Cutinase as well as coding gene and application thereof

Abstract

The invention relates to cutinase as well as a coding gene and application thereof. The amino acid sequence of the cutinase SCC is as shown in SEQ ID NO. 1, and the nucleotide sequence of the cutinase SCC is as shown in SEQ ID NO. 2. The inventor of the application finds out a novel cutinase SCC of a serine hydrolase family from Saccharothrix carnea, and the cutinase SCC is easy for heterologous expression and purification, and can efficiently and specifically catalyze and degrade mono (2-hydroxyethyl) terephthalate (MHET) and polyethylene glycol terephthalate (PET). And compared with the existing MHET degrading enzyme, the MHET degrading enzyme has the advantages that the catalytic efficiency is obviously improved, the use amount of the enzyme can be effectively reduced in the process of catalytically degrading MHET and PET, and the use cost of the enzyme is reduced.

Description

Technical Field

[0001] This invention relates to a keratinase, its encoding gene, and its applications, belonging to the field of biotechnology. Background Technology

[0002] Plastics are complex high-molecular polymers, and plastic products are obtained through both natural and synthetic methods. Natural plastics are typically composed of natural macromolecules such as starch, corn stalks, and other plant fibers, or macromolecules secreted when microorganisms cannot reproduce normally, such as poly(3-hydroxybutyrate) (PHB) and polycaprolactone (PCL). These plastics have excellent biodegradability and are environmentally friendly products. Synthetic plastics are produced from petroleum as a raw material, polymerizing small monomer molecules through addition or condensation reactions to form large-molecular polymers. They are diverse and widely used in human production and daily life. Polyethylene terephthalate (PET) is an aromatic synthetic polyester polymerized from terephthalic acid (TPA) and ethylene glycol (EG) through ester bonds. It is one of the most common plastic products in daily life, mainly existing in amorphous and semi-crystalline forms. Because of its lightweight, good insulation, stable chemical properties and low processing cost, it is widely used in packaging, materials, electrical and construction industries, such as beverage bottles, machine parts, control switch housings, mulch film and engineering plastics.

[0003] Biodegradation of PET is an eco-friendly method recognized by humans, typically referring to the degradation of PET polymers by microorganisms such as bacteria and fungi that exist in natural environments. Microorganisms play the role of decomposers in ecosystems, characterized by their small size, large numbers, and powerful metabolic functions. They can effectively break down waste plastics into environmentally friendly small molecules, playing a crucial role in promoting material cycling and energy transfer within ecosystems. Since the 1990s, the method of using microbial enzymes to degrade polymeric materials has received widespread attention from scientists. Many enzymes derived from bacteria and fungi have demonstrated the ability to degrade PET. In 2016, Yoshida et al. reported a Gram-negative strain, Ideonella sakaiensis 201-F6, which can efficiently degrade PET into bis(2-hydroxyethyl) terephthalate (BHET), mono(2-hydroxyethyl) terephthalate (MHET), and TPA using low-crystallinity PET film as the main carbon source. MHET is the main product. Finally, MHET is reabsorbed into the body by Ideonella sakaiensis 201-F6 bacteria and degraded into environmentally harmless end products TPA and EG by MHETases in the body.

[0004] MHET, as a major degradation product in the PET degradation process, reduces the degradation efficiency of the PET substrate. Therefore, effectively promoting the complete degradation of the intermediate MHET has become one of the key issues in the efficient biodegradation of PET. Thus, finding degradative enzymes that can specifically degrade MHET has significant practical implications. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a keratinase, its encoding gene, and its applications.

[0006] The technical solution of this invention is as follows:

[0007] A keratinase SCC, the amino acid sequence of which is shown in SEQ ID NO.1.

[0008] The nucleotide sequence of the gene encoding the aforementioned keratinase SCC is shown in SEQ ID NO.2.

[0009] According to a preferred embodiment of the present invention, the keratinase SCC is derived from Saccharothrix carnea and can be prepared by heterologous expression.

[0010] A recombinant expression vector is formed by inserting the coding gene for the aforementioned keratinase SCC into a plasmid vector.

[0011] According to a preferred embodiment of the present invention, the plasmid vector is pET-28a(+).

[0012] A recombinant strain is obtained by transforming the above-mentioned recombinant vector into a host cell.

[0013] According to a preferred embodiment of the present invention, the host cell is Escherichia coli.

[0014] The application of the above-mentioned keratinase SCC and / or the gene encoding keratinase SCC in the degradation of mono(2-hydroxyethyl) terephthalate and polyethylene terephthalate.

[0015] Beneficial effects:

[0016] 1. The inventors of this application have discovered a new serine hydrolase family of keratinase SCC from Saccharothrix carnea. This enzyme is easy to express and purify heterologously and can efficiently and specifically catalyze the degradation of mono(2-hydroxyethyl) terephthalate (MHET) and polyethylene terephthalate (PET).

[0017] 2. The keratinase SCC provided by this invention has significantly improved catalytic efficiency compared with existing MHET degrading enzymes. It can effectively reduce the amount of enzyme used and lower the cost of enzyme use during the catalytic degradation of MHET and PET.

[0018] 3. The keratinase SCC provided by this invention has simple and mild production and use conditions, and has great application potential in the large-scale industrial degradation of MHET and PET. Attached Figure Description

[0019] Figure 1 The structure diagram of the recombinant plasmid pET-28a(+)-SCC is shown.

[0020] Figure 2 The results are for the validation of the recombinant plasmid pET-28a(+)-SCC.

[0021] In the figure, a represents PCR verification, lane 1 is the PCR product of keratinase SCC, and lane M is the Maker; b represents double enzyme digestion verification; lanes 1-2 are the double enzyme digestion products of recombinant plasmid pET-28a(+)-SCC, and lane M is the Maker.

[0022] Figure 3 The results of temperature stability analysis are for the optimal enzyme activity temperature;

[0023] In the figure, a represents the optimal reaction temperature, and b represents the temperature stability.

[0024] Figure 4 The image shows the results of HPLC detection of the products of catalytic degradation of MHET. Detailed Implementation

[0025] The following embodiments and accompanying drawings are merely illustrative of specific implementation schemes for carrying out the present invention. These schemes and drawings should not be construed as limiting the present invention. Any changes made without departing from the principles and essence of the present invention shall fall within the protection scope of the present invention.

[0026] Unless otherwise specified, the experimental techniques and methods used in this embodiment are conventional. Unless otherwise specified, all materials and reagents used in this embodiment can be obtained through legitimate commercial channels.

[0027] In this invention, the enzyme activity of keratinase SCC is determined by high-performance liquid chromatography (HPLC). Specifically, the substrate MHET generates TPA during the enzymatic reaction of recombinant keratinase SCC protein. The enzyme activity of recombinant keratinase SCC protein can be calculated from the peak area of ​​TPA by HPLC and the standard curve.

[0028] Enzyme activity unit (U) is defined as: the amount of enzyme consumed to produce 1 μmol of terephthalic acid (TPA) per minute, with a specific enzyme activity of μmol / min. -1 mg -1 (U / mg -1 ).

[0029] Example 1: Construction of recombinant plasmid pET-28a(+)-SCC

[0030] 1. Based on the keratinase SCC gene (SEQ ID NO.2), design the following two PCR amplification primers:

[0031] SCC-F end primer: 5'-CGCGGATCC GCGGAAGAGGAATTTCGCCG-3'.

[0032] SCC-R end primer: 5'-CTGCCCGCATGGCGTGTAA AAGCTTGGG-3'.

[0033] The keratinase SCC gene, as shown in SEQ ID NO.2, was synthesized artificially by Genewiz. Then, using the SCC gene as a template and SCC-F / R as primers, PCR amplification was performed to obtain the PCR amplification product. The PCR amplification product was subjected to 1wt% agarose gel electrophoresis, and the results are as follows: Figure 2 As shown in Figure a, the amplified DNA fragments were then recovered using an Omega DNA Recovery Kit following its instructions.

[0034] The PCR amplification program was as follows: 98℃ pre-denaturation for 30 seconds; 98℃ denaturation for 10 seconds, 59℃ annealing for 15 seconds, 72℃ extension for 6 seconds, 32 cycles; 72℃ extension for 10 minutes.

[0035] The PCR amplification system (25 μL) is as follows: 9.5 μL sterile distilled water, 12.5 μL Primer star, 1 μL primer HoIDO-F, 1 μL primer HoIDO-R, and 1 μL template DNA.

[0036] Depend on Figure 2 As can be seen from a, the gene encoding keratinase SCC was successfully amplified.

[0037] 2. The pET-28a(+) vector and the DNA fragment amplified in step 2 were double-digested using restriction endonucleases BamHI and HindIII. Then, the amplified DNA fragment was ligated into the linearized pET-28a(+) vector using In-Fusion (TaKaRa). The expression vector was transformed into *E. coli* BL21(DE3) competent cells using the heat shock transformation method described in *Molecular Cloning: A Laboratory Manual*. Specifically, 2.5 μL of the ligation mixture was added to 50 μL of *E. coli* DH5α competent cells (TransGen), and incubated on ice for 30 min; heat-shocked at 42°C for 90 sec; rapidly transferred to ice and incubated for 10 min; 200 μL of LB broth was added, and the cells were incubated at 37°C for 1 h; the cells were then plated onto LB agar plates containing 100 μg / mL ampicillin and incubated overnight at 37°C.

[0038] The transformants were sent to BGI Genomics for sequencing verification. Sequencing results showed that the keratinase SCC coding gene fragment was successfully inserted between the BamHI and HindIII restriction sites of pET-28a(+), with the correct insertion direction and no base mutations, deletions, or additions. The recombinant plasmid vector was named pET-28a(+)-SCC, and its specific structure is shown in the figure below. Figure 1 As shown.

[0039] The recombinant plasmid pET-28a(+)-SCC was verified by double enzyme digestion. The specific method was as follows: Agarose gel was placed in an electrophoresis tank, completely immersed in 1×TAE buffer. 2 μL of the double-digested (BamHI and HindIII) recombinant plasmid pQE80-L-HoIDO was mixed with 5 μL of loading buffer. The prepared sample was then added to the gel wells, and DAN marker was added to a fresh gel well as a control. The electrophoresis conditions were 200V for 15 min. The results are shown below. Figure 2 As shown in b.

[0040] Depend on Figure 2 As shown in b, the two bands are approximately 800bp and 5300bp, respectively, corresponding to the size of the keratinase SCC encoding gene and the size of the plasmid pET-28a(+), indicating that the recombinant plasmid pET-28a(+)-SCC was successfully constructed.

[0041] Example 2: Heterologous expression and purification of keratinase SCC

[0042] 1. Heterologous expression of keratinase SCC in Escherichia coli BL21(DE3)

[0043] (1) The constructed recombinant plasmid vector pET-28a(+)-SCC was transformed into Escherichia coli BL21(DE3) competent cells according to the heat shock transformation method in Molecular Cloning Laboratory Manual, and plated on LB solid medium containing 100 μg / mL kanamycin in water and cultured overnight at 37°C.

[0044] (2) Pick a single colony from LB solid medium and inoculate it into LB liquid medium containing 100 μg / mL kanamycin. Incubate overnight at 37°C to obtain seed culture.

[0045] (3) Transfer the seed culture to LB liquid medium containing 100 μg / mL kanamycin at an inoculation rate of 1% (v / v), and culture at 37℃ and 180 rpm until the OD600nm is about 0.6 to 0.8. Then add IPTG (0.1 mM) and induce at 16℃ and 100 rpm for 16 h to obtain the bacterial culture.

[0046] (4) Centrifuge the bacterial solution at 4℃ and 4000rpm for 15min and collect the bacterial cells.

[0047] 2. Isolation and purification of keratinase SCC

[0048] (1) Preparation of crude enzyme solution: The bacterial cells were resuspended in Lysis buffer (50 mM Tris-HCl, 100 mM NaCl, pH 8.0), and the cells were broken using a pressure lysate. The cells were centrifuged at 4°C and 12,000 rpm for 60 min. The supernatant obtained was the crude enzyme solution.

[0049] (2) Nickel affinity chromatography

[0050] After pretreatment of the nickel affinity column with Lysis buffer, the crude enzyme solution was loaded onto the nickel affinity column, and then washed with Washbuffer (50 mM Tris-HCl, 100 mM NaCl, 10 mM Imidazole, pH 8.0) for 10 column volumes. Finally, the target protein was eluted with Elution buffer (50 mM Tris-HCl, 100 mM NaCl, 250 mM Imidazole, pH 8.0).

[0051] (3) Gel filtration chromatography

[0052] After equilibrating the Superdex 200 Prep grade gel filtration chromatography column with GF buffer (10 mM Tris-HCl, 100 mM NaCl, pH 8.0), the sample purified by nickel affinity chromatography was concentrated and loaded onto the column, eluted with GF buffer; the peak tip sample of the target protein was collected to obtain keratinase SCC, which was then stored at -80℃ after adding 10% glycerol for later use.

[0053] Example 3: Optimal enzyme activity temperature and stability analysis of keratinase SCC

[0054] The optimal reaction temperature of keratinase SCC was determined by placing diluted keratinase SCC in 20 mmol / L HEPES buffer solutions at a gradient temperature of 25–60 °C for 1 hour. The enzyme activity at each gradient temperature was calculated, with the highest enzyme activity defined as 100%. The results are shown below. Figure 3 As shown in a.

[0055] The stability of keratinase SCC was determined by the following method: Diluted keratinase SCC was placed in 20 mmol / L HEPES buffer solution at a gradient temperature of 20–70 °C and incubated for 1 hour. After the reaction, the precipitate was removed, and the residual enzyme activity was measured with enzyme activity at 4 °C as 100%. The results are shown below. Figure 3 As shown in b.

[0056] Depend on Figure 3 As shown in a and 3b, when the enzyme is incubated at different temperatures for a certain period of time, at the same temperature, the residual enzyme activity of the recombinant protein gradually decreases with increasing incubation time. At 25–30°C, after the incubation period, the residual enzyme activity can still be maintained at about 80%. After incubation at 35°C for a certain period, the residual enzyme activity can also be maintained at over 60%. Furthermore, when the temperature reaches 40°C, its activity rapidly decreases, with only about 20% of the residual enzyme activity remaining. The results indicate that this enzyme is a medium-to-low temperature enzyme.

[0057] Example 4: Keratinase SCC degrades MHET

[0058] A reaction system was constructed using MHET as the substrate. The system comprised MHET at a final concentration of 0.1 mmol / L, 0.2 mg / mL keratinase SCC, and 100 mmol / L Tris-HCl buffer (pH = 7.0). The reaction was then incubated at 30°C for 2 h, terminated by boiling in a water bath for 5 min, and analyzed by high-performance liquid chromatography (HPLC). Three parallel experiments were set up in the experimental group, and the control group (without keratinase SCC) was maintained under identical reaction conditions. The results are shown below. Figure 4 As shown.

[0059] Depend on Figure 4It can be seen that when the blank group does not add keratinase SCC enzyme solution for reaction, the substrate MHET will have a peak at 5.337 min. However, when the experimental group adds keratinase SCC enzyme solution for reaction under the same reaction conditions, it is found that the peak of substrate MHET decreases and the peak of product TPA appears at 4.773 min. This indicates that under the catalysis of keratinase SCC, MHET is effectively degraded into product TPA.

Claims

1. A keratinase SCC, characterized in that, The amino acid sequence is shown in SEQ ID NO.

1.

2. The gene encoding the keratinase SCC according to claim 1, characterized in that, The nucleotide sequence is shown in SEQ ID NO.

2.

3. A recombinant expression vector, characterized in that, The encoding gene for the keratinase SCC described in claim 2 is inserted into a plasmid vector.

4. The recombinant expression vector as described in claim 3, characterized in that, The plasmid vector is pET-28a(+).

5. A recombinant bacterial strain, characterized in that, It is obtained by converting the recombinant vector described in claim 3 into host cells.

6. The recombinant strain according to claim 5, characterized in that, The host cell is Escherichia coli.

7. The use of the gene encoding the keratinase SCC of claim 1 and / or the keratinase SCC of claim 2 in the degradation of mono(2-hydroxyethyl) terephthalate and polyethylene terephthalate.

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