Application of esterase PCEST in removing plasticizer from oils and fats

Abstract

The present invention discloses an application of an esterase PCEST in removing plasticizers from greases. The esterase PCEST can preferably remove plasticizers from greases by means of biological enzymatic hydrolysis. At an appropriate temperature, the esterase PCEST can effectively remove phthalates from woody oils. The esterase PCEST has strong specificity and mild action conditions, can reduce the loss of nutrients such as vitamin E in woody oils, and can reduce the formation of trans fatty acids. The esterase PCEST preferably retains the nutrients in the grease and has low energy consumption. The esterase PCEST can be well applied in industrial production applications for removing plasticizers from greases, and has the characteristics of being easy to operate, having a simple process, and not requiring the use of complex process equipment.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme engineering, and particularly relates to application of esterase PCEST in removing plasticizer from grease. Background Art

[0002] Phthalates (PAEs) are a class of plasticizers commonly used in the plastics industry and are widely used in food packaging, toys, medical supplies, cosmetics, electronics, building materials, and other industries. PAEs are ubiquitous in water, soil, air, and even food, and have become a major environmental pollutant. Global PAE consumption currently reaches millions of tons annually. Due to the increasing consumption of PAEs in recent years, their chronic toxicity has attracted widespread international attention. Studies have shown that PAEs may be associated with human neurological diseases, endocrine disorders, reproductive malformations, and other diseases. Humans are exposed to PAEs through three pathways: skin contact (e.g., cosmetics), inhalation (e.g., atmospheric dust), and diet (e.g., food and water). Diet is the primary route of human exposure to PAEs.

[0003] Since 2012, the Chinese government has developed a series of risk monitoring plans for PAEs in foods such as alcoholic beverages, beverages, candy, edible oils, condiments, infant formula, catering foods, convenience foods, special foods and food additives.

[0004] Research has shown that PAEs can transfer from plastic containers into packaged foods, such as bottled water, oily foods, milk, beverages, and cooking oil. Given the high consumption of bottled water and cooking oil, the potential risks posed by PAEs in these products to humans warrants significant attention. In recent years, risk assessments of PAEs in bottled water and cooking oil have been conducted both domestically and internationally. It is important to note that daily exposure to PAEs from cooking oil is significantly higher than from bottled water. In my country, residents' exposure to PAEs is primarily limited to dibutyl phthalate (DBP) and dibutyl phthalate (DEHP), with daily intake from cooking oil accounting for approximately 17% of the recommended daily intake. GB 5009.271-2016 sets limits for DBP and DEHP in food: ≤0.3 mg / kg and 1.5 mg / kg, respectively. Random inspections of 1,016 samples of cooking oil from 31 provinces and municipalities in China revealed positive detection rates for DBP and DEHP in 13.48% and 7.78%, respectively. Among them, the positive detection rate of DBP in 340 samples of rapeseed oil was as high as 21.28%, and the positive detection rate of DEHP in 121 samples of peanut oil was as high as 27.27%.

[0005] Camellia oil, a type of edible oil, boasts a clear color, fragrant flavor, and long shelf life. It is immune to aflatoxin contamination, resists rancidity, and is easily digested and absorbed. Long-term consumption offers excellent health benefits, making it a popular green and health-promoting edible vegetable oil. Its unsaturated fatty acid content exceeds 90%, the highest among edible oils, and it contains no harmful erucic acid. According to nearly 20 years of nutritional research, camellia oil is more effective than soybean and sunflower oils in reducing the risk of hyperlipidemia and cardiovascular disease. Animal and human studies have shown that camellia oil can lower total triglyceride and low-density lipoprotein (LDL) levels while increasing high-density lipoprotein (HDL). The Chinese Center for Disease Control and Prevention has confirmed that camellia oil is superior to olive oil. Research conducted by Shanghai Second Medical University shows that camellia oil can effectively improve cardiovascular and cerebrovascular disease, lower cholesterol and fasting blood sugar, and inhibit the rise of triglycerides, helping improve the health of patients with the "three highs." Camellia oil, extracted from camellia seeds, is of excellent quality and is an ideal edible oil. However, camellia oil seeds are stored in plastic containers such as plastic bags during the harvesting and storage process. PAEs can be transferred from the plastic containers to the camellia oil seeds, resulting in the presence of PAEs in the camellia oil after pressing and extraction. The potential impact on human health cannot be ignored.

[0006] Currently, research at home and abroad focuses on different detection methods for PAEs in environmental matrices, cosmetics, food packaging materials, and foods, as well as the migration patterns of PAEs, and the removal of PAEs from drinking water, wastewater, and liquor. Research on the removal of PAEs from edible oils has only been explored in recent years. Among the currently proposed methods for removing PAEs from edible oils, one is to study the removal effect of PAEs in edible oils through refining processes (deacidification, washing, decolorization, dewaxing, deodorization, etc.). This process is cumbersome and the removal effect is poor, also resulting in the loss of nutrients. A second method is to effectively remove PAEs from edible oils through steam distillation, two-stage dual-temperature steam distillation, and molecular distillation, which can effectively remove PAEs from edible oils to meet national standards. However, this method also has the disadvantages of partial loss of vitamin E or the formation of some trans fatty acids. Studies have shown that the main methods for removing PAEs are physical degradation, biological degradation and chemical degradation. Among them, bioenzymatic degradation has the characteristics of strong specificity, simple and mild action conditions, and is one of the most promising methods for removing PAEs from edible oils. However, at present, it is not easy to find suitable bioenzymes to specifically degrade PAEs in edible oils.

[0007] Extremophiles are microorganisms that can thrive in extreme environments, such as volcanic vents, polar regions, deep-sea sediments, and hot springs. The enzymes they produce can maintain excellent catalytic activity even under harsh reaction conditions. For example, the esterase Est (PCEST) from the thermophilic archaeon Pyrobaculum calidifontis VA1 showed no significant decrease in catalytic activity after being incubated at 100°C for two hours and in an 80% organic solvent for one hour. Existing technology demonstrates that this esterase, PCEST, is a thermophilic enzyme that maintains good catalytic activity at high temperatures. Summary of the Invention

[0008] The purpose of the present invention is to provide an application of esterase PCEST in removing plasticizers from oils and fats, which can achieve efficient degradation of plasticizers in oils and fats through biological enzymatic hydrolysis.

[0009] The above-mentioned objectives are achieved by the following technical solutions.

[0010] The invention discloses an esterase PCEST for use in removing plasticizers from oils and fats. The amino acid sequence of the esterase PCEST is shown in SEQ ID NO. 1, and the esterase PCEST has a sequence in which 0, 1, 2, or more amino acids are substituted or missing, but the biological activity remains unchanged.

[0011] In some embodiments, when the esterase PCEST is used to remove plasticizers from oils and fats, the reaction temperature of the reaction system is 25°C to 45°C.

[0012] In some embodiments, when the esterase PCEST is used to remove plasticizers from oils and fats, the reaction temperature of the reaction system is 30°C to 40°C.

[0013] In some embodiments, when the esterase PCEST is used to remove plasticizers from oils and fats, the reaction temperature of the reaction system is 33°C to 38°C.

[0014] In some embodiments, the nucleotide sequence encoding the esterase PCEST is shown as SEQ ID NO. 2 or its reverse complementary sequence.

[0015] In some embodiments, the oil comprises woody oil, preferably camellia oil.

[0016] In some embodiments, the plasticizer includes phthalates.

[0017] A recombinant expression vector capable of expressing esterase PCEST is inserted with a nucleotide sequence encoding esterase PCEST, wherein the nucleotide sequence is shown as SEQ ID NO. 2 or its reverse complementary sequence.

[0018] A host cell transformed with the recombinant expression vector as described above; preferably, the host cell is Escherichia coli.

[0019] A method for removing plasticizers from grease using esterase PCEST, the method comprising the following steps:

[0020] 1) Mixing oil and the esterase PCEST described above, stirring to obtain a reaction solution, the reaction temperature is 25°C to 45°C;

[0021] 2) adding an extractant to the reaction solution for extraction, and obtaining oil and fat after enzymatic decomposition of the plasticizer.

[0022] In some embodiments, in step 1), the reaction temperature is 30°C to 40°C.

[0023] In some embodiments, in step 1), the reaction temperature is 33°C to 38°C.

[0024] In some embodiments, in step 1), the weight ratio of the oil to the esterase PCEST is 1:2-10, preferably 1:6-8;

[0025] and / or, in step 1), the pH value is 6.0 to 10.0;

[0026] And / or, in step 1), the stirring speed is 200 rpm to 1000 rpm;

[0027] And / or, in step 2), the extractant comprises ethyl acetate.

[0028] The inventors of the present invention discovered that, in their application of the esterase PCEST, the known high-temperature properties of the esterase PCEST were not very effective in removing plasticizers from oils and fats. Unexpectedly, they discovered that although the esterase PCEST is a high-temperature esterase, high temperatures actually inhibit its activity, resulting in only one ester bond in the plasticizer being degraded, resulting in poor degradation of the plasticizer in the oil. However, when the reaction temperature was controlled to a lower temperature, such as 25°C to 45°C, both ester bonds in the plasticizer were degraded, resulting in better degradation of the plasticizer in the oil and fat. This allowed the esterase PCEST to fully degrade the plasticizer in the oil and fat, further improving the degradation effect and efficiency, and reducing the residual phthalates in the system to as low as 1.18%.

[0029] The present invention provides the use of an esterase PCEST in removing plasticizers from greases. The esterase PCEST can preferably remove plasticizers from greases by bio-enzymatic hydrolysis. At an appropriate temperature, it can effectively remove phthalates from woody oils. It has strong specificity and mild reaction conditions, can reduce the loss of nutrients such as vitamin E in woody oils, and can reduce the formation of trans fatty acids. It can also better retain the nutrients in the grease and has low energy consumption. It can be well applied to industrial production applications for removing plasticizers from greases, and has the characteristics of being easy to operate, simple in process, and not requiring the use of complex process equipment. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the result of reducing SDS-PAGE to detect the purity of PCEST protein. DETAILED DESCRIPTION

[0031] To facilitate understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the present disclosure more thorough and comprehensive.

[0032] In the following examples, the experimental methods without specific conditions are generally carried out under conventional conditions or conditions recommended by the manufacturers. The various commonly used chemical reagents used in the examples are all commercially available products.

[0033] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as those belonging to the art and

[0034] The terms "and / or" and "and / or" used in the present invention are intended to be used only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" as used in the present invention includes any and all combinations of one or more of the related listed items.

[0035] In some embodiments of the present invention, an application of esterase PCEST in removing plasticizers from oils and fats is provided. The amino acid sequence of the esterase PCEST is shown in SEQ ID NO.1, and has a sequence in which 0, 1, 2, or more amino acids are substituted or missing, but the biological activity remains unchanged.

[0036] It should be noted that, in practical applications, those skilled in the art may add one or more amino acids to one or both ends of the sequence shown in SEQ ID NO. 1, without affecting the activity of the esterase PCEST, and this is also within the scope of protection of this application. The above-mentioned esterase PCEST activity refers to the performance of enzymatically degrading plasticizers.

[0037] It can be understood that the present invention provides the use of esterase PCEST in removing plasticizers from oils and fats, whose amino acid sequence is shown in SEQ ID NO.1. It can effectively remove plasticizers from oils and fats by biological enzymatic hydrolysis. At the appropriate temperature, it can effectively remove phthalates from woody oils. It has strong specificity and mild action conditions. It can reduce the loss of nutrients such as vitamin E in woody oils and reduce the formation of trans fatty acids. It can better retain the nutrients in oils and fats, and has low energy consumption. It can be well applied to the industrial production application of removing plasticizers from oils and fats, and has the characteristics of simple operation, simple process, and no need to use complex process equipment.

[0038] In some embodiments of the present invention, a nucleotide sequence encoding the esterase PCEST for removing plasticizers from oils and fats or its reverse complementary sequence is provided. In some embodiments, the nucleotide sequence is shown as SEQ ID NO. 2 or its reverse complementary sequence.

[0039] The amino acid sequence of the esterase PCEST for removing plasticizers from oils and fats is shown in SEQ ID NO.1:

[0040]

[0041] The coding nucleotide sequence of the esterase PCEST for removing plasticizers from oils and fats is shown in SEQ ID NO.2:

[0042]

[0043] In some embodiments, when the esterase PCEST is used to remove plasticizers from oils and fats, the reaction temperature of the reaction system is 25°C to 45°C.

[0044] In some embodiments, when the esterase PCEST is used to remove plasticizers from oils and fats, the reaction temperature of the reaction system is 30°C to 40°C.

[0045] In some embodiments, when the esterase PCEST is used to remove plasticizers from oils and fats, the reaction temperature of the reaction system is 33°C to 38°C.

[0046] In some embodiments of the present invention, a recombinant expression vector capable of expressing the esterase PCEST is provided, into which is inserted a nucleotide sequence encoding an amino acid sequence such as SEQ ID NO.1 or a nucleotide sequence such as SEQ ID NO.2 or its reverse complementary sequence.

[0047] As described above, in the recombinant expression vector, the nucleotide sequence encoding the esterase PCEST for removing plasticizers from oils and fats is operably linked to an expression control sequence to achieve desired transcription and ultimately produce the esterase PCEST in the host cell. Suitable expression control sequences include, but are not limited to, promoters, enhancers, ribosome interaction sites such as ribosome binding sites, polyadenylation sites, transcriptional splicing sequences, transcriptional termination sequences, and sequences that stabilize mRNA.

[0048] The vectors used in the above-mentioned recombinant expression vectors include vectors that replicate autonomously in host cells, such as plasmid vectors, and vectors that can integrate into and replicate along with host cell DNA. In one embodiment, the recombinant expression vector is derived from pET-23a(+) from Novagen.

[0049] In some embodiments of the present invention, a host cell into which the above-mentioned recombinant expression vector is transferred is provided.

[0050] In certain embodiments, host cells for expressing the plasticizer hydrolase of the present invention include prokaryotes, yeasts, and higher eukaryotic cells. Preferred are prokaryotic hosts, such as bacteria of the genus Escherichia, Bacillus, Salmonella, and Pseudomonas and Streptomyces. In a further preferred embodiment, the host cell is an Escherichia cell, more preferably an Escherichia coli cell. In a specific embodiment of the present invention, the host cell used is an Escherichia coli BL21 (DE3) strain cell (Novagen).

[0051] Specifically, techniques for introducing recombinant expression vectors into host cells include, but are not limited to, heat shock transformation, electroporation, DEAE-dextran transfection, microinjection, liposome-mediated transfection, calcium phosphate precipitation, protoplast fusion, microprojectile bombardment, viral transformation, and the like.

[0052] The host cells transformed with the recombinant expression vector are cultured, and a large amount of the active enzyme protein is formed after expression in the host cells. The host cells are lysed, the precipitate is removed by centrifugation, and the supernatant is collected; the supernatant is purified using a HisTrap HP affinity chromatography column to obtain the esterase PCEST for removing plasticizers from oils and fats.

[0053] Specifically, the oil includes but is not limited to woody oil, preferably camellia oil.

[0054] Specifically, the plasticizer includes but is not limited to phthalates.

[0055] In some embodiments of the present invention, a method for removing plasticizers from oils and fats using esterase PCEST is provided, the method comprising the following steps:

[0056] 1) Mix the oil and the esterase PCEST described above and stir to obtain a reaction solution at a reaction temperature of 25°C to 45°C;

[0057] 2) adding an extractant to the reaction solution for extraction, and obtaining oil and fat after enzymatic decomposition of the plasticizer.

[0058] In some embodiments, in step 1), the reaction temperature is 30°C to 40°C.

[0059] In some embodiments, in step 1), the reaction temperature is 33°C to 38°C.

[0060] In some embodiments, in step 1), the weight ratio of the oil to the esterase PCEST is 1:2-10, preferably 1:6-9;

[0061] and / or, in step 1), the pH value is 6.0 to 10.0;

[0062] And / or, in step 1), the stirring speed is 200 rpm to 1000 rpm;

[0063] And / or, in step 2), the extractant comprises ethyl acetate.

[0064] The oil after enzymatic hydrolysis was added with ethyl acetate and fully extracted by vortex shaking. After membrane filtration, the residual amount of plasticizer was detected by HPLC.

[0065] As mentioned above, the inventors of the present invention discovered that when using the esterase PCEST to remove plasticizers from oils and fats, the known high-temperature properties of the esterase PCEST were not very effective when applied to the process of removing plasticizers from oils and fats. Unexpectedly, it was discovered that although the esterase PCEST is a high-temperature esterase, high temperatures actually inhibit its activity, resulting in only one ester bond in the plasticizer being degraded, resulting in poor degradation of the plasticizer in the oil. However, when the reaction temperature was controlled to a lower temperature, such as 25°C to 45°C, both ester bonds in the plasticizer were degraded, thereby better degrading the plasticizer in the oil and fat. This allowed the esterase PCEST to fully degrade the plasticizer in the oil and fat, further improving the degradation effect and efficiency, and reducing the residual phthalates in the system to as low as 1.18%.

[0066] The present invention is further described below by way of specific examples, but is not intended to limit the scope of protection of the present invention. The methods used in the following examples are all conventional methods unless otherwise specified. For specific steps, see, for example, "Molecular Cloning: A Laboratory Manual" (Sambrook, J., Russell, David W., Molecular Cloning: A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring Harbor). All primers used were synthesized by Invitrogen. The experimental reagents and instruments used in the following examples are all conventional reagents and instruments unless otherwise specified.

[0067] Example 1: Protein expression and purification of the target protein PCEST (hereinafter referred to as PCEST) for removing plasticizers from oils

[0068] Strain: Escherichia coli BL21(DE3)

[0069] Vector: pET-23a(+)-His-PCEST

[0070] Culture medium: LB / Amp, containing 10.0 g tryptone, 5.0 g yeast extract powder, 10.0 g NaCl, and 100.0 μg / mL ampicillin (Amp) per liter. For solid culture, add 15.0 g agar per liter.

[0071] The nucleotide and amino acid sequences of PCEST are shown in SEQ ID NO.2 and SEQ ID NO.1, respectively.

[0072] 1.1 Amplification of PCEST polynucleotide fragments

[0073] Using plasmid pET-23a(+)-CBD-PCEST-His as a template, oligonucleotide primers as shown in Table 1 were synthesized and the complete PCEST polynucleotide sequence was obtained by PCR amplification (Table 2).

[0074] Table 1 Primers for amplifying PCEST

[0075]

[0076] The underlined parts of the primers in Table 1 are the recognition sites of restriction endonucleases Nde I and Xho I, respectively.

[0077] Table 2 PCR reaction system (50 μL) and procedure

[0078] reactants Volume (μL) Reaction procedure 5× Q5reaction buffer 10.0 1.98℃, 2min dNTPs (10 mM) 1.0 2.98℃,10s PCEST-F / PCEST-R (20 μM) 2.5 / 2.5 3.63℃,30s pET-23a(+)-CBD-PCEST-His 1.0 4.72℃, 1min Q5DNA polymerase 0.5 5.Go to 2,29 cycles <![CDATA[ddH2O]]> 32.5 6.72℃, 2min

[0079] 1.2 Construction of PCEST expression vector pET-23a(+)-PCEST

[0080] Plasmid pET-23a(+)-CBD-PCEST-His was used as the vector backbone, and CBD-PCEST-His was replaced by the double restriction enzyme sites Nde I and Xho I to obtain pET-23a(+)-His-PCEST;

[0081] The ligation product was transformed into Escherichia coli BL21 (DE3) competent cells. The transformed cells were plated on LB plates containing 100.0 mg / mL Amp to screen for positive clones. The plasmid was extracted and sequenced. The sequencing results confirmed that the cloned PCEST gene sequence was correct. The reaction systems are shown in Tables 3 and 4 below.

[0082] Table 3 Plasmid and PCR product double enzyme digestion system (50 μL) and conditions

[0083]

[0084] Table 4 Ligation system (10 μL) and conditions

[0085]

[0086] 1.3 Expression and purification of PCEST

[0087] The constructed strain was inoculated into LB liquid medium containing 100.0 μg / mL Amp and cultured overnight at 37°C under shaking. The next day, the overnight culture was transferred to fresh LB liquid medium (100.0 μg / mL Amp) at an inoculum ratio of 1:50 and cultured at 37°C under shaking until the logarithmic phase (OD 600 =0.4-0.6). 0.2 mM IPTG was added, and expression was induced at 16°C for 24 hours, and the cells were harvested by centrifugation.

[0088] The cells were resuspended in lysis buffer (20 mM Tris, 500 mM NaCl, 1 mM Na2EDTA.2H2O, pH 8.0) to 10 OD / mL and disrupted by ultrasound in an ice-water bath (disruption conditions: power 200 W, ultrasound time 3 s, interval time 3 s, ultrasound 99 times). The cell lysate was centrifuged (12000 rpm, 4°C, 15 min), the supernatant and precipitate were separated, and the supernatant was purified using a HisTrap HP affinity chromatography column and analyzed by SDS-PAGE ( Figure 1 ).

[0089] 1.4 PCEST hydrolysis activity test of plasticizers

[0090] The hydrolysis reaction system of PCEST for plasticizer is shown in Table 5:

[0091] Table 5 Hydrolysis reaction system (1.0 mL) and conditions

[0092]

[0093] After reacting the reaction system in Table 5 at 50°C and 500rpm for 4h, 100.0μL 3M HCl was added to terminate the reaction. 1.0mL ethyl acetate was added, and after vortexing and extraction for 5.0min, the DBP content was detected by high performance liquid chromatography (HPLC) (no PCEST was added to the negative control group). The DBP content in the experimental group was N1, and the DBP content in the control group was N0. The residual DBP content was used to express the hydrolysis activity of PCEST on DBP: DBP (%) = N1 / N0×100% (N0 represents the DBP content of the negative control group, and N1 represents the DBP content of the experimental group). After reacting for 4.0h, the residual DBP content in the system was 3.51%.

[0094] Example 2: PCEST detection of DBP hydrolysis activity in camellia oil

[0095] Table 6 Hydrolysis reaction system (10g) and conditions

[0096]

[0097] The reaction system in Table 6 was reacted at 25°C and 800 rpm for 24 hours. 100 μL of sample was taken out under stirring, 1 mL of ethyl acetate was added, and the mixture was vortexed and extracted for 5 minutes. Centrifuged at 5000 r / min for 5 minutes, the supernatant was collected, and the DBP content was detected by high performance liquid chromatography (HPLC) after membrane filtration (no PCEST was added to the negative control group). The DBP content in the experimental group was N1, and the DBP content in the control group was N0. The residual DBP content was used to express the hydrolysis activity of PCEST on DBP: DBP (%) = N1 / N0 × 100% (N0 represents the DBP content of the negative control group, and N1 represents the DBP content of the experimental group). After 24 hours of reaction, the residual DBP content in the system was 3.46%.

[0098] Example 3: PCEST detection of DBP hydrolysis activity in camellia oil

[0099] Table 7 Hydrolysis reaction system (10g) and conditions

[0100]

[0101] The reaction system in Table 7 was reacted at 30°C and 800 rpm for 24 hours. A 100 μL sample was removed with stirring, 1 mL of ethyl acetate was added, and the mixture was vortexed for 5 minutes for extraction. The mixture was centrifuged at 5000 rpm for 5 minutes, the supernatant was collected, filtered through a membrane, and the DBP content was determined by high-performance liquid chromatography (HPLC) (no PCEST was added to the negative control group). The DBP content in the experimental group was designated N1, and the DBP content in the control group was designated N0. The residual DBP content was used to represent the DBP hydrolysis activity of PCEST: DBP (%) = N1 / N0 × 100% (N0 represents the DBP content in the negative control group, and N1 represents the DBP content in the experimental group). After 24 hours of reaction, the residual DBP content in the system was 2.20%.

[0102] Example 4: PCEST detection of DBP hydrolysis activity in camellia oil

[0103] Table 8 Hydrolysis reaction system (10g) and conditions

[0104]

[0105] The reaction system in Table 8 was reacted at 35°C and 800 rpm for 24 hours. A 100 μL sample was removed with stirring, 1 mL of ethyl acetate was added, and the mixture was vortexed for 5 minutes for extraction. The supernatant was collected by centrifugation at 5000 rpm for 5 minutes, filtered through a membrane, and then the DBP content was determined by high-performance liquid chromatography (HPLC). (No PCEST was added to the negative control group.) The DBP content in the experimental group was designated N1, and the DBP content in the control group was designated N0. The residual DBP content was used to represent the DBP hydrolysis activity of PCEST: DBP (%) = N1 / N0 × 100% (N0 represents the DBP content in the negative control group, and N1 represents the DBP content in the experimental group). After 24 hours of reaction, the residual DBP content in the system was 1.18%.

[0106] Example 5: Hydrolysis of DBP in Camellia Oil by PCEST

[0107] 5.1 PCEST hydrolysis activity test of camellia oil DBP

[0108] Table 9 Hydrolysis reaction system (10g) and conditions

[0109]

[0110] The reaction system in Table 9 was reacted at 40°C and 800 rpm for 24 hours. A 100 μL sample was removed with stirring, 1 mL of ethyl acetate was added, and the mixture was vortexed for 5 minutes for extraction. The mixture was centrifuged at 5000 rpm for 5 minutes, the supernatant was collected, filtered through a membrane, and the DBP content was determined using high-performance liquid chromatography (HPLC) (no PCEST was added to the negative control group). The DBP content in the experimental group was designated N1, and the DBP content in the control group was designated N0. The residual DBP content was used to represent the DBP hydrolysis activity of PCEST: DBP (%) = N1 / N0 × 100% (N0 represents the DBP content in the negative control group, and N1 represents the DBP content in the experimental group). After 24 hours of reaction, the residual DBP content in the system was 6.70%.

[0111] Example 6: Hydrolysis of DBP in Camellia Oil by PCEST

[0112] 6.1 PCEST hydrolysis activity test of camellia oil DBP

[0113] Table 10 Hydrolysis reaction system (10g) and conditions

[0114]

[0115]

[0116] The reaction system in Table 10 was reacted at 45°C and 800 rpm for 24 hours. A 100 μL sample was removed with stirring, 1 mL of ethyl acetate was added, and the mixture was vortexed for 5 minutes for extraction. The mixture was centrifuged at 5000 rpm for 5 minutes, the supernatant was collected, filtered through a membrane, and the DBP content was determined by high-performance liquid chromatography (HPLC) (no PCEST was added to the negative control group). The DBP content in the experimental group was designated N1, and the DBP content in the control group was designated N0. The residual DBP content was used to represent the DBP hydrolysis activity of PCEST: DBP (%) = N1 / N0 × 100% (N0 represents the DBP content in the negative control group, and N1 represents the DBP content in the experimental group). After 24 hours of reaction, the residual DBP content in the system was 9.63%.

[0117] Comparative Example 1: PCEST detection of DBP hydrolysis activity in camellia oil

[0118] Table 11 Hydrolysis reaction system (10g) and conditions

[0119]

[0120] The reaction system in Table 11 was reacted at 90°C and 800 rpm for 24 hours. A 100 μL sample was removed with stirring, 1 mL of ethyl acetate was added, and the mixture was vortexed for 5 minutes for extraction. The mixture was centrifuged at 5000 rpm for 5 minutes, the supernatant was collected, filtered through a membrane, and the DBP content was determined by high-performance liquid chromatography (HPLC) (no PCEST was added to the negative control group). The DBP content in the experimental group was N1, and the DBP content in the control group was N0. The residual DBP content was used to represent the hydrolysis activity of PCEST on DBP: DBP (%) = N1 / N0 × 100% (N0 represents the DBP content in the negative control group, and N1 represents the DBP content in the experimental group). After 24 hours of reaction, the residual DBP content in the system was 14.24%.

[0121] Comparative Example 2: PCEST detection of DBP hydrolysis activity in camellia oil

[0122] Table 12 Hydrolysis reaction system (10g) and conditions

[0123]

[0124]

[0125] The reaction system in Table 12 was reacted at 70°C and 800 rpm for 24 hours. A 100 μL sample was removed with stirring, 1 mL of ethyl acetate was added, and the mixture was vortexed for 5 minutes for extraction. The supernatant was collected by centrifugation at 5000 r / min for 5 minutes, filtered through a membrane, and the DBP content was determined by high-performance liquid chromatography (HPLC) (no PCEST was added to the negative control group). The DBP content in the experimental group was N1, and the DBP content in the control group was N0. The residual DBP content was used to represent the hydrolysis activity of PCEST on DBP: DBP (%) = N1 / N0 × 100% (N0 represents the DBP content in the negative control group, and N1 represents the DBP content in the experimental group). After 24 hours of reaction, the residual DBP content in the system was 13.30%.

[0126] Comparative Example 3: PCEST detection of DBP hydrolysis activity in camellia oil

[0127] Table 13 Hydrolysis reaction system (10g) and conditions

[0128]

[0129] The reaction system in Table 13 was reacted at 50°C and 800 rpm for 24 hours. A 100 μL sample was removed with stirring, 1 mL of ethyl acetate was added, and the mixture was vortexed for 5 minutes for extraction. The supernatant was collected by centrifugation at 5000 r / min for 5 minutes, filtered through a membrane, and the DBP content was determined by high-performance liquid chromatography (HPLC) (no PCEST was added to the negative control group). The DBP content in the experimental group was N1, and the DBP content in the control group was N0. The residual DBP content was used to represent the hydrolysis activity of PCEST on DBP: DBP (%) = N1 / N0 × 100% (N0 represents the DBP content in the negative control group, and N1 represents the DBP content in the experimental group). After 24 hours of reaction, the residual DBP content in the system was 10.18%.

[0130] As can be seen from the above, the present invention provides an application of esterase PCEST in removing plasticizers from greases, which can better remove plasticizers from greases according to the method of biological enzymatic hydrolysis. At the appropriate temperature, it can effectively remove phthalates from woody oils. It has strong specificity and mild action conditions. It can reduce the loss of nutrients such as vitamin E in woody oils and reduce the formation of trans fatty acids. It better retains the nutrients in the grease and has low energy consumption. It can be well applied to the industrial production application of removing plasticizers from greases, and has the characteristics of simple operation, simple process, and no need to use complex process equipment.

[0131] On this basis, when the above-mentioned esterase PCEST is used to remove plasticizers from oils and fats, although the esterase PCEST is a high-temperature esterase, according to its known high-temperature special effects, when it is used in the process of removing plasticizers from oils and fats, the effect is not very good. Under high temperature conditions (temperature ≥50°C), the activity of the esterase PCEST is inhibited. The esterase PCEST can only degrade one ester bond in the plasticizer. When its reaction temperature is controlled to a lower temperature such as 25°C to 45°C, it can degrade two ester bonds in the plasticizer, thereby better degrading the plasticizer in the oil and fat. Therefore, the esterase PCEST can fully degrade the plasticizer in the oil and fat, further improving the degradation effect and efficiency. The residual phthalates in the system can be as low as 1.18%.

[0132] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0133] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A method for removing plasticizers from oils and fats using esterase PCEST, characterized in that: The method comprises the following steps: 1) Mix the oil and esterase PCEST and stir to obtain a reaction solution at a temperature of 33°C to 38°C. 2) adding an extractant to the reaction solution for extraction, to obtain oil after enzymatic decomposition of the plasticizer; Wherein, the amino acid sequence of the esterase PCEST is shown in SEQ ID NO.1; The oil is wood oil, and the plasticizer is phthalate.

2. The method according to claim 1, characterized in that The oil is camellia oil.

3. The method according to claim 1, characterized in that In step 1), the weight ratio of the oil to the esterase PCEST is 1:2~10.

4. The method according to claim 3, characterized in that In step 1), the weight ratio of the oil to the esterase PCEST is 1:6-8.

5. The method according to claim 1, characterized in that In step 1), the pH value is 6.0-10.

0.

6. The method according to claim 1, characterized in that In step 1), the stirring speed is 200 rpm~1000 rpm.

7. The method according to claim 1, characterized in that In step 2), the extractant includes ethyl acetate.

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