Novel esterase as well as coding gene and new application thereof

Abstract

The invention discloses novel esterase as well as a coding gene and new application thereof. The amino acid sequence of the novel esterase provided by the invention is as shown in SEQ ID NO. 1, and the coding gene of the novel esterase is as shown in SEQ ID NO. 2. The novel esterase provided by the invention has excellent catalytic performance on hydrolysis of ester compounds, can be used for degrading plastic products containing plasticizers, and has a catalytic hydrolysis effect on phthalate compounds; and the method can be used for degrading phthalic acid ester compounds in food and other materials containing phthalic acid ester plasticizers.

Description

Technical Field

[0001] The invention relates to the field of bioengineering technology and environmental protection technology, in particular to a novel esterase and a coding gene thereof and a new use thereof. Background Art

[0002] Esterase is a class of enzymes that catalyze esterification and ester hydrolysis reactions, can degrade ester compounds, and are widely used in the chemical, food and biotechnology fields. In particular, esterase also has the potential for environmental remediation, especially in dealing with plasticizer pollution. Esterase can catalyze the hydrolysis of ester bonds and decompose plasticizers into corresponding alcohols and acids, thereby reducing the residual plasticizers in the environment and repairing the polluted environment. Phthalates are a common plasticizer used to enhance the softness and plasticity of polymers and are widely used in products such as toys, food packaging materials, medical blood bags and hoses. During the production, use and disposal of the above products, phthalates can enter the soil and water bodies through the infiltration of landfill leachate, rainwater washing, etc. In addition, phthalates can also enter the atmosphere through the volatilization of plasticizers in plastic products and the incineration of plastic waste. Some studies have shown that certain phthalates may have negative effects on the reproductive and endocrine systems, and may be carcinogenic and teratogenic, seriously threatening the health of the people and the environment.

[0003] For many years, people have been studying the method of remediating environmental pollution with microorganisms, and believe that this method has the potential to safely remove pollutants from the environment. However, microorganisms need a certain amount of time to adapt to the new environment before they can produce the required degradation enzymes. Therefore, the direct application of enzymes is more advantageous and is particularly suitable for rapid repair of polluted environments, especially low-concentration plasticizer pollution, such as vegetables, fruits and tea. This method can quickly degrade pollutants and reduce potential harm to the environment and food.

[0004] Therefore, it is necessary to find effective esterases to hydrolyze phthalates, thereby promoting the treatment of environments containing these compounds. In addition, it is necessary to have a deeper understanding of the interaction mechanism between phthalates and esterases, and to artificially modify and evolve esterases, in order to better utilize esterases for environmental remediation. Summary of the invention

[0005] In view of the above technical problems, the present invention provides a novel esterase and its encoding gene and new use.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] In one aspect, the present invention provides a novel esterase, the amino acid sequence of the novel esterase is shown as SEQ ID NO.1.

[0008] In another aspect, the present invention provides a gene encoding the novel esterase, the sequence of which is shown in SEQ ID NO.2.

[0009] In yet another aspect, the present invention provides a vector comprising the above encoding gene.

[0010] In another aspect, the present invention provides a genetically engineered bacterium comprising the above encoding gene.

[0011] In yet another aspect, the present invention provides use of the novel esterase, the encoding gene, the vector or the genetically engineered bacteria in catalyzing the hydrolysis of ester compounds.

[0012] Preferably, the invention is used for catalyzing the hydrolysis of ester compounds contained in water or soil.

[0013] Preferably, the ester compound is phthalate or p-nitrophenol ester.

[0014] In some specific embodiments, the phthalate esters include dibutyl phthalate, dihexyl phthalate, diethylhexyl phthalate and laurate phthalate.

[0015] In certain specific embodiments, the p-nitrophenol ester may be p-nitrophenol butyrate, p-nitrophenol hexanoate or p-nitrophenol octanoate.

[0016] In the technical solution of the present invention, the pH value at which the novel esterase catalyzes the hydrolysis of ester compounds is 8.0-9.0.

[0017] In the technical solution of the present invention, the temperature at which the novel esterase catalyzes the hydrolysis of ester compounds is 30-40°C.

[0018] The above technical solution has the following advantages or beneficial effects:

[0019] The present invention provides a novel esterase having excellent catalytic performance for the hydrolysis of ester compounds. The optimal pH for the novel esterase catalyzing the hydrolysis of ester compounds provided by the present invention is 8.0-9.0, and the optimal temperature is 30-40° C. In addition, the novel esterase has high degradation activity for common phthalate plasticizers, especially long side chain phthalates, and its degradation efficiency reaches 90%. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is a three-dimensional structure diagram of the novel esterase in Example 1;

[0021] Figure 2 This is a protein gel image of the novel esterase in Example 3;

[0022] Figure 3The catalytic activity diagram of the novel esterase in Example 4 at different pH values;

[0023] Figure 4 is a graph showing the catalytic activity of the novel esterase in Example 5 at different temperatures;

[0024] Figure 5 This is a statistical graph of the degradation rate of phthalate compounds by the new esterase in Example 6. DETAILED DESCRIPTION

[0025] The following embodiments are only some embodiments of the present invention, rather than all embodiments. Therefore, the detailed description in the embodiments of the present invention provided below is not intended to limit the scope of the present invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making creative work belong to the protection scope of the present invention.

[0026] In the present invention, unless otherwise specified, all equipment and raw materials can be purchased from the market or are commonly used in the industry. The methods in the following examples, unless otherwise specified, are conventional methods in the art or follow the commercial instructions of the kit.

[0027] The sources of materials in the following examples are as follows: Aspergillus polydophorus W1 preserved in the laboratory; 2×Rapid Taq PCR Master Mix, restriction endonucleases BamHI and Xho I are all from Novazon; expression plasmid pET-28a, rapid T4 DNA ligation kit, E. coli DH5α and E. coli BL21 (DE3) competent cells are from Anhui General Biotechnology; agarose gel DNA recovery kits are all purchased from Shanghai Biotech.

[0028] Example 1 Esterase gene cloning and analysis

[0029] First, the total RNA of Aspergillus polydorum W1 was extracted and reverse transcribed to obtain Aspergillus polydorum cDNA. The open reading frame of the new esterase was obtained according to the sequencing results, and the upstream and downstream primers were designed:

[0030] Primers1: 5'-CC GGATCC ATGGGTGACTACGGTTCTCCGG-3'; (SEQ IDNo.3)

[0031] Primers2: 5'-CC CTCGAG CAAAACTGCACTAATCTGCATCTCGAC-3'; (SEQ ID No. 4)

[0032] Among them, the underlined part of the upstream primer is the restriction endonuclease BamHI cleavage site, and the underlined part of the downstream primer is the restriction endonuclease XhoI cleavage site.

[0033] Using cDNA as a template, PCR amplification was performed to obtain the target gene. The PCR system was: 2×Rapid Taq PCRMaster Mix 25μL, 2μL of upstream primer and downstream primer (10μM), 1μL of genomic DNA of Aspergillus polydurans (100ng / μL), and 50μL of ddH2O. The PCR amplification program was: 95℃ pre-denaturation for 3min and then 30 cycles of the following: 95℃ denaturation for 15s, 60℃ annealing for 15s, and 72℃ extension for 90s; after the cycle, the final extension was 72℃ for 5min. After gel electrophoresis purification of the PCR amplification product, the target fragment was recovered using a DNA recovery kit. After DNA sequencing, the full length of the gene was 1169bp, and the ORF sequence encoding the esterase gene was 1169bp in length. Its base sequence is shown in SEQ ID NO.2, encoding a total of 377 amino acids, and its amino acid sequence is shown in SEQ ID NO.1. The molecular weight of the encoded translation protein is predicted to be 40.83kDa, and it is named M4.

[0034] In this example, the three-dimensional structure of protein M4 is as follows Figure 1 As shown, a is the active pocket of esterase M4, and b is the amino acid residue at the active pocket.

[0035] Example 2 Preparation of esterase recombinant expression plasmid and recombinant expression transformant

[0036] The esterase DNA fragment obtained by PCR amplification in Example 1 and the pET-28a empty plasmid were double-digested with restriction endonucleases BamHI and Xho I, and then purified by agarose gel electrophoresis and recovered by a DNA gel recovery kit. The recovered enzyme-digested target fragment and the empty vector were ligated at 4°C for 16 hours under the action of T4 DNA ligase to obtain a recombinant plasmid.

[0037] The obtained recombinant plasmid was transformed into E.coli DH5α, spread on LB medium plates containing 100 mg / L kanamycin, and cultured at 37°C for 1-2 hours. The grown colonies were verified by colony PCR, and positive clones that successfully amplified the target band of about 1169 bp were picked for sequencing verification. The plasmid was extracted using a plasmid extraction kit, electrotransformed into E.coliBL21 (DE3), the degraded bacteria were diluted and spread on LB plates (kanamycin 100 mg / L), cultured at 37°C overnight, and positive clones were picked to obtain recombinant expression transformants.

[0038] Example 3 Induced expression and purification of esterase

[0039] The recombinant expression transformant obtained in Example 2 was inoculated into LB liquid medium (containing 100 mg / L kanamycin) and cultured on a shaker at 37°C for 12 h; the inoculum was inoculated into a conical flask containing 100 mL of LB liquid medium (containing 100 mg / L kanamycin) at a 1% (v / v) inoculum amount and cultured at 37°C and 180 rpm; when the OD of the culture solution reached 600 When the concentration reached 0.6, IPTG was added to a final concentration of 1 mmol / L for induction, and the induction was carried out at 16°C for 24 h; the culture solution was centrifuged at 12000 rpm, the cell pellet was collected, and washed with physiological saline to obtain resting cells; the resting cells were resuspended in 50 mL of PBS buffer (20 mM, pH 7.4), ultrasonically disrupted in an ice water bath, centrifuged to collect the supernatant, and the enzyme solution was purified using a protein purifier. The enzyme solution was run on SDS-PAGE gel as shown in Figure 2 As shown in lanes b, c and d, there is a band with higher concentration below 50K.

[0040] Example 4 Effect of pH on esterase catalytic activity

[0041] Using the pure enzyme obtained in Example 3, the effect of different pH values ​​on esterase activity was determined in the range of pH 6.0 to 10.0 according to the standard method. The buffer solution was phosphate buffer (pH 6.0-8.0), Tris-Hcl buffer (pH 7.2-8.9) and glycine-sodium hydroxide buffer (pH 8.6-10.6). In 1 mL of the above buffer system, p-nitrophenol butyrate (pNPB) was added to a final concentration of 1 mmol / L, preheated to 30°C, and then an appropriate amount of esterase was added, mixed evenly, and after incubation at 30°C for 30 minutes, 1 mL of methanol was added to inactivate the enzyme. The absorbance change at 410 nm was detected on a spectrophotometer, and the p-nitrophenol concentration in the buffer solution of different pH and the activity difference of p-nitrophenol butyrate (pNPB) were determined. The results are as follows: Figure 3 As shown: The pH range of the enzymatic reaction is 8.0-9.0, and the enzyme activity is highest at pH 8.5.

[0042] Example 5 Effect of temperature on esterase catalytic activity

[0043] The pure enzyme solution obtained in Example 3 was used to determine the effect of different temperatures on esterase activity in the range of 30°C-40°C according to the standard method. In 1 mL of the buffer system, p-nitrophenol butyrate (pNPB) was added to a final concentration of 1 mmol / L, preheated for 2 min in an environment of 30-40°C, and then an appropriate amount of esterase was added and mixed evenly. After the reaction was kept in an environment at the same temperature as the preheating temperature for 30 min, 1 mL of methanol was added to inactivate the enzyme, and the absorbance change at 410 nm was detected on a spectrophotometer to analyze the difference in esterase activity under different temperature conditions. The results are as follows: Figure 4 As shown: the optimal temperature range for the enzymatic reaction is 30-40°C, and the esterase hydrolysis activity is the highest at 35°C, which is 410,000 U / g.

[0044] Example 6 Degradation spectrum of phthalate plasticizers by esterase

[0045] The degradation efficiency of esterase on several common phthalate plasticizer substrates was determined. The determination method is: appropriate amount of pure enzyme and substrate are placed in 1 mL of Tris-HCl buffer (pH 8.5, 2% dimethyl sulfoxide), kept in a water bath at 35°C, 1 mL of methanol is added after 10 minutes to terminate the reaction, and high performance liquid chromatography is used for detection. The results are shown in Figure 2. Figure 5 As shown, the esterase can degrade the tested phthalate plasticizers dibutyl phthalate, dihexyl phthalate, and diethylhexyl phthalate, and the degradation efficiency of long side chain phthalates is as high as 90%. Therefore, the esterase is a broad-spectrum hydrolytic esterase.

[0046] 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 scope of protection of the present invention.

Claims

1. A novel esterase, characterized in that: The amino acid sequence of the novel esterase is shown in SEQ ID NO.

1.

2. The gene encoding the novel esterase according to claim 1, characterized in that: The sequence of the coding gene is shown as SEQ ID NO.

2.

3. A vector comprising the encoding gene according to claim 2.

4. A genetically engineered bacterium comprising the coding gene according to claim 2.

5. Use of the novel esterase according to claim 1, the encoding gene according to claim 2, the vector according to claim 3 or the genetically engineered bacteria according to claim 4 in catalyzing the hydrolysis of ester compounds.

6. The use according to claim 5, characterized in that: Application in catalyzing the hydrolysis of ester compounds contained in water or soil.

7. The use according to claim 5, characterized in that: The ester compound is phthalate or p-nitrophenol ester.

8. The use according to claim 5, characterized in that: The phthalate is selected from any one of dibutyl phthalate, dihexyl phthalate, diethylhexyl phthalate and laurate phthalate; The p-nitrophenol ester is selected from any one of p-nitrophenol butyrate, p-nitrophenol hexanoate and p-nitrophenol octanoate.

9. The use according to claim 5, characterized in that: The pH value at which the novel esterase catalyzes the hydrolysis of ester compounds is 8.0-9.

0.

10. The use according to claim 5, characterized in that: The temperature at which the novel esterase catalyzes the hydrolysis of ester compounds is 30-40°C.

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