Recombinant bacillus subtilis for efficiently expressing plastic degrading enzyme LCCICCG as well as construction method and application of recombinant bacillus subtilis

Abstract

The invention discloses recombinant bacillus subtilis capable of efficiently expressing a plastic degrading enzyme LCCICCG as well as a construction method and application of the recombinant bacillus subtilis. The recombinant bacillus subtilis is used for overexpressing the plastic degrading enzyme LCCICCG and signal peptide. By constructing plasmids of different signal peptides and promoters, the biomass of the recombinant bacillus subtilis and the protein expression quantity of the LCCICCG in the fermentation culture process are improved, and the industrial production cost of the LCCICCG is reduced.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, and in particular to a method for efficiently expressing a plastic degrading enzyme LCC. ICCG Recombinant Bacillus subtilis and its construction method and application. Background Art

[0002] In recent years, the environmental accumulation of plastic waste, especially polyethylene terephthalate (PET), has become increasingly serious. Traditional physical and chemical treatment methods have limitations such as high cost and secondary pollution, and there is an urgent need for breakthroughs in green and efficient biodegradation technology. The biodegradation strategy based on enzyme catalysis has attracted much attention due to its environmental friendliness. Among them, LCC derived from metagenomic screening has attracted much attention. ICCG The enzyme exhibits excellent PET hydrolysis activity and thermal stability and is considered to be a promising tool enzyme for plastic degradation. ICCG The low expression efficiency of the enzyme seriously restricts its industrial application.

[0003] As a Gram-positive model strain, Bacillus subtilis has the advantages of efficient protein secretion, mature genetic manipulation, rapid growth and high safety, making it an ideal host for heterologous expression of industrial enzymes. Compared with intracellular expression, its secretory expression system can significantly reduce the aggregation and loss of recombinant proteins in cells, reduce the metabolic burden of the host, and avoid the interference of complex intracellular environment on enzyme activity, directly obtaining functional enzymes with natural conformations. In addition, protease-deficient strains in Bacillus subtilis can further reduce the extracellular degradation of enzyme proteins and improve the stability of target products.

[0004] Therefore, it is urgent to provide a method for efficiently and stably producing LCC. ICCG Enzyme biotechnology. Summary of the invention

[0005] The purpose of the present invention is to ICCG LCC in enzyme bioproduction technology ICCG To solve the problems of low enzyme expression efficiency and poor stability, we provide an efficient and stable subtilisin spore expression system and optimize the fermentation culture conditions to improve LCC ICCG Protein expression and biomass.

[0006] In order to achieve the above object, the technical solution provided by the present invention is as follows:

[0007] A LCC expressing plastic-degrading enzyme ICCG Recombinant Bacillus subtilis, the recombinant Bacillus subtilis overexpressing plastic degradation enzyme LCC ICCG and signal peptide.

[0008] Wherein, the plastic degrading enzyme LCC ICCG The nucleotide sequence of the coding gene is shown in SEQ ID NO.1.

[0009] Wherein, the signal peptide is SP Epr、 SP YjcM、 SP AprE、 SP Bpr、 SP BglS and SP YbdG Any one of the foregoing, whose amino acid sequences are shown as SEQ ID NOs. 2 to 7, and whose nucleotide sequences are shown as SEQ ID NOs. 9 to 14, respectively.

[0010] Wherein, the plastic degrading enzyme LCC ICCG The coding gene and the coding gene of the signal peptide are expressed under the control of the maltose promoter; the nucleotide sequence of the maltose promoter is shown in SEQ ID NO.8.

[0011] The present invention also provides the highly efficient expression of plastic degrading enzyme LCC ICCG Method for constructing recombinant Bacillus subtilis to transform plastic degrading enzyme LCC ICCG The coding gene is connected to a vector to construct a first recombinant expression vector, the original signal peptide coding gene in the first recombinant expression vector is replaced with the coding gene of the signal peptide by PCR technology, a second recombinant expression vector is constructed, the original promoter in the second recombinant expression vector is replaced with a maltose-inducible promoter by PCR technology, a third recombinant expression vector is constructed, and the third recombinant expression vector is introduced into Bacillus subtilis to obtain.

[0012] Preferably, the highly efficient expression of plastic degrading enzyme LCC ICCG The method for constructing the recombinant Bacillus subtilis is as follows: ICCG The coding gene is connected to the vector by a one-step cloning method to construct a first recombinant expression vector; by using PCR technology, primers are used to replace the original signal peptide coding gene in the first recombinant expression vector with the coding gene of the signal peptide to construct a second recombinant expression vector; by using PCR technology, primers malA-F / malA-R are used to replace the original promoter in the second recombinant expression vector with a maltose-inducible promoter to construct a third recombinant expression vector, and the third recombinant expression vector is introduced into Bacillus subtilis to obtain.

[0013] Wherein, the vector is a pBE-S Bacillus subtilis secretory expression vector plasmid; and the Bacillus subtilis is Bacillus subtilis MATE01.

[0014] The present invention also provides the highly efficient expression of plastic degrading enzyme LCC ICCGRecombinant Bacillus subtilis produces plastic-degrading enzyme LCC in fermentation ICCG The recombinant Bacillus subtilis is inoculated into a fermentation medium and cultured and fermented until the OD value of the recombinant Bacillus subtilis in the fermentation medium reaches 0. 600 is 0.6-0.8, and maltose is added to the fermentation medium to induce fermentation for 36-72 hours.

[0015] Wherein, the added amount of maltose is 0.01-0.05 g / mL.

[0016] Wherein, the culture fermentation and the induced fermentation are carried out at a temperature of 30-37° C. and a rotation speed of 200-250 rpm.

[0017] The formula of the fermentation medium is: 40-50 g / L yeast extract, 30-40 g / L tryptone, and 5-10 g / L dipotassium hydrogen phosphate.

[0018] Beneficial effects:

[0019] The present invention constructs an LCC based on the optimization of codon preference of Bacillus subtilis ICCG Expression strains encoding genes and optimization of LCC ICCG Encoding gene expression elements and strain culture conditions successfully improved LCC ICCG The expression efficiency and stability of the protein were improved. By optimizing the inducer concentration and fermentation time, the protein expression and biomass of the recombinant strain were significantly improved. In addition, through the optimized fermentation strategy, LCC was achieved in a 5L fermenter. ICCG The high-density fermentation production of protein provides a technical basis for the large-scale industrial production of the protein. These improvements not only improve production efficiency and reduce costs, but also provide strong support for the commercial application of plastic depolymerase. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0021] Figure 1 LCC produced by fermentation of recombinant strains expressing different signal peptides in Example 1 ICCG Enzyme activity, hydrophobic amino acid percentage and score statistics.

[0022] Figure 2 The biomass and extracellular enzyme activity statistics of the fermentation culture process of the recombinant strain containing two promoters in Example 1 are shown in FIG.

[0023] Figure 3This is a statistical diagram of the fermentation effect of the recombinant Bacillus subtilis of the present invention under the conditions of different concentrations of maltose inducer in Example 2.

[0024] Figure 4 LCC obtained from the fermentation supernatant of purified recombinant Bacillus subtilis in Example 3 ICCG Gel electrophoresis of purified protein; lane M is Maker with a molecular weight of 180 kDa, and lane 1 is LCC ICCG Crude enzyme, lane 2 is the flow-through, and lanes 3 to 7 are the eluates.

[0025] Figure 5 The PET film in Example 3 was subjected to LCC ICCG Scanning electron microscope images before and after degradation; among them, Figure a is a PET film magnified 20 times before degradation; Figure b is a PET film magnified 200 times after degradation; Figure c is a PET film magnified 20 times before degradation; Figure d is a PET film magnified 200 times after degradation. DETAILED DESCRIPTION

[0026] The present invention is further described below based on the following examples. It should be understood that the following embodiments are only used to illustrate the present invention, but not to limit the present invention.

[0027] If the specific techniques or conditions are not specified in the examples, they are carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased through regular channels.

[0028] The components of the fermentation medium described in the following examples are: 50 g / L yeast extract, 30 g / L tryptone, and 5 g / L dipotassium hydrogen phosphate.

[0029] Example 1 Construction of recombinant Bacillus subtilis and LCC ICCG Protein expression verification

[0030] Based on the LCC optimized based on the codon preference of Bacillus subtilis ICCG Protein coding gene (gene lcc ICCG , whose nucleotide sequence is shown in SEQ ID NO.1) as a template, using amplification primer lcc ICCG -F / lcc ICCG-R (Nde I and Xba I restriction sites were added to the 5′ ends of the forward primer and the reverse primer, respectively, and 15 bp homology arms were added to the 3′ ends of the forward primer and the reverse primer), and the gene fragment was obtained by PCR amplification; the vector plasmid pBE-S (Takara Bio, product number: 3380) was digested into a linearized vector using Nde I and Xba I enzymes; the gene fragment obtained by the above PCR amplification was connected with the linearized vector through one-step cloning, and the connection product was transformed into E. coli DH5α for amplification, and the single clone was screened using LB resistance plates (containing 100 μg / mL ampicillin) and sequenced to obtain the positive clone strain E. coli DH5α / PBE-S-LCC ICCG From the positive clone strain E. coli DH5α / PBE-S-LCC ICCG Extract plasmid PBE-S-LCC ICCG , PBE-S-LCC ICCG The plasmid was introduced into the competent cells of Bacillus subtilis MATE01 by using the Bacillus subtilis GM transformation agent (Tianjin Chunyao Biotechnology Co., Ltd., catalog number: CYY001). The monoclonal strain was screened by LB resistance plate (50 μg / mL kanamycin) and identified by colony PCR and sequencing to obtain the positive expression strain B. subtilis MATE01 / PBE-S-LCC. ICCG .

[0031] The constructed B. subtilis MATE01 / PBE-S-LCC ICCG Signal peptides were optimized and different signal peptide expression strains were constructed. ICCG As templates, primers PBE-S-Epr-F / PBE-S-Epr-R, PBE-S-YjcM-F / PBE-S-YjcM-R, PBE-S-AprE-F / PBE-S-AprE-R, PBE-S-Bpr-F / PBE-S-Bpr-R, PBE-S-BglS-F / PBE-S-BglS-R and PBE-S-YpdG-F / PBE-S-YpdG-R were used for PCR amplification to obtain the plasmid PBE-S-Epr-LCC ICCG 、PBE-S-YjcM-LCC ICCG 、PBE-S-AprE-LCC ICCG 、PBE-S-Bpr-LCC ICCG 、PBE-S-BglS-LCC ICCG and PBE-S-YbdG-LCC ICCG , so as to achieve the plasmid PBE-S-LCC ICCGThe original signal peptide coding gene in was replaced by the signal peptide SP Epr、 SP YjcM、 SP AprE、 SP Bpr、 SP BglS and SP YbdG The plasmids containing different signal peptide encoding genes were transformed into Bacillus subtilis MATE01, and monoclonal strains were screened by LB resistance plate (50 μg / mL kanamycin) and colony PCR and sequencing were performed to obtain the positive expression strain B. subtilis MATE01 / PBE-S-Epr-LCC ICCG , B.subtilis MATE01 / PBE-S-YjcM-LCC ICCG , B.subtilis MATE01 / PBE-S-AprE-LCC ICCG , B.subtilis MATE01 / PBE-S-Bpr-LCC ICCG , B.subtilis MATE01 / PBE-S-BglS-LCC ICCG and B. subtilis MATE01 / PBE-S-YbdG-LCC ICCG , and these strains were subjected to LCC ICCG Expressive ability test.

[0032] The above strains were inoculated into the fermentation medium at an inoculation rate of 5% v / v for shake flask fermentation experiments. The fermentation was carried out at 37°C and 200rpm for 36 hours. After the fermentation was completed, the fermentation liquid was centrifuged at 12000rpm to take the supernatant to detect the enzyme activity. The enzyme activity detection method is: 980μL PBS buffer (50mM, pH=8.0), 10μL 10M p-nitrophenol butyrate ethanol solution, and 10μL supernatant sample were mixed evenly, reacted at 37°C for 10 minutes, 200μL of the reaction solution was added to a 96-well plate, and the amount of p-nitrophenol generated was detected using an enzyme reader at a wavelength of 405nm. One enzyme activity unit (1U) is defined as the amount of enzyme required to catalyze 1μmol of p-nitrophenol butyrate to generate 1μmol of p-nitrophenol per minute at 37°C. The experimental results are as follows Figure 1 As shown, the experimental results show that the signal peptide SP AprE Demonstrated excellent LCC ICCG Secretion effect, the extracellular enzyme activity can reach 8.3U / mL.

[0033] Plasmid PBE-S-AprE-LCC ICCG As a template, PCR amplification was performed using primers malA-F / malA-R to obtain the plasmid malA-AprE-LCC ICCG, so as to achieve the plasmid PBE-S-AprE-LCC ICCG The original constitutive promoter aprE promoter in the plasmid was replaced with the maltose-inducible promoter malA promoter. ICCG Transformed into Bacillus subtilis, the monoclonal strain was screened by LB resistance plate (containing 50 μg / mL kanamycin) and identified by colony PCR and sequencing to obtain the positive expression strain B. subtilis MATE01 / malA-AprE-LCC ICCG , which is the recombinant Bacillus subtilis of the present invention. The primer sequences used in the construction process of the above recombinant Bacillus subtilis are shown in Table 1.

[0034] B. subtilis MATE01 / PBE-S-AprE-LCC ICCG and B.subtilis MATE01 / malA-AprE-LCC ICCG LCC ICCG The expression capacity was tested to compare the effects of the constitutive promoter aprE promoter and the maltose-inducible promoter malA promoter on LCC ICCG The strain B. subtilis MATE01 / PBE-S-AprE-LCC ICCG and B.subtilis MATE01 / malA-AprE-LCC ICCG The strain B. subtilis MATE01 / PBE-S-AprE-LCC was inoculated into the fermentation medium at a volume of 5% v / v for shake flask fermentation experiments. ICCG The fermentation was carried out at 37°C and 200 rpm for 48 h. The strain B. subtilis MATE01 / malA-AprE-LCC ICCG First, ferment and culture at 37°C and 200 rpm for 4 to 6 hours until the OD 600 =0.6, 0.05 g / mL maltose was added to the culture medium, and the fermentation was continued at 37°C and 200 rpm for 48 h. During the fermentation process, the bacterial biomass and extracellular enzyme activity in the fermentation broth were detected (the detection method is the same as above). The experimental results are as follows Figure 2 The results showed that the maltose-inducible malA promoter exhibited excellent LCC ICCG Expression promoter ability, strain B. subtilis MATE01 / malA-AprE-LCC ICCGThe extracellular enzyme activity reached 46.2U / mL, which is the strain B.subtilis MATE01 / PBE-S-AprE-LCC ICCG More than five times.

[0035] Table 1 Primer sequences used in the construction of recombinant Bacillus subtilis

[0036]

[0037]

[0038] Example 2 Optimization of the concentration of maltose as an inducer in the fermentation medium

[0039] The strain B. subtilis MATE01 / malA-AprE-LCC ICCG Inoculate into fermentation medium for shake flask fermentation experiment, ferment at 37℃, 200rpm for 4-6h. 600 =0.6, maltose with final concentrations of 0.01, 0.02, 0.03, 0.04 and 0.05 g / mL was added to the fermentation medium respectively, and fermentation was induced at 37°C, 200 rpm for 36 h. After the fermentation was completed, the bacteria were collected by centrifugation (12000 rpm, 10 min), dried at 60°C to measure the dry cell weight, and the extracellular enzyme activity in the supernatant was measured (the detection method was the same as in Example 1).

[0040] The experimental results are as follows Figure 3 As shown in the figure, with the increase of maltose inducer concentration, extracellular enzyme activity and cell dry weight gradually increased. When the concentration of maltose increased from 3% to 5%, the biomass of the recombinant strain increased significantly, but the extracellular enzyme activity only increased slightly. Therefore, it is speculated that when the concentration of inducer maltose exceeds 3%, most of the maltose is used to meet the growth needs of the recombinant strain itself. Considering the input-output ratio, the economic benefit can be maximized when the concentration of inducer maltose in the fermentation medium is 3%.

[0041] Example 3 LCC ICCG Characterization of PET film degradation by enzymes

[0042] The fermentation supernatant of 5% maltose-induced fermentation in Example 3 was purified by nickel column, and the pure enzyme was collected for PET film degradation verification. ICCG The enzyme was subjected to SDS-polyacrylamide gel electrophoresis, and the results were as follows Figure 4 0.2 g of PET film was placed in 50 mL of 50 mM phosphate buffer (pH 8.0), and 0.4 mg of LCC was added ICCGPure enzyme was reacted at 72°C and 200 rpm for 72 h. After the reaction was terminated, the residual PET film was rinsed with ultrapure water to remove the enzyme molecules and salt ions adsorbed on the surface, and then placed in a 50°C constant temperature drying oven to dry to constant weight. The morphology was characterized by scanning electron microscopy (SEM) and the mass loss rate was simultaneously measured. The results are shown in Figure 5 As shown, LCC ICCG The surface of the PET film treated with pure enzyme showed obvious erosion morphology, accompanied by the formation of a large number of microporous structures, and its mass loss rate reached 82%. SEM characterization and quantitative analysis results jointly confirmed that the LCC obtained by the Bacillus subtilis expression system ICCG It has significant hydrolase activity and can effectively degrade PET polymer matrix.

[0043] The present invention provides a method for efficiently expressing plastic degrading enzyme LCC ICCG There are many methods and approaches to realize the recombinant Bacillus subtilis and its construction method and application. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the protection scope of the present invention. All components not specified in this embodiment can be realized by existing technologies.

Claims

1. A LCC that efficiently expresses plastic-degrading enzymes ICCG The recombinant Bacillus subtilis is characterized in that The recombinant Bacillus subtilis overexpresses the plastic degradation enzyme LCC ICCG and signal peptide.

2. The highly efficient expression of plastic degrading enzyme LCC according to claim 1 ICCG The recombinant Bacillus subtilis is characterized in that The plastic degrading enzyme LCC ICCG The nucleotide sequence of the coding gene is shown in SEQ ID NO.

1.

3. The highly efficient expression of plastic degrading enzyme LCC according to claim 1 ICCG The recombinant Bacillus subtilis is characterized in that The signal peptide is SP Epr、 SP YjcM、 SP AprE、 SP Bpr、 SP BglS and SP YbdG Any one of the foregoing, whose amino acid sequences are shown in SEQ ID NOs. 2 to 7 respectively.

4. The highly efficient expression of plastic degrading enzyme LCC according to claim 1 ICCG The recombinant Bacillus subtilis is characterized in that The plastic degrading enzyme LCC ICCG The coding gene and the coding gene of the signal peptide are expressed under the control of the maltose promoter; the nucleotide sequence of the maltose promoter is shown in SEQ ID NO.

8.

5. The highly efficient expression plastic degrading enzyme LCC according to any one of claims 1 to 4 ICCG A method for constructing a recombinant Bacillus subtilis, characterized in that: Plastic degrading enzyme LCC ICCG The coding gene is connected to a vector to construct a first recombinant expression vector, the original signal peptide coding gene in the first recombinant expression vector is replaced with the coding gene of the signal peptide by PCR technology, a second recombinant expression vector is constructed, the original promoter in the second recombinant expression vector is replaced with a maltose-inducible promoter by PCR technology, a third recombinant expression vector is constructed, and the third recombinant expression vector is introduced into Bacillus subtilis to obtain.

6. The construction method according to claim 5, characterized in that: The vector is a pBE-S Bacillus subtilis secretory expression vector plasmid; the Bacillus subtilis is Bacillus subtilis MATE01.

7. The highly efficient expression plastic degrading enzyme LCC according to any one of claims 1 to 4 ICCG Recombinant Bacillus subtilis produces plastic-degrading enzyme LCC in fermentation ICCG Application in.

8. The use according to claim 7, characterized in that: The recombinant Bacillus subtilis is inoculated into a fermentation medium and cultured and fermented until the OD of the recombinant Bacillus subtilis in the fermentation medium reaches 0. 600 is 0.6-0.8, and maltose is added to the fermentation medium to induce fermentation for 36-72 hours.

9. The use according to claim 8, characterized in that: The added amount of maltose is 0.01-0.05 g / mL.

10. The use according to claim 8, characterized in that: The culture fermentation and the induced fermentation are carried out at a temperature of 30-37° C. and a rotation speed of 200-250 rpm.

Images