A marine-derived cutinase mutant, its preparation method and application

Abstract

The application discloses a marine-derived cutinase mutant and a preparation method and application thereof. Compared with a wild-type strain, the mutant of the marine-derived cutinase NSHC has an optimal temperature increased by 10 DEG C, an enzyme activity increased by 3.7 times, and the amount of a final product in the process of degrading PBAT is increased by 3.35 times. The mutant of the marine-derived cutinase NSHC enriches the types of marine-derived cutinases, can effectively degrade PBAT, improves plastic pollution in the environment, and has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of enzyme engineering, and particularly relates to a cutinase mutant of marine origin and a preparation method and application thereof. BACKGROUND

[0002] With the large-scale production of plastic products, while changing people's traditional production and lifestyle and bringing great convenience to people's life, its waste also accumulates in the environment in large quantities, causing serious burden to the global ecological environment. Among them, a small part can be recycled and reused, and most of them are treated by incineration and landfill, becoming one of the important sources of environmental pollution. The plastics in the environment are exposed to sunlight and rain, becoming microplastics (plastic particles with a particle size of less than 5 mm) that are smaller and more difficult to handle. Every year, a large amount of microplastic waste enters the marine ecosystem, and in the sampling of more than 20 kinds of common fish with high economic value, microplastics are found in 90% of the fish samples. Microplastics are enriched in the food chain through plankton, shrimps and crabs, and enter the human body, causing great harm to the human body.

[0003] Poly(butylene adipate-co-terephthalate) (PBAT) is a kind of aliphatic-aromatic copolyester formed by polycondensation reaction of butylene adipate (BA) and butylene terephthalate (BT), which has excellent biodegradability while maintaining the good performance of aromatic polyester. Compared with other biodegradable polyesters, PBAT has better ductility, elongation at break, heat resistance and impact performance, and is widely used in food packaging, agricultural films, textiles and other fields. However, with the wide application of PBAT, its pollution to the environment has become increasingly apparent, and the accumulation and degradation of PBAT have caused great pressure on the environment. All the time, the biological enzyme method is considered as one of the effective ways to degrade plastics because of its green environmental protection and no harmful pollutants to the environment. However, PBAT has a tight and regular crystalline molecular structure, and the polymer fibers are arranged very tightly, which makes the efficiency of enzyme degradation of PBAT poor.

[0004] In the current research on PBAT biodegradation method, PBAT is usually degraded under the condition of 70% humidity and 55℃ temperature. Since this condition is difficult to achieve in the natural environment, PBAT is difficult to degrade under the condition of the natural environment, and the weight loss rate is only 3% in 364 days in seawater, indicating that it has almost no degradability in seawater. Therefore, it is urgent to develop a biodegradable technology with good performance.

[0005] Cutinase can hydrolyze cutin macromolecules, soluble polyesters and various synthetic polyesters, belongs to the alpha / beta hydrolase family, and has the typical catalytic triad of esterase (serine, histidine, aspartic acid), and has a wide application in many fields such as textile industry, food industry and chemical industry, and has a good prospect in the current PBAT degradation process. At present, there are few reports on cutinase degrading PBAT, and PBAT can be degraded into intermediate product 4-((4-hydroxybutoxy)carbonyl)benzoic acid (1,4-benzenedicarboxylic acid, BTa) and final product terephthalic acid (TPA), but the degradation rate is generally low, and the effect in practical application is not ideal. Therefore, it is of great significance to further explore the cutinase with high efficiency of degrading PBAT. The patent constructs a marine source cutinase mutant with good activity to PBAT, and the degradation of PBAT in the environment has a certain application prospect. SUMMARY

[0006] The purpose of the present application is to overcome the shortcomings and deficiencies of the prior art, and to provide a marine source cutinase mutant.

[0007] Another purpose of the present application is to provide a preparation method of the marine source cutinase mutant.

[0008] Still another purpose of the present application is to provide the application of the marine source cutinase mutant.

[0009] The purpose of the present application is realized by the following technical scheme:

[0010] A marine source cutinase mutant is at least one of the following:

[0011] NSHC-60, the amino acid sequence of which is shown as SEQ ID NO. 3;

[0012] NSHC-120, the amino acid sequence of which is shown as SEQ ID NO. 5;

[0013] NSHC-180, the amino acid sequence of which is shown as SEQ ID NO. 7;

[0014] NSHC-240, the amino acid sequence of which is shown as SEQ ID NO. 9.

[0015] The marine source cutinase mutant is a chimeric body obtained by dividing the marine source cutinase NSHC into different length peptide segments from the N-terminal of the amino acid sequence according to the primary sequence, and replacing the peptide segments of different lengths with the peptide segments of corresponding length of the classic cutinase Cut2.

[0016] The nucleotide sequence of the coding gene of the marine-derived cutinase NSHC is shown as SEQ ID NO. 1.

[0017] The nucleotide sequence of the coding gene of the classical cutinase Cut2 is shown as SEQ ID NO. 2.

[0018] The coding gene of the marine-derived cutinase mutant has a nucleotide sequence obtained according to the codon usage.

[0019] Preferably, the coding gene of the marine-derived cutinase mutant is:

[0020] NSHC-60, the nucleotide sequence of the coding gene of which is shown as SEQ ID NO. 4;

[0021] NSHC-120, the nucleotide sequence of the coding gene of which is shown as SEQ ID NO. 6;

[0022] NSHC-180, the nucleotide sequence of the coding gene of which is shown as SEQ ID NO. 8;

[0023] NSHC-240, the nucleotide sequence of the coding gene of which is shown as SEQ ID NO. 10.

[0024] A recombinant expression vector comprising the coding gene of the marine-derived cutinase mutant.

[0025] The expression vector is pET24a.

[0026] An engineered bacterium comprising the recombinant expression vector.

[0027] The starting strain of the engineered bacterium is Escherichia coli BL21 (DE3).

[0028] The preparation method of the marine-derived cutinase mutant comprises the following steps:

[0029] (1) connecting the coding gene of the marine-derived cutinase mutant to a vector and introducing it into an engineered bacterium;

[0030] (2) culturing the engineered bacterium to express the protein, and obtaining the marine-derived cutinase mutant after purifying the product.

[0031] The nucleotide sequence of the coding gene of the marine-derived cutinase mutant in step (1) is shown as SEQ ID NO. 2.

[0032] The vector in step (1) is pET24a.

[0033] The engineered bacterium in step (1) is Escherichia coli BL21 (DE3).

[0034] The marine-derived cutinase mutant, the recombinant expression vector or the engineering bacteria described above are applied in degrading PBAT.

[0035] The marine-derived cutinase mutant, the recombinant expression vector or the engineering bacteria described above are applied in degrading biodegradable plastics.

[0036] The marine-derived cutinase mutant, the recombinant expression vector or the engineering bacteria described above are applied in the textile industry.

[0037] Compared with the prior art, the present application has the following beneficial effects:

[0038] (1) In the present application, the inventors, based on their rich experience, obtained the marine-derived cutinase NSHC mutant (hereinafter referred to as NSHC-120) engineering bacteria through a large number of experimental screening; then expressed the NSHC-120 recombinant protein engineering bacteria, and carried out separation and purification to obtain the protein, enzyme activity determination and optimum temperature determination; finally, the obtained NSHC-120 protein was applied to degrade PBAT to improve the yield of PBAT degradation as the end product TPA.

[0039] (2) The mutant NSHC-120 of the marine-derived cutinase of the present application has an optimum temperature 10℃ higher than that of the wild-type cutinase, an enzyme activity 3.7 times higher, and an amount of the end product of PBAT degradation 3.35 times higher. The mutant of the marine-derived cutinase NSHC of the present application enriches the types of marine-derived cutinases, can effectively degrade PBAT, improves the plastic pollution in the environment, and has a broad application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 SDS-PAGE protein map of the four marine-derived cutinase NSHC mutants in Example 2 of the present application.

[0041] Figure 2 Optimum temperature test results of the four marine-derived cutinase NSHC mutants and the wild type in Example 3 of the present application.

[0042] Figure 3 Enzyme activity analysis of the four marine-derived cutinase NSHC mutants and the wild type (wild type NSHC; mutants NSHC-60, NSHC-120, NSHC-180, NSHC-240) in Example 4 of the present application.

[0043] Figure 4 Comparison results of the amount of the end product (TPA) generated by the four marine-derived cutinase NSHC mutants and the wild type in degrading PBAT in Example 5 of the present application. DETAILED DESCRIPTION

[0044] The application will be described in further detail below with reference to the embodiments and drawings, but the embodiments of the application are not limited thereto.

[0045] Unless otherwise specified, the following experimental procedures in the embodiments are generally carried out according to conventional experimental conditions or according to the experimental conditions recommended by the reagent companies. The materials, reagents, etc. used are reagents and materials obtained from commercial channels, unless otherwise specified.

[0046] The experimental methods in the following embodiments of the application, for which no specific conditions are specified, are generally carried out according to conventional conditions, for example, the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Habor Laboratory Press 1989), or the conditions recommended by the manufacturer.

[0047] The materials and reagents used in the following embodiments: the plasmids pET24a-NSHC and pET24a-Cut2 were synthesized by Shenguo Bioengineering (Shanghai) Co., Ltd.; the empty plasmid pET24a was preserved in the laboratory of the applicant; the E. coli BL21 (DE3) competent cells were purchased from Weidi Biological Technology Co., Ltd.; the plasmid extraction kit was purchased from Shenguo Bioengineering (Shanghai) Co., Ltd.; the seamless cloning kit was purchased from Zhongmei Taihe Biotechnology (Beijing) Co., Ltd.; the PBAT powder (150 μm) was purchased from Shanghai Fentai Plasticizing Co., Ltd.; and all other chemical drugs were purchased from Sigma-Aldrich, TCI or Aldrich, which were of the highest purity and could be used without further purification.

[0048] Example 1 Construction of expression vectors of marine-derived cutinase mutants

[0049] In this embodiment, the cutinase NSHC is divided into four different length peptide segments (the first 60 amino acids, the first 120 amino acids, the first 180 amino acids, and the first 240 amino acids) according to the primary sequence from the N-terminal of the amino acid sequence. The four different length peptide segments are locally replaced with the corresponding length peptide segments of Cut2 to construct four chimeras, which are named NSHC-60, NSHC-120, NSHC-180, and NSHC-240, respectively. First, the different length peptide segments and the corresponding vectors are amplified by PCR technology, and the different length peptide segments are replaced into the corresponding positions of Cut2 by using the seamless cloning technology to obtain the cutinase mutant expression vectors.

[0050] Specifically, the following steps are included:

[0051] 1. Eight pairs of primers are designed for the sequence of the marine-derived cutinase NSHC and the sequence of the classic cutinase Cut2 (as shown in Table 1)

[0052] Table 1 primer pairs

[0053]

[0054] 2. Using the plasmid pET24a-NSHC of marine-derived cutinase as a template, primer pairs 1, 3, 5, and 7 shown in Table 1 as primers, different length NSHC fragments were amplified. Using the plasmid pET24a-Cut2 as a template, primer pairs 2, 4, 6, and 8 shown in Table 1 as primers, different length Cut2 fragments with pET24a were amplified. The PCR amplification reaction system is shown in Table 2.

[0055] Table 2 PCR reaction system

[0056]

[0057] The PCR reaction program is as follows: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 15 s, 55℃ annealing for 15 s, 72℃ extension for 3 min, 30 cycles; 72℃ extension for 5 min.

[0058] 3. The NSHC fragments and the Cut2 fragments with pET24a were connected by seamless cloning. The seamless cloning system is shown in Table 3. The program of seamless cloning is as follows: 50℃, 1 h. The pET24a-NSHC-120 positive plasmid was obtained, and then was transferred into the TOP10 cloning strain, which was cultured at 37℃ overnight, and then was verified by colony PCR. The bacterial liquid verified correctly was sequenced, and the plasmid with accurate sequencing was extracted to obtain the recombinant plasmids pET24a-NSHC-60, pET24a-NSHC-120, pET24a-NSHC-180, and pET24a-NSHC-240.

[0059] Table 3 seamless cloning system

[0060]

[0061] Example 2 Expression and purification of marine-derived cutinase mutants

[0062] 1. The recombinant plasmids pET24a-NSHC-60, pET24a-NSHC-120, pET24a-NSHC-180, and pET24a-NSHC-240 obtained in Example 1 were transformed into E. coli BL21 (DE3) by heat shock transformation. The strain was cultured at 37℃ in LB medium containing 50 μg / mL kanamycin until OD600 reached 0.4-0.6. 600When the OD600 is 0.6-0.8, isopropyl β-D-l-thiogalactopyranoside (IPTG) is added to a final concentration of 0.2 mM to induce expression, and the culture is incubated at 16°C for 20 hours. The bacteria are collected by centrifugation at 4000 rpm for 30 minutes at 4°C, and the NSHC-60, NSHC-120, NSHC-180, and NSHC-240 protein crude enzyme solution is obtained by ultrasonic disruption.

[0063] 2. The NSHC-60, NSHC-120, NSHC-180, and NSHC-240 crude enzyme solution is purified by affinity chromatography, and the target protein is exchanged into a 20 mM PBS buffer solution at pH 8.0 for stable storage. After the above steps, the protein with a purity of more than 95% is obtained, and the SDS-PAGE detection result is shown in FIG. 2. Figure 1 Figure 1 It can be seen that the four mutants have good purification effect. After the protein is collected, it is concentrated, divided, and frozen quickly with liquid nitrogen, and stored at -20°C for standby use.

[0064] 3. Determination of the concentration of the target protein: 20 μL of the protein solution to be tested is mixed with 200 μL of Bradford reagent, and the A595 is determined after reaction at room temperature for 5 minutes. The concentration of the mutant NSHC-60, NSHC-120, NSHC-180, and NSHC-240 is calculated to be 9.58, 7.05, 9.05, and 9.28 mg / mL (16 L of fermentation broth), respectively, in combination with the standard curve.

[0065] Example 3 Determination of the optimum temperature of the marine-derived cutinase mutant

[0066] 1. The cutinase can hydrolyze the p-nitrophenyl butyrate (pNPB) substrate to generate p-nitrophenol (pNP) and acid hydrolysis products. The pNP can be detected at 405 nm. Based on this principle, the marine-derived cutinase NSHC and its four mutants selected in the application are detected for enzyme activity using p-nitrophenyl butyrate as the reference substrate. The hydrolysis enzyme activity is defined as: the amount of enzyme required to hydrolyze 1 μmol of pNPB to generate p-nitrophenol per unit time (min) under certain conditions, which is one enzyme activity unit (U).

[0067] 2. The 100 μL reaction system includes 80 μL of PBS buffer solution, 10 μL of 10 mM pNPB, and 10 μL of enzyme solution. After reaction at the optimum reaction temperature for 1 minute, anhydrous ethanol is added in an equal volume to the system to terminate the reaction, and the OD value of pNP at 405 nm is determined using an enzyme marker. Three parallel groups are set for each protein sample, and the blank group is 10 μL of denatured protein enzyme solution. The average value of each parallel sample is recorded.

[0068] ​3. Determination of standard curve: 0, 1, 2, 4, 6, 8, 10 μL of p-nitrophenol with a concentration of 10 mM was added to a 96-well enzyme plate, respectively, 3 parallel groups were set for each concentration, and the solution in each well was supplemented to 100 μL with a buffer solution, after the reaction was completed, anhydrous ethanol was added to terminate the reaction, 3 technical replicates were made for each group of data, and the linear relationship between the OD value of pNP at 405 nm (Y axis) and the concentration of p-nitrophenol (X axis) was determined.

[0069] In this example, 5℃ was selected as the interval, and 20℃, 25℃, 30℃, 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, 70℃, 75℃, 80℃ were selected as the temperature gradient to determine the optimum reaction temperature. The determination system used pNPB as the substrate, and the enzyme activity was determined by the above method. Each group of data was made in 3 technical replicates, and the average value of the measured results was the relative enzyme activity.

[0070] The results are shown in Figure 2 The results are shown in

[0071] Example 4 Activity determination of marine-derived cutinase mutants

[0072] In this example, the specific enzyme activity of marine-derived cutinase NSHC wild type and its mutants NSHC-60, NSHC-120, NSHC-180, NSHC-240 was determined, and the experimental method was referred to Example 3, and the results are shown in Figure 3 The results are shown in

[0073] Example 5 Determination of the effect of mutant NSHC-120 on PBAT degradation

[0074] 1. 50 mg of PBAT powder (150 μm) purchased from Shanghai Foton Plastic Co., Ltd. was weighed and added to 2 mL of enzyme solution diluted to 0.2 mg / mL, and reacted at 16°C, 1500 rpm for 120 h, and sampled at 12, 24, 48, 72, 96, 120 h, respectively. 150 μL of the reaction product was taken, 143.5 μL of chromatographic grade methanol solution and 6.5 μL of 6N hydrochloric acid solution were added thereto, vortexed uniformly, filtered with a 0.22 μm membrane, and subjected to high performance liquid chromatography detection.

[0075] 2. HPLC detection conditions: Waters C18 column (250 mm x 4.6 mm, 5 μm) was used. Chromatographic system settings: flow rate 0.8 mL / min; injection volume 10 μL; detection wavelength 240 nm; column oven temperature 30°C; elution conditions: 40% chromatographic methanol and 60% 1 mM sulfuric acid aqueous solution for 3 min, then gradient increased to 50% chromatographic methanol and 50% 1 mM sulfuric acid aqueous solution within 10 min, and finally washed with 100% chromatographic methanol for 5 min.

[0076] 3. The amount of final product TPA generated by wild type NSHC and mutants NSHC-60, NSHC-120, NSHC-180, NSHC-240 in degrading PBAT was compared, and the results are shown in Table 1. Figure 4 As shown in Table 1, the wild type degrades PBAT to generate 4.70 mM of final product TPA; the mutants NSHC-60, NSHC-120, NSHC-180, NSHC-240 generate 12.84, 15.76, 13.18, 5.54 mM of final product TPA, respectively. Compared with other mutants, the TPA monomer in NSHC-120 degradation system is the highest, which is 3.35 times of the wild type cutinase NSHC. The experimental results prove that the mutant NSHC-120 has higher efficiency in degrading PBAT powder, and has a wider application prospect in this field.

[0077] The technical features of the above-described embodiments can be combined in any manner. To make the description concise, all possible combinations of the technical features in the above-described embodiments are not described, but as long as the combinations of the technical features do not contradict, they should be considered within the scope of the present disclosure.

[0078] The above-described embodiments only express several embodiments of the present application, which are described in a more specific and detailed manner, but should not be construed as limiting the scope of the patent. It should be noted that for ordinary skilled persons in the art, several modifications and improvements can be made without departing from the concept of the present application, which are within the scope of the present application. Therefore, the protection scope of the present application should be subject to the appended claims.

Claims

1. A marine-derived cutinase mutant, characterized in that at least one of the following: NSHC-60, the amino acid sequence of which is shown as SEQ ID NO. 3; NSHC-120, the amino acid sequence of which is shown as SEQ ID NO. 5; NSHC-180, the amino acid sequence of which is shown as SEQ ID NO. 7; NSHC-240, the amino acid sequence of which is shown as SEQ ID NO.

9.

2. The marine-derived cutinase mutant according to claim 1, characterized in that: the coding gene of the marine-derived cutinase mutant is obtained according to the codon usage rule.

3. The marine-derived cutinase mutant according to claim 1, characterized in that: the coding gene of the marine-derived cutinase mutant is: NSHC-60, the nucleotide sequence of the coding gene of which is shown as SEQ ID NO. 4; NSHC-120, the nucleotide sequence of the coding gene of which is shown as SEQ ID NO. 6; NSHC-180, the nucleotide sequence of the coding gene of which is shown as SEQ ID NO. 8; NSHC-240, the nucleotide sequence of the coding gene of which is shown as SEQ ID NO.

10.

4. A recombinant expression vector, characterized in that: the coding gene of the marine-derived cutinase mutant according to any one of claims 2 or 3 is included.

5. The recombinant expression vector according to claim 4, characterized in that: the expression vector is pET24a.

6. An engineered bacterium, characterized in that: the recombinant expression vector according to any one of claims 4 or 5 is included.

7. The engineered bacterium according to claim 6, characterized in that: the starting strain of the engineered bacterium is Escherichia coli BL21 (DE3).

8. Use of the marine-derived cutinase mutant according to any one of claims 1 to 3, the recombinant expression vector according to any one of claims 4 or 5, or the engineered bacterium according to any one of claims 6 or 7 in degrading PBAT.

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