Esterase Est909 sourced from bacillus subtilis and used for degrading phthalate, coding gene and application of esterase Est909

Abstract

The invention belongs to the technical field of biological genetic engineering, and particularly relates to a bacillus subtilis sourced esterase Est909 for degrading phthalate, a coding gene and application of the bacillus subtilis sourced esterase Est909. The amino acid sequence of the esterase Est909 is as shown in SEQ ID NO. 1, and the nucleotide sequence of the coding gene of the esterase Est909 is as shown in SEQ ID NO. 2. The invention specifically discloses a bacillus subtilis source esterase Est909 coding gene and construction of an escherichia coli expression vector carrying the gene, and also discloses application of the bacillus subtilis source esterase Est909 in hydrolysis of various diesters of phthalate and monoesters of phthalate. And the catalytic hydrolysis efficiencies of diisobutyl phthalate, dibutyl phthalate and bis (2-ethylhexyl) phthalate of the homologous enzyme PAEIIIase are respectively 1.26 times, 1.30 times and 2.17 times of the catalytic hydrolysis efficiencies of the homologous enzyme PAEIIIase, and the catalytic hydrolysis efficiencies of the homologous enzyme PAEIIIase are respectively 1.30 times, 1.30 times and 2.17 times of the catalytic hydrolysis efficiencies of the homologous enzyme PAEIIIase. The invention firstly provides the III-type enzyme Est909 for efficiently catalyzing and degrading the phthalate and the coding gene of the III-type enzyme Est909.

Description

Technical Field

[0001] The invention belongs to the technical field of biological gene engineering, and particularly relates to an esterase Est909 derived from Bacillus subtilis and a gene thereof, and application of the enzyme in catalyzing the hydrolysis of phthalates. Background Art

[0002] Phthalate esters (PAEs) are common plastic additives that can enhance the flexibility and plasticity of plastic products. PAEs are added to plastics and interact with the plastic skeleton in a non-covalent manner, so they are easily released into the environment as the plastic is used and discarded. Statistics show that the annual output of PAEs reaches 6 million tons, of which more than half are released into the environment as plastic products are discarded, and the concentration of PAEs in some surface water sources is as high as 500 mg / L. Plant growth has an enrichment effect on PAEs and can accumulate in the body. In the early stage of the study, tests on food crops across the country, including wheat, rice, corn and sorghum, found that PAEs were commonly found. Conventional food processing cannot eliminate PAEs. For example, PAEs are commonly detected in liquor, vinegar and tea produced by fermentation with plant-based raw materials, making it difficult to avoid foodborne human intake of PAEs. PAEs have estrogen-like effects, interfere with immune responses, cause neuropsychological diseases, cause reproductive toxicity and abnormal lipid metabolism, and induce childhood obesity. The European Food Safety Authority has assessed that the human body's tolerance limit for different types of PAEs is less than 50 μg / (Kg·d). However, due to the prevalence of PAEs pollution and the necessity of enrichment through the food chain, the potential harm of PAEs to the human body is extremely large. Therefore, the effective treatment of PAEs pollution will not only help protect the ecological environment and human health, but also promote sustainable development, which is in line with the long-term interests of society and the economy.

[0003] PAEs are very stable, and their natural degradation cycles range from tens to thousands of years. In view of the serious harm to human health, the effective treatment of PAEs has received research attention. Existing treatment methods include physical adsorption, chemical degradation, physical adsorption coupled with chemical catalytic degradation, and light-mediated catalytic degradation. Although the above methods are effective, their application in the food field has limitations. For example, PAEs are not completely degraded, and the intermediate products still have physiological toxicity. At the same time, food processing has its own particularity. While effectively reducing the toxicity of PAEs, it is necessary to maintain edibility. The color, aroma, taste and shape of the product cannot be significantly changed due to the treatment. Therefore, there is an urgent need for green and efficient processing technologies with mild conditions.

[0004] Microbial enzymatic treatment of PAEs has become the preferred method for treating food-derived PAEs due to its mild conditions, high efficiency, few by-products and environmental friendliness. Based on the characteristics of ester bond hydrolysis, microbial-derived PAEs ester bond hydrolases are divided into three categories: type I, type II and type III. Among them, type I hydrolases can only hydrolyze the single ester bond of the PAEs side chain; type II hydrolases can only hydrolyze the side chain ester bond of phthalic acid monoesters; and type III hydrolases can completely hydrolyze the diester bonds of the PAEs side chain. Due to the toxicity of phthalic acid monoesters and diesters, type III hydrolases have become the preferred enzyme resource for PAEs treatment. Exploring efficient type III enzymes has become the key to solving food-derived PAEs pollution by enzymatic methods. Summary of the invention

[0005] The present invention aims to provide an esterase Est909 derived from Bacillus subtilis for degrading phthalates and its gene, as well as application of the enzyme in the efficient catalytic degradation of phthalates.

[0006] The invention provides an esterase Est909 derived from Bacillus subtilis, which has the performance of efficiently catalyzing and hydrolyzing phthalates. The amino acid sequence of the esterase Est909 is shown in SEQ ID NO.1.

[0007] The present invention also provides an Escherichia coli expression vector containing the above-mentioned Bacillus subtilis-derived esterase Est909 encoding gene, and the nucleotide sequence of the vector cloning region is shown in SEQ ID NO.2.

[0008] The esterase Est909 from Bacillus subtilis of the present invention is obtained by using the metagenomic database IMG / M, using the sequence of the type III phthalate hydrolase PAEIIIase (GenBank accession no. GTW28_17760) with the highest hydrolysis efficiency reported in the literature as a probe, based on sequence similarity comparison and mining, and then predicting the kinetic parameters k by the catalytic kinetic parameter prediction website. cat and K m , indicating that its potential catalytic efficiency exceeds that of PAEIIIase. The coding sequence of the enzyme was then obtained through whole gene sequence synthesis, and an Escherichia coli heterologous expression engineering bacterium was constructed. The enzyme obtained by inducing expression was used to catalyze the degradation of phthalates.

[0009] The construction method of the E. coli engineering strain carrying the esterase Est909 is as follows: the sequence is synthesized by the whole gene synthesis platform of Beijing Boyi Huada Biogene Technology Co., Ltd., and the gene is integrated into the pET-28a (+) expression vector using the NdeI / XhoI restriction site. After sequencing verification, the plasmid is transformed into E. coli BL21 (DE3) by chemical transformation, and the expression is induced by IPTG to obtain the E. coli cells carrying the esterase Est909. The crude enzyme solution is obtained by breaking the cells, and the purified enzyme catalyzes the hydrolysis of the substrate phthalate ester, and the degradation of the substrate is detected by gas chromatography, and the hydrolysis of the monoester and the generation of phthalic acid (PA) are detected by thin layer chromatography.

[0010] The esterase Est909 of the present invention can be obtained by inducing expression of Escherichia coli engineering bacteria and is used for efficiently catalyzing the hydrolysis of phthalates.

[0011] Experiments have confirmed that the esterase Est909, which was induced and expressed and purified by Escherichia coli engineering bacteria, can hydrolyze diisobutyl phthalate (DiBP), dibutyl phthalate (DBP) and di(2-ethylhexyl)phthalate (DEHP) (the concentration of each substrate is 200 mg / L), and the hydrolysis amounts are 71.44±3.83 mg / L, 70.62±3.77 mg / L, and 47.05±0.99 mg / L, respectively, which are 1.26, 1.30, and 2.17 times higher than the efficiency of PAEIIIase in hydrolyzing the corresponding substrates under the same reaction conditions.

[0012] The beneficial effects of the present invention are as follows: the present invention utilizes biological genetic engineering technology to clone and induce expression to obtain esterase Est909 and its coding gene, and constructs an Escherichia coli expression plasmid containing the esterase Est909 coding gene, and the plasmid is transferred into Escherichia coli, and the enzyme is obtained by inducing expression, and the catalytic system verifies that the enzyme has the ability to efficiently catalyze and hydrolyze phthalates, and the efficiency of catalytic degradation of DiBP, DBP, and DEHP after purification is 1.26, 1.30, and 2.17 times that of PAEIIIase, respectively, and is the type III phthalate hydrolase with the highest hydrolysis efficiency known so far. The present invention is the first to report the known most efficient type III esterase Est909, which can be used to efficiently hydrolyze phthalates. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The amino acid sequence alignment result of esterase Est909 and PAEIIIase

[0014] Figure 2Results of induced expression of the gene encoding esterase Est909

[0015] Figure 3 The original gas chromatography spectrum (A) and thin layer chromatography results (B) of esterase Est909 catalyzing the hydrolysis of phthalates

[0016] Figure 4 Purification results of crude enzyme solution induced by esterase Est909 coding gene

[0017] Figure 5 The results of the quantitative comparison of the catalytic effect of esterase Est909 on the hydrolysis of phthalates (A) and PAEIIIase (BD) DETAILED DESCRIPTION

[0018] The present invention is further described below in conjunction with specific embodiments. The operating steps or conditions not described in detail in the following embodiments are all implemented according to conventional techniques and conditions in the art.

[0019] Example 1 Screening and cloning of an esterase Est909 from Bacillus subtilis that degrades phthalates

[0020] 1.1 Screening steps and selection basis of phthalate degrading enzymes

[0021] Using the sequence of type III hydrolase PAEHIase as a probe, all enzymes with a certain degree of similarity to the probe enzyme were obtained from the metagenomic database IMG / M. Then, dimethyl phthalate (DMP), diethyl phthalate (DEP), DiBP, DBP, DEHP, monomethyl phthalate (MMP), monoethyl phthalate (MEP), monoisobutyl phthalate (MiBP), monobutyl phthalate (MBP), and mono-(2-ethylhexyl) phthalate (MEHP) were used as substrates to predict the kinetic parameters k of these enzymes through the catalytic kinetic parameter prediction website. cat and K m Then calculate the k of all enzymes when they use the above substances as substrates. cat / K m The values ​​were compared to obtain the catalytic k values ​​of different substrates. cat / K mAmong them, esterase Est909 (IMG / M database accession number: 2670038909) catalyzed the k cat / K m The values ​​were higher than those of PAEIIIase, and the catalytic efficiency of the seven substrate reactions ranked among the top 15.

[0022] 1.2 Synthesis of the Est909 Encoding Gene and Construction of the Plasmid

[0023] The metagenomic database IMG / M database shows that the amino acid sequence of esterase Est909 is shown in SEQ ID NO.1, and the corresponding nucleic acid sequence is shown in SEQ ID NO.3. Beijing Boyi Huada Biogene Technology Co., Ltd. was commissioned to optimize the codons to obtain SEQ ID NO.3 and synthesize this sequence, and the gene was integrated into the pET-28a(+) expression vector using the NdeI / XhoI restriction site. The successfully constructed plasmid was transformed into Escherichia coli BL21(DE3) by chemical transformation, and the transformant was picked to obtain the Escherichia coli engineering bacteria carrying esterase Est909.

[0024] Example 2 Induced expression and purification of esterase Est909

[0025] 2.1 Inducible expression of esterase Est909

[0026] The engineered Escherichia coli bacteria carrying the esterase Est909 were transferred to LB liquid test tubes containing appropriate kanamycin sulfate antibiotics and cultured at 37±1°C overnight. Then, a 300mL Erlenmeyer flask containing 100mL LB medium was inoculated with 1% (v / v) inoculation volume and cultured at 37±1°C, 200±10rpm for 3h on a shaker. Then, the inducer IPTG was added at a final concentration of 0.5mM and cultured at 20±1°C, 200±10rpm for 20h.

[0027] 2.2 Preparation of crude enzyme solution of esterase Est909

[0028] After induction of expression in E. coli, the cells were collected by centrifugation at 6,000 rpm for 5 min, and then suspended and washed once with 0.05 M Tris-HCl buffer at pH 7.4, and then suspended. The cells were broken by ultrasonic cell disruptor and centrifuged at 12,000 rpm for 10 min. The supernatant was used as the crude enzyme solution and first subjected to SDS-PAGE electrophoresis to determine the expression of the target protein ( Figure 2 ), and then the phthalate hydrolysis characteristics of esterase Est909 were tested.

[0029] 2.3 Ester hydrolysis catalysis by crude esterase Est909

[0030] The 10mL diester reaction system is as follows: crude enzyme solution, 2mL; 0.05M Triris-HCl buffer (pH 7.5), 7.8mL; 0.2mL mixed diester substrates composed of DMP, DEP, DiBP, DBP, and DEHP, with a final concentration of each substrate of 200mg / L. Incubate in a water bath shaker at 37±1℃, 150±10rpm for 24h. Extract with 3mL of n-hexane, and detect the hydrolysis of phthalates by gas chromatography ( Figure 3 A). The 10mL monoester reaction system is as follows: crude enzyme solution, 2mL; 0.05M Tris-HCl buffer (pH 7.5), 7.9mL; 0.1mL of mixed monoester substrates composed of NMP, MBP, and MEHP, with the final concentration of each substrate being 100mg / L. Incubate in a water bath shaker at 37±1℃, 150±10rpm for 24h. Extract with 3mL of ethyl acetate, and detect the hydrolysis of phthalic acid monoester by thin layer chromatography ( Figure 3 B).

[0031] 2.4 Purification of esterase Est909

[0032] The crude enzyme solution was purified by nickel column, and the liquid obtained by eluting with different concentration gradients of imidazole solution was subjected to SDS-PAGE electrophoresis to determine the expression of the target protein. Then, the eluate containing only the target protein was removed by ultrafiltration centrifuge tube to concentrate the pure enzyme. The buffer required for ultrafiltration was Tris-HCl buffer, and the centrifugation conditions were 4°C, 6000r / min, 20min, and the pure enzyme solution of esterase Est909 was obtained ( Figure 4 ), and then the ester hydrolysis characteristics of esterase Est909 were detected.

[0033] Example 3. Determination of the efficiency of esterase Est909 pure enzyme solution in hydrolyzing phthalates

[0034] 3.1 The hydrolysis reaction system is as follows

[0035] 100 μL of pure enzyme solution; 880 μL of 0.05M Tris-HCl buffer (pH 7.5); 20 μL of mixed diester substrates composed of DMP, DEP, DiBP, DBP, and DEHP, with a final concentration of each substrate of 200 mg / L. Reaction in a water bath at 40±1℃ for 10 min. Extract with 1 mL of n-hexane, and quantitatively detect the amount of phthalate hydrolysis by gas chromatography.

[0036] 3.2 Gas chromatography quantitative detection

[0037] Chromatographic column: Agilent 19091N-213I. Detection conditions: 80℃, hold for 3min; increase to 250℃ at a rate of 20℃ / min, hold for 25min. Injection volume 1μl, no splitting. Carrier gas is nitrogen, flow rate is 1mL / min, FID detector. Standard curve quantitative method: set the concentration gradient of DMP, DEP, DiBP, DBP, DEHP to 0mg / L, 10mg / L, 20mg / L, 40mg / L, 80mg / L, 160mg / L, 220mg / L respectively, extract with 1mL of n-hexane containing internal standard 4-octanol, centrifuge and take the upper liquid for GC determination of the concentration of each substance, and draw a standard curve.

[0038] The results confirmed that the pure enzyme solution of the heterologous expression Escherichia coli engineered bacteria of the esterase Est909 constructed by the present invention can catalyze the hydrolysis of 63.65±0.61mg / L, 122.51±3.78mg / L, 71.44±3.83mg / L, 70.62±3.77mg / L, and 47.05±0.99mg / L of DMP, DEP, DiBP, DBP, and DEHP, respectively, within 10 minutes. Figure 5 A). The esterase Est909 differs from PAEIIIase by only 20 amino acids ( Figure 1 ), its efficiency of hydrolyzing DiBP, DBP, and DEHP was 1.26, 1.30, and 2.17 times higher than that of PAEIIIase, respectively ( Figure 5 B, 5C, 5D), indicating that the patent established based on enzyme sequence similarity and catalytic kinetic parameters k cat and K m It is technically feasible to predict the catalytic potential of enzymes and then discover efficient phthalate hydrolases through gene synthesis, enzyme-induced expression and property determination.

Claims

1. An esterase Est909 derived from Bacillus subtilis and its gene, characterized in that: The amino acid sequence of the esterase Est909 is shown in SEQ ID NO.1, and the DNA sequence of the gene encoding the esterase Est909 is shown in SEQ ID NO.2; the esterase Est909 is a protein composed of the amino acid sequence shown in Sequence 1 in the sequence table.

2. An Escherichia coli expression vector containing the gene of claim 1, characterized in that: The nucleotide sequence of the vector cloning region is shown in SEQ ID No.

2.

3. Use of the esterase Est909 described in claim 1 in the efficient catalytic hydrolysis of phthalates.

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