Esterase mutants and their use in the production of l-menthol

Abstract

The application discloses an esterase mutant and application thereof in preparation of L-menthol, wherein the esterase mutant is obtained by mutating a serine at the 389th position of an amino acid sequence shown in SEQ ID NO. 4 into proline and / or mutating a valine at the 421th position into isoleucine. A genetically engineered bacterium expressing a mutant pnbA-BS-A400P / S389P / V421I is used as a whole-cell catalyst, 1M DL-acetyl phenyl acetate is used as a substrate, 6% (v / v) n-octanol is added to construct a water / organic two-phase reaction system, reaction is carried out at 30 DEG C and 600 rpm for 10 hours, L-acetyl phenyl acetate is completely hydrolyzed, the conversion rate is increased from 17.43% of a wild type to 50%, and the e.e. p >99% is obtained, and the reaction space-time yield can reach 237.5 g / (L.d), which lays a foundation for efficient preparation of L-menthol by an enzyme method.

Description

(I) Technical Field

[0001] This invention belongs to the field of biocatalysis and relates to esterase mutants and their application in the enzymatic selective resolution of DL-menthyl acetate to prepare L-menthol. (II) Background Technology

[0002] Menthol is one of the world's three major flavorings and the main component of peppermint and peppermint essential oil. Because its molecule has three chiral centers, it has four pairs of enantiomers: DL-menthol, DL-isomenthol, DL-neomenthol, and DL-neoisomenthol. Due to the different molecular structures of menthol, its eight isomers have different physical properties, chemical properties, physiological effects, and industrial applications. Among them, L-menthol, with its refreshing minty aroma and unique physiological effects, is widely used in food, medicine, cosmetics, and flavorings, and its demand is constantly increasing, making it the isomer with the greatest industrial application value.

[0003] Currently, there are three main methods for producing L-menthol internationally: The first is an asymmetric synthesis using myrcene as a raw material. While this method is simple, the expensive catalysts and high costs can lead to environmental pollution. The second method uses thymol as a raw material and employs a resolution process, which is currently the highest-yielding method for L-menthol production. However, this method involves complex post-processing and cumbersome resolution and recovery processes. The third method uses natural chiral substances such as L-menthone as raw materials, but the yield is very low due to the limited availability of chiral raw materials. With increasing market demand, the limited-yield natural methods are gradually being replaced by chemical and biological methods. Chemical methods suffer from high costs, severe pollution, and low optical purity of the products. Biological enzymatic methods, on the other hand, lack specialized enzymes that simultaneously meet the requirements of high activity and high enantioselectivity, and therefore do not yet meet the requirements for practical application. Therefore, there is an urgent need to find enzymes and efficient catalytic methods capable of efficiently and selectively hydrolyzing and resolving L-menthol to produce it.

[0004] This invention focuses on pnbA-BS-A400P, a high L-enantioselectivity mutant of the esterase pnbA-BS derived from Bacillus subtilis 168. pnbA-BS-A400P exhibits reduced catalytic activity due to a "trade-off" effect, a negative correlation between enantioselectivity and activity. Through homology modeling, molecular docking, and mutagenesis, a mutant with high L-enantioselectivity and high catalytic activity in the selective resolution of DL-menthyl acetate was successfully obtained, denoted as pnbA-BS-A400P / S389P / V421I. The esterase pnbA-BS-A400P / S389P / V421I was expressed in *Escherichia coli* to obtain a genetically engineered bacterium. Using this engineered bacterium as a catalyst, and with the addition of 6% (v / v) of the water-insoluble organic solvent n-octanol to the reaction system, a highly efficient method for the preparation of 1-menthol was established, which effectively eliminates substrate and product inhibition and facilitates product separation via a two-phase catalytic process using water / organic solvent. (III) Summary of the Invention

[0005] The purpose of this invention is to provide an esterase mutant and its application in the preparation of L-menthol. This invention focuses on pnbA-BS-A400P, a high L-enantioselective esterase mutant derived from Bacillus subtilis 168. pnbA-BS-A400P exhibits reduced catalytic activity due to a "trade-off" effect, a negative correlation between enantioselectivity and activity. Through semi-rational design, homology modeling, molecular docking, and mutagenesis of the esterase pnbA-BS-A400P, a mutant with high L-enantioselectivity and high catalytic activity in the selective resolution of DL-menthyl acetate was successfully obtained, denoted as pnbA-BS-A400P / S389P / V421I. Using the wet cells of genetically engineered bacteria containing the esterase mutant as a catalyst, and adding 6% (v / v) of the water-insoluble organic solvent n-octanol to the reaction system, a highly efficient method for the preparation of l-menthol was established, which can eliminate substrate and product inhibition and facilitate product separation through a two-phase catalytic process of water / organic solvent.

[0006] The technical solution adopted in this invention is:

[0007] The present invention provides an esterase mutant, which is obtained by mutating serine at position 389 of the amino acid sequence shown in SEQ ID NO.4 to proline, and / or valine at position 421 to isoleucine.

[0008] Furthermore, the preferred esterase mutant is obtained by mutating serine at position 389 to proline and valine at position 421 to isoleucine in the amino acid sequence shown in SEQ ID NO.4, and is denoted as esterase mutant pnbA-BS-A400P / S389P / V421I, with the amino acid sequence shown in SEQ ID NO.6.

[0009] The present invention also provides a coding gene for the esterase mutant, the nucleotide sequence of which is shown in SEQ ID NO.5.

[0010] This invention also relates to a recombinant vector containing the esterase mutant encoding gene and a genetically engineered bacterium. The recombinant vector is based on the pET28a vector, and the genetically engineered bacterium uses E. coli BL21(DE3) as the host bacterium.

[0011] The genetically engineered bacteria were constructed as follows: the esterase mutant encoding gene was inserted between the Nco I and Xho I sites on the pET28a vector to obtain a recombinant vector; the recombinant vector was introduced into E. coli BL21(DE3) competent cells to obtain the genetically engineered bacteria.

[0012] Furthermore, this invention also provides the application of an esterase mutant in the resolution of DL-menthyl acetate to prepare L-menthol. The application involves: using the induced expression of a genetically engineered bacterium (preferably E. coli BL21(DE3) / / pET28a-pnbA-BS-A400P / S389P / V421I) containing the esterase mutant gene as a catalyst, DL-menthyl acetate as a substrate, and / or the addition of an organic solvent, and constructing a reaction system using 100mM PBS buffer (pH 7.0) as the reaction medium. The reaction is carried out completely at 25-35℃ and 500-800rpm (preferably at 30℃ and 600rpm for 12h) to obtain a reaction solution containing L-menthol. This solution is then separated and purified to obtain L-menthol. The organic solvent is ethyl acetate, chloroform, n-octanol, n-butanol, or n-hexane, preferably n-octanol.

[0013] Furthermore, in the reaction system, the catalyst dosage is 50-150 g / L based on wet bacterial cells, preferably 100 g / L; the substrate dosage is 250-2000 mM, preferably 1000 mM; and the organic solvent volume concentration is 0-12%, preferably 6%.

[0014] Further, the wet bacterial cells are prepared as follows: An engineered bacterium containing the esterase mutant encoding gene (preferably E. coli BL21(DE3) / pET28a-pnbA-BS-A400P / S389P / V421I) is inoculated into LB liquid medium containing 100 μg / mL kanamycin and cultured at 37°C and 200 rpm for 12 h to obtain a seed culture; the seed culture is then inoculated into fresh LB liquid medium containing 100 μg / mL kanamycin at a volume concentration of 2% and cultured at 37°C and 200 rpm until OD... 600 The concentration was 0.6–0.8, and then IPTG was added to a final concentration of 0.3 mM. The culture was induced at 24°C and 200 rpm for 16 h. The induction culture was then centrifuged at 4°C and 8000 rpm for 10 min. The supernatant was discarded and the wet cells were collected.

[0015] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in: (1) The esterase mutant pnbA-BS-A400P / S389P / V421I obtained by molecular modification has strict L-enantioselectivity and high catalytic activity in the selective hydrolysis of DL-menthyl acetate, and the product is optically pure L-menthyl alcohol. (2) Using the wet cells of the genetically engineered bacteria expressing the esterase mutant pnbA-BS-A400P / S389P / V421I as a catalyst and 1M DL-menthyl acetate as a substrate, a water / n-octanol two-phase catalytic reaction system is constructed. When the reaction is carried out at 30℃ and 600rpm for 10h, L-menthyl acetate is completely hydrolyzed, and ee p >99%, the conversion rate increased from 17.43% when the esterase pnbA-BS-A400P was used as a catalyst to 50%, and the space-time yield was 237.5 g / (L·d). (3) Adding n-octanol, an organic solvent that is not cosoluble with water and does not participate in the reaction, to the reaction system makes it easy to separate the product L-menthol, and also avoids substrate and product inhibition, which helps to improve the product yield. It is a new, efficient and simple method for preparing L-menthol. (iv) Description of the attached drawings

[0016] Figure 1 This is a schematic diagram of the reaction for the selective resolution of DL-menthyl acetate by the esterase pnbA-BS-A400P to synthesize the substrate L-menthol.

[0017] Figure 2 Gas chromatograms of the substrate and product standards in Example 3; solvent (2.997–3.987 min); standard D-menthol (20.615 min), standard L-menthol (20.774 min), standard L-menthyl acetate (21.097 min), standard D-menthyl acetate (21.638 min).

[0018] Figure 3 SDS-PAGE gel images of the pure enzyme solutions of esterase pnbA-BS-A400P and its mutant pnbA-BS-A400P / S389P / V421I; from left to right, lane M, Blue plus II protein marker; lane 1, pnbA-BS-A400P culture medium without IPTG; lane 2, crude pnbA-BS-A400P enzyme solution; lane 3, pure pnbA-BS-A400P enzyme; lane 4, pure pnbA-BS-A400P / S389P / V421I enzyme.

[0019] Figure 4 This is a gel electrophoresis image of the PCR products of the esterase pnbA-BS-A400P key site mutant in Example 4. (a) Lane M, 2K Plus II DNA marker; Lanes 1-15 are the PCR products encoding the following site mutants: T5I, H9Q, I23K, I34V, L37W, P65S, Q77R, R122K, D171E, K225Q, A230S, S240N, S242G, R246K, and N257K, respectively; (b) Lane M, 2K Plus II DNA. marker; lanes 1-14 are PCR products encoding the following site mutants: L270I, L274F, V278A, A286E, T296A, H298S, N307T, F327L, C370Y, S389P, V421I, T437I, D449A, and K451N.

[0020] Figure 5 This is an SDS-PAGE gel image of the esterase pnbA-BS-A400P key site mutant plasmid from Example 4. Lane M is Blue plus II protein marker; lanes 1-10 are I23K, P65S, Q77R, R122K, A230S, H298S, C370Y, S389P, V421I, and K451N, respectively.

[0021] Figure 6 This is a gel image of site-directed saturation mutant nucleic acid at S389 and V421 sites for esterase pnbA-BS-A400P in Example 5. From left to right, lane M is the 2K Plus II DNA marker; lane 1 is the PCR product of the saturation mutant at S389 site; and lane 2 is the PCR product of the saturation mutant at V421 site.

[0022] Figure 7This is an SDS-PAGE gel image of partial mutants of the site-directed saturation mutations at S389 and V421 sites of the esterase pnbA-BS-A400P in Example 5. Lane M is the 2K Plus II DNA marker; lane 1 is the control group; lanes 2-10 are the PCR products of the following site mutations: S389D, S389G, S389P, S389Y, V421A, V421E, V421F, V421K, and V421I, respectively.

[0023] Figure 8 In Example 5, the absorbance values ​​of the esterase pnbA-BS-A400P, pnbA-BS-A400P / S389P, and pnbA-BS-A400P / V421I were compared using a rapid screening method, with a culture medium in which pnbA-BS-A400P expression was not induced as a control.

[0024] Figure 9 The images show (a) and (b) SDS-PAGE gel images of the PCR product of the iterative mutant pnbA-BS-A400P / S389P / V421I in Example 6. (a) From left to right, lane M is the 2K Plus II DNA Marker; lane 1 is pnbA-BS-A400P / S389P / V421I. (b) From left to right, lane M is the Blue Plus II protein marker; lane 1 is the culture medium in which pnbA-BS-A400P / S389P / V421I is not induced as a control; lane 2 is the culture medium in which pnbA-BS-A400P / S389P / V421I is induced.

[0025] Figure 10 The effect of organic solvents on the catalytic performance of PNBA-BS-A400P / S389P / V421I was investigated. 1. No organic solvent added as a control; 2. Ethyl acetate; 3. Chloroform; 4. n-Octanol; 5. n-Butanol; 6. n-Hexane.

[0026] Figure 11 The effect of n-octanol concentration on the catalytic performance of pnbA-BS-A400P / S389P / V421I.

[0027] Figure 12This is a comparison of the whole-cell catalytic resolution of the 1M substrate DL-menthyl acetate to synthesize l-menthol under the optimal reaction system of Example 8. (a) Esterase pnbA-BS-A400P (without n-octanol); (b) Esterase pnbA-BS-A400P / S389P / V421I (without n-octanol); (c) Esterase pnbA-BS-A400P / S389P / V421I (with 6% n-octanol).

[0028] Figure 13 The reaction process at different substrate concentrations during the selective resolution of DL-menthyl acetate to synthesize L-menthol by the esterase mutant pnbA-BS-A400P / S389P / V421I in Example 9. (V) Detailed Implementation

[0029] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0030] LB liquid medium composition: tryptic peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, pH adjusted to 7.0–7.5 with 1M NaOH, solvent is water. Autoclave at 121℃ for 20 min, store at 4℃.

[0031] LB solid medium is LB liquid medium with 20 g / L agar added.

[0032] Example 1: Culture of genetically engineered E. coli BL21(DE3) / pET28a-pnbA-BS-A400P

[0033] Using the publicly disclosed pnbA-BS esterase encoding gene from Bacillus subtilis SB-14 (GenBank: PSI04734.1; nucleotide and amino acid sequences are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively), a recombinant genetically engineered bacterium, E. coli BL21(DE3) / pET-28a-pnbA-BS-A400P, was constructed and modified through patent application (application number: 2022107290122; application date: 2022.06.24). The nucleotide and amino acid sequences of the pnbA-BS-A400P esterase are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0034] The genetically engineered E. coli BL21(DE3) / pET-28a-pnbA-BS-A400P was removed from a -80°C freezer and thawed on ice. 10 μL of the preserved bacterial culture was streaked onto an LB agar plate containing 100 μg / mL kanamycin and incubated upside down at 37°C for 16 h. Single colonies were picked and transferred to 50 mL of LB liquid medium containing 100 μg / mL kanamycin resistance. The medium was then incubated overnight at 37°C and 200 rpm for 12 h. The bacterial culture was then sequenced and confirmed to be correct, thus obtaining the seed culture.

[0035] Example 2: Isolation, purification, and specific activity determination of wet cells and crude enzyme solution of esterase pnbA-BS-A400P.

[0036] 1. Preparation of wet mycelium

[0037] The seed culture of the genetically engineered E. coli BL21(DE3) / pET-28a-pnbA-BS-A400P from Example 1 was inoculated at a volume concentration of 2% into LB liquid medium containing a final concentration of 100 μg / mL kanamycin. The culture was incubated overnight at 37°C and 200 rpm. Then, it was transferred at a volume concentration of 2% into LB liquid medium containing 100 μg / mL kanamycin and incubated at 37°C and 200 rpm until the bacterial cell concentration reached OD500. 600 To obtain an induction culture medium, add IPTG to a final concentration of 0.3 mM to a pH of 0.6–0.8, and incubate at 24°C for 16 h. Under the same conditions, use the culture medium without added IPTG as the uninduced control culture medium (SDS-PAGE shown). Figure 3 (Middle lane 1) Then centrifuge the induction culture medium at 4℃ and 8000rpm for 10min, and discard the supernatant; then resuspend the bacterial cells in pH 7.0, 100mM PBS buffer, centrifuge at 4℃ and 8000rpm for 10min, discard the supernatant, and collect the wet bacterial cells.

[0038] 2. Sample preparation for SDS-PAGE detection

[0039] Take 1 mL each of the uninduced control culture medium and the induced culture medium and place them in 1.5 mL centrifuge tubes. Centrifuge at 12000 rpm for 1 min, discard the supernatant, and keep the bacterial cells. Resuspend the bacterial cells in 100 μL of ddH2O. Take 20 μL of the suspension and add 4 μL of 6×SDS PAGE loading buffer. Mix, boil in a water bath for 10 min, and centrifuge at 12000 rpm for 1 min. Take 15 μL of the supernatant from each culture for SDS-PAGE detection. The protein marker is BluePlus Protein Marker (14-120 kDa).

[0040] 3. Preparation of crude enzyme solution

[0041] Weigh 1g of the wet bacterial cells obtained in step 1, add 15mL of 100mM PBS buffer (pH 7.0) to fully resuspend the cells, and then sonicate the suspension under ice bath (0℃). Sonicate at 125W for 10min, with a 1s working and 3s rest interval, repeating this process 3 times. Centrifuge the lysed bacterial solution at 4℃ and 8000rpm for 10min to remove the precipitate. The resulting supernatant is the crude enzyme solution of the target protein (SDS-PAGE see [link]). Figure 3 Middle lane 2).

[0042] 4. Isolation and purification of the target protein

[0043] The crude enzyme solution obtained in step 3 was subjected to Ni-NTA metal chelate affinity chromatography (purchased from Bio-Rad, abbreviated as Ni). 2+ (Column, inner diameter 1.6 cm, height 15 cm) Instructions for use: Take 15 mL of crude enzyme solution and load it into the pre-equilibrated Ni... 2+ In the column, the target protein and other contaminating proteins were eluted sequentially with elution buffers containing 5 mM imidazole, 50 mM imidazole, 100 mM imidazole, 200 mM imidazole, and 500 mM imidazole (elution composition: imidazole of the corresponding concentration, 300 mM sodium chloride, 20 mM PBS buffer, pH 7.0). The elution rate was 2.0 mL / min for each concentration, and three column volumes were eluted with each concentration of elution buffer. The eluent corresponding to the 100 mM imidazole eluent was collected and desalted and concentrated by centrifugation at 4 °C and 5000 rpm for 30 min using an ultrafiltration tube with a molecular weight cutoff of 10 kDa. The retentate was taken as the pure enzyme solution and stored at 4 °C for later use.

[0044] The purity of the pnbA-BS-A400P esterase solution was verified by SDS-PAGE gel electrophoresis. The SDS-PAGE results are as follows: Figure 3 Lane 3 is shown. The esterase pnbA-BS showed a single band after SDS-PAGE electrophoresis, indicating that the isolated and purified esterase pnbA-BS was electrophoretically pure. The theoretical subunit size of the esterase pnbA-BS-A400P is 53.8 kDa, and its apparent size on SDS-PAGE electrophoresis is consistent with the expected results.

[0045] 5. Protein concentration determination

[0046] A protein concentration standard curve was plotted using the BCA protein concentration assay kit. The linear relationship was calculated as Y = 0.0633X + 0.066, where Y is the absorbance at 562 nm, X is the BSA solution concentration (mg / mL), and the standard deviation is R. 2=0.998. Then, calculate the protein concentration based on the standard protein concentration curve, and then calculate the specific enzyme activity. Perform three parallel experiments each time, and calculate the mean and standard error.

[0047] The protein concentration determination method is as follows: (1) Sample preparation: Dilute the protein sample to be tested with ultrapure water to the linear range that meets the standard curve, and add 200 μL of BCA working solution to 20 μL of sample; (2) Shake to mix, and place at 37℃ for 30 min; (3) Measure the absorbance at 562 nm using an enzyme-linked immunosorbent assay (ELISA) reader; (4) Substitute the measured absorbance into the standard curve equation to calculate the protein concentration of the sample.

[0048] Protein concentration determination showed that the pure enzyme solution of pnbA-BS-A400P esterase prepared in step 4 had a protein concentration of 9.62 mg / mL.

[0049] 6. Specific enzyme activity assay of esterase PnbA-BS-A400P

[0050] The enzyme activity of esterase PNBA-BS-A400P was calculated by measuring the change in absorbance at 405 nm using a single-factor kinetic method on a multi-functional microplate reader. A standard curve was plotted based on the concentration of p-nitrophenol and the absorbance at 405 nm, and the concentration of p-nitrophenol in the test sample was then calculated based on the standard curve, thereby calculating the specific enzyme activity.

[0051] Enzyme activity assay system: 1 mM p-nitrophenol butyrate, 1 μg pure enzyme, and 100 mM phosphate buffer (pH 7.0) to make up to 200 μL.

[0052] Enzyme activity assay: PBS buffer (pH 7.0) was added to the microplate and incubated at 30°C for 3 min. Then, 1 μg of purified enzyme and 1 μL of 200 mM p-nitrophenol butyrate were added sequentially. The volume was brought to 200 μL with PBS buffer (pH 7.0). Immediately after adding p-nitrophenol butyrate, the absorbance at 405 nm was measured using a microplate reader, with changes in absorbance measured every 10 s within 1 min. A 200 mM substrate solution of p-nitrophenol butyrate was prepared beforehand using dimethyl sulfoxide as the solvent.

[0053] The enzyme activity unit (U) is defined as the amount of enzyme required to generate 1.0 μmol of p-nitrophenol per minute under the above reaction conditions.

[0054] Perform three parallel experiments each time, and calculate the mean and standard error. The specific enzyme activity of esterase pnbA-BS is calculated using Formula 1:

[0055]

[0056] In the formula, X is the specific enzyme activity (U / mg); A is the concentration of p-nitrophenol (μM); V1 is the total volume of the reaction solution (L); t is the reaction time (min); V2 is the volume of the added enzyme solution (mL); and C is the protein concentration (mg / mL).

[0057] The enzyme activity of the pure enzyme solution of esterase pnbA-BS-A400P prepared in step 5 was determined to be 1.19 U / mg by enzyme activity assay.

[0058] Example 3: Construction of the initial reaction system for the selective resolution of DL-menthyl acetate by esterase pnbA-BS-A400P to synthesize L-menthol.

[0059] 1. Reaction system

[0060] The initial, unoptimized reaction system (10 mL) consisted of: a final concentration of 500 mM DL-menthyl acetate substrate, a final concentration of 40 g / L of wet pnbA-BS-A400P esterase cells prepared in step 1 of Example 2, added to 100 mM PBS buffer at pH 7.0 to form the 10 mL reaction system. The reaction was carried out at 600 rpm and 30°C for 6 h. Under the same conditions, a control without a catalyst was used.

[0061] After the reaction, 200 μL of the reaction solution was centrifuged at 12000 rpm for 3 min, and the supernatant was extracted with 500 μL of ethyl acetate. After extraction, the supernatant was centrifuged at 12000 rpm for 1 min, and 400 μL of the upper organic phase was added to an appropriate amount of anhydrous sodium sulfate for dehydration. Then, it was centrifuged at 12000 rpm for 1 min, and 200 μL of the supernatant was placed in a clean gas chromatography bottle. The peak areas of L-menthol, D-menthol, D-menthyl acetate and L-menthyl acetate in the supernatant were detected by gas chromatography. The concentrations were calculated according to their respective standard curves. Then, the corresponding catalytic reaction conversion rate C and the product ee of L-menthol were calculated according to formulas (2)-(5). p Values. Perform three parallel experiments each time, and calculate the mean and standard error.

[0062] The standard curve equation for L-menthol and D-menthol is: Y = 26233X - 25986, where Y is the peak area and X is the concentration of D-menthyl acetate or L-menthyl acetate, in mM.

[0063] The standard curve equation for D-menthyl acetate and L-menthyl acetate is: Y = 58608X + 20466, where Y is the peak area and X is the concentration of D-menthyl acetate or L-menthyl acetate, in mM.

[0064]

[0065]

[0066]

[0067]

[0068] In the formula, R1—concentration of d-menthyl acetate, mM; S1—concentration of L-menthyl acetate, mM; R2—concentration of D-menthol, mM; S2—concentration of L-menthol, mM. C—conversion rate; E—enantiomeric selectivity.

[0069] 2. Gas Chromatography Conditions

[0070] Gas chromatograph, Agilent 6890N; chiral column, chiral column CP7501 (50m×250μm×0.25μm); detector, FID, 225℃; carrier gas, N2; carrier gas flow rate, 50mL / min; split ratio: 1:50; injection volume: 1.0μL; injection port temperature: 225℃.

[0071] Temperature program for substrate and product: 100℃ for 8 min, 4℃ / min to 140℃ for 5 min, 40℃ / min to 180℃ for 1 min, for a total of 25 min.

[0072] Using ethyl acetate as solvent, standards of D-menthyl acetate (10 mM), L-menthyl acetate (10 mM), D-menthol (10 mM), and L-menthol (10 mM) were added respectively. The gas chromatograms are shown below. Figure 2 As shown.

[0073] Figure 2 The retention times of the substrate (DL-menthyl acetate), byproduct (D-menthol), and product (L-menthol) were as follows: D-menthol (20.615 min), L-menthol (20.774 min), L-menthyl acetate (21.097 min), and D-menthyl acetate (21.638 min).

[0074] Example 4: Single-point mutation of esterase pnbA-BS-A400P and construction of corresponding genetically engineered bacteria

[0075] Using the plasmid pET28a-pnbA-BS-A400P from Example 1 as a template, single-point mutations were performed on pnbA-BS-A400P using the primers in Table 1, and mutant plasmids were constructed using whole plasmid amplification PCR technology.

[0076] Table 1 shows the primer sequences used for single-point mutation of pnbA-BS-A400P.

[0077]

[0078]

[0079] The PCR amplification system is shown in Table 2.

[0080] Table 2 PCR amplification reaction system

[0081]

[0082] The PCR reaction process was as follows: pre-denaturation at 95℃ for 5 min; then, a cycle of denaturation at 95℃ for 15 s, annealing at 60℃ for 15 s, and holding at 72℃ for 1 min 20 s was performed, and the cycle was repeated 30 times; finally, the temperature was held at 72℃ for 5 min.

[0083] PCR products (T5I, H9Q, I23K, I34V, L37W, P65S, Q77R, R122K, D171E, K225Q, A230S, S240N, S242G, R246K, N257K, L270I, L274F, V278A, A286E, T296A, H298S, N307T, F327L, C370Y, S389P, V421I, T437I, K451N, D449A) were detected by 0.8% agarose gel electrophoresis. Under the same conditions, plasmid pET28a-pnbA-BS-A400P was used as a control. The results are shown in the figure. Figure 4 As shown. From Figure 4 A bright band can be seen at approximately 8000 bp, which is consistent with the theoretical value of the plasmid.

[0084] The PCR product was digested at 37°C for 1 hour to remove the methylated template. The digestion system is shown in Table 3.

[0085] Table 3. Digestion system for methylated template in PCR products

[0086]

[0087] The PCR product digested with DpnI was directly transformed into the host bacterium *E. coli* BL21(DE3) to obtain the genetically engineered *E. coli* BL21(DE3) / pET-28a-pnbA-BS-A400P-Mut, where Mut represents the mutant. After colony PCR verification, the transformants were inoculated into LB broth containing 100 μg / mL kanamycin and cultured at 37°C and 200 rpm for 12 h. The cells were then collected by centrifugation, plasmids were extracted, and sequencing was performed. The sequencing results were analyzed using software to determine the amino acid sequences, yielding the following mutants: T5I, H9Q, I23K, I34V, L37W, P65S, Q77R, R122K, D171E, K225Q, A230S, S240N, S242G, R246K, N257K, L270I, L274F, V278A, A286E, T296A, H298S, N307T, F327L, C370Y, S389P, V421I, T437I, K451N, and D449A. Some of these mutants are shown in gel electrophoresis images in Figure 5.

[0088] Catalysis and analysis were performed according to the method in Example 3, and the results are shown in Table 4. All mutants' ee... p All values ​​were >99%. Compared with the control group pnbA-BS-A400P (10.71%), the conversion rates of pnbA-BS-A400P / S389P and pnbA-BS-A400P / V421I were significantly improved, at 15.24% and 16.67%, respectively. Furthermore, the ee values ​​at these sites were >99%. p The values ​​are all above 99%.

[0089] Table 4 Catalytic results of the pnbA-BS-A400P mutant

[0090]

[0091] Example 5: Saturation mutations at positions 389 and 421 of the esterase pnbA-BS-A400P and construction of the corresponding genetically engineered bacteria.

[0092] Using plasmid pET28a-pnbA-BS-A400P from Example 1 as a template, saturation mutations were performed at positions 389 and 421 using primer pairs listed in Table 5. The mutant plasmid was then constructed using full plasmid amplification PCR technology. The nucleic acid gel image of the PCR product is shown below. Figure 6 As shown.

[0093] Table 5 Primer sequences for saturation mutations at positions 389 and 421 in pnbA-BS-A400P

[0094]

[0095] NNN represents all codons.

[0096] Under the same conditions, E. coli genetically engineered strains E. coli BL21(DE3) / pET-28a-pnbA-BS-A400P-S389X and E. coli BL21(DE3) / pET-28a-pnbA-BS-A400P-V421X were prepared according to the method in Example 4, where X represents 19 other amino acids that are different from the original amino acids.

[0097] 1. Seed culture: Add 1 mL of LB liquid medium containing 100 μg / mL kanamycin to each well of a sterile 96-well plate. Use a sterile toothpick to pick a single colony from the transformation plate and inoculate it into each well. Incubate the 96-well plate in a plate shaker at 37℃ and 600 rpm for 16 h to obtain the seed culture. The expression strain of esterase pnbA-BS-A400P was used as a control.

[0098] 2. Transfer and Induction: In a clean bench, use a pipette to add 900 μL of LB liquid medium containing 100 μg / mL kanamycin to each well of another sterile 96-well plate. Then, add 100 μL of the seed culture from step 1 to each well and incubate at 600 rpm and 37°C for 2-3 hours. When the OD of the bacterial culture... 600 When the pH reaches 0.6-0.8, add 0.2 mM IPTG to each well. Incubate at 200 rpm for 12 h at 24 °C.

[0099] 3. Cell collection: After the induction expression in step 2 is completed, take out the 96-well plate and centrifuge it at 3000 rpm for 10 min. Discard the supernatant, resuspend the precipitate in 100 mM PBS buffer (pH 7.0), centrifuge it again under the same conditions, discard the supernatant, and the precipitate is the desired wet cell. Store the wet cell at 4℃ for later use.

[0100] 4. Enzyme Activity Assay: The enzyme activity of pnbA-BS esterase was determined using a single-factor kinetic method with a microplate reader. p-Nitrophenol (pNPB) was used as the substrate for enzyme-catalyzed hydrolysis and detected colorimetrically. The wet bacterial cells (pnbA-BS-A400P, pnbA-BS-A400P-S389X, pnbA-BS-A400P-V421X) collected in step 3 were resuspended in 200 μL of PBS buffer (pH 7.0) and transferred to a microplate. After incubation at 30°C for 3 min, 1 μL of 100 mM p-nitrophenol butyrate was added, and the absorbance at 405 nm in each well was immediately measured using a microplate reader. The absorbance was measured every 10 s within 1 min. PBS buffer (pH 7.0) was used as a control group. Three parallel experiments were performed each time, and the mean and standard error were calculated.

[0101] Sequencing results of the mutants, analyzed using software, showed that positions 389 and 421 were successfully mutated from serine and valine, respectively, into 19 other amino acids different from the original amino acids. Gel images of some saturated mutants are shown below. Figure 7 As shown in the figure. Based on the enzyme activity assay in step 4, the optimal amino acid replacements for S389 and V421 were found to be S389P and V421I. The enzyme activity assay results for S389P and V421I are shown below. Figure 8 As shown, compared with pnbA-BS-A400P, the introduction of S389P and V421I mutations improves enzyme activity.

[0102] Example 6: Construction of genetically engineered bacteria expressing the iterative mutant pnbA-BS-A400P / S389P / V421I

[0103] Using the plasmid pET28a-pnbA-BS-A400P / S389P from Example 4 as a template, the V421I site on pnbA-BS-A400P / S389P was iteratively mutated using the primer pairs in Table 1. The mutant plasmid was then constructed using whole-plasmid amplification PCR. Under the same conditions, the genetically engineered *E. coli* BL21(DE3) / pET-28a-pnbA-BS-A400P / S389P / V421I was prepared using the method in Example 3. After colony PCR verification, the transformants were inoculated into LB broth containing 100 μg / mL kanamycin and cultured at 37°C and 200 rpm for 12 h. The cells were collected by centrifugation, the plasmid was extracted, and sequenced. Sequencing results, analyzed using software, showed that the V421I site mutation in pnbA-BS-A400P / S389P was successful. Nucleic acid gel image and protein gel image of PCR products of iterative mutants are as follows: Figure 9 As shown in a and b.

[0104] According to the catalysis and analysis method in Example 3, the conversion rate of the iterative mutant pnbA-BS-A400P / S389P / V421I increased from 10.71% of the wild-type pnbA-BS-A400P to 21.79% under the same catalytic conditions, as shown in Table 6. The conversion rates of both the mutants pnbA-BS-A400P / S389P and pnbA-BS-A400P / V421I were higher than those of pnbA-BS-A400P, consistent with the enzyme activity detection results in Example 5.

[0105] Table 6. Mutation results of the iterative mutant pnbA-BS-A400P / S389P / V421I

[0106]

[0107] Example 7: Isolation and purification of wet cells and crude enzyme solution of the esterase mutant pnbA-BS-A400P / S389P / V421I, and the activity and kinetic parameters of the purified enzyme.

[0108] Following the method in Example 2, wet cells of *E. coli* BL21(DE3) / pET28a-pnbA-BS-A400P and *E. coli* BL21(DE3) / pET28a-pnbA-BS-A400P / S389P / V421I were prepared. Under the same conditions, crude enzyme solution and pure enzyme solution of the esterase mutant pnbA-BS-A400P / S389P / V421I were prepared. A gel image of the pure enzyme solution of the esterase mutant pnbA-BS-A400P / S389P / V421I is shown below. Figure 3 As shown in lane 4. Protein concentration determination showed that the pure enzyme solution of the esterase mutant pnbA-BS-A400P / S389P / V421I had a protein concentration of 9.49 mg / mL. Specific enzyme activity determination showed that the pure enzyme solution of the esterase mutant pnbA-BS-A400P / S389P / V421I had a specific enzyme activity of 2.3 U / mg.

[0109] 3 μg of purified enzyme (pnbA-BS-A400P or pnbA-BS-A400P / S389P / V421I) was added to 100M phosphate buffer at pH 7.5. After incubation at 35°C for 3 min, different concentrations of the substrate p-nitrophenol butyrate (1 mM, 2 mM, 4 mM, 6 mM, 8 mM, 10 mM, 12 mM, 14 mM, 16 mM, 18 mM, 20 mM, and 22 mM) were added. Specific enzyme activity was measured immediately after substrate addition at 405 nm. The enzyme activity was calculated using PrismDemo software, with p-nitrophenol butyrate concentration as the x-axis and specific enzyme activity as the y-axis. As the substrate concentration increased, the enzyme activity initially increased and then stabilized, conforming to Michaelis-Menten kinetics. The kinetic parameters of the esterase pnbA-BS-A400P and its mutant pnbA-BS-A400P / S389P / V421I are shown in Table 7. The Kc of the wild-type esterase pnbA-BS-A400P is also shown in Table 7. m V max k cat and k cat / K m The concentrations were 2.02 mM, 2.58 U / mg, and 2.31 s, respectively. -1 and 1.15s -1 ·mM -1 K of the mutant pnbA-BS-A400P / S389P / V421I m V max k cat and k cat / Km The concentrations were 2.13 mM, 5.69 U / mg, and 5.11 s⁻¹, respectively. 1 and k cat / K m It is 2.40s -1 ·mM -1 The maximum reaction rate after mutation increased by 2.21 times compared to before mutation, K cat / K m It increased by 2.09 times.

[0110] Table 7 Kinetic parameters of pnbA-BS-A400P and the mutant pnbA-BS-A400P / S389P / V421I

[0111]

[0112] Example 8: Effect of the type and amount of water-insoluble organic solvent on whole-cell catalytic reaction

[0113] To explore a heterogeneous catalytic method that allows for easy separation of substrates and products from the reaction system, organic solvents such as ethyl acetate, n-hexane, and n-octanol were selected to perform whole-cell catalysis of the reaction of DL-menthyl acetate to prepare L-menthol.

[0114] 1. Selection of organic solvents

[0115] E. coli BL21(DE3) / pET28a-pnbA-BS-A400P / S389P / V421I wet cells were prepared according to the method in Example 2. 0.6 g of wet cells, 150 mM DL-menthyl acetate substrate, and 10% organic solvent (ethyl acetate, n-octanol, n-butanol, chloroform, n-hexane) were used as the reaction medium to form a 10 mL reaction system. The reaction was carried out at 30°C and 600 rpm for 6 h. The reaction solution was subjected to extraction, dehydration, and centrifugation as described in Example 3. The enantioselectivity and conversion rate were calculated by gas chromatography. A control was used without organic solvent. The results are as follows: Figure 10 As shown, compared with the control group without organic solvent, n-octanol had the most significant effect on increasing the conversion rate of bacterial enzymes catalyzing the hydrolysis of DL-menthyl acetate, increasing the conversion rate by 10.10% compared with the control group. Chloroform and n-hexane had no significant effect on catalytic activity, while ethyl acetate and n-butanol inhibited the catalytic activity of bacterial enzymes. Therefore, n-octanol was chosen as the optimal organic solvent to investigate the effect of n-octanol concentration on whole-cell catalytic activity.

[0116] 2. Selection of organic solvent concentration

[0117] After determining that n-octanol was the optimal organic solvent, the effect of different n-octanol concentrations on the catalytic reaction was further investigated. The organic solvent in step 1 was changed to n-octanol, with a concentration range of 0%-12% (v / v), specifically 0%, 2%, 4%, 6%, 8%, 10%, and 12%, while other procedures remained the same. The results are as follows: Figure 11 As shown, when the concentration of n-octanol is low, the conversion rate increases with increasing n-octanol concentration. At a n-octanol concentration of 6% (v / v), the conversion rate of L-menthol reaches as high as 36.1%, which is 13.07% higher than the control group. When the n-octanol concentration is further increased, the conversion rate decreases with increasing solubilizer concentration, exhibiting an inhibitory effect on bacterial enzyme activity. Therefore, the optimal concentration of n-octanol is 6% (v / v).

[0118] Example 8: Construction of the optimal reaction system for the whole-cell catalytic resolution of DL-menthyl acetate to synthesize L-menthol by the esterase mutant pnbA-BS-A400P / S389P / V421I

[0119] Using 100 g / L of wet E. coli BL21(DE3) / pET28a-pnbA-BS-A400P or E. coli BL21(DE3) / pET28a-pnbA-BS-A400P / S389P / V421I cells prepared according to the method in Example 2 as a catalyst, 1 M DL-menthyl acetate substrate was added, and a two-phase reaction system of 10 mL was constructed using phosphate buffer (pH 7.0, 100 mM) as the reaction medium. The reaction temperature was 30 °C, the pH was maintained at 7.0 by adding 1 M NaOH solution, the reaction speed was 600 rpm, and the reaction time was 12 h. The substrate conversion rate was determined using the method in Example 3.

[0120] Under the same conditions, the reaction process was enhanced by adding 6% (v / v) n-octanol to the esterase pnbA-BS-A400P / S389P / V421I.

[0121] The results are as follows Figure 12 As shown, at a substrate concentration of 1M and a reaction time of 10 h, the conversion rate of the original esterase pnbA-BS-A400P (a) to DL-menthyl acetate was 17.43%; the conversion rate of the modified esterase pnbA-BS-A400P / S389P / V421I (b) was 39.8%, an increase of 22.37% compared to the original esterase; the esterase pnbA-BS-A400P / S389P / V421I (c) with enhanced reaction process (6% n-octanol) achieved a conversion rate of 50% at 10 h, an increase of 10.2% compared to (b) before enhanced reaction process, and ee pThe conversion rate was above 99%. Considering both enzyme design modification and reaction process enhancement, the conversion rate increased from 11.43% of the wild type to 50%, and the space-time yield was 237.5 g / (L·d).

[0122] Example 9: Reaction process for the selective resolution of DL-menthyl acetate to synthesize L-menthol by the esterase mutant pnbA-BS-A400P / S389P / V421I

[0123] Using 100 g / L of wet E. coli BL21(DE3) / pET28a-pnbA-BS-A400P / S389P / V421I cells prepared according to the method in Example 2 as a catalyst, and DL-menthyl acetate at final concentrations of 250 mM, 500 mM, 750 mM, 1 M, 1.25 M, 1.5 M, and 2 M, along with 6% (v / v) n-octanol, a two-phase reaction system (10 mL) was constructed using phosphate buffer (pH 7.0, 100 mM) as the reaction medium. The reaction temperature was 30 °C, the pH was maintained at 7.0 by adding 1 M NaOH solution, the reaction speed was 600 rpm, and the reaction time was 12 h. The substrate conversion rate was determined using the method in Example 3, and the results are as follows. Figure 13 As shown, when the substrate concentration was below 1 M, the reaction was complete within 10 h; when the substrate concentration was 1 M, the conversion rate was 50% after 10 h. With increasing substrate concentration, high-concentration substrate inhibition occurred on the catalyst, inhibiting the enzyme's catalytic activity but not inactivating it, inevitably reducing the bioconversion efficiency. When the substrate concentration was 2 M, the conversion rate was only 22.13% within 12 h. Therefore, using 100 g / L wet bacterial cells to catalyze 1 M substrate in a 10 mL reaction system is the most economical. Under this reaction system, the optimal substrate concentration for catalyzing DL-menthyl acetate was 1 M, and the space-time yield of L-menthol was 237.5 g / (L·d), which is one of the highest levels currently achieved in the biocatalytic preparation of L-menthol, realizing the efficient conversion and utilization of the substrate DL-menthyl acetate with high enantioselectivity.

Claims

1. An esterase mutant, characterized in that, The esterase mutant was obtained by mutating serine at position 389 of the amino acid sequence shown in SEQ ID NO.4 to proline, and / or valine at position 421 to isoleucine.

2. The esterase mutant according to claim 1, characterized in that, The esterase mutant was obtained by mutating serine at position 389 of the amino acid sequence shown in SEQ ID NO.4 to proline and valine at position 421 to isoleucine.

3. The encoding gene of the esterase mutant of claim 1.

4. A recombinant vector containing the esterase mutant encoding gene of claim 1.

5. A genetically engineered bacterium constructed from the recombinant vector of claim 4.

6. The use of the esterase mutant of claim 1 in the preparation of L-menthol from the resolution of DL-menthyl acetate.

7. The application as described in claim 6, characterized in that, The application is as follows: using the wet cells of genetically engineered bacteria containing the esterase mutant encoding gene as a catalyst, DL-menthyl acetate as a substrate, and / or adding an organic solvent, a reaction system is constructed with pH 7.0 and 100mM PBS buffer as the reaction medium. The reaction is carried out at 25-35℃ and 500-800rpm until complete, and a reaction solution containing L-menthol is obtained. After separation and purification, L-menthol is obtained.

8. The application as described in claim 7, characterized in that, The organic solvent is ethyl acetate, chloroform, n-octanol, n-butanol, or n-hexane.

9. The application as described in claim 7, characterized in that, In the reaction system, the catalyst dosage is 50-150 g / L based on wet bacterial cells; the substrate dosage is 250-2000 mM; and the organic solvent volume concentration is 0-12%.

10. The application as described in claim 7, characterized in that, The wet bacterial cells were prepared as follows: Engineered bacteria containing the esterase mutant encoding gene were inoculated into LB liquid medium containing 100 μg / mL kanamycin and cultured at 37°C and 200 rpm for 12 h to obtain a seed culture; the seed culture was then inoculated into fresh LB liquid medium containing 100 μg / mL kanamycin at a volume concentration of 2% and cultured at 37°C and 200 rpm until OD... 600 The concentration was 0.6–0.8, and then IPTG was added to a final concentration of 0.3 mM. The culture was induced at 24°C and 200 rpm for 16 h. The induction culture was then centrifuged at 4°C and 8000 rpm for 10 min. The supernatant was discarded and the wet cells were collected.

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