Method for preparing (beta, S)-configuration glassine through enzyme catalysis and engineering bacteria

Abstract

The invention provides a method for preparing a (beta, S)-configuration glassine through enzyme catalysis and an engineering bacterium, and belongs to the field of biosynthesis, the engineering bacterium takes Escherichia coli BL21 (DE3) as an original strain, and an improved carbonyl reductase coding gene shown as SEQ ID No.1 and an improved formate dehydrogenase coding gene shown as SEQ ID No.2 are introduced. The enzyme produced by the strain is used for preparing carbonyl reductase LsADH and formate dehydrogenase PsFDHmut co-immobilized enzyme to carry out enzyme catalytic synthesis of (beta, S) configuration bose, the catalytic reaction is high in selectivity and can be carried out in a normal-temperature and normal-pressure neutral environment, the co-immobilized enzyme can be repeatedly used, and continuous production and cost reduction are facilitated. The conversion rate of the adopted catalytic reaction is greater than 99%, the used substrates beta-acetone xyloside and sodium formate are high in safety, and the synthesis efficiency of the product (beta, S) bose is remarkably improved.

Description

Technical Field

[0001] The present invention belongs to the field of biosynthesis, and specifically relates to a method and an engineering bacterium for preparing (β, S)-configuration boson through enzyme catalysis. Background Art

[0002] Bosin is a functional raw material commonly used in cosmetics, and its INCI name is hydroxypropyl tetrahydropyrantriol. 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, and are critical for maintaining the firmness, uniformity, smoothness, elasticity and health of the skin. The external manifestations of skin aging are thinning, sagging, loss of elasticity and deepening of wrinkles. In the study of skin aging, it was found that both naturally aged skin and photoaged skin have different levels of glycosaminoglycans and proteoglycans. The use of cosmetics with bosin can promote epidermal renewal, strengthen skin structure, tighten skin, improve wrinkles, and increase skin luster and elasticity. Therefore, bosin is known as one of the "three anti-aging giants" in the skin care industry, and is also widely used in creams, essences and other products of big brands.

[0003] Bosin is a β-xylose derivative originally developed by L'Oreal. 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 carbon atom at position 7 in the aglycone (hydroxypropane) produces two different configurations, namely (β, S) and (β, R), two diastereoisomers (such as Figure 1As shown). Studies have shown 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 bosylamine is chemical synthesis, using xylose as the raw material, condensing with acetylacetone in an alkaline aqueous solution, and then reducing the keto carbonyl with sodium borohydride to obtain bosylamine. Bosylamine produced by chemical synthesis is a mixture of two configurations. The ratio of (β,S) configuration to (β,R) configuration is usually 3:7~5:5, and the acetylacetone used is moderately toxic. Its vapor can form an explosive mixture when mixed with air. CN 117510446 A discloses a method for preparing bosense, which uses D-xylose as a starting material, first reacts with acetylacetone to obtain acetonyl tetrahydropyrantriol, and then performs a carbonyl reduction reaction in the presence of a cheap catalyst to obtain hydroxypropyl pyrantriol with 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)-configuration bosense, but the enzyme used cannot be recycled and reused, which is not conducive to continuous production. Therefore, the development of a safe, environmentally friendly, and continuously operable (β, S)-configuration bosense production process is of great significance for the large-scale production and market application of bosense. Summary of the invention

[0004] In order to solve the problems in the prior art, the present invention proposes a method and an engineering bacterium for preparing (β, S)-configuration boson by enzyme catalysis.

[0005] The present invention first provides a method for constructing an engineering bacterium for preparing (β, S)-configuration boson by enzyme catalysis, which comprises:

[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 positions 5070 and 5234 of the vector pET-28a(+), and the improved carbonyl reductase encoding gene is located upstream of the improved formate dehydrogenase encoding gene, to obtain a recombinant vector pET-28a(+)-Lsadh-Psfdh mut ;

[0007] S2: The recombinant vector pET-28a (+)-Lsadh-Psfdh mut Heat shock transformation of Escherichia coli BL21 (DE3) competent cells to obtain co-expression of carbonyl reductase Lsadh and formate dehydrogenase Psfdh mut The recombinant strain BL21 (DE3) / pET-28a (+)-Lsadh-Psfdh mut.

[0008] The present invention also provides an engineered bacterium constructed by the method, and further provides the use of the engineered bacterium or its fermentation product in the preparation of (β, S)-configuration boson by enzyme catalysis.

[0009] The present invention also provides a method for preparing (β, S)-configuration boson by enzyme catalysis, which comprises the following steps:

[0010] 1) Cultivating the engineered bacteria, breaking the cultured bacteria, and separating and purifying the carbonyl reductase LsADH and formate dehydrogenase PsFDH mut ;

[0011] 2) Prepare sodium alginate solution and add the obtained carbonyl reductase LsADH and formate dehydrogenase PsFDH 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;

[0012] The mixed enzyme solution was 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 were allowed to stand and solidify to obtain carbonyl reductase LsADH and formate dehydrogenase PsFDH. mut Co-immobilized enzymes;

[0013] 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;

[0014] 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 + .

[0015] Furthermore, in the step 3), the co-immobilized enzyme is repeatedly used to carry out the synthesis reaction of (β, S)-configuration boson.

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

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

[0018] (2) The catalytic reaction adopted in the present invention is carried out in a neutral environment at room 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 operation, and reducing equipment investment and energy consumption.

[0019] (3) The catalytic reaction conversion rate used in the present invention is greater than 99%, the substrates β-acetone xyloside and sodium formate used are highly safe, and the formate dehydrogenase is mutated and modified to enable it to specifically catalyze the coenzyme NADP ＋ The cyclic regeneration significantly improves the synthesis efficiency of the product (β, S) bosonine, and the byproduct is non-toxic and harmless carbon dioxide that is easily volatile and will not remain in the reaction system to affect the separation and purification of the product. Therefore, the bosonine preparation method proposed in the present invention has significant advantages in terms of cost, safety and environmental protection. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 are two configurations of Bose;

[0021] Figure 2 The present invention provides a reaction route for preparing (β, S)-configuration bosonine catalyzed by the enzyme;

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

[0023] Figure 4 Co-expression of carbonyl reductase LsADH and formate dehydrogenase PsFDH in recombinant strains mut SDS-PAGE of the samples, where lane 1 is the BL21 DE3 / pET-28a (+) empty control; lane 2 is the BL21 DE3 / pET-28a (+)-Lsadh-Psfdh mut ;

[0024] Figure 5 Carbonyl reductase LsADH and formate dehydrogenase PsFDH mut SDS-PAGE diagram; wherein: lane 1 is carbonyl reductase LsADH; lane 2 is formate dehydrogenase PsFDH mut . DETAILED DESCRIPTION

[0025] The present invention is further described and illustrated below in conjunction with specific embodiments. The embodiments are merely exemplary of the present disclosure and do not define the scope of limitation. The technical features of each embodiment of the present invention may be combined accordingly without conflicting with each other.

[0026] The culture medium used in the embodiment of the present invention is as follows:

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

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

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

[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 1M Tris-HCl solution (pH=6.8), dilute to 9.5 mL with deionized water, and add 0.5 mL of β-mercaptoethanol before use.

[0031] Colony PCR reaction system: 2× Rapid Taq Master Mix 10 μL, upstream primer and downstream primer 0.4 μL each, ddH2O 9.2 μL, appropriate amount of DNA template, total volume 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 (extension time calculated at 4 kb / min), 28 cycles; extension at 72°C for 2 min; storage at 4°C.

[0033] The HPLC detection method of β-acetone xyloside and (β, S)-configuration boson used in the present invention is:

[0034] Instrument: Shimadzu LC-20A high performance liquid chromatograph, detector: differential refractometer.

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

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

[0037] Method for establishing the standard curve of (β, S)-configuration Bose-like: Weigh 0.1g (β, S)-configuration Bose-like standard (accurate to 0.0001g), dissolve it in ultrapure water, and dilute to 50mL as the standard stock solution. Take 0.5mL, 1mL, 2mL, 4mL, and 8mL of the standard stock solution respectively, dilute to 10mL with ultrapure water, and prepare standard sample solutions with concentrations of 0.1mg / mL, 0.2mg / mL, 0.4mg / mL, 0.8mg / mL, and 1.6mg / mL, filter through a 0.22μm needle filter, and perform liquid chromatography analysis according to the above conditions. Use the standard sample concentration as the horizontal axis and the peak area as the vertical axis to draw the standard curve, calculate the regression equation, and the correlation coefficient should be greater than 0.99.

[0038] Method for establishing the standard curve of β-acetone xyloside: Weigh 0.1g of β-acetone xyloside standard (accurate to 0.0001g), dissolve it in ultrapure water, and dilute to 50mL as the standard stock solution. Take 0.5mL, 1mL, 2mL, 4mL, and 8mL of the standard stock solution respectively, dilute to 10mL with ultrapure water, and prepare standard sample solutions with concentrations of 0.1mg / mL, 0.2mg / mL, 0.4mg / mL, 0.8mg / mL, and 1.6mg / mL, filter through a 0.22μm needle filter, and perform liquid chromatography analysis according to the above conditions. Use the standard sample concentration as the horizontal axis and the peak area as the vertical axis to draw the standard curve, calculate the regression equation, and the correlation coefficient should be greater than 0.99.

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

[0040] The carbonyl reductase encoding gene Lsadh (GenBank accession number: AB213459) from Leifsonia sp. S749 was codon-optimized in E. coli, and histidine tag sequences were 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 formate dehydrogenase encoding gene Psfdh (GenBank accession number: P33160) from Pseudomonas sp. 101 was subjected to 5 site mutation design (A207G / D230Q / C264A / H388K / S389V) and E. coli codon optimization, 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 before 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 positions 5070 and 5234 of the vector pET-28a(+) (SEQ ID No. 1 is located upstream of SEQ ID No. 2), to obtain the recombinant vector pET-28a(+)-Lsadh-Psfdh mut (like Figure 3 as shown).

[0041] Pipette 2 μL of the recombinant vector pET-28a (+)-Lsadh-Psfdh mut , add 200μL of commercial Escherichia coli BL21 (DE3) competent cells, mix gently, and place on ice for 30 minutes. Place the competent cells after the ice bath in a 42℃ metal bath for heat shock for 90s, quickly return to the ice bath and let stand for 3min, then add 800μL of LB liquid culture medium and place at 37℃, 100rpm shaking culture for 1h. Pipette 100μL of bacterial solution and spread on LB solid culture medium plates supplemented with 50μg / mL kanamycin, and place at 37℃ for overnight culture. Use primers T7-F and T7-R to perform colony PCR verification on the obtained positive transformants, verify the correct transformants to prepare glycerol bacteria for preservation, and obtain co-expression of carbonyl reductase Lsadh and formate dehydrogenase Psfdh mut The recombinant strain BL21 (DE3) / pET-28a (+)-Lsadh-Psfdh mut .

[0042] Table 1 Primer names and sequences used in Example 1

[0043]

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

[0045] The frozen recombinant strain BL21 (DE3) / pET-28a (+)-Lsadh-Psfdh mut Streak on a plate of LB solid culture medium supplemented with 50 μg / mL kanamycin and invert and culture at 37°C for 12-16 hours. Pick a single colony that grows well on the plate and inoculate it with 5 mL of LB liquid culture medium supplemented with 50 μg / mL kanamycin. Culture it at 37°C and 200 rpm for 7.5 hours to prepare the seed solution. Transfer the seed solution to 100 mL of TB liquid culture medium at a transfer volume of 1% (V / V) and culture it at 37°C and 200 rpm until the OD reaches 0. 600 =0.6, add isopropyl-β-D-thiogalactoside (IPTG) with a final concentration of 0.2mM, change the culture temperature to 28℃, and continue to culture for 16h to obtain co-expressed carbonyl reductase LsADH and formate dehydrogenase PsFDH mut Another strain BL21 (DE3) / pET-28a (+) was activated and cultured according to the above conditions as an empty load control.

[0046] Take 20 mL of the cultured recombinant strain and control strain respectively, centrifuge at 12000 rpm for 5 min, and discard the supernatant. Wash the cells once with pure water, add 10 mL of pure water to resuspend the bacteria, and ultrasonically disrupt the cells in an ice water bath (150W, 15 min). Centrifuge the crude enzyme solution obtained after disruption at 4°C and 12000 rpm for 20 min to remove insoluble cell debris and protein inclusion bodies. Take 40 μL of the clear solution, add 10 μL of 5× protein loading buffer, mix well, place in a 100°C metal bath for 10 min, and detect the expression of the target protein by SDS-PAGE (5% concentrating gel and 12% separation gel). Figure 4 It can be shown that compared with the control strain, the recombinant strain had higher expression of carbonyl reductase LsADH (27.1 kDa) and formate dehydrogenase PsFDH mut (45.1kDa) were expressed in a good soluble form.

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

[0048] Carbonyl reductase LsADH was purified by Ni-NTA affinity chromatography column. Take 100mL of culture solution of Example 2, centrifuge for 5min at 4°C and 10000rpm to collect bacteria, and discard the supernatant. Take 20mL buffer (50mM Na2HPO4, 300mMNaCl, pH=8.0), resuspend the bacteria, and ultrasonically disrupt the cells in an ice water bath (150W, 15min). The crude enzyme solution obtained after disruption was centrifuged for 20min at 4°C and 12000rpm to remove insoluble cell debris and protein inclusion bodies, and the supernatant was filtered with a 0.22μm water system microporous filter membrane. First, balance 5 column volumes with buffer, and load the sample after baseline balance. After loading, balance 10 column volumes with washing buffer (50mM Na2HPO4, 300mM NaCl, 10mM imidazole, pH=8.0) until the penetration peak returns to the baseline. Finally, elution was performed with elution buffer (50 mM Na2HPO4, 300 mM NaCl, 250 mM imidazole, pH = 8.0) for 10 column volumes and the elution peak was collected. Figure 5 As shown, SDS-PAGE detection showed that the purity of the purified carbonyl reductase LsADH was high, and the protein concentration of the purified carbonyl reductase LsADH was 6.7 mg / mL as determined by the Coomassie Brilliant Blue method.

[0049] Formate dehydrogenase PsFDH mut Purify by StrepTrap HP affinity chromatography column. Centrifuge 100mL of culture medium from step 2 at 4°C and 10,000rpm for 5min to collect the bacteria and discard the supernatant. Take 20mL buffer (20mMNa2HPO4·12H2O, 280mM NaCl, 6mM KCl, pH=7.4), resuspend the bacteria, and ultrasonically disrupt the cells in an ice water bath (150W, 15min). The crude enzyme solution obtained after disruption was centrifuged at 4°C and 12,000rpm for 20min to remove insoluble cell debris and protein inclusion bodies, and filter the supernatant with a 0.22μm water microporous filter membrane. First, balance 5 column volumes with buffer, and load the sample after baseline balance. After loading, continue to balance with buffer for 20 column volumes until the penetration peak returns to the baseline. Finally, elution was performed with elution buffer (20 mM Na2HPO4·12H2O, 280 mM NaCl, 6 mM KCl, 2.5 mM d-dethiobiotin, pH = 7.4) for 10 column volumes, 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. Figure 5 As shown, the purified formate dehydrogenase PsFDH was detected by SDS-PAGE. mutThe purity of the purified formate dehydrogenase PsFDH was determined by the Coomassie brilliant blue method. mut The protein concentration was 9.5 mg / mL.

[0050] Example 4 Determination of Enzyme Activities of Carbonyl Reductase and Formate Dehydrogenase

[0051] In this example, the enzyme activity was 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 length (1 cm), and V is the volume of enzyme solution added (mL).

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

[0055] The enzyme activity of carbonyl reductase LsADH was determined by spectrophotometry, and the enzyme activity unit (U) was defined as the amount of enzyme required to convert 1µmoL NADPH per 1min at 37°C. The reaction system was 1mL, containing 750μL of dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer (10mmol / L, pH7.0), 100μL of 40mmol / L β-acetone xyloside, 100μL of 2mmol / L NADPH, and 50μL of the enzyme solution of carbonyl reductase LsADH purified in Example 3 (the protein concentration in the reaction system was 0.1mg / mL). The determination method was to incubate the reaction system at 37°C for 2min and use an ultraviolet spectrophotometer to measure the change in the absorbance value at 340nm. The enzyme activity of carbonyl reductase LsADH was calculated to be 4.5U / mL, and the specific enzyme activity was 0.67U / mg.

[0056] Spectrophotometric determination of formate dehydrogenase PsFDH mut The enzyme activity unit (U) is defined as the conversion of 1 µmol NADP per minute at 37°C. + The required amount of enzyme. The reaction system is 1 mL, including 750 μL of potassium dihydrogen 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 + , 50 μL of the formate dehydrogenase PsFDH purified in Example 3 mutThe determination method is to incubate the reaction system at 37°C for 2 minutes and use an ultraviolet spectrophotometer to measure the change in absorbance at 340nm. The formate dehydrogenase PsFDH was calculated. mut The enzyme activity was 5.2U / mL and the specific enzyme activity was 0.55U / mg.

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

[0058] A 3.0% sodium alginate solution was prepared and heated at 60°C until it was completely dissolved. After cooling to room temperature, the carbonyl reductase LsADH and formate dehydrogenase PsFDH obtained in Example 3 were added. mut Enzyme solution (enzyme activity ratio is 1:1, and the sum of the final concentrations of the two enzymes is 4 mg / mL). The mixed enzyme solution was added dropwise to a mixed solution of 2% chitosan and 2% calcium chloride of equal volume to the mixed enzyme solution using a syringe, and stirred at 25°C to form gel beads, which were then allowed to stand in the 2% calcium chloride solution for 30 minutes for solidification. After solidification, the gel beads were washed 3 times with ultrapure water to remove the calcium chloride solution on the surface, and carbonyl reductase LsADH and formate dehydrogenase PsFDH were obtained. mut The immobilized enzymes were stored at 4°C until use.

[0059] Example 6 Carbonyl reductase LsADH and formate dehydrogenase PsFDH mut Preparation of (β,S)-configuration Bose-like protein catalyzed by co-immobilized enzyme

[0060] The catalytic reaction system was 500 mL, including dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer (10 mM, pH 7.0), 20 g / L carbonyl reductase LsADH and formate dehydrogenase PsFDH obtained in Example 5. mut Co-immobilized enzyme, 1000mM β-acetone xyloside, 1.5mM sodium formate, 0.5mM coenzyme NADP + The reaction conditions are stirring speed of 400 rpm, reaction temperature of 37°C, and reaction time of 8 h. The reaction route of the enzyme-catalyzed preparation of (β, S)-configuration boson is as follows: Figure 2 shown.

[0061] Carbonyl reductase LsADH and formate dehydrogenase PsFDH mut Reuse of co-immobilized enzyme: After the reaction is completed, the gel beads are washed 3 times with ultrapure water. After the surface of the gel beads is dried, they are put into a new catalytic reaction system for the second reuse. The same method is used for 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) bosone in the reaction solution were measured by high performance liquid chromatography as shown in Table 2.

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

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

[0065] Table 2 (β,S) Bose yield and conversion rate of the catalytic reaction of Example 6

[0066]

[0067] Comparative Example 1

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

[0069] Table 3 (β,S) Bose yield and conversion rate of the catalytic reaction of Comparative Example 1

[0070]

[0071] The above-mentioned embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. For ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A method for constructing an engineered bacterium for enzyme-catalyzed preparation of (β, S)-configuration boson. It is characterized in that include: 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 .

2. The construction method according to claim 1, It is 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 construction method according to claim 1, It is 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. An engineered bacterium constructed according to the method according to any one of claims 1 to 3.

5. Use of the engineered bacteria or its fermentation product according to claim 4 in the enzyme-catalyzed preparation of (β, S)-configuration boson.

6. A method for preparing (β, S)-configuration boson by enzyme catalysis. It is characterized in that The steps include: 1) Cultivating the engineered bacteria according to claim 4, breaking the cultured bacteria, and separating and purifying the carbonyl reductase LS ADH and formate dehydrogenase Ps FDH mut ; 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 + .

7. The method according to claim 6, It is 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.

8. The method according to claim 6, It is 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.

9. The method according to claim 6, It is 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.

10. The method according to claim 9, It is 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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