A method for biocatalytic synthesis of carboxylic acid esters

Abstract

The application discloses a method for directly catalyzing synthesis of carboxylic acid ester compounds by using biological enzymes, and belongs to the technical field of biology. The method uses carboxylic acid ester compounds as raw materials, and in the presence of alcohol, uses salicylic acid binding protease 2 (SABP2) as a catalyst to catalyze ester exchange reaction, so as to convert the carboxylic acid ester compounds into corresponding alcohol and corresponding ester compounds. Since biological enzymes are used for catalysis, the method has the advantages of mild conditions, simple operation, small pollution, green safety and high product purity, and is a very promising biological enzyme route for synthesizing spices and essences.

Description

Technical Field

[0001] This invention belongs to the field of bio-enzyme catalytic synthesis technology, specifically relating to a method for bio-enzyme catalytic synthesis of carboxylic acid ester compounds. Background Technology

[0002] Carboxylic acid esters are an important class of organic compounds with wide applications in chemical synthesis, food industry, pharmaceuticals, lubricants, and fragrances.

[0003] Carboxylic acid esters, synthesized from carboxylic acids and alcohols, are a significant class of carboxylic acid esters in fragrances. For example, ethyl butyrate, with its pineapple-like aroma, is widely used in various food, beverage, alcoholic, and tobacco flavorings, primarily for formulating pineapple, grape, rum, and strawberry flavors. Methyl propionate, as a solvent, is widely used in the coatings and inks industries due to its strong dissolving power and moderate evaporation rate. It is particularly suitable for dissolving resins such as nitrocellulose and cellulose acetate, adjusting the viscosity and drying speed of coatings to create a smooth and even surface. In printing inks, methyl propionate can replace some benzene-based solvents, reducing VOC emissions and meeting environmental standards.

[0004] In industry, carboxylic acid esters are mostly produced on a large scale using inorganic catalysts for the esterification of carboxylic acids and alcohols. Catalysts used in the synthesis of carboxylic acid esters include zirconium sulfate, strong acid ion exchange resins, and p-toluenesulfonic acid. However, zirconium sulfate catalysts are complex to prepare, require large quantities, are costly, and have long production cycles; the preparation of solid superacids is cumbersome, time-consuming, and has low reusability; and p-toluenesulfonic acid has a low esterification rate and easily introduces impurities. Therefore, these three catalysts are not commonly used in actual production. Currently, sulfuric acid catalysis is commonly used to synthesize carboxylic acid esters. However, the traditional sulfuric acid catalysis method has disadvantages such as low yield, low product purity, excessive alcohol consumption, strong corrosiveness posing safety hazards, and potential environmental pollution.

[0005] Therefore, it is particularly important to develop a method for synthesizing carboxylic acid esters that has mild reaction conditions, is easy to operate, and is environmentally friendly. Summary of the Invention

[0006] To address the aforementioned technical deficiencies, the technical problem this invention aims to solve is how to develop a mild, simple, and environmentally friendly method for synthesizing carboxylic acid esters.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is: a method for the bio-enzymatic synthesis of carboxylic acid ester compounds, which uses carboxylic acid esters and alcohols as raw materials and SABP2 as a catalyst to catalyze the conversion of carboxylic acid esters into the corresponding methyl ester, ethyl ester and propyl ester.

[0008] The present invention is achieved through the following technical solution: Step 1, providing salicylic acid-binding protein 2 (SABP2) with high conversion rate, catalytic activity and a very broad substrate spectrum.

[0009] Furthermore, the gene encoding SABP2 is provided.

[0010] Furthermore, a vector carrying the aforementioned genes is provided.

[0011] Furthermore, microbial cells expressing the aforementioned genes are provided.

[0012] In one embodiment, the recombinant bacteria are expressed using pET-21a(+) as an expression vector.

[0013] In one embodiment, the recombinant bacteria uses BL21(DE3) as the host bacteria.

[0014] Step 2: Using methyl propionate as a model, investigate the optimal ethanol concentration for the synthesis of ethyl carboxylate from methyl carboxylate by SABP2 obtained in Step 1, preferably 5% (v / v); the optimal propanol concentration for the synthesis of propyl carboxylate from methyl carboxylate is preferably 5% (v / v); using ethyl propionate as a model, investigate the optimal methanol concentration for the synthesis of methyl carboxylate from ethyl carboxylate by SABP2, preferably 1% (v / v); the optimal propanol concentration for the synthesis of propyl carboxylate from ethyl carboxylate is preferably 5% (v / v); using propyl propionate as a model, investigate the optimal methanol concentration for the synthesis of methyl carboxylate from propyl carboxylate by SABP2, preferably 5% (v / v); the optimal ethanol concentration for the synthesis of ethyl carboxylate from propyl carboxylate is preferably 5% (v / v).

[0015] Step 3: Using methyl propionate as a model, investigate the optimal temperature for the synthesis of carboxylic acid esters catalyzed by SABP2 obtained in Step 1 and its stability at different temperatures. The optimal temperature is 40℃.

[0016] Step 4: Using methyl propionate as a model, investigate the optimal pH for the synthesis of carboxylic acid esters catalyzed by SABP2 obtained in Step 1. The preferred pH is 7.4.

[0017] The specific implementation process of SABP2-catalyzed synthesis of carboxylic acid esters is as follows: a certain concentration of carboxylic acid ester, a certain amount of corresponding alcohol, and a certain amount of 0.1M PH7.4 PB are first added to the reaction vessel. After shaking and mixing, a certain concentration of SABP2 is added. The reaction vessel is then immediately placed in a 40℃ water bath for a certain period of time, and the conversion rate is detected by HPLC.

[0018] Step 5: SABP2 at different enzyme concentrations (0.5µM, 1µM, 2µM, 4µM) catalyzes the conversion of methyl propionate to ethyl propionate.

[0019] In a preferred embodiment 1 of the present invention, the preparation of bacterial culture based on laboratory conditions is described in detail.

[0020] In a preferred embodiment 2 of the present invention, the acquisition of the SABP2 recombinant plasmid is described in detail.

[0021] In a preferred embodiment 3 of the present invention, the process of prokaryotic expression of SABP2 in Escherichia coli is described in detail.

[0022] In a preferred embodiment 4 of the present invention, the process of purifying SABP2 using Ni-agarose gel 6FF (His-tagged purification resin) is described in detail.

[0023] In a preferred embodiment 5 of the present invention, the process of identifying the purity of SABP2 by SDS-PAGE is described in detail.

[0024] In a preferred embodiment 6 of the present invention, the determination of SABP2 protein concentration is described in detail.

[0025] In a preferred embodiment 7 of the present invention, the optimal ethanol concentration for SABP2-catalyzed conversion of methyl propionate to ethyl propionate is described in detail.

[0026] In a preferred embodiment 8 of the present invention, the optimal temperature for HPLC determination of the conversion of methyl propionate to ethyl propionate catalyzed by SABP2 is described in detail.

[0027] In a preferred embodiment 9 of the present invention, the stability of SABP2-catalyzed conversion of methyl propionate to ethyl propionate at different temperatures is described in detail by HPLC.

[0028] In a preferred embodiment 10 of the present invention, the optimal pH for determining the conversion of methyl propionate to ethyl propionate catalyzed by SABP2 is described in detail.

[0029] In a preferred embodiment 11 of the present invention, the conversion rate of methyl propionate to ethyl propionate catalyzed by SABP2 with different enzyme concentrations (0.5µM, 1µM, 2µM, 4µM) is described in detail by HPLC.

[0030] In a preferred embodiment 12 of the present invention, the conversion rate of 20 mM methyl propionate to ethyl propionate catalyzed by 1 µM SABP2 at different times under optimal conditions is described in detail by HPLC determination.

[0031] In a preferred embodiment 13 of the present invention, the conversion rate of 20 mM methyl butyrate to ethyl butyrate catalyzed by 1 µM SABP2 at different times under optimal conditions is described in detail by HPLC determination.

[0032] In a preferred embodiment 14 of the present invention, the conversion rate of 20 mM methyl valerate to ethyl valerate catalyzed by 1 µM SABP2 at different times under optimal conditions is described in detail by HPLC.

[0033] In a preferred embodiment 15 of the present invention, the conversion rate of 20 mM methyl hexanoate to ethyl hexanoate catalyzed by 1 µM SABP2 at different times under optimal conditions is described in detail by HPLC determination.

[0034] In a preferred embodiment 16 of the present invention, the conversion rate of 20 mM isobutyrate to ethyl isobutyrate catalyzed by 1 µM SABP2 at different times under optimal conditions is described in detail by HPLC.

[0035] In a preferred embodiment 17 of the present invention, the conversion rate of 20 mM methyl isovalerate to ethyl isovalerate catalyzed by 1 µM SABP2 at different times under optimal conditions is described in detail by HPLC.

[0036] In a preferred embodiment 18 of the present invention, the conversion rate of 20 mM methyl 2-methylbutyrate to ethyl 2-methylbutyrate catalyzed by 1 µM SABP2 at different times under optimal conditions is described in detail by HPLC.

[0037] In a preferred embodiment 19 of the present invention, the conversion rate of 20 mM methyl 2-methylvalerate to ethyl 2-methylvalerate under optimal conditions catalyzed by 1 µM SABP2 at different times is described in detail by HPLC.

[0038] In a preferred embodiment 20 of the present invention, the conversion rate of 20 mM trimethylacetate to trimethyl ethyl ester catalyzed by 1 µM SABP2 at different times under optimal conditions is described in detail by HPLC.

[0039] In a preferred embodiment 21 of the present invention, the conversion rate of 20 mM methyl acetate to ethyl acetate catalyzed by 1 µM SABP2 at different times under optimal conditions is described in detail by HPLC.

[0040] In a preferred embodiment 22 of the present invention, the conversion rate of 20 mM ethyl propionate to methyl propionate catalyzed by 1 µM SABP2 at different times under optimal conditions is described in detail by HPLC determination.

[0041] In a preferred embodiment 23 of the present invention, the conversion rate of 20 mM ethyl butyrate to methyl butyrate catalyzed by 1 µM SABP2 at different times under optimal conditions is described in detail by HPLC determination.

[0042] In a preferred embodiment 24 of the present invention, the conversion rate of 20 mM ethyl valerate to methyl valerate catalyzed by 1 µM SABP2 at different times under optimal conditions is described in detail by HPLC determination.

[0043] In a preferred embodiment 25 of the present invention, the conversion rate of 20 mM ethyl hexanoate to methyl hexanoate catalyzed by 1 µM SABP2 at different times under optimal conditions is described in detail by HPLC determination.

[0044] In a preferred embodiment 26 of the present invention, the conversion rate of 20 mM ethyl isobutyrate to methyl isobutyrate catalyzed by 1 µM SABP2 at different times under optimal conditions is described in detail by HPLC.

[0045] In a preferred embodiment 27 of the present invention, the conversion rate of 20 mM ethyl isovalerate to methyl isovalerate catalyzed by HPLC under optimal conditions at different times using 1 µM SABP2 is described in detail.

[0046] In a preferred embodiment 28 of the present invention, the conversion rate of 20 mM ethyl 2-methylbutyrate to methyl 2-methylbutyrate catalyzed by 1 µM SABP2 at different times under optimal conditions is described in detail by HPLC.

[0047] In a preferred embodiment 29 of the present invention, the conversion rate of 20 mM ethyl 2-methylvalerate to methyl 2-methylvalerate under optimal conditions catalyzed by 1 µM SABP2 at different times is described in detail by HPLC.

[0048] In a preferred embodiment 30 of the present invention, the conversion rate of 20 mM trimethylethyl acetate to trimethylacetate catalyzed by 1 µM SABP2 at different times under optimal conditions is described in detail by HPLC.

[0049] In a preferred embodiment 31 of the present invention, the conversion rate of 20 mM ethyl acetate to methyl acetate catalyzed by 1 µM SABP2 at different times under optimal conditions is described in detail by HPLC.

[0050] In a preferred embodiment 32 of the present invention, the conversion rate of 20 mM methyl propionate to propyl propionate catalyzed by 1 µM SABP2 at different times under optimal conditions is described in detail by HPLC determination.

[0051] In a preferred embodiment 33 of the present invention, the conversion rate of methyl butyrate to propyl butyrate catalyzed by 1 µM SABP2 at different times under optimal conditions is described in detail by HPLC.

[0052] In a preferred embodiment 34 of the present invention, the conversion rate of 20 mM methyl valerate to propyl valerate catalyzed by 1 µM SABP2 at different times under optimal conditions is described in detail by HPLC.

[0053] In a preferred embodiment 35 of the present invention, the conversion rate of 20 mM methyl hexanoate to propyl hexanoate catalyzed by 1 µM SABP2 at different times under optimal conditions is described in detail by HPLC determination.

[0054] In a preferred embodiment 36 of the present invention, the conversion rate of 20 mM methyl isobutyrate to propyl isobutyrate catalyzed by 1 µM SABP2 at different times under optimal conditions is described in detail by HPLC determination.

[0055] In a preferred embodiment 37 of the present invention, the conversion rate of 20 mM methyl isovalerate to propyl isovalerate catalyzed by HPLC under optimal conditions at different times using 1 µM SABP2 is described in detail.

[0056] In a preferred embodiment 38 of the present invention, the conversion rate of 20 mM methyl 2-methylbutyrate to propyl 2-methylbutyrate catalyzed by 1 µM SABP2 at different times under optimal conditions is described in detail by HPLC.

[0057] In a preferred embodiment 39 of the present invention, the conversion rate of 20 mM methyl 2-methylvalerate to propyl 2-methylvalerate under optimal conditions catalyzed by 1 µM SABP2 at different times is described in detail by HPLC.

[0058] In a preferred embodiment 40 of the present invention, the conversion rate of 20 mM trimethylacetate to trimethylacetate by HPLC determination of 1 µM SABP2 at different times under optimal conditions is described in detail.

[0059] In a preferred embodiment 41 of the present invention, the conversion rate of 20 mM methyl acetate to propyl acetate catalyzed by 1 µM SABP2 at different times under optimal conditions is described in detail by HPLC.

[0060] In a preferred embodiment 42 of the present invention, the conversion rate of 20 mM ethyl propionate to propyl propionate catalyzed by 1 µM SABP2 at different times under optimal conditions is described in detail by HPLC determination.

[0061] In a preferred embodiment 43 of the present invention, the conversion rate of 20 mM ethyl butyrate to propyl butyrate catalyzed by 1 µM SABP2 at different times under optimal conditions is described in detail by HPLC determination.

[0062] In a preferred embodiment 44 of the present invention, the conversion rate of 20 mM ethyl valerate to propyl valerate catalyzed by 1 µM SABP2 at different times under optimal conditions is described in detail by HPLC.

[0063] In a preferred embodiment 45 of the present invention, the conversion rate of 20 mM ethyl hexanoate to propyl hexanoate catalyzed by 1 µM SABP2 at different times under optimal conditions is described in detail by HPLC determination.

[0064] In a preferred embodiment 46 of the present invention, the conversion rate of 20 mM ethyl isobutyrate to propyl isobutyrate catalyzed by 1 µM SABP2 at different times under optimal conditions is described in detail by HPLC determination.

[0065] In a preferred embodiment 47 of the present invention, the conversion rate of 20 mM ethyl isovalerate to propyl isovalerate catalyzed by HPLC under optimal conditions at different times using 1 µM SABP2 is described in detail.

[0066] In a preferred embodiment 48 of the present invention, the conversion rate of 20 mM ethyl 2-methylbutyrate to propyl 2-methylbutyrate catalyzed by 1 µM SABP2 at different times under optimal conditions is described in detail by HPLC.

[0067] In a preferred embodiment 49 of the present invention, the conversion rate of 20 mM ethyl 2-methylvalerate to propyl 2-methylvalerate under optimal conditions catalyzed by 1 µM SABP2 at different times is described in detail by HPLC.

[0068] In a preferred embodiment 50 of the present invention, the conversion rate of 20 mM trimethylethyl acetate to trimethylacetate catalyzed by 1 µM SABP2 at different times under optimal conditions is described in detail by HPLC.

[0069] In a preferred embodiment 51 of the present invention, the conversion rate of 20 mM ethyl acetate to propyl acetate catalyzed by 1 µM SABP2 at different times under optimal conditions is described in detail by HPLC determination.

[0070] In a preferred embodiment 52 of the present invention, the conversion rate of 20 mM propyl propionate to methyl propionate catalyzed by 1 µM SABP2 at different times under optimal conditions is described in detail by HPLC determination.

[0071] In a preferred embodiment 53 of the present invention, the conversion rate of 20 mM propyl butyrate to methyl butyrate catalyzed by 1 µM SABP2 at different times under optimal conditions is described in detail by HPLC determination.

[0072] In a preferred embodiment 54 of the present invention, the conversion rate of 20 mM propyl isobutyrate to methyl isobutyrate catalyzed by 1 µM SABP2 at different times under optimal conditions is described in detail by HPLC.

[0073] In a preferred embodiment 55 of the present invention, the conversion rate of 20 mM propyl isovalerate to methyl isovalerate catalyzed by HPLC determination at different times under optimal conditions using 1 µM SABP2 isovaronate isovaronate.

[0074] In a preferred embodiment 56 of the present invention, the conversion rate of 20 mM propyl 2-methylbutyrate to methyl 2-methylbutyrate catalyzed by 1 µM SABP2 at different times under optimal conditions is described in detail by HPLC.

[0075] In a preferred embodiment 57 of the present invention, the conversion rate of 20 mM propyl acetate to methyl acetate catalyzed by 1 µM SABP2 at different times under optimal conditions is described in detail by HPLC.

[0076] In a preferred embodiment 58 of the present invention, the conversion rate of 20 mM propyl propionate to ethyl propionate catalyzed by 1 µM SABP2 at different times under optimal conditions is described in detail by HPLC determination.

[0077] In a preferred embodiment 59 of the present invention, the conversion rate of 20 mM propyl butyrate to ethyl butyrate catalyzed by 1 µM SABP2 at different times under optimal conditions is described in detail by HPLC determination.

[0078] In a preferred embodiment 60 of the present invention, the conversion rate of 20 mM propyl isobutyrate to ethyl isobutyrate catalyzed by HPLC under optimal conditions at different times using 1 µM SABP2 is described in detail.

[0079] In a preferred embodiment 61 of the present invention, the conversion rate of 20 mM propyl isovalerate to ethyl isovalerate catalyzed by HPLC under optimal conditions at different times using 1 µM SABP2 is described in detail.

[0080] In a preferred embodiment 62 of the present invention, the conversion rate of 20 mM propyl 2-methylbutyrate to ethyl 2-methylbutyrate catalyzed by 1 µM SABP2 at different times under optimal conditions is described in detail by HPLC.

[0081] In a preferred embodiment 63 of the present invention, the conversion rate of 20 mM propyl acetate to methyl acetate catalyzed by 1 µM SABP2 at different times under optimal conditions is described in detail by HPLC determination. Attached Figure Description

[0082] Figure 1 The purification results of SABP2 were analyzed by SDS-PAGE, where M represents the marker, and A, B, and C all represent purified SABP2.

[0083] Figure 2 This is a standard curve of SABP2 protein concentration.

[0084] Figure 3 The conversion rates of methyl propionate to ethyl propionate catalyzed by SABP2 at different ethanol concentrations are shown.

[0085] Figure 4 The content of methyl propionate converted from ethyl propionate to methyl propionate under different methanol concentrations catalyzed by SABP2.

[0086] Figure 5 The values ​​represent the amounts of methyl propionate converted to propyl propionate by SABP2 under different propanol concentrations.

[0087] Figure 6 The values ​​represent the amount of ethyl propionate converted to propyl propionate by SABP2 under different propanol concentrations.

[0088] Figure 7 The content of methyl propionate converted from propyl propionate to methyl propionate under different methanol concentrations catalyzed by SABP2.

[0089] Figure 8 The values ​​represent the amounts of ethyl propionate converted from propyl propionate to ethyl propionate under different ethanol concentrations catalyzed by SABP2.

[0090] Figure 9 The conversion rates of SABP2-catalyzed methyl propionate to ethyl propionate at different temperatures are shown.

[0091] Figure 10 To investigate the stability of SABP2-catalyzed conversion of methyl propionate to ethyl propionate at different temperatures.

[0092] Figure 11 The conversion rates of SABP2-catalyzed methyl propionate to ethyl propionate under different pH conditions are shown.

[0093] Figure 12 The conversion rate of methyl propionate to ethyl propionate catalyzed by SABP2 at different enzyme concentrations over time.

[0094] Figure 13 This is a schematic diagram of the reaction mechanism for the enzyme-catalyzed synthesis of carboxylic acid esters. Detailed Implementation

[0095] The technical solution of the present invention will be further described in detail below with specific embodiments, so that those skilled in the art can implement it based on the description. However, the scope of protection of the present invention is not limited to these examples.

[0096] Example 1: Preparation of bacterial culture medium and experimental reagents.

[0097] LB medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15 g / L agar powder (for solid medium), autoclaved at 121℃ for 20 min before use.

[0098] Ampicillin (Amp): The recommended concentration of Amp is 100 mg / mL. -1 Dissolve 1g of ampicillin in 10mL of sterile deionized water, filter sterilize using a 0.22 μm filter membrane, dispense into sterile EP tubes, and store at -20℃ for later use.

[0099] Isopropyl-β-D-thiogalactoside (IPTG): Dissolve 2g IPTG in sterile deionized water, filter sterilize using a 0.22μm filter membrane, dispense into sterile EP tubes, and store at -20℃ for later use.

[0100] Ultrasonic lysis buffer: 998 ml of 1×PBS, add 2 mL of 0.5 M EDTA solution, and store at 4 °C for later use.

[0101] Lysozyme: The concentration of lysozyme solution used is 1 mg·mL⁻¹. It should be dissolved in deionized water and stored at 4°C.

[0102] Lysis Buffer: 50 mmol / L NaH2PO4, 300 mmol / L NaCl, 10 mmol / L imidazole, using NaOH Adjust the pH to 8.0, filter using a 0.22µm filter membrane, and store at 4℃ for later use.

[0103] Elution Buffer: 50 mmol / L NaH2PO4, 300 mmol / L NaCl, 250 mmol / L imidazole. Adjust the pH to 8.0 with NaOH, filter using a 0.22µm filter membrane, and store at 4℃ for later use.

[0104] Dialysis buffer (0.1M pH 7.4 PB): 138.6g Na2HPO4·12H2O and 17.63g NaH2PO4 dissolved in 5L deionized water, autoclaved at 121℃ for 20min and then used.

[0105] 12% SDS-PAGE separating gel: 1.6 mL deionized water, 2 mL 30% acrylamide buffer, 1.3 mL 1.5 mol / L pH 8.7 Tris, 50 µL 10% SDS, 50 µL 10% APS, 2 µL TEMED.

[0106] 5% SDS-PAGE stacking gel: 2.1 mL deionized water, 0.5 mL 30% acrylamide buffer, 0.38 mL 1.0 mol / L pH 6.8 Tris, 30 µL 10% SDS, 30 µL 10% APS, 3 µL TEMED.

[0107] 5× Protein Electrophoresis Buffer (500mL: 7.5 g Tris, 36 g glycine, 2.5 g SDS, dissolved in 500 mL deionized water, diluted five times).

[0108] SDS-PAGE staining solution: 45 mL methanol, 10 mL glacial acetic acid, 0.25 g Coomassie Brilliant Blue R250, 45 mL deionized water, store at room temperature.

[0109] SDS-PAGE decolorization solution: 150 mL methanol, 30 mL glacial acetic acid, 300 mL deionized water. Store at room temperature to prevent evaporation.

[0110] Example 2: Obtaining the salicylic acid-binding proteinase 2 (SABP2) recombinant plasmid.

[0111] The salicylate-binding protease gene (Sequence ID: NP_001312442.1 Length: 260, SEQ ID NO.6) was retrieved from the NCBI gene database, sent to the company for synthesis, and the sequence was cloned into the pET-21α(+) vector in one step to construct a recombinant plasmid.

[0112] Example 3: Prokaryotic expression of SABP2 in Escherichia coli.

[0113] The plasmid obtained in Example 2 was transformed into competent cells expressing host Escherichia coli BL21(DE3) and plated on LB solid medium containing Amp.

[0114] Single clones of bacteria were picked from LB solid medium and placed into 15 mL EP tubes. 3 mL of LB liquid medium containing Amp was added, and the culture was incubated overnight at 37°C and 225 rpm. 1 mL of the overnight culture was added to 100 mL of LB medium containing Amp, and the culture was incubated at 37°C and 250 rpm with shaking for approximately 4 hours until the OD600 nm value reached 1.6–1.8. 2 μL of 500 mM IPTG was added to the culture to bring the final concentration to 10 μM, and expression was induced at 17°C and 225 rpm for 20 hours.

[0115] After the induction of expression, the induced bacterial culture was transferred to a 50 mL EP tube and centrifuged at 3000 rpm for 20 min at 4 °C. The supernatant was discarded, and the bacterial cells were collected. The bacterial cells were resuspended in 4.45 mL of sonication lysis buffer, and 500 μL of lysozyme was added. The bacterial suspension was placed on ice and sonicated for 8 min (3 s sonication, 4 s interval). After sonication, the fragmented sample was aliquoted into 1.5 mL EP tubes and centrifuged at 12000 rpm for 20 min at 4 °C. The supernatant was collected and filtered through a 0.22 µm filter membrane to obtain the crude enzyme solution.

[0116] Example 4: Purification of SABP2 by Ni-agarose gel 6FF (His-tagged purification resin).

[0117] The recombinant bacteria contain a His-tag, therefore they can be purified by affinity chromatography using a nickel column. The specific purification process is as follows.

[0118] Equilibration: First, rinse the column with at least 5 column volumes of deionized water, then equilibrate the column with Lysis Buffer until the conductivity is stable.

[0119] Sample loading: The crude enzyme solution obtained in Example 3 was fed into the chromatography column at a low flow rate (0.5 ml / min).

[0120] Reequilibration: After the sample loading is complete, equilibrate the chromatography column with Lysis Buffer until the UV value returns to near normal.

[0121] Elution of contaminating proteins: The ratio of Lysis Buffer and Elution Buffer (4%) was adjusted to an imidazole concentration of 20 mmol / L on the computer. This imidazole concentration was used to wash the contaminating proteins in the chromatography column. At this time, the UV first rose and then fell to a stable state.

[0122] Elution of target protein: Elute the target protein on the chromatography column with Elution Buffer, and collect the sample when the UV begins to rise.

[0123] Dialysis: Transfer the collected protein sample into a dialysis bag, place it in 1L of dialysis solution, and dialyze three times at 4°C. Change the dialysis solution every 4 hours, and dialyze overnight for the last time to remove imidazole and salt from the protein sample.

[0124] Example 5: SDS-PAGE identification of SABP2 purity.

[0125] According to the reagent preparation method in Example 1, the reagents required for SDS-PAGE, as well as the separating gel and stacking gel, were prepared.

[0126] The purified and dialyzed protein was analyzed by SDS-PAGE. 10 μL of sample was mixed with 73 μL PBS and 17 μL Protein Buffer, boiled for 10 min, cooled, centrifuged at 10000 rpm for 3 min, loaded onto a plate, and electrophoresed at 200 V for 41 min. The SDS-PAGE analysis results are as follows: Figure 1 As shown.

[0127] Example 6: Determination of SABP2 protein concentration.

[0128] The concentration of the purified SABP2 protein obtained in Example 4 was determined using the Bradford method.

[0129] Prepare a 5 mg / ml bovine serum albumin (BSA) standard solution and dilute it with PBS according to Table 1 to construct a protein standard gradient solution.

[0130] Table 1. BSA Standard Protein Concentration Curve Serial Number BSA PBS (μL) Final concentration (mg / mL) A 30 μL 70 1.5 B Take 60 μL from tube A 30 1.0 C Take 60 μL from tube B. 20 0.75 D Take 60 μL from tube C. 30 0.50 E Take 60 μL from tube D. 60 0.25 F Take 60 μL from tube E. 60 0.125 G Take 60 μL from tube F. 60 0.0625 H 0 60 0 .

[0131] Use a pipette to aspirate 5 μL of LA-H protein standard solution and sample protein into a 96-well plate.

[0132] Add 250 μL of Coomassie Brilliant Blue G250 staining solution to each well.

[0133] The absorbance at OD595nm was measured using an ELISA reader.

[0134] A standard curve is plotted based on the absorbance values ​​of the protein standards, such as... Figure 2 As shown in the figure. Substituting the absorbance values ​​of the sample protein into the curve and calculating, the concentration of the purified SABP2 protein was found to be 74 µM.

[0135] Example 7: Determination of ethanol concentration in the conversion of methyl propionate to ethyl propionate catalyzed by SABP2 using high performance liquid chromatography (HPLC).

[0136] The HPLC conditions were as follows: chromatographic separation was performed using Hyper ODS2 C18 (5 μm particle size, 250 mm × 4.6 mm); UV detection wavelength was 220 nm; injection volume was 20 μL; column temperature was 25 °C; and the mobile phase was a mixture of 20 mM PB buffer (adjusted to pH 2.5 with phosphoric acid) and methanol (volume ratio 25:75). The mobile phase flow rate was 1.0 mL / min.

[0137] Reaction system and conditions: The total volume of the reaction system was 1 mL. First, methyl propionate (final concentration 20 mM), ethanol of different concentrations (2.5%, 5%, 10%, 20%, and 30%), and 0.1 M pH 7.4 PB were added. Then, SABP2 (final concentration 1 μM) was added. The reaction was then carried out in a water bath at 40℃ for 10 min. HPLC was used to detect the conversion of methyl propionate to ethyl propionate at different ethanol concentrations. Figure 3 The figure shows the conversion rate of ethyl propionate at different ethanol concentrations, with the optimal ethanol concentration being 5%.

[0138] Example 8: Determination of methanol concentration in the conversion of ethyl propionate to methyl propionate catalyzed by SABP2 using high performance liquid chromatography (HPLC).

[0139] The HPLC conditions were as follows: chromatographic separation was performed using Hyper ODS2 C18 (5 μm particle size, 250 mm × 4.6 mm); UV detection wavelength was 220 nm; injection volume was 20 μL; column temperature was 25 °C; and the mobile phase was a mixture of 20 mM PB buffer (adjusted to pH 2.5 with phosphoric acid) and methanol (volume ratio 25:75). The mobile phase flow rate was 1.0 mL / min.

[0140] Reaction system and conditions: The total volume of the reaction system was 1 mL. First, methyl propionate at a final concentration of 20 mM was added, along with methanol at different concentrations (0.25%, 0.5%, 1%, 5%, 10%, and 20%), and 0.1 M pH 7.4 PB. Then, SABP2 at a final concentration of 1 μM was added. The reaction was then carried out in a water bath at 40 °C for 10 min. HPLC was used to detect the conversion of ethyl propionate to methyl propionate at different methanol concentrations. Figure 4 The figure shows the conversion rate of methyl propionate at different methanol concentrations, with the optimal methanol concentration being 1%.

[0141] Example 9: Determination of propanol concentration in the SABP2-catalyzed conversion of methyl propionate to propyl propionate by high performance liquid chromatography (HPLC).

[0142] The HPLC conditions were as follows: chromatographic separation was performed using Hyper ODS2 C18 (5 μm particle size, 250 mm × 4.6 mm); UV detection wavelength was 220 nm; injection volume was 20 μL; column temperature was 25 °C; and the mobile phase was a mixture of 20 mM PB buffer (adjusted to pH 2.5 with phosphoric acid) and methanol (volume ratio 25:75). The mobile phase flow rate was 1.0 mL / min.

[0143] Reaction system and conditions: The total volume of the reaction system was 1 mL. First, methyl propionate at a final concentration of 20 mM, propanol at different concentrations (1%, 5%, 10%, and 20%), and 0.1 M pH 7.4 PB were added. Then, SABP2 at a final concentration of 1 μM was added. The reaction was then carried out in a water bath at 40 °C for 10 min. HPLC was used to detect the conversion of methyl propionate to propyl propionate at different propanol concentrations. Figure 5 The figure shows the conversion rate of propyl propionate at different propanol concentrations, with the optimal propanol concentration being 5%.

[0144] Example 10: Determination of propanol concentration in the SABP2-catalyzed conversion of ethyl propionate to propyl propionate by high performance liquid chromatography (HPLC).

[0145] The HPLC conditions were as follows: chromatographic separation was performed using Hyper ODS2 C18 (5 μm particle size, 250 mm × 4.6 mm); UV detection wavelength was 220 nm; injection volume was 20 μL; column temperature was 25 °C; and the mobile phase was a mixture of 20 mM PB buffer (adjusted to pH 2.5 with phosphoric acid) and methanol (volume ratio 25:75). The mobile phase flow rate was 1.0 mL / min.

[0146] Reaction system and conditions: The total volume of the reaction system was 1 mL. First, methyl propionate (final concentration 20 mM), propanol at different concentrations (1%, 5%, and 10%), and 0.1 M pH 7.4 PB were added. Then, SABP2 (final concentration 1 μM) was added. The reaction was carried out in a water bath at 40 °C for 10 min. HPLC was used to detect the conversion of ethyl propionate to propyl propionate at different propanol concentrations. Figure 6 The figure shows the conversion rate of ethyl propionate at different propanol concentrations, with the optimal propanol concentration being 5%.

[0147] Example 11: Determination of methanol concentration in the SABP2-catalyzed conversion of propyl propionate to methyl propionate.

[0148] The HPLC conditions were as follows: chromatographic separation was performed using Hyper ODS2 C18 (5 μm particle size, 250 mm × 4.6 mm); UV detection wavelength was 220 nm; injection volume was 20 μL; column temperature was 25 °C; and the mobile phase was a mixture of 20 mM PB buffer (adjusted to pH 2.5 with phosphoric acid) and methanol (volume ratio 25:75). The mobile phase flow rate was 1.0 mL / min.

[0149] Reaction system and conditions: The total volume of the reaction system was 1 mL. First, methyl propionate at a final concentration of 20 mM, methanol at different concentrations (0.5%, 1%, 5%, and 10%), and 0.1 M pH 7.4 PB were added. Then, SABP2 at a final concentration of 1 μM was added. The reaction was then carried out in a water bath at 40 °C for 10 min. HPLC was used to detect the conversion of propyl propionate to methyl propionate at different methanol concentrations. Figure 7 The figure shows the conversion rate of methyl propionate at different methanol concentrations, with the optimal methanol concentration being 5%.

[0150] Example 12: Determination of ethanol concentration in the conversion of propyl propionate to ethyl propionate catalyzed by SABP2 using high performance liquid chromatography (HPLC).

[0151] The HPLC conditions were as follows: chromatographic separation was performed using Hyper ODS2 C18 (5 μm particle size, 250 mm × 4.6 mm); UV detection wavelength was 220 nm; injection volume was 20 μL; column temperature was 25 °C; and the mobile phase was a mixture of 20 mM PB buffer (adjusted to pH 2.5 with phosphoric acid) and methanol (volume ratio 25:75). The mobile phase flow rate was 1.0 mL / min.

[0152] Reaction system and conditions: The total volume of the reaction system was 1 mL. First, methyl propionate at a final concentration of 20 mM, ethanol at different concentrations (0.5%, 1%, 5%, and 10%), and 0.1 M pH 7.4 PB were added. Then, SABP2 at a final concentration of 1 μM was added. The reaction was then carried out in a water bath at 40 °C for 10 min. HPLC was used to detect the conversion of propyl propionate to ethyl propionate at different ethanol concentrations. Figure 8 The figure shows the conversion rate of ethyl propionate at different ethanol concentrations, with the optimal ethanol concentration being 5%.

[0153] Example 13: HPLC determination of the optimal temperature for the conversion of methyl propionate to ethyl propionate catalyzed by SABP2.

[0154] Reaction system and conditions: The total volume of the reaction system was 1 mL. First, methyl propionate (final concentration 20 mM), 5% ethanol, and 0.1 M pH 7.4 PB were added. Then, SABP2 (final concentration 1 μM) was added. The reaction was carried out at 20℃, 30℃, 40℃, 50℃, and 60℃ for 10 min each. HPLC was used to detect the conversion rate of methyl propionate to ethyl propionate. Figure 9 The figure shows the conversion rate of ethyl propionate at different temperatures, with the optimum temperature being 50℃.

[0155] Example 14: HPLC determination of the temperature stability of SABP2-catalyzed conversion of methyl propionate to ethyl propionate.

[0156] Reaction system and conditions: The total volume of the reaction system was 1 mL. First, methyl propionate (final concentration 20 mM), 5% ethanol, and 0.1 M pH 7.4 PB were added. Then, SABP2 (final concentration 1 μM) was added and incubated at 30℃, 40℃, 50℃, and 60℃ for 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h, and 24 h, respectively. The reaction was then carried out for 10 min at the corresponding temperature conditions. HPLC was used to detect the conversion rate of methyl propionate to ethyl propionate. Figure 10 The figure shows the conversion rate of ethyl propionate at different temperatures. The optimal temperature stability and conversion rate are found at 40℃.

[0157] Taking into account both Examples 13 and 14, the optimal temperature for the conversion of methyl propionate to ethyl propionate catalyzed by SABP2 was determined to be 40°C.

[0158] Example 15: HPLC determination of the optimal pH for the conversion of methyl propionate to ethyl propionate catalyzed by SABP2.

[0159] Prepare 0.1M PB solutions with pH values ​​of 5, 6, 7.4, 8, and 9.

[0160] Reaction system and conditions: The total volume of the reaction system was 1 mL. First, methyl propionate (final concentration 20 mM), 5% ethanol, and 0.1 M PB at different pH values ​​(5, 6, 7.4, 8, 9) were added. Then, SABP2 (final concentration 1 μM) was added. The reaction was carried out at 40 °C for 10 min. HPLC was used to detect the conversion rate of methyl propionate to ethyl propionate. Figure 11 The figure shows the conversion rate of ethyl propionate at different pH values, with the optimal pH being 7.4.

[0161] Example 16: HPLC determination of the conversion rate of methyl propionate to ethyl propionate catalyzed by different concentrations of SABP2.

[0162] Reaction system and conditions: The total volume of the reaction system was 1 mL. First, methyl propionate (final concentration 20 mM), 5% ethanol, and 0.1 M pH 7.4 PB were added. Then, SABP2 was added at final concentrations of (0.5 µM, 1 µM, 2 µM, 4 µM). The reaction was then carried out at 40 °C for 10 min. HPLC was used to detect the conversion rate of methyl propionate to ethyl propionate. Figure 12 The figure shows the conversion rate of ethyl propionate at different enzyme concentrations, with the optimal enzyme concentration being 1 µM.

[0163] Example 17: HPLC determination of the conversion rate of 20mM methyl propionate to ethyl propionate catalyzed by 1µM SABP2 at different time points under optimal conditions.

[0164] Reaction system and conditions: The total volume of the reaction system was 1 mL. First, methyl propionate (final concentration 20 mM), 5% ethanol, and 0.1 M pH 7.4 PB were added. Then, SABP2 (final concentration 1 μM) was added. The reaction was carried out at 40℃ for 10 min, 20 min, 30 min, 40 min, 60 min, 80 min, and 120 min, respectively. The conversion rate of methyl propionate to ethyl propionate was detected by HPLC. The conversion rates at different times are shown below. Figure 13 As shown, the conversion rate of methyl propionate to ethyl propionate after 80 minutes of reaction was 78.08%.

[0165] Example 18: HPLC determination of the conversion rate of 20mM methyl butyrate to ethyl butyrate catalyzed by 1µM SABP2 at different times under optimal conditions.

[0166] Reaction system and reaction conditions: The total volume of the reaction system was 1 mL. First, methyl butyrate (final concentration 20 mM), 5% ethanol, and 0.1 M pH 7.4 PB were added. Then, SABP2 (final concentration 1 μM) was added. The reaction was carried out at 40 °C for 2 min, 5 min, 10 min, 20 min, and 30 min, respectively. The conversion rate of methyl butyrate to ethyl butyrate was detected by HPLC. The conversion rate of methyl butyrate to ethyl butyrate after 20 min of reaction was 82.28%.

[0167] Example 19: HPLC determination of the conversion rate of 20mM methyl valerate to ethyl valerate by 1µM SABP2 at different times under optimal conditions.

[0168] Reaction system and reaction conditions: The total volume of the reaction system was 1 mL. First, methyl valerate with a final concentration of 20 mM, 5% ethanol, and 0.1 M pH 7.4 PB were added. Then, SABP2 with a final concentration of 1 μM was added. The reaction was carried out at 40 °C for 2 min, 5 min, 10 min, and 20 min, respectively. The conversion rate of methyl valerate to ethyl valerate was detected by HPLC. The conversion rate of methyl valerate to ethyl valerate after 2 min of reaction was 80.30%.

[0169] Example 20: HPLC determination of the conversion rate of 20mM methyl hexanoate to ethyl hexanoate catalyzed by 1µM SABP2 at different times under optimal conditions.

[0170] Reaction system and reaction conditions: The total volume of the reaction system was 1 mL. First, methyl hexanoate with a final concentration of 20 mM, 5% ethanol, and 0.1 M pH 7.4 PB were added. Then, SABP2 with a final concentration of 1 μM was added. The reaction was carried out at 40 °C for 2 min, 5 min, 10 min, 20 min, and 30 min, respectively. The conversion rate of methyl hexanoate to ethyl hexanoate was detected by HPLC. The conversion rate of methyl hexanoate to ethyl hexanoate after 10 min of reaction was 34.25%.

[0171] Example 21: HPLC determination of the conversion rate of 20mM methyl isobutyrate to ethyl isobutyrate catalyzed by 1µM SABP2 at different time points under optimal conditions.

[0172] Reaction system and reaction conditions: The total volume of the reaction system was 1 mL. First, methyl isobutyrate (final concentration 20 mM), 5% ethanol, and 0.1 M pH 7.4 PB were added. Then, SABP2 (final concentration 1 μM) was added. The reaction was carried out at 40 °C for 0.5 h, 1 h, 1.5 h, 2 h, and 3 h, respectively. The conversion rate of methyl isobutyrate to ethyl isobutyrate was detected by HPLC. The conversion rate of methyl isobutyrate to ethyl isobutyrate after 1.5 h of reaction was 83.77%.

[0173] Example 22: HPLC determination of the conversion rate of 20mM methyl isovalerate to ethyl isovalerate catalyzed by 1µM SABP2 at different time points under optimal conditions.

[0174] Reaction system and reaction conditions: The total volume of the reaction system was 1 mL. First, methyl isovalerate with a final concentration of 20 mM, 5% ethanol, and 0.1 M pH 7.4 PB were added. Then, SABP2 with a final concentration of 1 μM was added. The reaction was carried out at 40 °C for 10 min, 20 min, 30 min, 40 min, and 50 min, respectively. The conversion rate of methyl isovalerate to ethyl isovalerate was detected by HPLC. The conversion rate of methyl isovalerate to ethyl isovalerate after 40 min of reaction was 77.40%.

[0175] Example 23: HPLC determination of the conversion rate of 20 mM methyl 2-methylbutyrate to ethyl 2-methylbutyrate catalyzed by 1 µM SABP2 at different time points under optimal conditions.

[0176] Reaction system and reaction conditions: The total volume of the reaction system was 1 mL. First, methyl 2-methylbutyrate (20 mM), 5% ethanol, and 0.1 M pH 7.4 PB were added. Then, SABP2 (1 μM) was added. The reaction was carried out at 40 °C for 10 min, 20 min, 30 min, 40 min, and 50 min, respectively. The conversion rate of methyl 2-methylbutyrate to ethyl 2-methylbutyrate was detected by HPLC. After 30 min of reaction, the conversion rate of methyl 2-methylbutyrate to ethyl 2-methylbutyrate was 71.25%.

[0177] Example 24: HPLC determination of the conversion rate of 20 mM methyl 2-methylvalerate to ethyl 2-methylvalerate under optimal conditions using 1 µM SABP2 at different time points.

[0178] Reaction system and reaction conditions: The total volume of the reaction system was 1 mL. First, methyl 2-methylvalerate (20 mM), 5% ethanol, and 0.1 M pH 7.4 PB were added. Then, SABP2 (1 μM) was added. The reaction was carried out at 40 °C for 10 min, 20 min, 30 min, 40 min, and 50 min, respectively. The conversion rate of methyl 2-methylvalerate to ethyl 2-methylvalerate was detected by HPLC. After 30 min of reaction, the conversion rate of methyl 2-methylvalerate to ethyl 2-methylvalerate was 56.21%.

[0179] Example 25: HPLC determination of the conversion of 20 mM trimethylacetate to trimethylethyl ester by 1 µM SABP2 at different time points under optimal conditions.

[0180] Reaction system and reaction conditions: The total volume of the reaction system was 1 mL. First, methyl trimethylacetate (20 mM), 5% ethanol, and 0.1 M pH 7.4 PB were added. Then, SABP2 (1 μM) was added. The reaction was carried out at 40 °C for 1 h, 1.5 h, 2 h, 3 h, 4 h, and 6 h, respectively. The conversion rate of methyl trimethylacetate to ethyl trimethylacetate was detected by HPLC. After 4 h of reaction, the conversion rate of methyl trimethylacetate to ethyl trimethylacetate was 72.41%.

[0181] Example 26: HPLC determination of the conversion rate of 20mM methyl acetate to ethyl acetate catalyzed by 1µM SABP2 at different time points under optimal conditions.

[0182] Reaction system and reaction conditions: The total volume of the reaction system was 1 mL. First, methyl acetate (20 mM), 5% ethanol, and 0.1 M pH 7.4 PB were added. Then, SABP2 (1 μM) was added. The reaction was carried out at 40 °C for 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h, respectively. The conversion rate of methyl acetate to ethyl acetate was detected by HPLC. After 10 h of reaction, the conversion rate of methyl acetate to ethyl acetate was 75.40%.

[0183] Example 27: HPLC determination of the conversion rate of 20mM ethyl propionate to methyl propionate catalyzed by 1µM SABP2 at different time points under optimal conditions.

[0184] Reaction system and reaction conditions: The total volume of the reaction system was 1 mL. First, ethyl propionate with a final concentration of 20 mM, 1% methanol, and 0.1 M pH 7.4 PB were added. Then, SABP2 with a final concentration of 1 μM was added. The reaction was carried out at 40 °C for 10 min, 20 min, 30 min, 40 min, 50 min, and 70 min, respectively. The conversion rate of ethyl propionate to methyl propionate was detected by HPLC. After 50 min of reaction, the conversion rate of ethyl propionate to methyl propionate was 78.17%.

[0185] Example 28: HPLC determination of the conversion rate of 20mM ethyl butyrate to methyl butyrate catalyzed by 1µM SABP2 at different times under optimal conditions.

[0186] Reaction system and reaction conditions: The total volume of the reaction system was 1 mL. First, ethyl butyrate with a final concentration of 20 mM, 1% methanol, and 0.1 M pH 7.4 PB were added. Then, SABP2 with a final concentration of 1 μM was added. The reaction was carried out at 40 °C for 2 min, 5 min, 10 min, 20 min, and 30 min, respectively. The conversion rate of ethyl butyrate to methyl butyrate was detected by HPLC. After 50 min of reaction, the conversion rate of ethyl butyrate to methyl butyrate was 70.68%.

[0187] Example 29: HPLC determination of the conversion rate of 20mM ethyl valerate to methyl valerate catalyzed by 1µM SABP2 at different time points under optimal conditions.

[0188] Reaction system and reaction conditions: The total volume of the reaction system was 1 mL. First, ethyl valerate with a final concentration of 20 mM, 1% methanol, and 0.1 M pH 7.4 PB were added. Then, SABP2 with a final concentration of 1 μM was added. The reaction was carried out at 40 °C for 0.2 min, 0.5 min, 2 min, 5 min, and 10 min, respectively. The conversion rate of ethyl valerate to methyl valerate was detected by HPLC. The conversion rate of ethyl valerate to methyl valerate after 2 min of reaction was 61.68%.

[0189] Example 30: HPLC determination of the conversion rate of 20mM ethyl hexanoate to methyl hexanoate catalyzed by 1µM SABP2 at different time points under optimal conditions.

[0190] Reaction system and reaction conditions: The total volume of the reaction system was 1 mL. First, ethyl hexanoate with a final concentration of 20 mM, 1% methanol, and 0.1 M pH 7.4 PB were added. Then, SABP2 with a final concentration of 1 μM was added. The reaction was carried out at 40 °C for 1 min, 2 min, 5 min, 10 min, and 30 min, respectively. The conversion rate of ethyl hexanoate to methyl hexanoate was detected by HPLC. The conversion rate of ethyl hexanoate to methyl hexanoate after 2 min of reaction was 39.02%.

[0191] Example 31: HPLC determination of the conversion rate of 20mM ethyl isobutyrate to methyl isobutyrate catalyzed by 1µM SABP2 at different time points under optimal conditions.

[0192] Reaction system and reaction conditions: The total volume of the reaction system was 1 mL. First, ethyl isobutyrate with a final concentration of 20 mM, 1% methanol, and 0.1 M pH 7.4 PB were added. Then, SABP2 with a final concentration of 1 μM was added. The reaction was carried out at 40 °C for 10 min, 20 min, 30 min, 40 min, and 60 min, respectively. The conversion rate of ethyl isobutyrate to methyl isobutyrate was detected by HPLC. The conversion rate of ethyl isobutyrate to methyl isobutyrate after 40 min of reaction was 77.18%.

[0193] Example 32: HPLC determination of the conversion rate of 20mM ethyl isovalerate to methyl isovalerate catalyzed by 1µM SABP2 at different times under optimal conditions.

[0194] Reaction system and reaction conditions: The total volume of the reaction system was 1 mL. First, ethyl isovalerate with a final concentration of 20 mM, 1% methanol, and 0.1 M pH 7.4 PB were added. Then, SABP2 with a final concentration of 1 μM was added. The reaction was carried out at 40 °C for 5 min, 10 min, 20 min, 30 min, 40 min, and 60 min, respectively. The conversion rate of ethyl isovalerate to methyl isovalerate was detected by HPLC. After 40 min of reaction, the conversion rate of ethyl isovalerate to methyl isovalerate was 66.27%.

[0195] Example 33: HPLC determination of the conversion rate of 20 mM ethyl 2-methylbutyrate to methyl 2-methylbutyrate catalyzed by 1 µM SABP2 at different time points under optimal conditions.

[0196] Reaction system and reaction conditions: The total volume of the reaction system was 1 mL. First, 20 mM ethyl 2-methylbutyrate, 1% methanol, and 0.1 M pH 7.4 PB were added. Then, SABP2 with a final concentration of 1 μM was added. The reaction was carried out at 40 °C for 5 min, 10 min, 20 min, 30 min, and 40 min, respectively. The conversion rate of ethyl 2-methylbutyrate to methyl 2-methylbutyrate was detected by HPLC. After 30 min of reaction, the conversion rate of ethyl 2-methylbutyrate to methyl 2-methylbutyrate was 60.35%.

[0197] Example 34: HPLC determination of the conversion rate of 20 mM ethyl 2-methylvalerate to methyl 2-methylvalerate under optimal conditions using 1 µM SABP2 at different time points.

[0198] Reaction system and reaction conditions: The total volume of the reaction system was 1 mL. First, 20 mM ethyl 2-methylvalerate, 1% methanol, and 0.1 M pH 7.4 PB were added. Then, SABP2 with a final concentration of 1 μM was added. The reaction was carried out at 40 °C for 2 min, 5 min, 10 min, 20 min, and 30 min, respectively. The conversion rate of ethyl 2-methylvalerate to methyl 2-methylvalerate was detected by HPLC. After 10 min of reaction, the conversion rate of ethyl 2-methylvalerate to methyl 2-methylvalerate was 37.58%.

[0199] Example 35: HPLC determination of the conversion of 20 mM trimethylethyl acetate to methyl trimethylacetate by 1 µM SABP2 at different times under optimal conditions.

[0200] Reaction system and reaction conditions: The total volume of the reaction system was 1 mL. First, ethyl trimethylacetate with a final concentration of 20 mM, 1% methanol, and 0.1 M pH 7.4 PB were added. Then, SABP2 with a final concentration of 1 μM was added. The reaction was carried out at 40 °C for 20 min, 30 min, 60 min, 120 min, and 180 min, respectively. The conversion rate of ethyl trimethylacetate to methyl trimethylacetate was detected by HPLC. After 120 min of reaction, the conversion rate of ethyl trimethylacetate to methyl trimethylacetate was 75.66%.

[0201] Example 36: HPLC determination of the conversion rate of 20 mM ethyl acetate to methyl acetate catalyzed by 1 µM SABP2 at different time points under optimal conditions.

[0202] Reaction system and reaction conditions: The total volume of the reaction system was 1 mL. First, ethyl acetate with a final concentration of 20 mM, 1% methanol, and 0.1 M pH 7.4 PB were added. Then, SABP2 with a final concentration of 1 μM was added. The reaction was carried out at 40 °C for 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h, respectively. The conversion rate of ethyl acetate to methyl acetate was detected by HPLC. After 8 h of reaction, the conversion rate of ethyl acetate to methyl acetate was 55.94%.

[0203] Example 37: HPLC determination of the conversion rate of 20mM methyl propionate to propyl propionate catalyzed by 1µM SABP2 at different time points under optimal conditions.

[0204] Reaction system and reaction conditions: The total volume of the reaction system was 1 mL. First, methyl propionate with a final concentration of 20 mM, 5% propanol, and 0.1 M pH 7.4 PB were added. Then, SABP2 with a final concentration of 1 μM was added. The reaction was carried out at 40 °C for 10 min, 30 min, 6 min, 120 min, 360 min, and 480 min, respectively. The conversion rate of methyl propionate to propyl propionate was detected by HPLC. After 360 min of reaction, the conversion rate of methyl propionate to propyl propionate was 50.49%.

[0205] Example 38: HPLC determination of the conversion rate of 20mM methyl butyrate to propyl butyrate catalyzed by 1µM SABP2 at different time points under optimal conditions.

[0206] Reaction system and reaction conditions: The total volume of the reaction system was 1 mL. First, methyl butyrate (20 mM), 5% propanol, and 0.1 M pH 7.4 PB were added. Then, SABP2 (1 μM) was added. The reaction was carried out at 40 °C for 5 min, 10 min, 30 min, 60 min, and 90 min, respectively. The conversion rate of methyl butyrate to propyl butyrate was detected by HPLC. After 30 min of reaction, the conversion rate of methyl butyrate to propyl butyrate was 45.92%.

[0207] Example 39: HPLC determination of the conversion rate of 20 mM methyl valerate to propyl valerate catalyzed by 1 µM SABP2 at different time points under optimal conditions.

[0208] Reaction system and reaction conditions: The total volume of the reaction system was 1 mL. First, methyl valerate (20 mM), 5% propanol, and 0.1 M pH 7.4 PB were added. Then, SABP2 (1 μM) was added. The reaction was carried out at 40 °C for 1 min, 5 min, 8 min, 10 min, and 20 min, respectively. The conversion rate of methyl valerate to propyl valerate was detected by HPLC. After 8 min of reaction, the conversion rate of methyl valerate to propyl valerate was 48.84%.

[0209] Example 40: HPLC determination of the conversion rate of 20 mM methyl hexanoate to propyl hexanoate catalyzed by 1 µM SABP2 at different time points under optimal conditions.

[0210] Reaction system and reaction conditions: The total volume of the reaction system was 1 mL. First, methyl hexanoate (final concentration 20 mM), 5% propanol, and 0.1 M pH 7.4 PB were added. Then, SABP2 (final concentration 1 μM) was added. The reaction was carried out at 40 °C for 5 min, 15 min, 30 min, 45 min, and 60 min, respectively. The conversion rate of methyl hexanoate to propyl hexanoate was detected by HPLC. After 45 min of reaction, the conversion rate of methyl hexanoate to propyl hexanoate was 15.99%.

[0211] Example 41: HPLC determination of the conversion rate of 20mM methyl isobutyrate to propyl isobutyrate catalyzed by 1µM SABP2 at different times under optimal conditions.

[0212] Reaction system and reaction conditions: The total volume of the reaction system was 1 mL. First, methyl isobutyrate at a final concentration of 20 mM, 5% propanol, and 0.1 M pH 7.4 PB were added. Then, SABP2 at a final concentration of 1 μM was added. The reaction was carried out at 40 °C for 0.5 h, 1 h, 1.5 h, 2 h, and 3 h, respectively. The conversion rate of methyl isobutyrate to propyl isobutyrate was detected by HPLC. After 1.5 h of reaction, the conversion rate of methyl isobutyrate to propyl isobutyrate was 38.59%.

[0213] Example 42: HPLC determination of the conversion rate of 20mM methyl isovalerate to propyl isovalerate catalyzed by 1µM SABP2 at different time points under optimal conditions.

[0214] Reaction system and reaction conditions: The total volume of the reaction system was 1 mL. First, methyl isovalerate with a final concentration of 20 mM, 5% propanol, and 0.1 M pH 7.4 PB were added. Then, SABP2 with a final concentration of 1 μM was added. The reaction was carried out at 40 °C for 5 min, 30 min, 60 min, 90 min, and 120 min, respectively. The conversion rate of methyl isovalerate to propyl isovalerate was detected by HPLC. After 90 min of reaction, the conversion rate of methyl isovalerate to propyl isovalerate was 33.22%.

[0215] Example 43: HPLC determination of the conversion rate of 20 mM methyl 2-methylbutyrate to propyl 2-methylbutyrate catalyzed by 1 µM SABP2 at different time points under optimal conditions.

[0216] Reaction system and reaction conditions: The total volume of the reaction system was 1 mL. First, methyl 2-methylbutyrate (20 mM), 5% propanol, and 0.1 M pH 7.4 PB were added. Then, SABP2 (1 μM) was added. The reaction was carried out at 40 °C for 10 min, 30 min, 60 min, 120 min, and 180 min, respectively. The conversion rate of methyl 2-methylbutyrate to propyl 2-methylbutyrate was detected by HPLC. After 120 min of reaction, the conversion rate of methyl 2-methylbutyrate to propyl 2-methylbutyrate was 23.51%.

[0217] Example 44: HPLC determination of the conversion rate of 20 mM methyl 2-methylvalerate to propyl 2-methylvalerate under optimal conditions using 1 µM SABP2 at different time points.

[0218] Reaction system and reaction conditions: The total volume of the reaction system was 1 mL. First, methyl 2-methylvalerate (20 mM), 5% propanol, and 0.1 M pH 7.4 PB were added. Then, SABP2 (1 μM) was added. The reaction was carried out at 40 °C for 10 min, 30 min, 60 min, and 120 min, respectively. The conversion rate of methyl 2-methylvalerate to propyl 2-methylvalerate was detected by HPLC. After 60 min of reaction, the conversion rate of methyl 2-methylvalerate to propyl 2-methylvalerate was 22.45%.

[0219] Example 45: HPLC determination of the conversion rate of 20 mM trimethylacetate to trimethylacetate by 1 µM SABP2 at different times under optimal conditions.

[0220] Reaction system and reaction conditions: The total volume of the reaction system was 1 mL. First, methyl trimethylacetate (20 mM), 5% propanol, and 0.1 M pH 7.4 PB were added. Then, SABP2 (1 μM) was added. The reaction was carried out at 40 °C for 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, and 8 h, respectively. The conversion rate of methyl trimethylacetate to propyl trimethylacetate was detected by HPLC. After 5 h of reaction, the conversion rate of methyl trimethylacetate to propyl trimethylacetate was 13.55%.

[0221] Example 46: HPLC determination of the conversion rate of 20mM methyl acetate to propyl acetate catalyzed by 1µM SABP2 at different time points under optimal conditions.

[0222] Reaction system and reaction conditions: The total volume of the reaction system was 1 mL. First, methyl acetate (20 mM), 5% propanol, and 0.1 M pH 7.4 PB were added. Then, SABP2 (1 μM) was added. The reaction was carried out at 40 °C for 4 h, 12 h, 24 h, 48 h, and 72 h, respectively. The conversion rate of methyl acetate to propyl acetate was detected by HPLC. After 24 h of reaction, the conversion rate of methyl acetate to propyl acetate was 24.61%.

[0223] Example 47: HPLC determination of the conversion rate of 20mM ethyl propionate to propyl propionate catalyzed by 1µM SABP2 at different time points under optimal conditions.

[0224] Reaction system and reaction conditions: The total volume of the reaction system was 1 mL. First, ethyl propionate with a final concentration of 20 mM, 5% propanol, and 0.1 M pH 7.4 PB were added. Then, SABP2 with a final concentration of 1 μM was added. The reaction was carried out at 40 °C for 1 h, 2 h, 6 h, 8 h, 10 h, 12 h, and 16 h, respectively. The conversion rate of ethyl propionate to propyl propionate was detected by HPLC. After 10 h of reaction, the conversion rate of ethyl propionate to propyl propionate was 39.51%.

[0225] Example 48: HPLC determination of the conversion rate of 20mM ethyl butyrate to propyl butyrate catalyzed by 1µM SABP2 at different time points under optimal conditions.

[0226] Reaction system and reaction conditions: The total volume of the reaction system was 1 mL. First, ethyl butyrate (final concentration 20 mM), 5% propanol, and 0.1 M pH 7.4 PB were added. Then, SABP2 (final concentration 1 μM) was added. The reaction was carried out at 40 °C for 5 min, 10 min, 20 min, 30 min, 40 min, and 60 min, respectively. The conversion rate of ethyl butyrate to propyl butyrate was detected by HPLC. After 30 min of reaction, the conversion rate of ethyl butyrate to propyl butyrate was 43.96%.

[0227] Example 49: HPLC determination of the conversion rate of 20mM ethyl valerate to propyl valerate catalyzed by 1µM SABP2 at different time points under optimal conditions.

[0228] Reaction system and reaction conditions: The total volume of the reaction system was 1 mL. First, ethyl valerate (final concentration 20 mM), 5% propanol, and 0.1 M pH 7.4 PB were added. Then, SABP2 (final concentration 1 μM) was added. The reaction was carried out at 40 °C for 2 min, 5 min, 8 min, 10 min, and 20 min, respectively. The conversion rate of ethyl valerate to propyl valerate was detected by HPLC. After 5 min of reaction, the conversion rate of ethyl valerate to propyl valerate was 44.07%.

[0229] Example 50: HPLC determination of the conversion rate of 20 mM ethyl hexanoate to propyl hexanoate catalyzed by 1 µM SABP2 at different time points under optimal conditions.

[0230] Reaction system and reaction conditions: The total volume of the reaction system was 1 mL. First, ethyl hexanoate (final concentration 20 mM), 5% propanol, and 0.1 M pH 7.4 PB were added. Then, SABP2 (final concentration 1 μM) was added. The reaction was carried out at 40 °C for 5 min, 15 min, 30 min, 45 min, 60 min, and 120 min, respectively. The conversion rate of ethyl hexanoate to propyl hexanoate was detected by HPLC. After 15 min of reaction, the conversion rate of ethyl hexanoate to propyl hexanoate was 23.35%.

[0231] Example 51: HPLC determination of the conversion rate of 20mM ethyl isobutyrate to propyl isobutyrate catalyzed by 1µM SABP2 at different times under optimal conditions.

[0232] Reaction system and reaction conditions: The total volume of the reaction system was 1 mL. First, ethyl isobutyrate (final concentration 20 mM), 5% propanol, and 0.1 M pH 7.4 PB were added. Then, SABP2 (final concentration 1 μM) was added. The reaction was carried out at 40 °C for 0.5 h, 1 h, 1.5 h, 2 h, 3 h, and 4 h, respectively. The conversion rate of ethyl isobutyrate to propyl isobutyrate was detected by HPLC. After 3 h of reaction, the conversion rate of ethyl isobutyrate to propyl isobutyrate was 37.32%.

[0233] Example 52: HPLC determination of the conversion rate of 20mM ethyl isovalerate to propyl isovalerate catalyzed by 1µM SABP2 at different time points under optimal conditions.

[0234] Reaction system and reaction conditions: The total volume of the reaction system was 1 mL. First, ethyl isovalerate with a final concentration of 20 mM, 5% propanol, and 0.1 M pH 7.4 PB were added. Then, SABP2 with a final concentration of 1 μM was added. The reaction was carried out at 40 °C for 5 min, 30 min, 60 min, 90 min, and 120 min, respectively. The conversion rate of ethyl isovalerate to propyl isovalerate was detected by HPLC. After 90 min of reaction, the conversion rate of ethyl isovalerate to propyl isovalerate was 37.26%.

[0235] Example 53: HPLC determination of the conversion rate of 20 mM ethyl 2-methylbutyrate to propyl 2-methylbutyrate catalyzed by 1 µM SABP2 at different time points under optimal conditions.

[0236] Reaction system and reaction conditions: The total volume of the reaction system was 1 mL. First, 20 mM ethyl 2-methylbutyrate, 5% propanol, and 0.1 M pH 7.4 PB were added. Then, SABP2 with a final concentration of 1 μM was added. The reaction was carried out at 40 °C for 10 min, 30 min, 60 min, 120 min, and 180 min, respectively. The conversion rate of ethyl 2-methylbutyrate to propyl 2-methylbutyrate was detected by HPLC. After 120 min of reaction, the conversion rate of ethyl 2-methylbutyrate to propyl 2-methylbutyrate was 16.98%.

[0237] Example 54: HPLC determination of the conversion rate of 20 mM ethyl 2-methylvalerate to propyl 2-methylvalerate under optimal conditions using 1 µM SABP2 at different time points.

[0238] Reaction system and reaction conditions: The total volume of the reaction system was 1 mL. First, 20 mM ethyl 2-methylvalerate, 5% propanol, and 0.1 M pH 7.4 PB were added. Then, SABP2 with a final concentration of 1 μM was added. The reaction was carried out at 40 °C for 10 min, 30 min, 60 min, and 90 min, respectively. The conversion rate of ethyl 2-methylvalerate to propyl 2-methylvalerate was detected by HPLC. After 30 min of reaction, the conversion rate of ethyl 2-methylvalerate to propyl 2-methylvalerate was 13.84%.

[0239] Example 55: HPLC determination of the conversion of 20 mM trimethylethyl acetate to trimethylacetate by 1 µM SABP2 at different time points under optimal conditions.

[0240] Reaction system and reaction conditions: The total volume of the reaction system was 1 mL. First, ethyl trimethylacetate with a final concentration of 20 mM, 5% propanol, and 0.1 M pH 7.4 PB were added. Then, SABP2 with a final concentration of 1 μM was added. The reaction was carried out at 40 °C for 1 h, 2 h, 3 h, 4 h, 5 h, and 6 h, respectively. The conversion rate of ethyl trimethylacetate to propyl trimethylacetate was detected by HPLC. After 5 h of reaction, the conversion rate of ethyl trimethylacetate to propyl trimethylacetate was 24.16%.

[0241] Example 56: HPLC determination of the conversion of 20 mM ethyl acetate to propyl acetate catalyzed by 1 µM SABP2 at different time points under optimal conditions.

[0242] Reaction system and reaction conditions: The total volume of the reaction system was 1 mL. First, ethyl acetate with a final concentration of 20 mM, 5% propanol, and 0.1 M pH 7.4 PB were added. Then, SABP2 with a final concentration of 1 μM was added. The reaction was carried out at 40 °C for 12 h, 24 h, 36 h, 48 h, and 72 h, respectively. The conversion rate of ethyl acetate to propyl acetate was detected by HPLC. After 48 h of reaction, the conversion rate of ethyl acetate to propyl acetate was 29.92%.

[0243] Example 57: HPLC determination of the conversion rate of 20mM propyl propionate to methyl propionate catalyzed by 1µM SABP2 at different time points under optimal conditions.

[0244] Reaction system and reaction conditions: The total volume of the reaction system was 1 mL. Propionate with a final concentration of 20 mM, 5% methanol, and 0.1 M pH 7.4 PB were added first, followed by SABP2 with a final concentration of 1 μM. The reaction was then carried out at 40 °C for 1 h, 3 h, 4 h, 6 h, 8 h, 12 h, 18 h, and 24 h. The conversion rate of propyl propionate to methyl propionate was detected by HPLC. After 18 h of reaction, the conversion rate of propyl propionate to methyl propionate was 39.05%.

[0245] Example 58: HPLC determination of the conversion rate of 20mM propyl butyrate to methyl butyrate catalyzed by 1µM SABP2 at different times under optimal conditions.

[0246] Reaction system and reaction conditions: The total volume of the reaction system was 1 mL. First, propyl butyrate at a final concentration of 20 mM, 5% methanol, and 0.1 M pH 7.4 PB were added. Then, SABP2 at a final concentration of 1 μM was added. The reaction was carried out at 40 °C for 10 min, 20 min, 30 min, 40 min, and 50 min, respectively. The conversion rate of propyl butyrate to methyl butyrate was detected by HPLC. After 30 min of reaction, the conversion rate of propyl butyrate to methyl butyrate was 28.78%.

[0247] Example 59: HPLC determination of the conversion rate of 20mM propyl isobutyrate to methyl isobutyrate catalyzed by 1µM SABP2 at different times under optimal conditions.

[0248] Reaction system and reaction conditions: The total volume of the reaction system was 1 mL. First, propyl isobutyrate at a final concentration of 20 mM, 5% methanol, and 0.1 M pH 7.4 PB were added. Then, SABP2 at a final concentration of 1 μM was added. The reaction was carried out at 40 °C for 1 h, 2 h, 3 h, 5 h, 7 h, 10 h, and 12 h, respectively. The conversion rate of propyl isobutyrate to methyl isobutyrate was detected by HPLC. After 10 h of reaction, the conversion rate of propyl isobutyrate to methyl isobutyrate was 53.47%.

[0249] Example 60: HPLC determination of the conversion rate of 20mM propyl isovalerate to methyl isovalerate catalyzed by 1µM SABP2 at different times under optimal conditions.

[0250] Reaction system and reaction conditions: The total volume of the reaction system was 1 mL. Propyl isovalerate (final concentration 20 mM), 5% methanol, and 0.1 M pH 7.4 PB were added first, followed by SABP2 (final concentration 1 μM). The reaction was then carried out at 40 °C for 10 min, 30 min, 60 min, 120 min, and 180 min, respectively. The conversion rate of propyl isovalerate to methyl isovalerate was detected by HPLC. After 60 min of reaction, the conversion rate of propyl isovalerate to methyl isovalerate was 36.14%.

[0251] Example 61: HPLC determination of the conversion rate of 20 mM propyl 2-methylbutyrate to methyl 2-methylbutyrate catalyzed by 1 µM SABP2 at different time points under optimal conditions.

[0252] Reaction system and reaction conditions: The total volume of the reaction system was 1 mL. 2-methylbutyrate propyl ester (20 mM), 5% methanol, and 0.1 M pH 7.4 PB were added first, followed by SABP2 (1 μM). The reaction was carried out at 40 °C for 10 min, 30 min, 60 min, 120 min, and 180 min, respectively. The conversion rate of 2-methylbutyrate propyl ester to 2-methylbutyrate methyl ester was detected by HPLC. After 120 min of reaction, the conversion rate of 2-methylbutyrate propyl ester to 2-methylbutyrate methyl ester was 39.31%.

[0253] Example 62: HPLC determination of the conversion rate of 20mM propyl acetate to methyl acetate catalyzed by 1µM SABP2 at different time points under optimal conditions.

[0254] Reaction system and reaction conditions: The total volume of the reaction system was 1 mL. Propyl acetate (final concentration 20 mM), 5% methanol, and 0.1 M pH 7.4 PB were added first, followed by SABP2 (final concentration 1 μM). The reaction was then carried out at 40 °C for 6 h, 16 h, 24 h, 36 h, 72 h, and 96 h, respectively. The conversion rate of propyl acetate to methyl acetate was detected by HPLC. After 96 h of reaction, the conversion rate of propyl acetate to methyl acetate was 34.5%.

[0255] Example 63: HPLC determination of the conversion rate of 20mM propyl propionate to ethyl propionate catalyzed by 1µM SABP2 at different times under optimal conditions.

[0256] Reaction system and reaction conditions: The total volume of the reaction system was 1 mL. Propyl propionate with a final concentration of 20 mM, 5% ethanol, and 0.1 M pH 7.4 PB were added first, followed by SABP2 with a final concentration of 1 μM. The reaction was then carried out at 40 °C for 1 h, 3 h, 6 h, 8 h, 12 h, 18 h, and 24 h, respectively. The conversion rate of propyl propionate to ethyl propionate was detected by HPLC. After 18 h of reaction, the conversion rate of propyl propionate to ethyl propionate was 24.67%.

[0257] Example 64: HPLC determination of the conversion rate of 20mM propyl butyrate to ethyl butyrate catalyzed by 1µM SABP2 at different time points under optimal conditions.

[0258] Reaction system and reaction conditions: The total volume of the reaction system was 1 mL. First, propyl butyrate at a final concentration of 20 mM, 5% ethanol, and 0.1 M pH 7.4 PB were added. Then, SABP2 at a final concentration of 1 μM was added. The reaction was carried out at 40 °C for 10 min, 20 min, 30 min, 40 min, and 50 min, respectively. The conversion rate of propyl butyrate to ethyl butyrate was detected by HPLC. After 40 min of reaction, the conversion rate of propyl butyrate to ethyl butyrate was 34.03%.

[0259] Example 65: HPLC determination of the conversion rate of 20mM propyl isobutyrate to ethyl isobutyrate catalyzed by 1µM SABP2 at different times under optimal conditions.

[0260] Reaction system and reaction conditions: The total volume of the reaction system was 1 mL. First, propyl isobutyrate at a final concentration of 20 mM, 5% ethanol, and 0.1 M pH 7.4 PB were added. Then, SABP2 at a final concentration of 1 μM was added. The reaction was carried out at 40 °C for 0.5 h, 1 h, 2 h, 3 h, and 5 h, respectively. The conversion rate of propyl isobutyrate to ethyl isobutyrate was detected by HPLC. After 10 h of reaction, the conversion rate of propyl isobutyrate to ethyl isobutyrate was 26.36%.

[0261] Example 66: HPLC determination of the conversion rate of 20mM propyl isovalerate to ethyl isovalerate catalyzed by 1µM SABP2 at different time points under optimal conditions.

[0262] Reaction system and reaction conditions: The total volume of the reaction system was 1 mL. Propyl isovalerate (final concentration 20 mM), 5% ethanol, and 0.1 M pH 7.4 PB were added first, followed by SABP2 (final concentration 1 μM). The reaction was then carried out at 40 °C for 10 min, 20 min, 30 min, 40 min, and 60 min, respectively. The conversion rate of propyl isovalerate to ethyl isovalerate was detected by HPLC. The conversion rate of propyl isovalerate to ethyl isovalerate after 40 min of reaction was 26.76%.

[0263] Example 67: HPLC determination of the conversion rate of 20 mM propyl 2-methylbutyrate to ethyl 2-methylbutyrate catalyzed by 1 µM SABP2 at different time points under optimal conditions.

[0264] Reaction system and reaction conditions: The total volume of the reaction system was 1 mL. 2-methylbutyrate propyl ester (final concentration 20 mM), 5% ethanol, and 0.1 M pH 7.4 PB were added first, followed by SABP2 (final concentration 1 μM). The reaction was then carried out at 40 °C for 10 min, 20 min, 30 min, 4 min, and 60 min, respectively. HPLC was used to detect the conversion rate of 2-methylbutyrate propyl ester to 2-methylbutyrate ethyl ester. After 20 min of reaction, the conversion rate of 2-methylbutyrate propyl ester to 2-methylbutyrate ethyl ester was 23.77%.

[0265] Example 68: HPLC determination of the conversion of 20mM propyl acetate to ethyl acetate catalyzed by 1µM SABP2 at different time points under optimal conditions.

[0266] Reaction system and conditions: The total volume of the reaction system was 1 mL. Propyl acetate (final concentration 20 mM), 5% ethanol, and 0.1 M pH 7.4 PB were added first, followed by SABP2 (final concentration 1 μM). The reaction was carried out at 40 °C for 12 h, 24 h, 36 h, 48 h, 72 h, and 96 h, respectively. The conversion rate of propyl acetate to ethyl acetate was determined by HPLC. After 72 h of reaction, the conversion rate of propyl acetate to ethyl acetate was 21%.

Claims

1. A method for synthesizing carboxylic acid ester compounds by bio-enzyme catalysis, characterized in that... Using carboxylic acid esters and alcohols as raw materials, transesterification occurs under the catalysis of SABP2 to produce methyl carboxylic acid ester, ethyl carboxylic acid ester, and propyl carboxylic acid ester.

2. The method for synthesizing carboxylic acid esters using a biological enzyme as a catalyst according to claim 1, wherein the carboxylic acid ester compound is selected from: methyl acetate, methyl propionate, methyl butyrate, methyl hexanoate, methyl isobutyrate, methyl valerate, methyl isovalerate, methyl 2-methylbutyrate, methyl 2-methylvalerate, methyl trimethylacetate, ethyl acetate, ethyl propionate, ethyl butyrate, ethyl hexanoate, ethyl isobutyrate, ethyl valerate, ethyl isovalerate, ethyl 2-methylbutyrate, ethyl 2-methylvalerate, ethyl trimethylacetate, propyl acetate, propyl propionate, propyl butyrate, propyl isobutyrate, propyl isovalerate, and propyl 2-methylbutyrate.

3. The method for synthesizing carboxylic acid ester compounds by bio-enzyme catalysis according to claim 1, wherein the alcohol is methanol, ethanol, or n-propanol.

4. The method for synthesizing carboxylic acid ester compounds by bio-enzyme catalysis according to claim 1, characterized in that, The SABP2 was expressed and synthesized in Escherichia coli using pET-21a(+) as the expression vector.

5. The method for synthesizing carboxylic acid ester compounds by bio-enzyme catalysis according to claim 1, characterized in that, The reaction was carried out at a temperature of 40°C and a pH of 7.

4.

6. The method for synthesizing ethyl carboxylate compounds by bio-enzyme catalysis of methyl carboxylate conversion to ethyl carboxylate as described in claim 1, characterized in that, The preferred concentration of ethanol in the reaction is 5% (v / v); the method for catalyzing the conversion of ethyl carboxylate to methyl carboxylate is characterized in that the preferred concentration of methanol in the reaction is 1% (v / v); the preferred concentration of methanol in the reaction is 5% (v / v); the preferred concentration of ethanol in the reaction is 5% (v / v); the preferred concentration of propanol in the reaction is 5% (v / v); the preferred concentration of propanol in the reaction is 5% (v / v); the preferred concentration of propanol in the reaction is 5% (v / v).

7. The method for synthesizing carboxylic acid esters using a biological enzyme as a catalyst according to claim 1, characterized in that, The specific implementation process is as follows: 20 mM carboxylic acid ester, the corresponding alcohol concentration, and 0.1 M pH 7.4 phosphate buffer were added to the reactor, shaken to mix, and 1 µM SABP2 was added to start the reaction. The reaction progress was detected by HPLC.

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