Hydrolase Hyl507 sourced from genwuella and used for degrading phthalic acid ester, coding gene and application of hydrolase Hyl507
Through genetic engineering technology, the hydrolase Hyl507 from Brucea origin was cloned and induced to express, which solved the problem of incomplete degradation of PAEs in food sources, and achieved efficient and green PAEs hydrolysis and degradation effects.
Patent Information
- Application Number
- CN202510279737.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-11
- Publication Date
- 2025-05-13
AI Technical Summary
The prior art is difficult to effectively degrade and treat phthalate (PAEs) in food sources, and traditional methods have problems such as incomplete degradation and physiological toxicity of intermediate products, which are difficult to meet the special requirements of food treatment.
Through genetic engineering technology, a hydrolase Hyl507, derived from Brucea japonicus, was cloned and induced to express, and an E. coli expression vector was constructed to achieve the efficient catalytic hydrolysis of phthalate of this enzyme.
This enzyme can efficiently hydrolyze a variety of PAEs, significantly reducing their toxicity, and provides a green treatment technology with mild conditions, high efficiency and few by-products, suitable for the effective treatment of food-derived PAEs.
Smart Images
Figure CN119979506A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of biological genetic engineering, and particularly relates to a hydrolase Hyl507 derived from Bacillus subtilis and a gene thereof, as well as 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 food-derived PAEs pollution has become an urgent need to focus on "food safety" and a major national strategic need that affects the national economy and people's livelihood.
[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 byproducts and environmental friendliness. In the degradation of phthalates, the hydrolysis of ester bonds plays a key role. Studies have found that the hydrolysis of PAEs side chain ester bonds can significantly reduce the toxicity of PAEs. Therefore, the discovery of enzymes that can hydrolyze PAEs has become the key to solving food-derived PAEs pollution by enzymatic methods. Summary of the invention
[0005] The purpose of the present invention is to first provide a hydrolase Hyl507 derived from Bacillus subtilis and its gene, as well as the application of the enzyme in the efficient catalytic degradation of phthalates.
[0006] The present invention provides a hydrolase Hyl507 derived from Bacillus subtilis, which has the performance of efficiently catalyzing the hydrolysis of phthalates. The amino acid sequence of the hydrolase Hyl507 is shown in SEQ ID NO.1.
[0007] The present invention also provides an Escherichia coli expression vector containing the gene encoding the hydrolase Hyl507 derived from Bacillus subtilis. The nucleotide sequence of the cloning region of the vector is shown in SEQ ID NO.2.
[0008] The hydrolase Hyl507 from Bacillus subtilis of the present invention is obtained by using the metagenomic database IMG / M, using the sequence of the β-lactamase hydrolase C family (GenBank retrieval number KP113669) as a probe, and mining based on sequence similarity comparison. The kinetic parameters K m and k cat , indicating that it has a high potential to catalyze the hydrolysis of PAEs. Subsequently, the coding sequence of the enzyme was obtained by synthesizing the whole gene sequence, 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] Method for constructing an engineered strain of Escherichia coli carrying the hydrolase Hyl507: The sequence was synthesized by the whole gene synthesis platform of Beijing Boyi Huada Biogene Technology Co., Ltd., and the gene was integrated into the pET-28a(+) expression vector using the NdeI / XhoI restriction site. After sequencing verification, the plasmid was transformed into Escherichia coli BL21(DE3) by chemical transformation, and the expression was induced by IPTG to obtain Escherichia coli cells carrying the hydrolase Hyl507. The crude enzyme solution was obtained by crushing the cells, and the purified enzyme catalyzed the hydrolysis of the substrate phthalate, and the degradation of the substrate was detected by gas chromatography.
[0010] The hydrolase Hyl507 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 hydrolase Hyl507, which was induced and expressed and purified by Escherichia coli engineering bacteria, can hydrolyze dimethyl phthalate (DMP), diethyl phthalate (DEP), diisobutyl phthalate (DiBP), dibutylphthalate (DBP) and di(2-ethylhexyl)phthalate (DEHP) (the concentration of each substrate is 200 mg / L), and the hydrolysis amounts are 131.61±2.27 mg / L, 45.61±2.21 mg / L, 6.32±1.85 mg / L, 35.29±2.88 mg / L and 19.31±1.39 mg / L, respectively.
[0012] The invention has the beneficial effects that: the invention uses biological genetic engineering technology to clone and induce expression to obtain the hydrolase Hyl507 and its coding gene, and constructs an Escherichia coli expression plasmid containing the hydrolase Hyl507 coding gene, transfers the plasmid into Escherichia coli, obtains the enzyme through induced expression, and verifies through a catalytic system that it has the ability to efficiently catalyze and hydrolyze phthalates. The invention first reports that a hydrolase Hyl507 derived from Raoultella sp. can be used to efficiently hydrolyze phthalates. BRIEF DESCRIPTION OF THE DRAWINGS
[0013] Figure 1 The amino acid sequence alignment result of hydrolase Hyl507 and family C β-lactamase KP113669
[0014] Figure 2 The results of induced expression of the gene encoding hydrolase Hyl507
[0015] Figure 3 The original gas chromatographic spectrum of phthalate hydrolysis catalyzed by hydrolase Hyl507
[0016] Figure 4 The results of purification of crude enzyme solution induced by the gene encoding hydrolase Hyl507
[0017] Figure 5 Quantitative results of hydrolysis of phthalates catalyzed by hydrolase Hyl507 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 a hydrolase Hyl507 from Sphaeroides rapae that degrades phthalates
[0020] 1.1 Screening steps and selection basis of phthalate degrading enzymes
[0021] The sequence of hydrolase C family β-lactamase (GenBank retrieval number KP113669) was used as a probe, and all enzymes with a certain degree of similarity to the probe enzyme were obtained from the metagenomic database IMG / M. Then, DMP, DEP, DiBP, DBP, and DEHP were used as substrates to predict the kinetic parameters K of these enzymes through the catalytic kinetic parameter prediction website. m and k cat Then calculate the k of all enzymes when they use the above substances as substrates. cat / K m The values of catalytic activity of different substrates were obtained. cat / K m Among them, the hydrolase Hyl507 (IMG / M database accession number is 2915822507) catalyzes the k cat / K m The values are all in the top 15.
[0022] 1.2 Synthesis of the gene encoding the hydrolase Hyl507, construction of the plasmid, and sequencing verification results
[0023] The metagenomic database IMG / M database shows that the amino acid sequence of the hydrolase Hyl507 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.2 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 engineered bacteria carrying the hydrolase Hyl507.
[0024] Example 2 Inducible expression and purification of hydrolase Hyl507
[0025] 2.1 Inducible expression of hydrolase Hyl507
[0026] The engineered Escherichia coli bacteria carrying the hydrolase Hyl507 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 hydrolase Hyl507
[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 the hydrolase Hyl507 were tested.
[0029] 2.3 Ester hydrolysis catalysis of crude enzyme Hyl507
[0030] The 10mL reaction system is as follows: crude enzyme solution, 2mL; 0.05MTris-HCl buffer (pH7.5), 7.8mL; 0.2mL mixed diester substrate composed of DMP, DEP, DiBP, DBP, and DEHP, with the final concentration of each substrate being 200mg / L. Incubate in a water bath shaker at 37±1℃ and 150±10rpm for 24h. Extract with 3mL of n-hexane, and detect the hydrolysis of phthalates by gas chromatography ( Figure 3 ).
[0031] 2.4 Purification of hydrolase Hyl507
[0032] The crude enzyme solution was purified using a 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. The eluate containing only the target protein was then removed of imidazole using an ultrafiltration centrifuge tube, and the pure enzyme was concentrated at the same time. The buffer required for ultrafiltration was Tris-HCl buffer, and the centrifugation conditions were 4°C, 6000r / min, 20min, to obtain the pure enzyme solution of hydrolase Hyl507 ( Figure 4 ), and then the phthalate hydrolysis characteristics of the hydrolase Hyl507 were tested.
[0033] Example 3. Determination of the efficiency of hydrolyzing phthalates by pure enzyme solution of hydrolase Hyl507
[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 engineering bacteria of the hydrolase Hyl507 constructed by the present invention can catalyze the hydrolysis of 131.61±2.27mg / L, 45.61±2.21mg / L, 6.32±1.85mg / L, 35.29±2.88mg / L, and 19.31±1.39mg / L of DMP, DEP, DiBP, DBP, and DEHP, respectively, within 10 minutes. Figure 5 ), indicating that the patent established based on enzyme sequence similarity and catalytic kinetic parameter K m and k cat Predicting the catalytic potential of enzymes and then exploring efficient phthalate hydrolases through gene synthesis, enzyme induced expression and property determination has good technical feasibility. The present invention first reports that a hydrolase Hyl507 from Raoultella sp. can be used to efficiently hydrolyze phthalates.
Claims
1. A hydrolase Hyl507 derived from Bacillus subtilis and its gene, characterized in that: The amino acid sequence of the hydrolase Hyl507 is shown in SEQ ID NO.1, and the DNA sequence of the gene encoding the hydrolase Hyl507 is shown in SEQ ID NO.2; the hydrolase Hyl507 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 hydrolase Hyl507 described in claim 1 in the efficient catalytic hydrolysis of phthalates.