Mutant of esterase E7 and application of mutant in preparation of EPAMG

Abstract

The invention discloses a plurality of mutants of esterase E7, comprising: esterase E7-F145L, the amino acid sequence of which is as shown in SEQ ID NO.5; the amino acid sequence of the esterase E7-F145A is as shown in SEQ ID NO. 4; the amino acid sequence of the esterase E7-Q199F is as shown in SEQ ID NO. 8. The mutant of the esterase E7 is applied to preparation of glyceride, preferably EPAMG. Through semi-rational design, site-directed mutagenesis is carried out on a bridge-shaped structure of esterase E7 from stenotrophomonas maltophilia, a plurality of mutants with remarkably improved catalytic activity are obtained, the effect of the mutant E7-F145L is optimal, the reaction efficiency is greatly improved, the high specificity of a catalytic product is reserved, and the application potential is huge.

Description

Technical Field

[0001] This invention relates to mutants of esterase E7 and their application in the preparation of EPAMG, belonging to the field of genetic engineering technology. Background Technology

[0002] As a representative of polyunsaturated fatty acids, eicosapentaenoic acid (EPA) can improve mood and prevent and treat cardiovascular diseases such as hypertension and coronary heart disease, earning it the nickname "vascular cleanser." EPA in food mainly exists in the form of glycerides, which are further divided into triglycerides (TG), diglycerides (DG), and monoglycerides (MG). Compared to EPA diglycerides (EPADG) and EPA triglycerides (EPATG), EPA monoglycerides (EPAMG) can be absorbed by cells via passive diffusion, exhibiting better digestive and absorptive properties. Therefore, the efficient synthesis of EPAMG is of great significance for the development of novel functional foods.

[0003] Lipid hydrolases (including lipases and esterases) can catalyze various reactions such as hydrolysis, transesterification, and esterification, enabling lipid structural modification under mild conditions. They have been applied in various industries, including food and pharmaceuticals. Enzymatic esterification can synthesize glycerides from free EPA and glycerol as substrates. However, the synthetic products of lipid hydrolases often contain a high proportion of DG and TG, which is detrimental to the targeted synthesis of high-purity EPAMG. Furthermore, the long reaction time required for esterification catalyzed by lipid hydrolases may cause oxidative degradation of EPA, thereby affecting the quality of EPAMG products.

[0004] Chinese invention patent CN 108913674 A discloses a lipid hydrolase and its application in the synthesis of EPA / DHA glycerides. It discloses esterase E7 derived from *Stenotrophomonas maltophilia*, which catalyzes the reaction of ethyl EPA / DHA with glycerol to synthesize EPA / DHA monoglycerides. After 48 hours of catalytic synthesis, it specifically catalyzes the conversion of ethyl EPA / DHA to EPA / DHA monoglycerides. Esterase E7 can be used to synthesize EPAMG, but the reaction efficiency is low and the reaction time is long, indicating room for improvement. Summary of the Invention

[0005] In view of the above-mentioned prior art, the present invention provides several mutants of esterase E7 and their application in the preparation of EPAMG.

[0006] This invention is achieved through the following technical solution: The mutant of esterase E7: esterase E7-F145L, with the amino acid sequence shown in SEQ ID NO.5.

[0007] The mutant of esterase E7: esterase E7-F145A, with the amino acid sequence shown in SEQ ID NO.4.

[0008] The mutant of esterase E7: esterase E7-Q199F, with the amino acid sequence shown in SEQ ID NO.8.

[0009] The use of the mutant of esterase E7 in the preparation of glycerides. Preferably, the glycerides are selected from EPAMG.

[0010] Furthermore, in specific applications, glycerol esters are synthesized using fatty acids and glycerol as substrates under the catalysis of a mutant esterase E7; the fatty acids are selected from any one or more of oleic acid (OA), linoleic acid (LA), EPA, and docosahexaenoic acid (DHA).

[0011] Furthermore, in specific applications, recombinant engineered bacteria expressing esterase E7 mutants are constructed, and the mutants expressing esterase E7 are cultured and induced to produce bacterial culture. The culture is then centrifuged, resuspended, and a liquid enzyme preparation is obtained. This liquid enzyme preparation is then freeze-dried to obtain a solid enzyme preparation. Glycerol, fatty acids, and buffer solutions are mixed, and the liquid or solid enzyme preparation is added. The mixture is homogenized to form an emulsion and reacted at 65°C for 1–2 hours. After the reaction, isopropanol and n-hexane are added for extraction.

[0012] A method for preparing EPAMG is as follows: using EPA and glycerol as substrates, EPAMG is synthesized under the catalysis of a mutant esterase E7.

[0013] Further, a recombinant engineered bacterium expressing a mutant of esterase E7 was constructed, and the mutant expressing esterase E7 was cultured and induced to obtain a bacterial culture. The culture was centrifuged, resuspended, and a liquid enzyme preparation was obtained. The liquid enzyme preparation was then freeze-dried to obtain a solid enzyme preparation. Glycerol, EPA, and buffer were mixed, and the liquid or solid enzyme preparation was added. The mixture was homogenized to form an emulsion and reacted at 65°C for 1–2 hours. After the reaction, isopropanol and n-hexane were added for extraction.

[0014] Furthermore, after extraction, isopropanol and n-hexane are removed by rotary evaporation, the ethanol solution is diluted, and n-hexane is added for extraction; the number of extractions with n-hexane is 1 to 5 times; high-purity EPAMG is obtained.

[0015] This invention utilizes semi-rational design to perform site-directed mutagenesis on the bridge-like structure of esterase E7 derived from *Stenotrophomonas maltophilia*, resulting in several mutants with significantly enhanced catalytic activity. These mutants exhibited significantly improved catalytic efficiency in the preparation of EPAMG. Among them, mutant E7-F145L showed the best performance, not only greatly improving reaction efficiency but also retaining the high specificity of the catalytic product, demonstrating great application potential. This invention also compared the esterification effects of esterase E7 and mutant E7-F145L on different types of fatty acids, finding that mutant E7-F145L significantly improved the catalytic efficiency for EPA and DHA, indicating that this mutant can also be better applied to the targeted synthesis of other long-chain polyunsaturated fatty acid monoglycerides, achieving broader applications.

[0016] The various terms and phrases used in this invention have their general meanings known to those skilled in the art. Attached Figure Description

[0017] Figure 1 Schematic diagram of the bridge-like structure of esterase E7.

[0018] Figure 2 Esterase E7, E7 A Results of conversion rate determination during catalytic synthesis of glycerides.

[0019] Figure 3 Results of conversion rate determination when various mutants of esterase E7 catalyze the synthesis of glycerol esters.

[0020] Figure 4 Results of conversion rate determination during the synthesis of glycerol esters catalyzed by esterase E7-F145L.

[0021] Figure 5 Results of EPAMG purity determination.

[0022] Figure 6 The esterification effects of esterases E7 and E7-F145L on different fatty acids. Detailed Implementation

[0023] The present invention will be further described below with reference to embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art will understand that various changes and modifications can be made to the present invention without departing from the spirit and scope thereof.

[0024] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.

[0025] Example 1: Functional Study of the Bridge Structure of Esterase E7 The wild-type esterase E7 is derived from *Stenotrophomonas maltophilia*, as described in Chinese invention patent CN 108913674 A (i.e., the lipase E7 described in that patent). The amino acid sequence of esterase E7 is shown in SEQ ID NO.1, containing 617 amino acids, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.2, containing 1854 nucleotides.

[0026] The amino acid sequence of esterase E7 is shown in SEQ ID NO.1, as follows: MLLSKRPIRSLMAAAIALAAVPAMAGESPYSRAVFFGDSLTDAGYFRPLLDPGVRPVTGQFTTNPGLVWSQQLANYYGLDGTPNGNGQNGDNYAVGGARVSVDEAGGLGAIPSLKSQAARYLAANGGKADANALYTVWGGANDLFAATRAAAGGASQAQVQGIIGAAVTDQIALVGALKQAGAQYVLVPNLPDVGITPQFRGPNAAAATALSAGYNKALYGGLKQAGIEFIPLDTFSILREVTANPAMYGFTNVTSTACKIDPNNSTASIIGCNPTSYVSPDAANTYLFADGVHPTTAGHQLLGQYAVSVLEGPRLQQVLSHSAQTIGRSRADQVSMHLGGRPADGLSWWGGVRGDLQRYDHADLYDGLAPAGLFGIDWARDGMVFGGFAGFGRLNADFGNSRGDFTQKDTTAGLFAGWYHDRIWVNGQVSYTWLSYDVNRKVQLGPATREHGGSPDGSNLTAALNAGYEFGTEGGFRHGPIASVIWQKVKIDGYTESAAAGTLATALGYDRQNVDSTVGRIGWQARFDGGTVKPYAQLTYDHEFEDTKQASAWLQTLPELGSYRVPGLNFDKNYATVVLGARTELFGLQSNFGLSASAGQKRAQDATLFANFSGSF。

[0027] The nucleotide sequence of the encoding gene of esterase E7 is shown in SEQ ID NO.2, as follows (orientation 5'-3'):

[0028] The enzymatic properties of esterase E7 and its application in the synthesis of EPA / DHA monoglycerides have been described in CN 108913674 A. Further structural modeling and comparative studies revealed a bridge-like structure above the catalytic site of esterase E7, such as... Figure 1 As shown, this structure ( Figure 1 (shown in red) is composed of side chain groups of F145, Q199, and F200, dividing its catalytic pocket into an acyl acceptor binding pocket ( Figure 1 (shown in yellow) and fatty acid binding pocket ( Figure 1 The two parts (shown in blue) hinder the contact between large acyl receptors (such as MG) and fatty acids, thus inhibiting the formation of DG. To clarify the effect of this bridging structure on the catalytic effect of esterase E7, this invention constructed a bridging structure-deficient mutant E7. A (The amino acids at positions 145, 199, and 200 were mutated to alanine), and their catalytic effect was measured. The specific implementation process is as follows: (1) Construction of esterase E7 bridge structure deletion mutant expression strain Using the original pET28a-E7 plasmid as a template (refer to CN 108913674 A), the plasmid was linearized using F145A-F / R primers, and then... Dpn After treatment, the cells were transformed into *E. coli* DH5α competent cells and screened on plates containing 50 μg / mL kanamycin sulfate. After positive clone verification and gene sequencing, mutations were performed on amino acids 199 and 200 sequentially using primers Q199A-F / R and F200A-F / R, respectively. Finally, *E. coli* BL21(DE3) competent cells were transformed in the same manner. This *E. coli* can be used to express E7. A .

[0029] E7 A The amino acid sequence is shown in SEQ ID NO.3, as follows: MLLSKRPIRSLMAAAIALAAVPAMAGESPYSRAVFFGDSLTDAGYFRPLLDPGVRPVTGQFTTNPGLVWSQQLANYYGLDGTPNGNGQNGDNYAVGGARVSVDEAGGLGAIPSLKSQAARYLAANGGKADANALYTVWGGANDLAAATRAAAGGASQAQVQGIIGAAVTDQIALVGALKQAGAQYVLVPNLPDVGITPAARGPNAAAATALSAGYNKALYGGLKQAGIEFIPLDTFSILREVTANPAMYGFTNVTSTACKIDPNNSTASIIGCNPTSYVSPDAANTYLFADGVHPTTAGHQLLGQYAVSVLEGPRLQQVLSHSAQTIGRSRADQVSMHLGGRPADGLSWWGGVRGDLQRYDHADLYDGLAPAGLFGIDWARDGMVFGGFAGFGRLNADFGNSRGDFTQKDTTAGLFAGWYHDRIWVNGQVSYTWLSYDVNRKVQLGPATREHGGSPDGSNLTAALNAGYEFGTEGGFRHGPIASVIWQKVKIDGYTESAAAGTLATALGYDRQNVDSTVGRIGWQARFDGGTVKPYAQLTYDHEFEDTKQASAWLQTLPELGSYRVPGLNFDKNYATVVLGARTELFGLQSNFGLSASAGQKRAQDATLFANFSGSF。

[0030] The primers used for PCR amplification are as follows: F145A-F: 5'-AATGACCTGGCCGCGGCCACGCGCGCAG-3', as shown in SEQ ID NO.11; F145A-R: 5'-GTGGCCGCGGCCAGGTCATTGGCACCGCCCCAG-3', as shown in SEQ ID NO.12; Q199A-F: 5'-TCACTCCGGCGTTCCGCGGCCCCAACGCCG-3', as shown in SEQ ID NO.15; Q199A-R: 5'-GCCGCGGAACGCCGGAGTGATGCCCACGTCCGG-3', as shown in SEQ ID NO.16; F200A-F: 5'-CACTCCGCAGGCTCGCGGCCCCAACGCCGCTGC-3', as shown in SEQ ID NO.21; F200A-R: 5'-GGGGCCGCGAGCCTGCGGAGTGATGCCCACGTCC-3', as shown in SEQ ID NO.22.

[0031] (2) E7 A Heterologous expression in Escherichia coli and preparation of enzyme preparations Transformants were inoculated into 5 mL LB medium and cultured at 37°C for 12 h to obtain a seed culture. Then, 1% of the seed culture was inoculated into ZYP-5052 medium (a standard commercial medium) and cultured at 20°C and 200 rpm for 48 h to induce E7 expression in *E. coli*. A After induction of expression, E. coli cells were collected by centrifugation at 8000 r / min for 5 min, resuspended and washed with Tris-HCl buffer (50 mM, pH 8.0), and then freeze-dried to obtain E. coli powder.

[0032] The surface display properties of esterase E7 have been described in Chinese invention patent CN 118562764 A. The enzyme activity assay of esterase E7 and its mutant *E. coli* in whole cells in this invention was performed according to Example 2 of that invention patent, specifically as follows: A 20 mM substrate solution of p-nitrophenol butyrate was prepared using isopropanol and dimethyl sulfoxide (DMSO) in a volume ratio of 3:1. 180 μL of Tris-HCl buffer (50 mM, pH 8.0) was added to 10 μL of bacterial suspension, mixed, and incubated at 37°C for 2 min. 10 μL of the substrate solution was then added to the reaction system, and the reaction was timed precisely for 5 min. 200 μL of sodium dodecyl sulfate (SDS) solution (1% concentration, g / mL) was added to terminate the reaction. After centrifugation, the supernatant was collected, and the absorbance was measured at 405 nm. The concentration of p-nitrophenol in the reaction system was determined based on the standard curve, and the enzyme activity was calculated. Enzyme activity is defined as the amount of enzyme required to catalyze the hydrolysis of p-nitrophenol butyrate to produce 1 μmol of p-nitrophenol within 1 min, which is one enzyme activity unit, i.e., 1 U.

[0033] (3) Esterification synthesis of EPAMG Take 120 U to express esterase E7 or E7 AWhole cells of *E. coli* were added sequentially with 150 μL Tris-HCl buffer (50 mM, pH 8.0), 800 μL glycerol, and 200 μL free EPA. The mixture was homogenized using a high-speed homogenizer (IKA, T10 Basic, Germany) at 30,000 r / min for 30 s to form an emulsion. The emulsion reaction system was reacted in a water bath at 65°C for different times (1 h, 2 h, 3 h, 4 h, 6 h), followed by the addition of 1 mL isopropanol and thorough mixing. Then, 3 mL of n-hexane was added and the mixture was shaken to extract the oil phase. Centrifuge at 8000 r / min for 5 min to separate the extraction system into layers. Take the upper organic phase and remove trace amounts of water using anhydrous sodium sulfate. Then, use liquid chromatography (equipped with a differential refractive index detector) to detect the lipid components. The chromatographic column used is a Phenomenex Luna silica gel column (250 mm × 4.6 mm, 5 μm), and the mobile phase is n-hexane:isopropanol:acetic acid = 92:8:0.003 (volume ratio).

[0034] Esterase E7, E7 A The conversion rate during the catalytic synthesis of glycerides was measured as follows: Figure 2 As shown, E7 A The conversion rate of EPAMG reached 45.58% after 1 h of catalytic reaction, higher than the conversion rate of E7 esterase after 6 h (38.85%), indicating a significant improvement in reaction efficiency. This is likely due to the reduced steric hindrance. Meanwhile, E7... A After 6 h of catalytic reaction, 44.10% EPAMG and 21.15% EPADG were generated, and the catalytic specificity was significantly reduced, demonstrating that the bridging structure of esterase E7 is crucial for maintaining its catalytic specificity.

[0035] Example 2 Construction and catalytic effect of a bridge-like point mutant of esterase E7 To maintain the catalytic specificity of esterase E7 and improve its reaction efficiency, unit point mutants of alanine, leucine, and phenylalanine, the amino acids that make up its bridging structure, were constructed using the original pET28a-E7 plasmid as a template, aiming to reduce steric hindrance while retaining its bridging structure. The specific construction and expression process is described in Example 1.

[0036] Seven mutants were constructed: E7-F145A, E7-F145L, E7-Q199A, E7-Q199L, E7-Q199F, E7-F200A, and E7-F200L, with amino acid sequences shown in SEQ ID NO.4 to 10.

[0037] The amino acid sequence of E7-F145A is shown in SEQ ID NO.4, as follows: MLLSKRPIRSLMAAAIALAAVPAMAGESPYSRAVFFGDSLTDAGYFRPLLDPGVRPVTGQFTTNPGLVWSQQLANYYGLDGTPNGNGQNGDNYAVGGARVSVDEAGGLGAIPSLKSQAARYLAANGGKADANALYTVWGGANDLAAATRAAAGGASQAQVQGIIGAAVTDQIALVGALKQAGAQYVLVPNLPDVGITPQFRGPNAAAATALSAGYNKALYGGLKQAGIEFIPLDTFSILREVTANPAMYGFTNVTSTACKIDPNNSTASIIGCNPTSYVSPDAANTYLFADGVHPTTAGHQLLGQYAVSVLEGPRLQQVLSHSAQTIGRSRADQVSMHLGGRPADGLSWWGGVRGDLQRYDHADLYDGLAPAGLFGIDWARDGMVFGGFAGFGRLNADFGNSRGDFTQKDTTAGLFAGWYHDRIWVNGQVSYTWLSYDVNRKVQLGPATREHGGSPDGSNLTAALNAGYEFGTEGGFRHGPIASVIWQKVKIDGYTESAAAGTLATALGYDRQNVDSTVGRIGWQARFDGGTVKPYAQLTYDHEFEDTKQASAWLQTLPELGSYRVPGLNFDKNYATVVLGARTELFGLQSNFGLSASAGQKRAQDATLFANFSGSF。

[0038] The amino acid sequence of E7-F145L is shown in SEQ ID NO.5 as follows: MLLSKRPIRSLMAAAIALAAVPAMAGESPYSRAVFFGDSLTDAGYFRPLLDPGVRPVTGQFTTNPGLVWSQQLANYYGLDGTPNGNGQNGDNYAVGGARVSVDEAGGLGAIPSLKSQAARYLAANGGKADANALYTVWGGANDLLAATRAAAGGASQAQVQGIIGAAVTDQIALVGALKQAGAQYVLVPNLPDVGITPQFRGPNAAAATALSAGYNKALYGGLKQAGIEFIPLDTFSILREVTANPAMYGFTNVTSTACKIDPNNSTASIIGCNPTSYVSPDAANTYLFADGVHPTTAGHQLLGQYAVSVLEGPRLQQVLSHSAQTIGRSRADQVSMHLGGRPADGLSWWGGVRGDLQRYDHADLYDGLAPAGLFGIDWARDGMVFGGFAGFGRLNADFGNSRGDFTQKDTTAGLFAGWYHDRIWVNGQVSYTWLSYDVNRKVQLGPATREHGGSPDGSNLTAALNAGYEFGTEGGFRHGPIASVIWQKVKIDGYTESAAAGTLATALGYDRQNVDSTVGRIGWQARFDGGTVKPYAQLTYDHEFEDTKQASAWLQTLPELGSYRVPGLNFDKNYATVVLGARTELFGLQSNFGLSASAGQKRAQDATLFANFSGSF。

[0039] The amino acid sequence of E7-Q199A is shown in SEQ ID NO.6 as follows: MLLSKRPIRSLMAAAIALAAVPAMAGESPYSRAVFFGDSLTDAGYFRPLLDPGVRPVTGQFTTNPGLVWSQQLANYYGLDGTPNGNGQNGDNYAVGGARVSVDEAGGLGAIPSLKSQAARYLAANGGKADANALYTVWGGANDLFAATRAAAGGASQAQVQGIIGAAVTDQIALVGALKQAGAQYVLVPNLPDVGITPAFRGPNAAAATALSAGYNKALYGGLKQAGIEFIPLDTFSILREVTANPAMYGFTNVTSTACKIDPNNSTASIIGCNPTSYVSPDAANTYLFADGVHPTTAGHQLLGQYAVSVLEGPRLQQVLSHSAQTIGRSRADQVSMHLGGRPADGLSWWGGVRGDLQRYDHADLYDGLAPAGLFGIDWARDGMVFGGFAGFGRLNADFGNSRGDFTQKDTTAGLFAGWYHDRIWVNGQVSYTWLSYDVNRKVQLGPATREHGGSPDGSNLTAALNAGYEFGTEGGFRHGPIASVIWQKVKIDGYTESAAAGTLATALGYDRQNVDSTVGRIGWQARFDGGTVKPYAQLTYDHEFEDTKQASAWLQTLPELGSYRVPGLNFDKNYATVVLGARTELFGLQSNFGLSASAGQKRAQDATLFANFSGSF。

[0040] The amino acid sequence of E7-Q199 is shown in SEQ ID NO.7 as follows: MLLSKRPIRSLMAAAIALAAVPAMAGESPYSRAVFFGDSLTDAGYFRPLLDPGVRPVTGQFTTNPGLVWSQQLANYYGLDGTPNGNGQNGDNYAVGGARVSVDEAGGLGAIPSLKSQAARYLAANGGKADANALYTVWGGANDLFAATRAAAGGASQAQVQGIIGAAVTDQIALVGALKQAGAQYVLVPNLPDVGITPLFRGPNAAAATALSAGYNKALYGGLKQAGIEFIPLDTFSILREVTANPAMYGFTNVTSTACKIDPNNSTASIIGCNPTSYVSPDAANTYLFADGVHPTTAGHQLLGQYAVSVLEGPRLQQVLSHSAQTIGRSRADQVSMHLGGRPADGLSWWGGVRGDLQRYDHADLYDGLAPAGLFGIDWARDGMVFGGFAGFGRLNADFGNSRGDFTQKDTTAGLFAGWYHDRIWVNGQVSYTWLSYDVNRKVQLGPATREHGGSPDGSNLTAALNAGYEFGTEGGFRHGPIASVIWQKVKIDGYTESAAAGTLATALGYDRQNVDSTVGRIGWQARFDGGTVKPYAQLTYDHEFEDTKQASAWLQTLPELGSYRVPGLNFDKNYATVVLGARTELFGLQSNFGLSASAGQKRAQDATLFANFSGSF。

[0041] The amino acid sequence of E7-Q199F is shown in SEQ ID NO.8 as follows: MLLSKRPIRSLMAAAIALAAVPAMAGESPYSRAVFFGDSLTDAGYFRPLLDPGVRPVTGQFTTNPGLVWSQQLANYYGLDGTPNGNGQNGDNYAVGGARVSVDEAGGLGAIPSLKSQAARYLAANGGKADANALYTVWGGANDLFAATRAAAGGASQAQVQGIIGAAVTDQIALVGALKQAGAQYVLVPNLPDVGITPFFRGPNAAAATALSAGYNKALYGGLKQAGIEFIPLDTFSILREVTANPAMYGFTNVTSTACKIDPNNSTASIIGCNPTSYVSPDAANTYLFADGVHPTTAGHQLLGQYAVSVLEGPRLQQVLSHSAQTIGRSRADQVSMHLGGRPADGLSWWGGVRGDLQRYDHADLYDGLAPAGLFGIDWARDGMVFGGFAGFGRLNADFGNSRGDFTQKDTTAGLFAGWYHDRIWVNGQVSYTWLSYDVNRKVQLGPATREHGGSPDGSNLTAALNAGYEFGTEGGFRHGPIASVIWQKVKIDGYTESAAAGTLATALGYDRQNVDSTVGRIGWQARFDGGTVKPYAQLTYDHEFEDTKQASAWLQTLPELGSYRVPGLNFDKNYATVVLGARTELFGLQSNFGLSASAGQKRAQDATLFANFSGSF。

[0042] The amino acid sequence of E7-F200A is as shown in SEQ ID NO.9 and is as follows: MLLSKRPIRSLMAAAIALAAVPAMAGESPYSRAVFFGDSLTDAGYFRPLLDPGVRPVTGQFTTNPGLVWSQQLANYYGLDGTPNGNGQNGDNYAVGGARVSVDEAGGLGAIPSLKSQAARYLAANGGKADANALYTVWGGANDLFAATRAAAGGASQAQVQGIIGAAVTDQIALVGALKQAGAQYVLVPNLPDVGITPQARGPNAAAATALSAGYNKALYGGLKQAGIEFIPLDTFSILREVTANPAMYGFTNVTSTACKIDPNNSTASIIGCNPTSYVSPDAANTYLFADGVHPTTAGHQLLGQYAVSVLEGPRLQQVLSHSAQTIGRSRADQVSMHLGGRPADGLSWWGGVRGDLQRYDHADLYDGLAPAGLFGIDWARDGMVFGGFAGFGRLNADFGNSRGDFTQKDTTAGLFAGWYHDRIWVNGQVSYTWLSYDVNRKVQLGPATREHGGSPDGSNLTAALNAGYEFGTEGGFRHGPIASVIWQKVKIDGYTESAAAGTLATALGYDRQNVDSTVGRIGWQARFDGGTVKPYAQLTYDHEFEDTKQASAWLQTLPELGSYRVPGLNFDKNYATVVLGARTELFGLQSNFGLSASAGQKRAQDATLFANFSGSF。

[0043] The amino acid sequence of E7-F200L is shown in SEQ ID NO.10 and is as follows: MLLSKRPIRSLMAAAIALAAVPAMAGESPYSRAVFFGDSLTDAGYFRPLLDPGVRPVTGQFTTNPGLVWSQQLANYYGLDGTPNGNGQNGDNYAVGGARVSVDEAGGLGAIPSLKSQAARYLAANGGKADANALYTVWGGANDLFAATRAAAGGASQAQVQGIIGAAVTDQIALVGALKQAGAQYVLVPNLPDVGITPQLRGPNAAAATALSAGYNKALYGGLKQAGIEFIPLDTFSILREVTANPAMYGFTNVTSTACKIDPNNSTASIIGCNPTSYVSPDAANTYLFADGVHPTTAGHQLLGQYAVSVLEGPRLQQVLSHSAQTIGRSRADQVSMHLGGRPADGLSWWGGVRGDLQRYDHADLYDGLAPAGLFGIDWARDGMVFGGFAGFGRLNADFGNSRGDFTQKDTTAGLFAGWYHDRIWVNGQVSYTWLSYDVNRKVQLGPATREHGGSPDGSNLTAALNAGYEFGTEGGFRHGPIASVIWQKVKIDGYTESAAAGTLATALGYDRQNVDSTVGRIGWQARFDGGTVKPYAQLTYDHEFEDTKQASAWLQTLPELGSYRVPGLNFDKNYATVVLGARTELFGLQSNFGLSASAGQKRAQDATLFANFSGSF。

[0044] The primers used for PCR amplification are newly added as follows based on Example 1: F145L-F: 5'-ATGACCTGCTGGCGGCCACGCGCGCAGCG-3', as shown in SEQ ID NO.13; F145L-R: 5'-GTGGCCGCCAGCAGGTCATTGGCACCGCCCCAGACGGTG-3', as shown in SEQ ID NO.14; Q199L-F: 5'-TCACTCCGTTGTTCCGCGGCCCCAACGC-3', as shown in SEQ ID NO.17; Q199L-R: 5'-GCCGCGGAACAACGGAGTGATGCCCACGTCCG-3', as shown in SEQ ID NO.18; Q199F-F: 5'-TCACTCCGTTCTTCCGCGGCCCCAACGC-3', as shown in SEQ ID NO.19; Q199F-R: 5'-GCCGCGGAAGAACGGAGTGATGCCCACGTCCG-3', as shown in SEQ ID NO.20; F200L-F: 5'-CACTCCGCAGCTACGCGGCCCCAACGCCGCTGC-3', as shown in SEQ ID NO.23; F200L-R: 5'-GGGGCCGCGTAGCTGCGGAGTGATGCCCACGTCC-3', as shown in SEQ ID NO.24.

[0045] After obtaining whole cells of E. coli expressing each E7 mutant, the esterification reaction process was carried out as described in Example 1. The lipid components in the system were measured after reacting at 65°C for 3 h, with esterase E7 as a control.

[0046] The conversion rates of various mutants of esterase E7 during the synthesis of glycerol esters were measured as follows: Figure 3 As shown in the figure, the conversion rate of wild-type E7 reached 23.75%, while the conversion rates of E7-F145A and E7-F145L reached 52.48% and 58.23%, respectively, showing a significant improvement in reaction efficiency. The conversion rates of E7-Q199L and E7-Q199F were 29.12% and 37.35%, respectively, showing a relatively small improvement, while the conversion rate of E7-Q199A decreased significantly. The conversion rates of E7-F200A and E7-F200L were 25.09% and 17.05%, respectively, showing no significant advantage compared to wild-type E7.

[0047] Overall, this invention yielded several mutants with improved reaction efficiency, among which E7-F145L exhibited the highest reaction efficiency and did not generate significant diesters (of the 58.23% conversion rate, EPAMG accounted for 55.51% and EPADG only accounted for 2.72%), thus maintaining good catalytic specificity.

[0048] Example 3: Synthesis and purification of EPAMG catalyzed by mutant E7-F145L Referring to Example 1, the esterification reaction was catalyzed by E7-F145L, and samples were taken hourly to determine the composition of the esterification product. The conversion rate of the esterase E7-F145L catalyzing the synthesis of glycerol esters is shown in the figure below. Figure 4As shown in the figure. It was determined that at 2 h of reaction, the product was EPAMG, with a conversion rate of 56.93%; at 3 h, 4 h, 5 h and 6 h of reaction, the conversion rate of EPAMG did not change significantly, and EPADG appeared in the product.

[0049] A large-scale reaction was conducted (reaction time 2 h). One volume of isopropanol and three volumes of n-hexane were added to the reacted emulsion, and the mixture was vigorously shaken to extract the lipid components. The extraction system was centrifuged at 8000 r / min for 5 min to separate the layers. The supernatant was collected and the isopropanol and n-hexane were removed using a rotary evaporator (Tokyo Rika, EYELA N-1300V) to obtain a mixture of EPA and EPAMG. The mixture was diluted 10-fold with 85% ethanol solution (volume percentage), and then 10 volumes of n-hexane were added. The mixture was vigorously shaken and extracted to dissolve the free EPA in the n-hexane, while the EPAMG remained in the ethanol. After centrifugation at 8000 r / min for 5 min, the n-hexane layer was removed. The extraction was repeated with n-hexane to completely remove the EPA. The lipid components in the crude sample and the ethanol layer after each extraction were analyzed by liquid chromatography, and the purity of EPAMG was calculated.

[0050] The results of the purity determination of EPAMG are as follows: Figure 5 As shown in the figure. Measurements showed that the purity of EPAMG in the ethanol layer was 93.66% during the third extraction and reached 97.09% during the fifth extraction. This purity is sufficient for the application of EPAMG in various fields such as food and medicine.

[0051] Example 4: Esterification effects of esterases E7 and E7-F145L on different fatty acids In this embodiment, different fatty acids were used as substrates for esterification reaction to evaluate the applicability of esterases E7 and E7-F145L to OA, LA, EPA and DHA. The esterification reaction and product detection process were the same as in Example 1. In order to make the reaction effect show obvious differentiation, the reaction time was shortened to 1 h.

[0052] The esterification effects of esterases E7 and E7-F145L on different fatty acids are as follows: Figure 6As shown in the figure, the conversion rates of wild-type E7 for OA and LA reached 21.52% and 23.76%, respectively, significantly higher than its conversion rates for EPA (9.95%) and DHA (2.03%). E7-F145L showed a significantly enhanced catalytic effect on long-chain polyunsaturated fatty acids, achieving a conversion rate of 53.48% for EPA and 12.70% for DHA, while the conversion rates for OA and LA were 42.58% and 46.03%, respectively. This indicates that E7-F145L can not only efficiently synthesize EPAMG, but also, compared to wild-type E7, it can be better applied to the esterification synthesis of other 20-carbon and higher long-chain polyunsaturated fatty acid monoglycerides, demonstrating significant potential for industrial applications.

[0053] The above embodiments are provided to those skilled in the art to fully disclose and describe how the claimed implementations can be carried out and used, and are not intended to limit the scope of the disclosure herein. Modifications that will be obvious to those skilled in the art will be within the scope of the appended claims.

Claims

1. A mutant of esterase E7, characterized in that: It is an esterase E7-F145L, and its amino acid sequence is shown in SEQ ID NO.

5.

2. A mutant of esterase E7, characterized in that: It is esterase E7-F145A, and its amino acid sequence is shown in SEQ ID NO.

4.

3. A mutant of esterase E7, characterized in that: It is an esterase E7-Q199F, and its amino acid sequence is shown in SEQ ID NO.

8.

4. The use of the mutant of esterase E7 according to claim 1, 2 or 3 in the preparation of glycerides.

5. The application according to claim 4, characterized in that: The glycerides are selected from EPAMG.

6. The application according to claim 4, characterized in that: In practical applications, glycerol esters are synthesized using fatty acids and glycerol as substrates under the catalysis of a mutant esterase E7; the fatty acids are selected from any one or more of oleic acid, linoleic acid, EPA, and docosahexaenoic acid.

7. The application according to claim 6, characterized in that: In practical applications, recombinant engineered bacteria expressing esterase E7 mutants are constructed, and the mutants expressing esterase E7 are cultured and induced to produce bacterial culture. The culture is centrifuged, resuspended, and a liquid enzyme preparation is obtained. The liquid enzyme preparation is then freeze-dried to obtain a solid enzyme preparation. Glycerol, fatty acids, and buffer are mixed, and the liquid or solid enzyme preparation is added. The mixture is homogenized to form an emulsion and reacted at 65°C for 1–2 hours. After the reaction, isopropanol and n-hexane are added for extraction.

8. A method for preparing EPAMG, characterized in that: EPAMG was synthesized using EPA and glycerol as substrates under the catalysis of a mutant of the esterase E7 as described in claim 1, 2 or 3.

9. The method for preparing EPAMG according to claim 8, characterized in that: Recombinant engineered bacteria expressing esterase E7 mutants were constructed, and the mutants expressing esterase E7 were cultured and induced to produce bacterial culture. The culture was centrifuged, resuspended, and liquid enzyme preparations were obtained. The liquid enzyme preparations were freeze-dried to obtain solid enzyme preparations. Glycerol, EPA, and buffer were mixed, and liquid or solid enzyme preparations were added. The mixture was homogenized to form an emulsion and reacted at 65°C for 1–2 hours. After the reaction, isopropanol and n-hexane were added for extraction.

10. The method for preparing EPAMG according to claim 9, characterized in that: After extraction, isopropanol and n-hexane are removed by rotary evaporation. The ethanol solution is diluted, and n-hexane is added for extraction. The number of extractions with n-hexane is 1 to 5 times to obtain high-purity EPAMG.

Images