Method for preparing (β,S)-configuration boswellic acid by enzymatic catalysis and engineering bacteria

Abstract

The present invention provides a method for preparing (β,S)-configuration boswellic acid by enzymatic catalysis and an engineered bacterium, belonging to the field of biosynthesis. The engineered bacterium of the present invention uses Escherichia coli BL21(DE3) as the starting strain, and introduces the improved carbonyl reductase encoding gene shown in SEQ ID No.1 and the improved formate dehydrogenase encoding gene shown in SEQ ID No.2. The present invention uses the enzymes produced by this strain to prepare carbonyl reductase Ls ADH and formate dehydrogenase Ps FDH mut The co-immobilized enzymes are used for the enzymatic synthesis of (β,S)-configuration boswellic acid. The catalytic reaction has high selectivity and can be carried out in a neutral environment at normal temperature and pressure. The co-immobilized enzymes can be reused, which is beneficial to continuous production and cost reduction. The conversion rate of the catalytic reaction adopted by the present invention is >99%. The substrates β-acetoxyl xyloside and sodium formate used have high safety, significantly improving the synthesis efficiency of the product (β,S)-boswellic acid.

Description

Technical Field

[0001] The present invention belongs to the field of biosynthesis, and particularly relates to a method for preparing (β,S)-configuration boswellic acid by enzymatic catalysis and an engineered bacterium. Background Art

[0002] Boswellic acid is a functional raw material commonly used in cosmetics, and its INCI name is hydroxypropyltetrahydropyrantriol. It can promote the synthesis of glycosaminoglycans in the human dermis, promote the production of proteoglycans and the regeneration of collagen. Glycosaminoglycans, proteoglycans and collagen are all important structural components of the extracellular matrix, which are crucial for maintaining the firmness, uniformity, smoothness and elasticity of the skin and keeping the skin in a healthy state. The external manifestations of skin aging are thinning of the skin, skin laxity, loss of elasticity and deepening of wrinkles. In the study of skin aging, it was found that there are different levels of decline in glycosaminoglycans and proteoglycans in both naturally aged skin and photoaged skin. Using cosmetics containing boswellic acid can promote epidermal renewal, strengthen the skin structure, tighten the skin, improve wrinkles, increase skin luster and elasticity. Therefore, boswellic acid is known as one of the "big three anti-aging giants" in the skincare industry and is also widely used in products such as creams and serums of big-name products.

[0003] Boswellic acid is a β-xylose derivative, initially developed by L'Oréal. Its molecular structure contains multiple chiral carbon atoms. The spatial arrangement of the chiral carbon atoms on its parent nucleus is relatively fixed, while the 7th carbon atom in the glycosyl ligand (hydroxypropane) has two different configurations, namely (β,S) and (β,R), these two diastereoisomers (as Figure 1As shown). Research shows that the biological activity of the (β,S) configuration is better than that of the (β,R) configuration, and the cytotoxicity of the (β,S) configuration is weaker than that of the (β,R) configuration. The conventional production process of pro-xylane is chemical synthesis. Using xylose as the raw material, it undergoes a condensation reaction with acetylacetone in an alkaline aqueous solution, and then the ketone carbonyl is reduced by the action of sodium borohydride to obtain pro-xylane. The pro-xylane produced by the chemical synthesis process is a mixture of two configurations, and the ratio of the (β,S) configuration to the (β,R) configuration is usually between 3:7 and 5:5. Moreover, the acetylacetone used has medium toxicity, and its vapor mixed with air can form an explosive mixture. CN 117510446 A discloses a preparation method of pro-xylane. Starting from D-xylose as the raw material, it first reacts with acetylacetone to obtain acetonyltetrahydropyran triol, and then undergoes a carbonyl reduction reaction in the presence of an inexpensive catalyst to obtain hydroxypropylpyran triol with an S configuration:R configuration = 50:50 to 55:45. The catalytic reaction process requires the use of hydrogen and high temperature and high pressure conditions. There are also reports in the prior art on the synthesis of (β,S)-configured pro-xylane by enzymatic methods, but the enzymes used cannot be recycled and reused, which is not conducive to continuous production. Therefore, developing a safe, environmentally friendly, and continuously operable production process for (β,S)-configured pro-xylane is of great significance for the large-scale production and market application of pro-xylane. Summary of the Invention

[0004] To solve the problems in the prior art, the present invention proposes a method and engineering bacteria for preparing (β,S)-configured pro-xylane by enzymatic catalysis.

[0005] The present invention first provides a method for constructing an engineering bacterium for enzymatic catalysis to prepare (β,S)-configured pro-xylane, which includes:

[0006] S1: Design an improved carbonyl reductase encoding gene with a sequence as shown in SEQ ID No.1; design an improved formate dehydrogenase encoding gene with a sequence as shown in SEQ ID No.2; insert the improved carbonyl reductase encoding gene and the improved formate dehydrogenase encoding gene between the 5070th and 5234th positions of the vector pET-28a(+), and the improved carbonyl reductase encoding gene is located upstream of the improved formate dehydrogenase encoding gene to obtain the recombinant vector pET-28a(+)-Lsadh-Psfdh mut ;

[0007] S2: Heat shock transform the recombinant vector pET-28a(+)-Lsadh-Psfdh mut into Escherichia coli BL21(DE3) competent cells to obtain a recombinant strain BL21(DE3) / pET-28a(+)-Lsadh-Psfdh co-expressing carbonyl reductase Lsadh and formate dehydrogenase Psfdh mut mut ​。

[0008] The present invention also provides an engineered bacterium constructed by the above method. Further provided is the use of the engineered bacterium or its fermentation product in the enzymatic preparation of (β,S)-configuration boswellic acid.

[0009] The present invention also provides a method for enzymatically preparing (β,S)-configuration boswellic acid, which comprises the following steps:

[0010] 1) Culturing the engineered bacterium, disrupting the cultured bacterial cells, and separating and purifying to obtain carbonyl reductase LsADH and formate dehydrogenase PsFDH mut ;

[0011] 2) Preparing a sodium alginate solution, and adding the obtained carbonyl reductase LsADH and formate dehydrogenase PsFDH mut enzyme solution, wherein the enzyme activity ratio of carbonyl reductase to formate dehydrogenase is 1:1 to 1:2, and the sum of the final concentrations of the two enzymes is 2 to 5 mg / mL;

[0012] Adding the above mixed enzyme solution dropwise into a chitosan and calcium chloride mixed solution with the same volume as the mixed enzyme solution, stirring to form gel beads, and then standing and curing the gel beads to obtain a co-immobilized enzyme of carbonyl reductase LsADH and formate dehydrogenase PsFDH mut ;

[0013] 3) Using a reaction system containing the co-immobilized enzyme to prepare (β,S)-configuration boswellic acid, and the reaction conditions are stirring speed of 300 to 500 rpm, reaction temperature of 35 to 40 °C, and reaction time of 6 to 8 h;

[0014] The reaction system contains 10 to 50 mM potassium hydrogen phosphate-potassium dihydrogen phosphate buffer, 15 to 30 g / L co-immobilized enzyme, 800 to 1000 mM β-acetoxyl xyloside, 1.5 to 2.5 mM sodium formate, and 0.5 to 1 mM coenzyme NADP + 。

[0015] Further, in step 3), the co-immobilized enzyme is reused for the synthesis reaction of (β,S)-configuration boswellic acid.

[0016] Compared with the prior art, the present invention has at least the following beneficial effects:

[0017] (1) The present invention uses a co-immobilized carbonyl reductase LsADH and formate dehydrogenase PsFDH mut dual-enzyme system to catalyze the preparation of boswellic acid, and the catalytic reaction has high selectivity. The obtained product is pure (β,S)-configuration, making the product superior to the existing chemical synthesis process in terms of efficacy activity and safety. The co-immobilized carbonyl reductase LsADH and formate dehydrogenase PsFDH mutIt has good stability and can be reused, which is beneficial to continuous production and cost reduction.

[0018] (2) The catalytic reaction adopted in the present invention is carried out in a neutral environment at normal temperature and pressure, avoiding the high-temperature and high-pressure reaction conditions and alkaline environment in the chemical synthesis process, reducing the requirements for reaction equipment and process operations, and reducing equipment investment and energy consumption.

[0019] (3) The conversion rate of the catalytic reaction adopted in the present invention is >99%. The substrates β-acetoxyxylose and sodium formate used have high safety, and by mutating and modifying formate dehydrogenase, it can specifically catalyze the coenzyme NADP ＋ The recycling of, significantly improves the synthesis efficiency of the product (β,S)-boswellic acid. The by-product is carbon dioxide, which is non-toxic, harmless and easy to volatilize, and will not remain in the reaction system to affect the separation and purification of the product. Therefore, the method for preparing boswellic acid proposed by the present invention has significant advantages both in terms of cost and safety and environmental protection. Description of the Drawings

[0020] Figure 1 are two configurations of boswellic acid;

[0021] Figure 2 is the reaction route for the enzymatic preparation of (β,S)-configured boswellic acid in the present invention;

[0022] Figure 3 is the recombinant vector pET-28a(+)-Lsadh-Psfdh mut ;

[0023] Figure 4 is the SDS-PAGE diagram of the recombinant strain co-expressing carbonyl reductase LsADH and formate dehydrogenase PsFDH mut Among them: Lane 1 is the BL21 DE3 / pET-28a(+) empty vector control; Lane 2 is BL21 DE3 / pET-28a(+)-Lsadh-Psfdh mut ;

[0024] Figure 5 are the SDS-PAGE diagrams of carbonyl reductase LsADH and formate dehydrogenase PsFDH mut Among them: Lane 1 is carbonyl reductase LsADH; Lane 2 is formate dehydrogenase PsFDH mut . Detailed Embodiments

[0025] The present invention will be further described and explained below in conjunction with the specific embodiments. The described embodiments are only demonstrations of the present disclosure content and do not delimit the scope of limitation. Without conflict, the technical features of each embodiment in the present invention can be combined accordingly.

[0026] The culture media used in the embodiments of the present invention are as follows:

[0027] LB liquid medium: 5 g / L of OXOID yeast extract, 10 g / L of OXOID tryptone, 10 g / L of sodium chloride.

[0028] LB solid medium: 5 g / L of OXOID yeast extract, 10 g / L of OXOID tryptone, 10 g / L of sodium chloride, 15 g / L of agar.

[0029] TB liquid medium: 24 g / L of OXOID yeast extract, 12 g / L of OXOID tryptone, 4 mL / L of glycerol, 17 mM of potassium dihydrogen phosphate, 72 mM of dipotassium hydrogen phosphate.

[0030] 5×SDS-PAGE protein loading buffer: 1 mL of 1% bromophenol blue solution, 5 mL of 50% glycerol solution, 2 mL of 10% SDS solution, 0.6 mL of 1 M Tris-HCl solution (pH = 6.8), made up to 9.5 mL with deionized water, and 0.5 mL of β-mercaptoethanol was added before use.

[0031] Colony PCR reaction system: 10 μL of 2×Rapid Taq Master Mix, 0.4 μL each of the upstream primer and the downstream primer, 9.2 μL of ddH2O, an appropriate amount of DNA template, and the total volume is 20 μL.

[0032] Colony PCR reaction conditions: Pre-denaturation at 95°C for 10 min; denaturation at 95°C for 15 sec; annealing at 58°C for 15 sec; extension at 72°C (the extension time is calculated at 4 kb / min), 28 cycles; extension at 72°C for 2 min; stored at 4°C.

[0033] The high performance liquid chromatography detection method for β-acetoxyl xyloside and (β,S)-configuration boswellic acid used in the present invention is as follows:

[0034] Instrument: Shimadzu LC-20A high performance liquid chromatograph, and the detector is a refractive index detector.

[0035] Chromatographic column model: CAPCELL PAK ADME, 250×4.6 mm, 5 μm.

[0036] Method parameters: The mobile phase is 100% aqueous solution, the flow rate is 1 mL / min, the detection cell temperature is 40°C, the column temperature is 35°C, and the injection volume is 10 μL.

[0037] Method for establishing standard curve of (β,S)-pro-xylane: Weigh 0.1 g of (β,S)-pro-xylane standard (accurate to 0.0001 g), dissolve it with ultrapure water, and make up the volume to 50 mL to obtain the standard stock solution. Take 0.5 mL, 1 mL, 2 mL, 4 mL, and 8 mL of the standard stock solution respectively, make up the volume to 10 mL with ultrapure water to prepare standard sample solutions with concentrations of 0.1 mg / mL, 0.2 mg / mL, 0.4 mg / mL, 0.8 mg / mL, and 1.6 mg / mL. Filter through a 0.22-μm syringe filter, and perform liquid chromatography analysis according to the above conditions. Use the standard sample concentration as the abscissa and the peak area as the ordinate to draw the standard curve, calculate the regression equation, and the correlation coefficient should be > 0.99.

[0038] Method for establishing standard curve of β-acetonylxyloside: Weigh 0.1 g of β-acetonylxyloside standard (accurate to 0.0001 g), dissolve it with ultrapure water, and make up the volume to 50 mL to obtain the standard stock solution. Take 0.5 mL, 1 mL, 2 mL, 4 mL, and 8 mL of the standard stock solution respectively, make up the volume to 10 mL with ultrapure water to prepare standard sample solutions with concentrations of 0.1 mg / mL, 0.2 mg / mL, 0.4 mg / mL, 0.8 mg / mL, and 1.6 mg / mL. Filter through a 0.22-μm syringe filter, and perform liquid chromatography analysis according to the above conditions. Use the standard sample concentration as the abscissa and the peak area as the ordinate to draw the standard curve, calculate the regression equation, and the correlation coefficient should be > 0.99.

[0039] Example 1 Construction of recombinant strain co-expressing carbonyl reductase and formate dehydrogenase

[0040] The gene Lsadh encoding a carbonyl reductase derived from Leifsonia sp. S749 (GenBank accession number: AB213459) was optimized for E. coli codons, and a histidine tag sequence was added to the N-terminus and C-terminus of its corresponding amino acid sequence. The designed gene sequence is shown in SEQ ID No.1. The gene Psfdh encoding a formate dehydrogenase derived from Pseudomonas sp. 101 (GenBank accession number: P33160) was designed with 5-site mutations (A207G / D230Q / C264A / H388K / S389V) and optimized for E. coli codons, and a Strep tag was added to the N-terminus of its corresponding amino acid sequence, and an RBS sequence and a spacer sequence were added in front of the start codon of its corresponding amino acid sequence. The designed gene sequence is shown in SEQ ID No.2. SEQ ID No.1 and SEQ ID No.2 were delivered to a gene synthesis company for gene synthesis, and SEQ ID No.1 and SEQ ID No.2 were inserted between the 5070th and 5234th positions of the vector pET-28a(+) (SEQ ID No.1 is upstream of SEQ ID No.2) to obtain the recombinant vector pET-28a(+)-Lsadh-Psfdh mut (as Figure 3 shown).

[0041] 2 μL of the recombinant vector pET-28a(+)-Lsadh-Psfdh was taken mut and added to 200 μL of commercially available E. coli BL21(DE3) competent cells, gently mixed, and ice-bathed for 30 min. The ice-bathed competent cells were placed in a 42°C metal bath for heat shock for 90 s, quickly returned to the ice bath and allowed to stand for 3 min, and then 800 μL of LB liquid medium was added and cultured with shaking at 37°C and 100 rpm for 1 h. 100 μL of the bacterial solution was taken and spread on an LB solid medium plate supplemented with 50 μg / mL kanamycin, and cultured overnight at 37°C. The obtained positive transformants were verified by colony PCR using primers T7-F and T7-R, and the correctly verified transformants were prepared for glycerol bacteria preservation, and the recombinant strain BL21(DE3) / pET-28a(+)-Lsadh-Psfdh co-expressing the carbonyl reductase Lsadh and the formate dehydrogenase Psfdh was obtained mut mut was obtained.

[0042] Table 1 Names and sequences of primers used in Example 1

[0043]

[0044] Example 2 Co-expression of carbonyl reductase and formate dehydrogenase in recombinant strains​

[0045] The cryopreserved recombinant strain BL21(DE3) / pET-28a(+)-Lsadh-Psfdh mut was streaked on an LB solid medium plate supplemented with 50 μg / mL kanamycin and cultured inverted at 37 °C for 12 - 16 h. Well-grown single colonies on the plate were picked and inoculated into 5 mL of LB liquid medium supplemented with 50 μg / mL kanamycin, and cultured at 37 °C and 200 rpm for 7.5 h to prepare a seed solution. The seed solution was transferred to 100 mL of TB liquid medium at a transfer volume of 1% (V / V) and cultured at 37 °C and 200 rpm until OD 600 = 0.6, isopropyl-β-D-thiogalactoside (IPTG) with a final concentration of 0.2 mM was added, the culture temperature was changed to 28 °C, and the culture was continued for 16 h to obtain cells co-expressing carbonyl reductase LsADH and formate dehydrogenase PsFDH mut Another strain BL21(DE3) / pET-28a(+) was activated and cultured according to the above method conditions as an empty vector control.

[0046] 20 mL of the cultured recombinant strain and control strain bacterial solutions were each taken, centrifuged at 12000 rpm for 5 min, and the supernatant was discarded. The cells were washed once with pure water, then 10 mL of pure water was added to resuspend the cells, and the cells were ultrasonically broken in an ice-water bath (150 W, 15 min). The crude enzyme solution obtained after breaking was centrifuged at 4 °C and 12000 rpm for 20 min to remove insoluble cell debris and protein inclusion bodies. 40 μL of the supernatant was taken, 10 μL of 5× protein loading buffer was added, mixed well, and then placed in a 100 °C metal bath for 10 min. The expression of the target protein was detected by SDS-PAGE (5% stacking gel and 12% separating gel). It can be shown by Figure 4 that compared with the control strain, the carbonyl reductase LsADH (27.1 kDa) and formate dehydrogenase PsFDH mut (45.1 kDa) of the recombinant strain were both well solubly expressed.

[0047] Example 3 Purification of carbonyl reductase and formate dehydrogenase

[0048] The carbonyl reductase LsADH was purified by Ni-NTA affinity chromatography column. 100 mL of the culture solution from Example 2 was centrifuged at 4 °C and 10,000 rpm for 5 min to collect the bacterial cells, and the supernatant was discarded completely. 20 mL of buffer (50 mM Na2HPO4, 300 mM NaCl, pH = 8.0) was taken to resuspend the bacterial cells, and the cells were ultrasonically disrupted in an ice-water bath (150 W, 15 min). The crude enzyme solution obtained after disruption was centrifuged at 4 °C and 12,000 rpm for 20 min to remove insoluble cell debris and protein inclusion bodies, and the supernatant was filtered through a 0.22 μm aqueous microporous membrane. First, 5 column volumes were equilibrated with the buffer, and the sample was loaded after baseline equilibration. After loading, 10 column volumes were equilibrated with the washing buffer (50 mM Na2HPO4, 300 mM NaCl, 10 mM imidazole, pH = 8.0) until the breakthrough peak returned to the baseline. Finally, 10 column volumes were eluted with the elution buffer (50 mM Na2HPO4, 300 mM NaCl, 250 mM imidazole, pH = 8.0), and the elution peak was collected. As Figure 5 shown, the purity of the purified carbonyl reductase LsADH was detected by SDS-PAGE, and the protein concentration of the purified carbonyl reductase LsADH was determined by the Coomassie brilliant blue method to be 6.7 mg / mL.

[0049] Formate dehydrogenase PsFDH mut was purified by StrepTrap HP affinity chromatography column. 100 mL of the culture solution from Step 2 was centrifuged at 4 °C and 10,000 rpm for 5 min to collect the bacterial cells, and the supernatant was discarded completely. 20 mL of buffer (20 mM Na2HPO4·12H2O, 280 mM NaCl, 6 mM KCl, pH = 7.4) was taken to resuspend the bacterial cells, and the cells were ultrasonically disrupted in an ice-water bath (150 W, 15 min). The crude enzyme solution obtained after disruption was centrifuged at 4 °C and 12,000 rpm for 20 min to remove insoluble cell debris and protein inclusion bodies, and the supernatant was filtered through a 0.22 μm aqueous microporous membrane. First, 5 column volumes were equilibrated with the buffer, and the sample was loaded after baseline equilibration. After loading, 20 column volumes were continuously equilibrated with the buffer until the breakthrough peak returned to the baseline. Finally, 10 column volumes were eluted with the elution buffer (20 mM Na2HPO4·12H2O, 280 mM NaCl, 6 mM KCl, 2.5 mM d-biotin, pH = 7.4), and the elution peak was collected. The purification effect of the target protein was detected by SDS-PAGE, and the concentration of the target protein was determined by the Coomassie brilliant blue method. As Figure 5 shown, the purified formate dehydrogenase PsFDH was detected by SDS-PAGE mutThe purified formate dehydrogenase PsFDH has a relatively high purity, which is determined by the Coomassie brilliant blue method. mut The protein concentration is 9.5 mg / mL.

[0050] Example 4 Determination of the enzyme activities of carbonyl reductase and formate dehydrogenase

[0051] In this example, the enzyme activity is calculated according to the following formula:

[0052] Enzyme activity (U / mL) = (ΔA × V 总 × n) / (6.22 × d × V);

[0053] Where: ΔA is the difference in absorbance per minute within the initial reaction rate range (min -1 ), V 总 is the total reaction volume (mL), n is the dilution factor of the enzyme solution, 6.22 is the extinction coefficient of NADPH (L / (mmol·cm)), d is the optical path (1 cm), and V is the added volume of the enzyme solution (mL).

[0054] The specific enzyme activity (U / mg) is defined as the number of enzyme activity units per milligram of pure enzyme.

[0055] The enzyme activity of carbonyl reductase LsADH is determined by spectrophotometry. The enzyme activity unit (U) is defined as the amount of enzyme required to convert 1 μmol of NADPH per 1 min at 37°C. The reaction system is 1 mL, containing 750 μL of potassium hydrogen phosphate - potassium dihydrogen phosphate buffer (10 mmol / L, pH 7.0), 100 μL of 40 mmol / L β - acetoxyl xyloside, 100 μL of 2 mmol / L NADPH, and 50 μL of the enzyme solution of carbonyl reductase LsADH purified in Example 3 (the protein concentration in the reaction system is 0.1 mg / mL). The determination method is to incubate the reaction system at 37°C for 2 min and measure the change in absorbance at 340 nm using an ultraviolet spectrophotometer. The calculated enzyme activity of carbonyl reductase LsADH is 4.5 U / mL, and the specific enzyme activity is 0.67 U / mg.

[0056] The enzyme activity of formate dehydrogenase PsFDH is determined by spectrophotometry. mut The enzyme activity unit (U) is defined as the amount of enzyme required to convert 1 μmol of NADP + per 1 min at 37°C. The reaction system is 1 mL, containing 750 μL of potassium hydrogen phosphate - potassium dihydrogen phosphate buffer (10 mmol / L, pH 7.0), 100 μL of 40 mmol / L sodium formate, 100 μL of 2 mmol / L NADP + , and 50 μL of the formate dehydrogenase PsFDH purified in Example 3 mutThe enzyme solution. The measurement method is to incubate the reaction system at 37 °C for 2 min, and use an ultraviolet spectrophotometer to measure the change in absorbance at 340 nm. The formate dehydrogenase PsFDH mut has an enzyme activity of 5.2 U / mL and a specific enzyme activity of 0.55 U / mg.

[0057] Example 5 Co-immobilization of carbonyl reductase LsADH and formate dehydrogenase PsFDH mut of

[0058] Prepare a 3.0% sodium alginate solution and heat it to a completely dissolved state at 60 °C. After cooling to room temperature, add the carbonyl reductase LsADH and formate dehydrogenase PsFDH mut enzyme solution obtained in Example 3 (the ratio of enzyme activities is 1:1, and the sum of the final concentrations of the two enzymes is 4 mg / mL). Drop the above mixed enzyme solution into a 2% chitosan and 2% calcium chloride mixed solution with the same volume as the mixed enzyme solution using a syringe, stir at 25 °C to form gel beads, and then let the gel beads stand in a 2% calcium chloride solution for 30 min for curing. After curing, wash the gel beads 3 times with ultrapure water to remove the calcium chloride solution on the surface, and prepare the co-immobilized enzyme of carbonyl reductase LsADH and formate dehydrogenase PsFDH mut for storage at 4 °C for later use.

[0059] Example 6 Preparation of (β,S)-configuration boswellic acid by co-immobilized enzyme of carbonyl reductase LsADH and formate dehydrogenase PsFDH mut The catalytic reaction system is 500 mL, containing potassium hydrogen phosphate-potassium dihydrogen phosphate buffer (10 mM, pH 7.0), 20 g / L of the co-immobilized enzyme of carbonyl reductase LsADH and formate dehydrogenase PsFDH

[0060] obtained in Example 5, 1000 mM β-acetoxyxylose, 1.5 mM sodium formate, and 0.5 mM coenzyme NADP mut . The reaction conditions are a stirring speed of 400 rpm, a reaction temperature of 37 °C, and a reaction time of 8 h. Among them, the reaction route for preparing (β,S)-configuration boswellic acid by the enzyme-catalyzed reaction of the present invention is as + shown. Figure 2 shown.

[0061] Reusability of the co-immobilized enzyme of carbonyl reductase LsADH and formate dehydrogenase PsFDH mut After the reaction, wash the gel beads 3 times with ultrapure water. After the surface of the gel beads is dried, put them into a new catalytic reaction system again for the second reuse, and handle them in the same way during subsequent reuse.

[0062] The reaction was repeated 5 times using the co-immobilized enzyme. After the reaction, the yield and conversion rate of (β,S)-boswellic acid in the reaction solution were measured by high performance liquid chromatography as shown in Table 2.

[0063] Among them, the conversion rate (%) = (initial addition amount of β-acetoxyxylose - remaining amount of β-acetoxyxylose in the reaction system) / initial addition amount of β-acetoxyxylose × 100%.

[0064] It can be seen from the data in the table that the co-immobilized enzyme has good stability, can be reused, and can carry out multiple consecutive catalytic reactions.

[0065] Table 2 Yield and conversion rate of (β,S)-boswellic acid in the catalytic reaction of Example 6

[0066]

[0067] Comparative Example 1

[0068] The strain construction process was the same as that in Example 1, except that the 5-site mutation design was not carried out on the formate dehydrogenase-encoding gene Psfdh involved in this comparative example. The co-expression, co-immobilization and catalytic reaction of the carbonyl reductase and formate dehydrogenase were carried out according to the methods described in Examples 2 to 6. The reaction was repeated 5 times using the co-immobilized enzyme. After the catalytic reaction, the yield and conversion rate of (β,S)-boswellic acid in the reaction solution were detected by high performance liquid chromatography as shown in Table 3. Combining with Example 6, it can be shown that the 5-site mutation of the formate dehydrogenase-encoding gene Psfdh in the present invention effectively improves the synthesis efficiency of (β,S)-boswellic acid.

[0069] Table 3 Yield and conversion rate of (β,S)-boswellic acid in the catalytic reaction of Comparative Example 1

[0070]

[0071] The above-described embodiments only represent several embodiments of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the patent of the present invention. For those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention.

Claims

1. A method for preparing (β, S)-configuration boson by enzyme catalysis, characterized in that: The steps include: 1) Cultivate the engineered bacteria, break the cultured bacteria, separate and purify the carbonyl reductase LS ADH and formate dehydrogenase Ps FDH mut ; The engineering bacteria are constructed by the following method: S1: Design an improved carbonyl reductase encoding gene with a sequence as shown in SEQ ID No. 1; Design an improved formate dehydrogenase encoding gene with a sequence as shown in SEQ ID No. 2; The improved carbonyl reductase encoding gene and the improved formate dehydrogenase encoding gene were inserted between positions 5070 and 5234 of the vector pET-28a(+), and the improved carbonyl reductase encoding gene was located upstream of the improved formate dehydrogenase encoding gene to obtain the recombinant vector pET-28a(+)- Lsadh - PcqI mut ; S2: The recombinant vector pET-28a (+)- Lsadh - PcqI mut Heat shock transformation of E. coli BL21 (DE3) competent cells to obtain co-expressed carbonyl reductase Lsadh and formate dehydrogenase PcqI mut The recombinant strain BL21 (DE3) / pET-28a (+)- Lsadh - PcqI mut , i.e., the engineered bacteria; 2) Prepare sodium alginate solution and add the obtained carbonyl reductase LS ADH and formate dehydrogenase Ps FDH mut Enzyme solution, wherein the ratio of the enzymatic activities of carbonyl reductase to formate dehydrogenase is 1:1-1:2, and the sum of the final concentrations of the two enzymes is 2-5 mg / mL; The mixed enzyme solution is added dropwise to a mixed solution of chitosan and calcium chloride of equal volume to the mixed enzyme solution, stirred to form gel beads, and then the gel beads are allowed to stand and solidify to obtain carbonyl reductase LS ADH and formate dehydrogenase Ps FDH mut Co-immobilized enzymes; 3) using the reaction system containing the co-immobilized enzyme to prepare (β, S)-configuration boson, the reaction conditions are stirring speed 300-500 rpm, reaction temperature 35-40° C., and reaction time 6-8 h; The reaction system contains 10~50mM potassium dihydrogen phosphate-potassium dihydrogen phosphate buffer, 15~30g / L co-immobilized enzyme, 800~1000mM β-acetone xyloside, 1.5~2.5mM sodium formate, 0.5~1mM coenzyme NADP + .

2. The method according to claim 1, characterized in that The improved carbonyl reductase encoding gene is designed by: Leifsonia sp. S749-derived carbonyl reductase encoding gene Lsadh E. coli codon optimization was performed, and histidine tag sequences were added to the N-terminus and C-terminus of the corresponding amino acid sequence. Lsadh The GenBank accession number of the gene is AB213459.

3. The method according to claim 1, characterized in that The improved formate dehydrogenase encoding gene is designed by: Pseudomonas sp. 101-derived formate dehydrogenase encoding gene PcqI Mutation design of 5 sites and optimization of E. coli codons were performed, and a Strep tag was added to the N-terminus of the corresponding amino acid sequence, and an RBS sequence and a spacer sequence were added in front of the start codon of the corresponding amino acid sequence; wherein the mutation designs of the 5 sites were A207G, D230Q, C264A, H388K, S389V, respectively, PcqI Gene GenBank accession number: P33160.

4. The method according to claim 1, characterized in that: In the step 1), the engineered bacteria are cultured, including: The engineered bacteria BL21 (DE3) / pET-28a (+)- Lsadh - PcqI mut Streak on a LB solid medium plate supplemented with 50 μg / mL kanamycin, and invert and culture at 37°C for 12-16 hours; pick a single colony with good growth, inoculate 5 mL LB liquid medium supplemented with 50 μg / mL kanamycin, and culture at 37°C and 200 rpm for 7-8 hours to prepare seed solution; Transfer the seed solution to TB liquid medium at a volume transfer rate of 1-2% and culture until OD 600 = 0.6~0.8, add isopropyl-β-D-thiogalactoside with a final concentration of 0.1~0.2mM, change the culture temperature to 25~30℃, continue to culture for 12~16h, and obtain co-expressed carbonyl reductase LS ADH and formate dehydrogenase Ps FDH mut of cells.

5. The method according to claim 1, characterized in that In the step 1), carbonyl reductase LS ADH was purified by Ni-NTA affinity chromatography, and formate dehydrogenase Ps FDH mut Purification was performed by StrepTrap HP affinity chromatography.

6. The method according to claim 1, characterized in that The step 2) specifically comprises: preparing a sodium alginate solution with a concentration of 1.0-4.0%, heating it at 50-60°C until it is completely dissolved; after cooling it to room temperature, adding the obtained carbonyl reductase LS ADH and formate dehydrogenase Ps FDH mut enzyme solution; adding the mixed enzyme solution to a mixed solution of 2 wt % chitosan and 2 wt % calcium chloride of equal volume to the mixed enzyme solution, stirring at 20-30° C. to form gel beads, and then standing the gel beads in the 2 wt % calcium chloride solution for 30-40 min to solidify; after solidification, washing with ultrapure water to remove the calcium chloride solution on the surface, to obtain carbonyl reductase LS ADH and formate dehydrogenase Ps FDH mut Co-immobilized enzymes.

7. The method according to claim 1, characterized in that In the step 3), the co-immobilized enzyme is repeatedly used to carry out the synthesis reaction of (β, S)-configuration boson.

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