pET28a-XXII-G655 was transformed into the Escherichia coli Rosetta-gami 2 (DE3) strain, and the specific operation was as follows:

[0067] (1) Extraction of recombinant plasmid. The transformant of Example 4 was inoculated in 3 mL of LB liquid culture medium containing 50 μg / mL of kanamycin and cultured overnight at 37°C and 220 rpm (aseptic operation). The culture medium was then used to extract the plasmid using the Omega Plasmid Extraction Kit D6943 Plasmid Mini Kit I, following the steps in its instruction manual. The solution product was a purified recombinant vector, which was directly used to transform E. coli Rosetta gami 2.

[0068] (2) Transformation of E. coli Rosetta gami 2. Aseptic operation (except for the recombinant vector solution). The transformation steps are similar to those in Example 4. The object to be transformed is E. coli Rosetta gami2, and the amount of plasmid solution is 10 μL. The prepared transformant solution is seeded in LB solid medium containing 50 μg / mL kanamycin and 10 μg / mL tetracycline hydrochloride, and cultured at 37°C for 18 hours. Select a single colony and inoculate it in LB liquid medium containing 50 μg / mL kanamycin and 10 μg / mL tetracycline hydrochloride, and culture it overnight at 37°C and 220 rpm to obtain a seed solution for freezing (-80°C) or use.

[0069] The transformed strain is cultured and fermented. The specific steps are as follows:

[0070] (1) Preparation of fermentation seed liquid. Aseptic operation. Thaw the frozen seed liquid on ice and spread it on solid LB medium containing antibiotics corresponding to the strain resistance (the strain culture medium contains 50μg / mL kanamycin and 10μg / mL tetracycline hydrochloride), and culture it at 37℃ overnight. Select a single colony and inoculate it into 100mL liquid LB medium containing antibiotics corresponding to the strain resistance, and culture it at 37℃ and 220rpm overnight to obtain fermentation seed liquid.

[0071] (2) Fermentation of PTTaseXXII-G655. Aseptic operation. Inoculate 8 mL of seed solution into 800 mL of liquid LB medium (containing antibiotics corresponding to the strain resistance). Cultivate at 37°C and 220 rpm until the fermentation liquid OD reaches 0. 600 When the pH value was greater than 0.6 and less than 1.0, 100 μL IPTG solution was added to every 500 mL of bacterial solution to induce protein expression, and the temperature was switched to 18°C ​​for 18 hours.

[0072] (3) Collect the bacteria and freeze them. Centrifuge the final fermentation broth at -4°C and 6500 rcf for 15 min. Collect the precipitated bacteria and freeze them at -80°C or use them directly for protein purification.

[0073] After the fermentation is completed, the protein is purified. During the purification process, all systems are kept at a low temperature of 4°C. The specific steps are as follows:

[0074] ① Resuspension: Take out the frozen expressed bacteria and thaw them at 4℃. After thawing, add 25-30mL of Lysis Buffer. Resuspend by shaking and keep the temperature low. After reselection, add Lysis Buffer to the system to 50mL.

[0075] ②Ultrasound: Ultrasonicate the resuspended bacterial solution on ice using an ultrasonic cell disruptor. Insert the ultrasonic probe about 1.5 cm into the liquid surface. Set the power to 280W, ultrasonicate for 5 seconds, interval 10 seconds, time 30 minutes, and the alarm will be paused when the temperature exceeds 8°C.

[0076] ③ Centrifugation: After ultrasonication, use a large centrifuge to centrifuge at 4°C, 10,000 rcf, and 1.5 h.

[0077] ④ Incubation: After centrifugation, transfer the supernatant to a new centrifuge tube and discard the precipitate. Equilibrate the nickel column with 50 mL Lysis Buffer in advance, pour it into the above new centrifuge tube, and place it horizontally. Incubate in an ice bath with horizontal shaking for 1 hour.

[0078] ⑤ Washing: Add the mixture of the incubated supernatant and the nickel column into the nickel column tube and filter out. Use Lysis Buffer twice and Wash Buffer once by gravity filtration. For proteins that do not need Wash Buffer, use Lysis Buffer to filter and wash again.

[0079] ⑥ Elution: Use 3.5 mL of elution buffer to gravity elute the protein on the nickel column into the ultrafiltration tube, and then centrifuge at 4°C and 7500 rcf to a volume of about 2.5 mL.

[0080] ⑦ Desalting: Use a pipette to blow the ultrafiltration tube, and then draw the solution in the ultrafiltration tube to the PD-10 desalting column that has been filtered and washed four times with desalting buffer in advance. After the filtrate is drained, add 3.5mL Desalting Buffer, filter out the protein in a clean ultrafiltration tube, centrifuge at 4℃ and 7500rcf to the appropriate concentration, and freeze it in aliquots.

[0081] The purified PTTaseXXII-G655 protein was obtained. The buffer formula for purification is as follows:

[0082] All buffer solutions had a pH of 8.0; lysis buffer: 25 mM Tris, 100 mM sodium chloride solution; cleaning buffer: 25 mM Tris, 100 mM sodium chloride, 40 mM imidazole solution; elution buffer: 25 mM Tris, 100 mM sodium chloride, 250 mM imidazole solution; desalting buffer: 25 mM Tris, 10% volume fraction glycerol solution.

[0083] Example 6

[0084] This example provides the use of the PTT hydrolase of Examples 1-2, the recombinant expression vector of Example 4 or the recombinant expression transformant of Example 5 for degrading PTT plastic.

[0085] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A PTT hydrolase, characterized in that The amino acid sequence is shown in SEQ ID NO:

1.

2. The PTT hydrolase according to claim 1, characterized in that The PTT hydrolase is derived from a Brevundimonas sp. strain, and its deposit number is: CCTCCNO: M2021791.

3. A PTT hydrolase encoding gene, characterized in that: The coding gene encodes the PTT hydrolase described in any one of claims 1-2.

4. The PTT hydrolase encoding gene according to claim 3, characterized in that The nucleotide sequence of the PTT hydrolase encoding gene is shown in SEQ ID NO:

2.

5. A recombinant expression vector comprising the PTT hydrolase encoding gene according to any one of claims 3-4.

6. The recombinant expression vector according to claim 5, characterized in that The recombinant expression vector has resistance to kanamycin and tetracycline.

7. The method for constructing a recombinant expression vector according to any one of claims 5 to 6, characterized in that: The method comprises the following steps: removing all the sequences from the ATG start codon to the second His·Tag on the Nco I site after the T7 promoter of pET28a, and then inserting the PTT hydrolase encoding gene.

8. A recombinant expression transformant comprising the PTT hydrolase encoding gene according to any one of claims 3-4 or the recombinant expression vector according to any one of claims 5-6.

9. The recombinant expression transformant according to claim 8, characterized in that: The host cell is Escherichia coli Rosetta-gami 2 (DE3).

10. Use of the PTT hydrolase according to any one of claims 1 to 2, the recombinant expression vector according to any one of claims 5 to 6, or the recombinant expression transformant according to any one of claims 8 to 9, characterized in that: The PTT hydrolase described in any one of claims 1-2, the recombinant expression vector described in any one of claims 5-6, or the recombinant expression transformant described in any one of claims 8-9 is used to degrade PTT plastic.

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