Genetically engineered bacterium for high yield of menthol glucoside and application of genetically engineered bacterium

Abstract

The invention discloses a genetically engineered bacterium for high yield of menthol glucoside and application thereof, and belongs to the technical field of genetic engineering. According to the recombinant escherichia coli containing the alpha-glucosidase gene for producing menthol glucoside, alpha-glucosidase can be efficiently synthesized in cells, menthol and maltose monohydrate are used as auxiliary substrates, the glycosylation reaction of menthol is efficiently catalyzed in a whole-cell mode, the reaction lasts for 10-24 hours, the product concentration reaches up to 200 g / L, and the yield is high. And the molar conversion rate of the substrate is stabilized at 98%, so that the menthol glucoside can be recovered and purified. The menthol glucoside can be combined with lactose, mannose and other glycosyls to serve as a cosmetic additive with cooling and moisturizing effects.

Description

TECHNICAL FIELD

[0001] The present application relates to a genetically engineered bacteria with high yield of menthol glucoside and its application, belonging to the technical field of genetic engineering. BACKGROUND

[0002] L-menthol (chemical formula: C 10 H 20 O) is a colorless transparent needle crystal, with the following physical properties: relative molecular mass of 156.4, melting point of 44℃, boiling point of 216.4℃. The compound has low solubility in water, but has good solubility in conventional organic solvents such as cyclohexane, ethanol and benzene. L-menthol is widely used in cigarettes, cosmetics, toothpaste, mouthwash, chewing gum, sweets and other products due to its fresh and brisk odor and unique fragrance. In addition, L-menthol can activate the cold receptors on the skin without causing actual temperature changes, and has pharmacological effects such as local antipruritic, analgesic, vasodilative, mild local anesthetic and promoting drug penetration, so it is also used in drug rubbing and local anesthesia. In view of the wide application of L-menthol in the fields of medicine and health, food industry and daily fine chemical products, its market demand continues to grow, and improving the quality and use effect of L-menthol has significant economic and social benefits.

[0003] Traditional methods of producing glucosides, such as direct extraction and purification from plants or microorganisms, have high technical requirements, high cost and low yield. Chemical synthesis is often limited by low yield, poor selectivity and complicated functional group protection and deprotection steps. In contrast, glycosyltransferase (GT) is highly efficient in catalyzing the formation of glucosides, and shows good regioselectivity and stereochemical selectivity, and has become a promising tool for biosynthesis.

[0004] Alpha-glucosidase (EC 3.2.1.20) is a hydrolytic enzyme that can hydrolyze alpha-glucosidic bonds and release glucose. Some alpha-glucosidases also exhibit transglycosylation activity, i.e., in the presence of high concentrations of substrates, they can transfer glucose groups from donor substrates to the hydroxyl groups of acceptor substrates. This glucose group transfer reaction is believed to be accomplished by a double displacement mechanism, in which a sugar enzyme intermediate plays a key role. The intermediate is composed of a nucleophile (e.g., aspartic acid, Asp) and an acid-base catalyst (e.g., glutamic acid, Glu), which is responsible for transferring protons to the glycosidic bond, facilitating the separation of the leaving group. At the same time, the nucleophile forms a covalent intermediate with the anomeric carbon, which can be attacked by a water molecule and decomposed, releasing glucose. If the intermediate is attacked by an alcohol (including sugar) instead of water, a transglycosylation reaction occurs, producing a glycosylated product.

[0005] L-menthol has some limitations in practical applications, such as poor thermal stability, gradual weakening of its fragrance and cooling effect over time, poor solubility in water, etc. In order to overcome these shortcomings, a glycosylation modification method can be used. The glycosylation product of L-menthol, i.e. menthol glucoside, breaks through the application limitations of native menthol by structural modification, improves its water solubility and stability, and becomes a functional raw material across fields. At present, L-menthol itself has the problem of poor water solubility, which needs to be dissolved using an organic solvent, hindering its scale application; moreover, L-menthol has poor stability and is easy to volatilize, which makes it inconvenient to store. At present, L-menthol is modified into menthol glucoside by enzyme catalysis to increase its water solubility and stability, however, the enzyme purification step is complicated and increases the production cost, and the enzyme catalysis conditions are limited; moreover, the glycosylation enzyme of L-menthol has the problem of low conversion efficiency, which limits the glycosylation efficiency. SUMMARY

[0006] In view of the above problems of the prior art, the present application provides a genetically engineered bacterium for high-yield production of menthol glucoside and its application, aiming to solve the technical problems that L-menthol is modified into menthol glucoside by enzyme catalysis to increase its water solubility and stability, however, the enzyme purification step is complicated and increases the production cost, and the enzyme catalysis conditions are limited; moreover, the glycosylation enzyme of L-menthol has the problem of low conversion efficiency, which limits the glycosylation efficiency.

[0007] The first technical solution provided by the present application is the application of an alpha-glucosidase in the preparation of menthol glucoside, wherein the amino acid sequence of the alpha-glucosidase is shown in SEQ ID NO. 2.

[0008] The second technical solution provided by the present application is a method for synthesizing menthol glucoside, which comprises using L-menthol and maltose as substrates, using an alpha-glucosidase or a recombinant cell expressing the alpha-glucosidase as a catalyst to form a catalytic system, and reacting to obtain a catalytic liquid containing menthol glucoside, wherein the amino acid sequence of the alpha-glucosidase is shown in SEQ ID NO. 2.

[0009] In some embodiments, in the catalytic system, the final concentration of the menthol is 10 mM.

[0010] In some embodiments, in the catalytic system, the addition amount of the maltose is 10-40 mM.

[0011] In some embodiments, the reaction temperature is 30-45℃, and the reaction time is at least 10 h.

[0012] In some embodiments, the addition amount of the recombinant cell is 500-1500 mg / mL.

[0013] In some embodiments, the recombinant cell is E. coli with PET32a(+) plasmid as an expression vector to express the alpha-glucosidase.

[0014] In some embodiments, the catalyst construction method of the recombinant cell comprises the following steps: (1) inserting the alpha-glucosidase gene into the PET32a(+) plasmid to obtain a recombinant plasmid, transforming the recombinant plasmid into E. coli Rosetta (DE3), and obtaining a recombinant cell;

[0015] (2) culturing the recombinant cell to induce the expression of the exogenous recombinant protein;

[0016] (3) collecting the bacterial cells to obtain a whole-cell catalyst.

[0017] In some embodiments, in step (2), the specific steps of induction are as follows: inoculating the transformant into LB medium (with 100 mg / mL ampicilin (Amp) added) and culturing at 200 rpm and 25-37°C until the OD 600 = 0.8-1.8, adding IPTG to a final concentration of 0.1-1.5 mM to continue the induction expression culture for 6-18 h.

[0018] In some embodiments, in step (3), the bacterial cell collection conditions are as follows: centrifuging at 4°C and 5000 rpm for 10 min to collect the bacterial cells, washing twice with BSP buffer, and placing in an ice box for standby.

[0019] In some embodiments, the obtained catalytic liquid is centrifuged, filtered, and the mixed product is obtained by freeze-drying, an organic reagent is added, the filtrate is collected by filtering membrane, and the pure menthol glucoside is obtained by rotary evaporation concentration, and high performance liquid chromatography detection is performed.

[0020] Further, the centrifugation conditions are 4000-12000 rpm for 10-30 min.

[0021] Further, the filter membrane pore size is 0.22 μm.

[0022] Further, the vacuum freeze-drying conditions are a vacuum degree < 10 Pa, a temperature of -70°C, and a drying time of 24 h.

[0023] Further, the organic solvent is ethanol.

[0024] Further, the rotary evaporator is used at a temperature of 45-60°C.

[0025] Further, the high performance liquid chromatography detection conditions are as follows: the chromatographic column is Agilent, ZORBAX SB-C18, 4.6*250mm, 5um; the mobile phase is methanol: water: trifluoroacetic acid = 75:25:0.1, the flow rate is 1mL / min, the injection volume is 20ul, the differential refractive index detector, the column temperature is 30 DEG C, and the running time is 20min. The peak time of menthol glucoside is 9-10min; the peak time of the substrate menthol is 15-16min; and the peak time of the substrate maltose is 2-3min.

[0026] The third technical solution provided by the application is application of a recombinant plasmid containing alpha-glucosidase or a recombinant bacterium in preparation of menthol glucoside or a product containing menthol glucoside, and the amino acid sequence of the alpha-glucosidase is shown in SEQ ID NO. 2.

[0027] The fourth technical solution provided by the application is application of alpha-glucosidase, a recombinant plasmid containing alpha-glucosidase or a recombinant bacterium in preparation of oral care products or makeup products, and the amino acid sequence of the alpha-glucosidase is shown in SEQ ID NO. 2.

[0028] The technical effects of the application are as follows:

[0029] 1. The application uses an exogenous expression vector system to induce expression of alpha glA gene in E. coli, so that it can efficiently utilize menthol to catalytically produce a product of menthol glucoside with better water solubility, and the molar conversion rate of the substrate is maintained at 98%.

[0030] 2. The enzyme activity is improved by 300% through optimization of the induction conditions.

[0031] 3. The application separates and purifies the whole cell catalytic liquid to obtain a pure product of menthol glucoside with a purity of up to 99.5%, and the reagents used for purification are non-toxic and harmless, which meets the environmental protection requirements. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 It is a flow chart for PET32a-alpha glA vector construction;

[0033] Figure 2 It is a schematic diagram of PET32a-alpha glA recombinant plasmid;

[0034] Figure 3 It is a standard curve of p-nitrophenol;

[0035] Figure 4 It is an optimization of induction time conditions for E. coli expressing alpha-glA;

[0036] Figure 5 It is an optimization of induction concentration conditions for E. coli expressing alpha-glA;

[0037] Figure 6 Optimization of OD for E. coli expressing α-glA 600 Optimization of conditions;

[0038] Figure 7 Optimization of temperature conditions for E. coli expressing α-glA

[0039] Figure 8 Schematic diagram for whole cell catalysis of menthol glucoside Figure 9 Schematic diagram for HPLC detection of menthol glucoside DETAILED DESCRIPTION

[0040] The following describes preferred embodiments of the present application, and it should be understood that the embodiments are for better explaining the present application and are not used to limit the present application.

[0041] Example 1 Construction of PET32a-aglA recombinant plasmid

[0042] (1) The gene aglA encoding α-glucosidase was chemically synthesized (GenScript), and its nucleic acid sequence is shown in SEQ ID NO. 1 and its amino acid sequence is shown in SEQ ID NO. 2.

[0043] (2) The plasmid PET32a preserved in the laboratory was used as a template, and the purpose gene was double-digested with the restriction enzymes EcoR I and EcoR V (purchased from GenScript) without a cutting point, and the circular plasmid was cut into a linearized vector by the double-digestion method, and the recovery method was performed according to the OMEGA agarose gel recovery kit (item number D2500).

[0044] (3) The plasmid skeleton and the α-glA gene fragment were connected by the method of seamless cloning, and the PET32a (+) outer skeleton and the gene fragment were mixed at a molar ratio of 1:3 and treated in a 50°C water bath for 20 min. The ligation product was transformed into E. coli DH5α competent cells, and the transformed cells were plated on agar plates containing 100 μg / mL ampicilin (Amp) and cultured overnight.

[0045] (4) Single colonies growing on the plates were picked and cultured in 5 mL LB medium containing 100 μg / mL ampicilin (Amp) overnight. Bacterial liquid PCR identification was performed using the primers in the table, and sequencing was performed. The sequencing results showed that the constructed PET32a-aglA sequence was correct. Figure 1 A schematic flowchart for plasmid construction is shown in Figure 2 The primers used are shown in Table 1.

[0046] Table 1 Primer Table

[0047]

[0048] Example 2: Induced expression of protein

[0049] The recombinant vector of Example 1 which was sequenced correctly was transformed into E. coli DH5a for plasmid amplification, and E. coli Rosetta (DE3) was used as the host bacteria for plasmid expression. After the E. coli Rosetta was cultured overnight from a single colony at 37°C, a single colony was picked and inoculated into 3 ml of LB (containing ampicilin (Amp) resistance) culture medium and cultured overnight at 37°C, 200 rpm. 0.5 ml of the bacterial solution was inoculated into 50 ml of LB culture medium and cultured at 37°C, 200 rpm, until the OD 600 After reaching about 0.6-0.8, IPTG (final concentration 0.5 mM) was added, and the culture was continued for 6-24 h. After 24 h, the bacterial cells were collected by centrifugation under the following conditions: 4000 rpm, 4°C, 12 min. The collected bacterial cells were washed with BSP buffer, and the protein expression level was detected by α-glA enzyme activity detection. Enzyme activity detection analysis: according to the Solabio α-glA enzyme activity detection kit (BC2550). The enzyme activity detection product standard curve is shown in Figure 3 The R-square of the enzyme activity detection product standard curve was as high as 0.9998, and was within the confidence interval; the protein expression level was analyzed by enzyme activity detection, and the average expression enzyme activity of α-glA was 0.74 x 10 4 U / g.

[0050] Example 3: Optimization of induced expression conditions

[0051] (1) Optimization of the induction time for recombinant E. coli expressing αglA

[0052] After sequencing, the PET32a-αglA transformant was inoculated into 5 mL of LB liquid culture medium containing ampicilin (Amp) resistance, and 50 mL of LB liquid was added at a ratio of 1%, respectively, and induced at 220 rpm for 6 h, 12 h, 18 h, and 24 h, respectively. The samples were prepared by centrifugation, and the enzyme activity was verified by the α-glA enzyme activity detection kit.

[0053] As shown in Figure 4 , the optimal expression αglA induction time was 12 h, and the enzyme activity was 1.42 x 10 4 U / g.

[0054] (2) Optimization of the induction concentration for recombinant E. coli expressing αglA

[0055] After sequencing, pick the PET32a-αglA transformants and inoculate in 5 mL LB liquid medium containing ampicilin (Amp) resistance, add 50 mL LB liquid according to 1% proportion, and then add IPTG to the final concentration of 0.0 mM, 0.5 mM, 1 mM, 1.5 mM and 2.0 mM under the optimal induction time condition, and induce at 220 rpm for 12 h. Centrifuge to prepare samples, and verify the enzyme activity by α-glA enzyme activity test kit.

[0056] As shown in Figure 5 , the optimal expression αglA induction concentration is 1.5 mM, and the enzyme activity is 1.6 x 10 4 U / g.

[0057] (3) Recombinant E. coli expression αglA induction OD 600 Optimization

[0058] After sequencing, pick the PET32a-αglA transformants and inoculate in 5 mL LB liquid medium containing ampicilin (Amp) resistance, add 50 mL LB liquid according to 1% proportion, and then add IPTG to the final concentration of 0.0 mM, 0.5 mM, 1 mM, 1.5 mM and 2.0 mM under the optimal induction time condition, and induce at 220 rpm for 12 h. Centrifuge to prepare samples, and verify the enzyme activity by α-glA enzyme activity test kit. 600 respectively, add IPTG to the final concentration of 1.5 mM, and put all into a shaker for induction at 25°C and 220 rpm for 12 h. Centrifuge to prepare samples, and verify the enzyme activity by α-glA enzyme activity test kit.

[0059] As shown in Figure 6 , the optimal expression αglA induction OD 600 is 1.8, and the enzyme activity is 1.9 x 10 4 U / g.

[0060] (4) Recombinant E. coli expression αglA induction temperature optimization

[0061] After sequencing, pick the PET32a-αglA transformants and inoculate in 5 mL LB liquid medium containing ampicilin (Amp) resistance, add 50 mL LB liquid according to 1% proportion, and then add IPTG to the final concentration of 0.0 mM, 0.5 mM, 1 mM, 1.5 mM and 2.0 mM under the optimal induction time condition, and induce at 220 rpm for 12 h. Centrifuge to prepare samples, and verify the enzyme activity by α-glA enzyme activity test kit. 600 respectively, add IPTG to the final concentration of 1.5 mM, and put all into a shaker for induction at 25°C and 220 rpm for 12 h. Centrifuge to prepare samples, and verify the enzyme activity by α-glA enzyme activity test kit.

[0062] As shown in Figure 7As shown, the optimal αglA expression induction temperature is 30℃, and the enzyme activity is 2.2 × 10⁻⁶. 4 U / g.

[0063] In summary, the optimal induction conditions for recombinant Escherichia coli are: culturing recombinant Escherichia coli to OD200. 600 The value was 1.8, and IPTG was added to a final concentration of 1.5 mM. The mixture was then incubated at 37°C and 200 rpm for 12 h.

[0064] Example 4: Whole-cell catalytic production of menthol glucoside

[0065] The α-glA Escherichia coli strain obtained in Example 2 was cultured in LB medium to expand OD. 600 The pH value was 1.8, and IPTG was added to a final concentration of 1.5 mM. The cells were incubated at 37°C and 200 rpm for 12 h. The cells were then centrifuged at 10,000 rpm at 4°C for 20 min to collect the cells. The cells were washed twice with BSP. The bacterial cells were then cultured at a concentration of 500 mg / mL. -1 The amount added is increased to a concentration of 15.6 g·L. -1 Menthol, 72 g·L -1 Whole-cell catalysis was performed in an aqueous solution of maltose monohydrate for 10-48 h at a catalytic temperature of 35-45℃ and a shaking speed of 150-250 rpm. After whole-cell catalysis, the molar conversion of menthol remained relatively stable at 98%, and the highest concentration of menthol glucoside could reach 204 g / L after scaling up the catalytic system. A schematic diagram of the whole-cell catalytic reaction is shown below. Figure 8 .

[0066] Example 5: Isolation and purification of menthol glucoside

[0067] The catalytic solution containing menthol glucoside obtained in Example 4 was centrifuged at 8000 rpm for 30 min. The supernatant was collected and filtered through a 0.22 μm pore size filter membrane to obtain a sterile supernatant. The sterile supernatant was then freeze-dried under vacuum (<10 Pa) at -70°C for 24 h to obtain a catalytic mixture. This catalytic mixture was dissolved in anhydrous ethanol at a mass ratio of 1:10. The substrate sugar was removed by passing the solution through a 0.22 μm organic filter membrane to obtain an ethanol solution containing menthol glucoside. The ethanol solution of menthol glucoside was then subjected to rotary evaporation in a 50°C water bath to obtain pure menthol glucoside. The HPLC results are shown below. Figure 9 Menthol glucoside has a purity of over 99.5% after purification.

[0068] Comparative Example 1

[0069] A whole cell catalysis method with terpinen-4-ol as a substrate, the specific implementation same as example 4, the difference from example 4 is that in the present comparative example, terpinen-4-ol with similar structure of menthol is selected as a substrate for whole cell catalysis, and the final concentration of the substrate is 10 mM.

[0070] Comparative example 2:

[0071] A whole cell catalysis method with terpinen-4-ol as a substrate, the specific implementation same as example 4, the difference from example 4 is that in the present comparative example, terpinen-4-ol with similar structure of menthol is selected as a substrate for whole cell catalysis, and the final concentration of the substrate is 10 mM.

[0072] Comparative example 3:

[0073] A whole cell catalysis method with terpinen-4-ol as a substrate, the specific implementation same as example 4, the difference from example 4 is that in the present comparative example, terpinen-4-ol with similar structure of menthol is selected as a substrate for whole cell catalysis, and the final concentration of the substrate is 10 mM.

[0074] Table 1

[0075]

[0076] From the above table 1, it can be seen that the expressed alpha glA enzyme protein has strong selectivity to the substrate alcohol, and has high substrate activity to menthol. Through whole cell catalysis, a relatively stable product can be obtained, which is convenient for subsequent purification.

[0077] Although the present application has been disclosed with the preferred embodiments as above, it is not intended to limit the present application, and anyone skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application. Therefore, the protection scope of the present application should be defined by the claims.

Claims

1. Use of α-glucosidase for the preparation of menthol glucoside, characterized in that, The amino acid sequence of the alpha-glucosidase is shown as SEQ ID NO.

2.

2. A method for synthesizing menthol glucoside, which comprises using L-menthol and maltose as substrates, using alpha-glucosidase or recombinant cells expressing alpha-glucosidase as a catalyst to form a catalytic system, and reacting to obtain a catalytic liquid containing menthol glucoside, and the amino acid sequence of the alpha-glucosidase is shown as SEQ ID NO.

2.

3. The method of claim 2, wherein, In the catalytic system, the final concentration of the menthol is 10 mM; and the addition amount of the maltose is 10-40 mM.

4. The method of claim 2, wherein, The addition amount of the recombinant cells is 500-1500 mg / mL.

5. The method of claim 2, wherein, The reaction temperature is 30-45 DEG C, and the reaction time is at least 10 h.

6. The method of claim 2, wherein, The recombinant cells are E. coli as a host, PET32a(+) plasmid as an expression vector, and the alpha-glucosidase is expressed.

7. The method of claim 2, wherein, The catalyst construction method of the recombinant cells comprises the following steps: (1) inserting the alpha-glucosidase gene into the PET32a(+) plasmid to obtain a recombinant plasmid, transforming the recombinant plasmid into E. coli Rosetta (DE3), and obtaining recombinant cells; (2) culturing the recombinant cells to induce the expression of exogenous recombinant proteins; (3) collecting bacterial cells to obtain whole-cell catalysts; Optionally, in step (2), the specific steps of induction are as follows: the transformants are inoculated into LB medium, cultured at 200 rpm, 25-37°C to OD 600 = 0.8-1.8, IPTG is added to a final concentration of 0.1-1.5 mM to continue the induction expression culture for 6-18 h.

8. The method of claim 2, wherein, The obtained catalytic liquid is centrifuged, filtered, and freeze-dried to obtain a mixed product, organic reagents are added, the filtrate is collected by filtering membrane, and rotary evaporation is used for concentration to obtain pure menthol glucoside, which is detected by high performance liquid chromatography.

9. Use of a recombinant plasmid or a recombinant bacteria containing α-glucosidase for the preparation of menthol glucoside or a product containing menthol glucoside, characterized in that, The amino acid sequence of the alpha-glucosidase is shown as SEQ ID NO.

2.

10. Use of α-glucosidase, a recombinant plasmid containing α-glucosidase or a recombinant bacteria in the preparation of an oral care product or a makeup product, characterized in that, The amino acid sequence of the alpha-glucosidase is shown as SEQ ID NO. 2.

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