Lipolytic enzyme MfLPE and application of lipolytic enzyme MfLPE in synthesis of ester perfume

Abstract

The invention discloses lipolytic enzyme MfLPE and application of the lipolytic enzyme MfLPE to synthesis of ester spices, and belongs to the technical fields of enzyme engineering, new enzyme resource mining, biological catalysis and green chemical engineering. According to the method disclosed by the invention, lipolytic enzyme MfLPE obtained by heterologous expression and purification is used as a biocatalyst, and cheap and easily available acyl donors (methyl p-nitrobenzoate, methyl p-nitrobenzene butyrate or methyl p-nitrobenzene caprylate) and common aromatic alcohols (benzyl alcohol and phenethyl alcohol) are subjected to catalytic reaction in pure water or a small amount of organic solvent; typical ester perfumes such as benzyl acetate, phenethyl acetate and phenethyl caprylate can be efficiently synthesized, the conversion rate reaches 75% or above, the product selectivity is high, the technological conditions are mild, environment friendliness is achieved, amplification to a continuous flow reaction is easy, and organic solvent dependence and production cost are remarkably reduced. The invention provides a new enzyme source and process scheme for green biosynthesis of ester spices, and has a good industrial application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of enzyme engineering, new enzyme resource mining, biological catalysis and green chemical industry, and particularly relates to a lipolytic enzyme MfLPE and application thereof in synthesis of ester perfumes. BACKGROUND

[0002] Ester compounds are important perfumes and essence raw materials, and are widely used in food, cosmetics, medicine and daily chemical industry fields. Traditional ester synthesis methods usually adopt chemical esterification or acyl transfer reaction, which needs high temperature, strong acid catalysis and a large amount of organic solvent, and has problems of high energy consumption, harsh conditions and environmental pollution.

[0003] Lipase is widely studied in organic phase or ionic liquid for catalyzing “reverse hydrolysis” or transesterification reaction due to its good stereoselectivity and regioselectivity. However, the existing lipase catalytic system still needs a high proportion of organic solvent or surfactant to maintain the activity and stability of the enzyme, and the production cost is high, which limits its industrial application, and the conversion rate of part of the reaction is low.

[0004] Although typical acyltransferase can catalyze esterification reaction in water phase, it is still dependent on complex and expensive activated donor (such as acyl-CoA, pentafluorophenol ester, etc.), and the substrate spectrum is narrow, which makes it difficult to realize green synthesis of diversified ester products, and limits its application in perfume synthesis.

[0005] In recent years, promiscuous hydrolases / acyltransferases with dual activity of hydrolytic enzyme and acyltransferase have attracted attention from academia and industry. This kind of enzyme can directly utilize cheap acyl donor to efficiently catalyze ester synthesis in water phase, and the process is green and the raw material is easy to obtain, which meets the requirements of sustainable development. However, the number of known enzymes of this type is limited, and the catalytic efficiency and stability for common aromatic alcohols are not ideal, and the activity needs to be improved, which makes it difficult to meet the needs of industrial scale production. Therefore, it is urgent to develop a promiscuous hydrolases / acyltransferases with dual activity of hydrolytic enzyme and acyltransferase, which can greenly synthesize ester perfumes in water phase or low solvent system, so as to overcome some deficiencies in the prior art. SUMMARY

[0006] In view of the deficiencies in the prior art, the application provides a lipolytic enzyme MfLPE and application thereof in synthesis of ester perfumes, which overcomes the dependence of traditional esterification on organic solvent and high temperature, and significantly reduces environmental pollution and production cost.

[0007] To achieve the above technical purposes, the technical solutions adopted by the application are as follows:

[0008] The present application provides a lipolytic enzyme MfLPE with hydrolytic enzyme and acyltransferase dual activity, the amino acid sequence of the lipolytic enzyme MfLPE is shown as SEQ ID No: 2.

[0009] Further, the lipolytic enzyme MfLPE is derived from Candidatus Moduliflexus flocculans.

[0010] The present application also provides a polynucleotide encoding the lipolytic enzyme MfLPE, the polynucleotide sequence is shown as SEQ ID No: 1.

[0011] The present application also provides a recombinant expression vector comprising the polynucleotide.

[0012] The present application also provides a recombinant engineering bacterium comprising the polynucleotide or the recombinant expression vector.

[0013] The present application also provides the application of the lipolytic enzyme MfLPE or the polynucleotide or the recombinant expression vector or the recombinant engineering bacterium in the synthesis of ester perfumes, the ester perfumes including benzyl acetate, phenethyl acetate or phenethyl octanoate.

[0014] The present application also provides a method for synthesizing ester perfumes, the method comprising:

[0015] In a pure water system or a system containing a low proportion of organic solvents, the acyl donor is mixed with the acyl acceptor, the lipolytic enzyme MfLPE is added, and the catalytic reaction is carried out at 20-40℃ for 0.5-24h to generate ester perfumes.

[0016] The molar ratio of the acyl donor to the acyl acceptor is 1:0.5-50.

[0017] The acyl acceptor includes benzyl alcohol or phenethyl alcohol.

[0018] The acyl donor includes 4-nitrophenyl acetate, 4-nitrophenyl butyrate or 4-nitrophenyl octanoate.

[0019] The lipolytic enzyme MfLPE is used in continuous flow synthesis of ester perfumes in the form of immobilization, the immobilization carrier is sodium alginate gel beads, and the enzyme loading is 10-30mg / g, preferably 20mg / g.

[0020] The present application also provides the application of the lipolytic enzyme MfLPE or the polynucleotide or the recombinant expression vector or the recombinant engineering bacterium or the method for preparing ester perfumes in the preparation of essence perfumes, the essence perfumes including floral, fruity, honey sweet or milk fragrances.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] This invention is the first to propose that the lipolytic enzyme Candidatus Moduliflexus flocculans MfLPE possesses dual catalytic functions of hydrolysis and acyl transfer. Furthermore, based on functional verification of Candidatus Moduliflexus flocculans MfLPE, a green synthesis technique for ester-based fragrances under all-aqueous conditions is proposed. This technique eliminates organic solvents and expensive enzyme activators, reducing production costs and significantly minimizing environmental pollution. Benefiting from the unique SGNH superfamily structure and excellent enzymatic properties of MfLPE, the product exhibits high selectivity and is easily scaled up to continuous flow or immobilized reactors, realizing a complete technical path from enzyme source innovation to process engineering, providing a novel green synthesis platform for the fragrance and flavor industry. Verification through examples shows that the method described in this invention can prepare floral, fruity, honey-sweet, and creamy ester-based fragrances. Specifically, the conversion rate of phenethyl acetate (with a honey-sweet aroma) prepared using phenylethanol and methyl p-nitrophenylacetate (pNPA) can reach 91%. Attached Figure Description

[0023] Figure 1 A schematic diagram of the expression plasmid constructed for the lipase MfLPE gene of Candidatus Moduliflexus flocculans.

[0024] Figure 2 A schematic diagram showing the results of double enzyme digestion verification of the expression plasmid constructed for the Candidatus Moduliflexus flocculans lipase MfLPE gene.

[0025] Figure 3 The results show the effects of different temperatures, different induction times, and different IPTG concentrations on the expression concentration of the lipase MfLPE protein.

[0026] in, Figure 3 SDS-PAGE images of crude enzyme supernatant and crude enzyme precipitate of lipase MfLPE obtained under IPTG conditions of 0.5 mM A at different temperatures and induction times. Figure 3 B uses ImageJ software to... Figure 3 A histogram of the target band obtained in A, analyzed for grayscale. Figure 3 C is an SDS-PAGE image of crude enzyme supernatant and crude enzyme precipitate of lipase MfLPE induced by different IPTG concentrations under the condition of induction at 37℃ for 4h. Figure 3 D is using ImageJ software to... Figure 3The bar chart of gray scale analysis of the target band obtained in C.

[0027] Figure 4 SDS-PAGE analysis chart of different components in the purification process of Candidatus Moduliflexus flocculans lipolytic enzyme MfLPE protein, and the protein obtained after purification Figure 4 A) and the bar chart of gray scale analysis of protein expression level Figure 4 B).

[0028] Figure 5 Analysis chart of the influence of different substrate types and concentrations on the acyltransferase activity of Candidatus Moduliflexus flocculans lipolytic enzyme MfLPE; wherein, Figure 5 A, 5B, 5C respectively represent the reaction of pNPA, pNPB and pNPO with a final concentration of 2mM and 0-100mM of benzyl alcohol, Figure 5 D, 5E, 5F respectively represent the reaction of pNPA, pNPB and pNPO with a final concentration of 2mM and 0-100mM of phenethyl alcohol.

[0029] Figure 6 Gas chromatogram of the reaction product of Candidatus Moduliflexus flocculans lipolytic enzyme MfLPE (left) and enzyme kinetics curve chart (right).

[0030] Figure 7 Gas chromatogram of benzyl acetate synthesized by lipolytic enzyme MfLPE in aqueous phase.

[0031] Figure 8 Gas chromatogram of phenethyl acetate synthesized by lipolytic enzyme MfLPE in low solvent system.

[0032] Figure 9 Gas chromatogram of octanoic acid phenethyl ester synthesized by lipolytic enzyme MfLPE in continuous flow of immobilized enzyme. DETAILED DESCRIPTION

[0033] The technical solutions of the present application are further described below through specific embodiments. It should be understood that the embodiments are preferred solutions of the present application, and are intended to assist in understanding the technical content of the present application, but not to limit the protection scope of the present application. Without deviating from the basic concept of the present application, those skilled in the art can make various substitutions, equivalent improvements or combined applications, which should be considered to fall within the protection scope of the present application.

[0034] In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present application. However, it will be apparent to one skilled in the art that the present application can be practiced without the specific details set forth in this description, that the present application can be practiced with different specific embodiments and variations, and that the present application is not limited to the specific embodiments described herein.

[0035] Example 1: Heterologous expression and purification of Candidatus Moduliflexus flocculans lipolytic enzyme MfLPE

[0036] (1) Gene synthesis and vector construction:

[0037] This example is based on the gene sequence of Candidatus Moduliflexus flocculans lipolytic enzyme MfLPE published in NCBI database (Sequence Accession No: GAK52576.1), and the gene sequence of lipolytic enzyme MfLPE is obtained after optimization according to the codon bias of E. coli (see SEQ ID No: 1). The gene is synthesized by Nanjing Kingsriver Biotechnology Co., Ltd., and NdeI / XhoI enzyme cutting sites are added at both ends. The fragment is cloned into pET-28a(+) vector to construct recombinant plasmid pET28a-MfLPE (plasmid map see Figure 1 ), and double enzyme digestion verification is performed (verification results see Figure 2 ).

[0038] The codon-optimized gene sequence of Candidatus Moduliflexus flocculans lipolytic enzyme MfLPE (SEQ ID No: 1) is as follows:

[0039] ATGAAAACAATACTATGTTATGGAGATTCAAACACCTGGGGTTACAATCCGCGTCTTAAGTCTCGTTATAGCAAAGAGGAGCGCTGGGCAGGAGTTTTGCGCAAACAGTTGGGCGAGGGCTATTTCGTCATCGAGGAGGGCCTGAATGGCCGTACCACGGTTTGGGATGATCCGATCGAAGGTGAGCACAAAAACGGCAAAACCTATCTGTTTCCGTGCCTGGAAAGCCATGCACCGCTGGACCTGGTAGTCGTGATGCTGGGCACGAACGATTTGAAGAACCGTTTTGGTTTATCTGCGTACGACATCGCGAAGGGTGCTGGCACTCTGGTGCAGATTATCCAGAAATCCGGTTTCGGCCAAGGTGGTCAATCGCCGAAGGTGTTGCTGATTGCGCCACCGTCCCTGGCGCAGTTGGCTGGCACCGATTTCGCCGACATGTTTGCGGGTGGTGAAGATAAAAGCCGTCAGCTGGCCGACGCGTACCGCCGTGTTGCGGCAGAACTGGACTGTGAATTCCTGGACGCGGGTAGCGTTATCACCAGCTCCCCGGTTGATGCGATTCATTTTGACCCGGAAGAGCACCAAACCCTCGCTGTGGCTGTGGCCGCAAAGATCAGAAGCATTCTGCTCGAGCACCACCACCACCACCACTGA

[0040] (2) E. coli transformation and expression:

[0041] The recombinant plasmid was transformed into E. coli BL21(DE3) competent cells, which were spread on LB plates containing 50 μg / mL kanamycin. A single colony was inoculated into LB medium (containing 50 μg / mL kanamycin) and incubated at 37°C until the OD600 was 0.6. IPTG was then added at a final concentration of 0.05 mM, 0.1 mM, and 0.5 mM, respectively, and the cells were induced at 15°C, 30°C, or 37°C for 4-16 h. The induced bacterial solution was centrifuged at 4°C and 8000 rpm for 12 min, and the supernatant was discarded. The precipitate was resuspended in 1 mL lysis buffer (0.9 g NaH2PO4·2H2O, 1.75 g NaCl, ddH2O to 90 mL, and NaOH to adjust the pH to 8.0, then ddH2O to 100 mL), transferred to a 2 mL centrifuge tube, and subjected to ultrasonic disruption at 50% power for 3 s, with a 4 s interval, for a total of 3 min. After treatment, the mixture was centrifuged at 4°C and 15000 rpm for 12 min to separate the crude enzyme supernatant and the crude enzyme precipitate, and the protein concentrations of the two fractions were determined.

[0042] (3) Lipolytic enzyme MfLPE protein expression analysis:

[0043] The total protein concentrations of the samples were determined using Nano-300, and each sample was diluted to 10 μg / μL with the lysis buffer to ensure that the total protein amounts of the different samples were equal when loaded onto the SDS-PAGE. Then, 40 μL of each diluted sample was taken and mixed with 10 μL loading buffer (5×), and the mixture was heated in a 100°C water bath for 10 min and then centrifuged at 15000 rpm for 5 min.

[0044] SDS-PAGE analysis was performed using FuturePAGETM precast gels (4-20%, 12 wells), with a sample loading amount of 15 μL and a protein marker loading amount of 6 μL. The electrophoresis voltage was set to 170 V, and the electrophoresis time was about 1 h. After electrophoresis, the gel was stained and destained, and the target bands were analyzed for gray scale using ImageJ software. The results are shown in Figure 3 .

[0045] In this example, the effects of 15°C induction for 16 h, 30°C induction for 4 h, and 37°C induction for 4 h on protein expression were studied at an IPTG concentration of 0.5 mM (results shown in Figure 3A, wherein, Lane M: Protein maker; Lane 1: supernatant of cell lysate induced at 15°C for 16h; Lane 2: supernatant of cell lysate induced at 30°C for 4h; Lane 3: supernatant of cell lysate induced at 37°C for 4h; Lane 4: precipitate of cell lysate induced at 15°C for 16h; Lane 5: precipitate of cell lysate induced at 30°C for 4h; Lane 6: precipitate of cell lysate induced at 37°C for 4h; Lane 7: supernatant of cell lysate without IPTG, cultured at 15°C for 16h; Lane 8: supernatant of cell lysate without IPTG, cultured at 30°C for 4h; Lane 9: supernatant of cell lysate without IPTG, cultured at 37°C for 4h.)

[0046] In this example, the effect of 0mM, 0.05mM, 0.1mM and 0.5mM IPTG on protein expression was studied under the condition of 37°C induction for 4h (results as shown in Figure 3 C, wherein, Lane M: Protein maker; Lane 1: supernatant of cell lysate with IPTG concentration of 0.5mM; Lane 2: supernatant of cell lysate with IPTG concentration of 0.1mM; Lane 3: supernatant of cell lysate with IPTG concentration of 0.05mM; Lane 4: supernatant of cell lysate without IPTG; Lane 5: precipitate of cell lysate without IPTG; Lane 6: precipitate of cell lysate with IPTG concentration of 0.05mM; Lane 7: precipitate of cell lysate with IPTG concentration of 0.1mM; Lane 8: precipitate of cell lysate with IPTG concentration of 0.5mM.)

[0047] By comparing the concentration of the target protein under different induction conditions, the optimal induction temperature of the enzyme was found to be 37°C, the optimal induction time was 4h, and the IPTG concentration was 0.5mM.

[0048] (4) Protein purification of lipolytic enzyme MfLPE

[0049] After inducing and culturing at 37°C for 4h, the bacterial cells were collected by centrifugation, and the supernatant was obtained after ultrasonic disruption. The target protein was eluted from the Ni-NTA affinity chromatography column (His-Tag label) with PBS buffer containing 20mM imidazole, and then desalted by Superdex 75 gel filtration chromatography. The MfLPE enzyme powder with a purity of >95% was obtained by freeze-drying (amino acid sequence as shown in SEQ ID No: 2), and the purification results are shown in Figure 4 ( Figure 4Lane M: Protein marker; Lane 1: purified protein 1; Lane 2: purified protein 2; Lane 3: crude enzyme supernatant; Lane 4: crude enzyme precipitate; Lane 5: flow-through; Lane 6: flow-through); wherein the purified protein 1 and the purified protein 2 are parallel samples.

[0050] The results show that the optimal induction condition for the recombinant E. coli strain expressing the lipolytic enzyme MfLPE is 37°C under induction culture for 4h Figure 3 A and Figure 3 B), the IPTG concentration is 0.5mM Figure 3 C and Figure 3 D); the effect of purifying the lipolytic enzyme MfLPE by using the Ni-NTA affinity column is good, the SDS-PAGE analysis shows that the target band in the purified sample is obvious and almost does not contain other impurity bands Figure 4 A), the molecular weight of the lipolytic enzyme MfLPE is about 24kDa, the gray scale analysis shows that the protein concentration of the purified lipolytic enzyme MfLPE is 1.6-1.8mg / mL Figure 4 B).

[0051] The amino acid sequence of the recombinant protein of Candidatus Moduliflexus flocculans lipolytic enzyme MfLPE (SEQ ID No: 2) is as follows:

[0052] MKTILCYGDSNTWGYNPRLKSRYSKEERWAGVLRKQLGEGYFVIEEGLNGRTTVWDDPIEGEHKNGKTYLFPCLESHAPLDLVVVMLGTNDLKNRFGLSAYDIAKGAGTLVQIIQKSGFGQGGQSPKVLLIAPPSLAQLAGTDFADMFAGGEDKSRQLADAYRRVAAELDCEFLDAGSVITSSPVDAIHFDPEEHQTLAVAVAAKIRSILHHHHHH*

[0053] Example 2: Influence of the type and concentration of the reaction substrate on the promiscuous hydrolase and acyltransferase activities of the lipolytic enzyme MfLPE

[0054] (1) The purified lipolytic enzyme MfLPE of Example 1 is diluted with 0.2M potassium phosphate buffer (pH 7.5) to prepare 50μL of a 50ng / μL lipolytic enzyme MfLPE solution;

[0055] (2) Select different acyl acceptors: benzyl alcohol (BeOH), phenethyl alcohol (PEA); different acyl donors: 4-nitrophenyl acetate (pNPA), 4-nitrophenyl butyrate (pNPB) and 4-nitrophenyl octanoate (pNPO), according to the reaction system in Table 1. The OD was measured by an enzyme marker. 405 The absorbance was measured, the incubator temperature was set to 20°C, the total reaction time was 30 min, and a value was read once every 20 s, the data was analyzed, and the optimal acyl acceptor type and the best concentration ratio of acceptor and donor were determined.

[0056] Table 1. Effect of type and concentration of reaction substrate on MfLPE activity Reaction system

[0057]

[0058] The results show that lipase MfLPE exhibits higher catalytic activity and tolerance in the benzyl alcohol and pNPA system, and the relative activity is as high as 1.56. The optimal concentration ratio of benzyl alcohol and pNPA reaction is 15:1 (results are shown in Figure 5 , Figure 5 A, 5B, 5C respectively represent the reaction of pNPA, pNPB and pNPO with a final concentration of 2mM and 0-100mM of benzyl alcohol, Figure 5 D, 5E, 5F respectively represent the reaction of pNPA, pNPB and pNPO with a final concentration of 2mM and 0-100mM of phenethyl alcohol.

[0059] Example 3: Enzyme kinetic properties of lipase MfLPE acyltransferase activity

[0060] (1) Dilute the lipase MfLPE purified in Example 1 with 0.2M potassium phosphate buffer (pH 7.5) to prepare 50μL, 1800ng / μL of lipase MfLPE solution;

[0061] (2) Select benzyl alcohol and pNPA as reaction substrates, add according to Table 2 system, add 500μL diethyl ether after reaction for 30min to quickly vortex quench the enzyme, fix the sample on the oscillator at 14000rpm for 2h to extract the product. After extraction, stand for stratification, carefully transfer the upper organic phase to a new centrifuge tube, add excess magnesium sulfate powder to adsorb residual moisture, mix thoroughly, then centrifuge at 15000rpm for 5min, discard the lower layer containing magnesium sulfate precipitate. Take the upper clear organic phase, and determine the product concentration by gas chromatograph.

[0062] Table 2. Enzymatic reaction system under different substrate concentrations

[0063]

[0064] Gas chromatography parameters and method settings: the initial temperature of the chromatographic column was 70℃, maintained for 2 min; the temperature was increased to 90℃ at a rate of 15℃ / min, maintained for 2 min; then the temperature was increased to 100℃ at a rate of 15℃ / min, maintained for 2 min; then the temperature was increased to 115℃ at a rate of 10℃ / min, maintained for 2 min; then the temperature was increased to 125℃ at a rate of 10℃ / min, maintained for 1 min; finally, the temperature was increased to 300℃ at a rate of 30℃ / min; the temperatures of the injection port and the detector were 250℃ and 275℃, respectively.

[0065] According to the product yield and reaction time, the initial reaction rate V of each substrate concentration was calculated. The obtained data was imported into GraphPad Prism software, the Michaelis equation curve was fitted by using nonlinear regression, the relationship graph of substrate concentration and reaction rate was obtained, and the Michaelis constant (Km), the maximum reaction rate (Vmax) and the catalytic constant (Kcat) were further calculated.

[0066] The results show that the maximum reaction rate V of the acyl transfer activity of the lipase MfLPE to the substrate benzyl alcohol is 80.38 μM / min, the Michaelis constant K is 61.26 mM, the catalytic constant K is 1.71 s max m cat -1 - 1 -1 The specific enzyme activity is 4.47 U / mg (the results are shown in Table 2). Figure 6 The above kinetic parameters for characterizing the enzyme properties show that the hydrolytic enzyme of the application has acyl transfer activity and can catalyze the synthesis of ester compounds in water phase.

[0067] Example 4: Catalytic synthesis of benzyl acetate (floral fragrance) in water phase

[0068] 5 mL of pure water was added with 250 ng of MfLPE enzyme powder obtained in Example 1, 150 mM of benzyl alcohol and 15 mM of methyl p-nitrophenylacetate (pNPA), and the reaction was carried out at 20℃ with 200 rpm oscillation for 30 min. 500 μL of ether was added to quickly quench the enzyme, and the sample was fixed on the oscillator for 14000 rpm continuous oscillation for 2 h to make the organic phase and the water phase fully contact to extract the product. After the extraction was completed, the sample was allowed to stand and stratify, and the upper organic phase was carefully sucked and transferred to a new centrifuge tube, and an excess of magnesium sulfate powder was added to adsorb the residual moisture, and after being fully mixed, the sample was centrifuged at 15000 rpm for 5 min, and the lower layer containing the magnesium sulfate precipitate was discarded. The upper layer of clear organic phase was taken, and the product concentration was determined by using a gas chromatograph. The gas chromatography analysis showed that the product was benzyl acetate (jasmine fragrance), and the gas chromatography results of the product are shown in Table 2. Figure 7 . ​​​​​

[0069] Example 5: Synthesis of phenyl ethyl acetate (fruity flavor) in low solvent system

[0070] 3 mL of 10% DMSO-water mixed solution was added with 150 ng MfLPE, 100 mM phenyl ethanol, 9 mM methyl p-nitrophenyl acetate (pNPA), and the reaction was carried out at 30°C with 200 rpm oscillation for 30 min, and the conversion rate reached 91%. The reaction solution was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 10:1) to obtain phenyl ethyl acetate (with honey sweet smell), and the gas chromatography result of the product is shown in Table 2. Figure 8 .

[0071] Example 6: Continuous flow synthesis of phenyl octanoate (buttery flavor) by immobilized enzyme

[0072] (1) Immobilized enzyme preparation: the MfLPE enzyme powder obtained in Example 1 was mixed with a sodium alginate solution (2%), and was dropped into a 0.1M CaCl2 solution to solidify, to prepare immobilized enzyme particles with a diameter of 1.5 mm (enzyme loading capacity 20 mg / g).

[0073] (2) Continuous flow reaction: the immobilized enzyme was filled into a column reactor (10 cm x 1 cm), and the substrate solution (50 mM phenyl ethanol + 10 mM methyl p-nitrophenyl octanoate (pNPO) dissolved in pH 7.0 phosphate buffer) was fed at a flow rate of 0.5 mL / min, and the reaction was carried out at 40°C. After 24 h of reaction, the effluent was collected, extracted and purified to obtain phenyl octanoate (buttery flavor), and the gas chromatography result of the product is shown in Table 3. Figure 9 .

[0074] In summary, the enzyme provided by the present application is suitable for application in water phase or low solvent system to prepare ester flavor, and is further applied in the field of preparation of essence and flavor, and the shortcoming of adding organic solvent in the traditional enzymatic method is improved, and is suitable for large-scale popularization and application.

[0075] Although the present application has been described in detail in the foregoing description with general principles and specific embodiments, some modifications or improvements can be made on the basis of the present application, which is obvious to those skilled in the art. Therefore, these modifications or improvements made on the basis of not deviating from the spirit of the present application, all belong to the scope of protection claimed by the present application.

Claims

1. A lipolytic enzyme MfLPE, characterized in that, The lipolytic enzyme has both hydrolytic enzyme and acyltransferase dual activities, and the amino acid sequence of the lipolytic enzyme MfLPE is shown in SEQ ID No:

2.

2. A polynucleotide encoding the lipolytic enzyme MfLPE according to claim 1, characterized in that, The polynucleotide sequence is shown in SEQ ID No:

1.

3. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the polynucleotide of claim 2.

4. A recombinant engineered bacterium characterized in that, The recombinant engineering bacteria comprise the polynucleotide of claim 2 or the recombinant expression vector of claim 3.

5. The lipolytic enzyme MfLPE of claim 1, or the polynucleotide of claim 2, or the recombinant expression vector of claim 3, or the recombinant engineering bacteria of claim 4, is applied in synthesis of ester perfumes.

6. Use according to claim 5, characterized in that, The ester perfume comprises benzyl acetate, phenethyl acetate or phenethyl octanoate.

7. A method of synthesizing an ester-based fragrance, characterized by, The ester perfume is synthesized in water or a low solvent system, and the method comprises: In a pure water system or a system containing a low proportion of organic solvent, an acyl donor is mixed with an acyl acceptor, the lipolytic enzyme MfLPE is added, and the reaction is catalyzed at 20-40℃ for 0.5-24h to generate the ester perfume.

8. The method of claim 7, wherein, The acyl acceptor comprises benzyl alcohol or phenethyl alcohol; the acyl donor comprises any one of 4-nitrophenyl acetate, 4-nitrophenyl butyrate or 4-nitrophenyl octanoate.

9. The method of claim 7, wherein, The molar ratio of the acyl donor to the acyl acceptor is 1:0.5-50; the lipolytic enzyme MfLPE is used in continuous flow synthesis of ester perfume in a fixed form, the fixed carrier is sodium alginate gel beads, the enzyme loading is 10-30mg / g, and preferably the enzyme loading is 20mg / g.

10. The lipolytic enzyme MfLPE of claim 1, or the polynucleotide of claim 2, or the recombinant expression vector of claim 3, or the recombinant engineering bacteria of claim 4, or the ester perfume synthesized by the method of any one of claims 7-9, is applied in preparation of a fragrance, which comprises a floral, fruity, honey sweet or milky fragrance.

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