A mutant of Microbacterium dextranolyticum β-glucosidase, method and application thereof

Abstract

The present invention belongs to the technical field of enzyme engineering, and discloses a Microbacterium dextranolyticum β-glucosidase mutant, which is obtained by mutating the 329th position of the β-glucosidase of the amino acid sequence as shown in SEQ ID NO.1 from glutamic acid to glycine; or, mutating the 329th position of the β-glucosidase of the amino acid sequence as shown in SEQ ID NO.1 from glutamic acid to asparagine; or, mutating the 329th position of the β-glucosidase of the amino acid sequence as shown in SEQ ID NO.1 from glutamic acid to lysine, and mutating the 72nd position from arginine to asparagine. The present invention solves the technical problems existing in the current enzymatic conversion production of high-value natural products, and obtains a β-glucosidase mutant with improved enzyme activity by site-directed mutagenesis technology.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme engineering, in particular to a mutant of Microbacterium dextranolyticum beta-glucosidase, a method and an application thereof. Background Art

[0002] Microbacterium dextranolyticum β-glucosidase is a hydrolase from the GH3 family, catalyzing the hydrolysis and cleavage of glycosidic bonds. Its ability to efficiently hydrolyze glycosides can solve the problems of environmental pollution and high energy consumption caused by the industrial hydrolysis of glycosides. Its conservative hydrolysis mechanism can also hydrolyze specific types of glycosides specifically and specifically, and ensure that the molecular structure of the original aglycone is not changed. Therefore, β-glucosidase has great advantages in the process of transformation and synthesis of natural products.

[0003] Many natural products are widely used as additives in medicines and health foods because of their good physiological activity. There are many glucose units in the structure of natural products, such as ginsenoside Rb1, polydatin, astragaloside IV, etc. Some studies have shown that the hydrolysis of these glucose units can improve the solubility, bioavailability and even physiological activity of these natural products. However, these natural products lacking glucose units are currently mainly obtained by plant extraction or chemical hydrolysis, which has the disadvantages of low efficiency and high pollution. In addition, due to the non-specificity of the hydrolysis reaction, many other by-products are easily formed, making the separation and extraction work very difficult. In view of the above problems, the use of enzymatic methods to obtain these natural products has become a good way. Due to the strict requirements of industrial production, there is still an urgent need for more efficient and stable enzyme resources. After digging out suitable enzyme resources, the activity of the enzyme can be improved through site-directed mutagenesis technology. For example, CN107142254A mutated the amino acid at position 315 of the β-glucosidase from the thermophilic and acidophilic Alicyclobacillus sp.A4 from histidine to arginine, and obtained a β-glucosidase mutant with higher glucose tolerance. Under the same conditions, glucose promoted the enzyme activity of the mutant and the wild type by 212% and 163%, respectively. CN115011582B mutated the alanine at position 354 of the wild-type β-glucosidase bglHi from Humicola insolens to proline, which increased the activity by 40.28% compared with the wild type.

[0004] Currently, many existing β-glucosidases are mainly used to hydrolyze small molecular substrates such as salicin and polydatin, but there are few reports on the hydrolysis of natural products with larger molecular weights such as ginsenosides and astragaloside IV. Summary of the invention

[0005] The purpose of the present invention is to overcome the deficiencies in the prior art and provide a mutant of Microbacterium dextranolyticum β-glucosidase and a method and application thereof.

[0006] The technical solution adopted by the present invention to solve its technical problem is:

[0007] A Microbacterium dextranolyticum β-glucosidase mutant, wherein the β-glucosidase mutant is obtained by: mutating the 329th position of the β-glucosidase with the amino acid sequence as shown in SEQ ID NO.1 from glutamic acid to glycine;

[0008] Alternatively, the amino acid sequence of β-glucosidase as shown in SEQ ID NO.1 is obtained by mutating position 329 from glutamic acid to asparagine;

[0009] Alternatively, the amino acid sequence of the β-glucosidase shown in SEQ ID NO. 1 is obtained by mutating the 329th position from glutamic acid to lysine and the 72nd position from arginine to asparagine.

[0010] A gene encoding a Microbacterium dextranolyticum β-glucosidase mutant as described above.

[0011] The recombinant plasmid carrying the gene as described above. Further, the vector plasmid used is pETDuet-1.

[0012] A microbial cell carrying the gene as described above or the recombinant plasmid as described in claim 3. Furthermore, the expression strain used is Escherichia coli BL21 (DE3).

[0013] A method for preparing a Microbacterium dextranolyticum β-glucosidase mutant enzyme liquid using the above-mentioned microbial cells, wherein the recombinant cells are inoculated into a culture medium for culturing, an inducer is added and the culturing is continued to obtain a bacterial liquid containing the β-glucosidase mutant; the bacterial liquid is centrifuged to collect the bacterial cells, and after ultrasonic disruption, the solution obtained by the disruption is centrifuged to obtain a supernatant to obtain the Microbacterium dextranolyticum β-glucosidase mutant enzyme liquid.

[0014] A method for preparing ginsenoside CK using the mutant as described above, the method comprising adding a Microbacterium dextranolyticum β-glucosidase mutant enzyme solution to a reaction system containing ginsenoside Rb1 to react to obtain a reaction solution, and the reaction solution is separated to obtain the prepared ginsenoside CK;

[0015] The volume of the mutant enzyme solution added to the reaction system is 30-50% of the volume of the reaction system, the concentration of the substrate ginsenoside Rb1 in the reaction system is 5-10 g / L, the reaction pH is 6.5-7.5, and the temperature is 40-50°C.

[0016] A method for preparing cycloastragenol-β-D-xyloside using the mutant as described above, characterized in that: the method comprises adding the Microbacterium dextranolyticum β-glucosidase mutant to a reaction system containing astragaloside IV to react to obtain a reaction solution, and the reaction solution is separated to obtain the prepared ginsenoside CK;

[0017] The volume of the mutant enzyme solution added to the reaction system is 30-50% of the volume of the reaction system, the concentration of the substrate ginsenoside Rb1 in the reaction system is 5-10 g / L, the reaction pH is 6.5-7.5, and the temperature is 40-50°C.

[0018] A method for preparing resveratrol using the mutant as described above, the method comprising adding the Microbacterium dextranolyticum β-glucosidase mutant to a reaction system containing ginsenoside Rb1 to react to obtain a reaction solution, and the reaction solution can be separated to obtain the prepared polydatin;

[0019] The volume of the mutant enzyme solution added to the reaction system is 30-50% of the volume of the reaction system, the concentration of the substrate ginsenoside Rb1 in the reaction system is 5-10 g / L, the reaction pH is 6.5-7.5, and the temperature is 40-50°C.

[0020] Use of the mutant as described above in the preparation of ginsenoside CK and / or ginsenoside 75 and / or resveratrol and / or cycloastragenol-β-D-xyloside.

[0021] A method for converting polydatin into resveratrol using the mutant as described above, wherein the obtained β-glucosidase mutant is added to a reaction system containing polydatin for reaction, and the reaction solution obtained after the reaction is completed is extracted to obtain resveratrol;

[0022] Wherein, in the reaction system, the addition amount of the β-glucosidase mutant is 10-100 U / mL;

[0023] Alternatively, the reaction system contains a β-glucosidase mutant, polydatin and an appropriate buffer system;

[0024] Alternatively, in the reaction system, the buffer used is a phosphate buffer with a concentration of 0.02-0.1 M;

[0025] Alternatively, the reaction is carried out at 30-40°C and pH 6-7.

[0026] The advantages and positive effects achieved by the present invention are:

[0027] 1. The β-glucosidase from Microbacterium dextranolyticum obtained by the present invention is obtained by site-directed mutagenesis technology, and the β-glucosidase mutant is obtained, and heterologous expression is performed in the Escherichia coli protein expression system. Under the same reaction conditions, the β-glucosidase mutant E329K / R72N provided by the present invention has an ability to specifically hydrolyze polydatin and convert it into resveratrol by about 25%. The ability of the mutant E329N to hydrolyze ginsenoside Rb1 to obtain ginsenoside CK and ginsenoside 75 is improved. The ability of the mutant E329G to specifically hydrolyze astragaloside Ⅳ to convert it into cycloastragenol-6-O-xyloside is increased by about 20%. The mutant of the present invention has a high application value in the fields of medical health, food health care and biosynthesis.

[0028] 2. The hydrolysis activity of the β-glucosidase mutants E329G, E329N, and E329K / R72N obtained by site-directed mutagenesis is improved, and the hydrolysis activity is 3.90 times, 3.71 times, and 15.06 times that of the wild type, respectively. Under the optimal conditions, the yield of cycloastragenol-β-D-xyloside prepared by E329G is 99.8%, which is 1.2 times that of the wild type, the yield of ginsenoside CK and ginsenoside 75 prepared by E329N is 40.8% and 59.2%, respectively, which are 1.1 times and 3.2 times that of the wild type, respectively, and the yield of resveratrol prepared by E329K / R72N is 99.7%, which is 1.25 times that of the wild type, and has good potential for industrial application.

[0029] 3. The present invention solves the technical problems existing in the current enzymatic conversion production of high-value natural products, and obtains a β-glucosidase mutant with improved enzyme activity through site-directed mutagenesis technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1The figure is a full plasmid PCR amplification electrophoresis diagram of the construction of the β-glucosidase gene mutant from Microbacterium dextranolyticum in the embodiment of the present invention; wherein: M is DNAMarker, 1 is the amplification result of the E329K mutant, 2 is the amplification result of the E329G mutant, 3 is the amplification result of the E329N mutant, and 4 is the amplification result diagram of the E329K / R72N mutant;

[0031] Figure 2 The figure is a SDS-PAGE analysis result diagram of the wild type and mutants of β-glucosidase after purification in the embodiment of the present invention; wherein, M is DNA Marker, 1 is the wild type, 2 is the purification result of mutant E329K / R72N, 3 is the amplification result of mutant E329G, and 4 is the purification result diagram of mutant E329N;

[0032] Figure 3 This is a liquid chromatography detection result diagram of polydatin conversion of the mutant E329K / R72N in the embodiment of the present invention;

[0033] Figure 4 This is a liquid chromatography test result diagram of the mutant E329N in the embodiment of the present invention transforming ginsenoside Rb1;

[0034] Figure 5 This is a liquid chromatography detection result diagram of the conversion of astragaloside IV by the mutant E329G in the example of the present invention. DETAILED DESCRIPTION

[0035] The present invention will be further described below in conjunction with the embodiments. The following embodiments are descriptive rather than restrictive, and the protection scope of the present invention cannot be limited by the following embodiments.

[0036] The various experimental operations involved in the specific embodiments are all routine techniques in the art. For parts not specially annotated in this document, ordinary technicians in the art can implement them by referring to various commonly used reference books, scientific and technological literature or related instructions, manuals, etc. before the filing date of this invention.

[0037] A Microbacterium dextranolyticum β-glucosidase mutant, wherein the β-glucosidase mutant is obtained by: mutating the 329th position of the β-glucosidase with the amino acid sequence as shown in SEQ ID NO.1 from glutamic acid to glycine;

[0038] Alternatively, the amino acid sequence of β-glucosidase as shown in SEQ ID NO.1 is obtained by mutating position 329 from glutamic acid to asparagine;

[0039] Alternatively, the amino acid sequence of the β-glucosidase shown in SEQ ID NO. 1 is obtained by mutating the 329th position from glutamic acid to lysine and the 72nd position from arginine to asparagine.

[0040] A gene encoding a Microbacterium dextranolyticum β-glucosidase mutant as described above.

[0041] The recombinant plasmid carrying the gene as described above. Further, the vector plasmid used is pETDuet-1.

[0042] A microbial cell carrying the gene as described above or the recombinant plasmid as described in claim 3. Furthermore, the expression strain used is Escherichia coli BL21 (DE3).

[0043] A method for preparing a Microbacterium dextranolyticum β-glucosidase mutant enzyme liquid using the above-mentioned microbial cells, wherein the recombinant cells are inoculated into a culture medium for culturing, an inducer is added and the culturing is continued to obtain a bacterial liquid containing the β-glucosidase mutant; the bacterial liquid is centrifuged to collect the bacterial cells, and after ultrasonic disruption, the solution obtained by the disruption is centrifuged to obtain a supernatant to obtain the Microbacterium dextranolyticum β-glucosidase mutant enzyme liquid.

[0044] A method for preparing ginsenoside CK using the mutant as described above, the method comprising adding a Microbacterium dextranolyticum β-glucosidase mutant enzyme solution to a reaction system containing ginsenoside Rb1 to react to obtain a reaction solution, and the reaction solution is separated to obtain the prepared ginsenoside CK;

[0045] The volume of the mutant enzyme solution added to the reaction system is 30-50% of the volume of the reaction system, the concentration of the substrate ginsenoside Rb1 in the reaction system is 5-10 g / L, the reaction pH is 6.5-7.5, and the temperature is 40-50°C.

[0046] A method for preparing cycloastragenol-β-D-xyloside using the mutant as described above, characterized in that: the method comprises adding the Microbacterium dextranolyticum β-glucosidase mutant to a reaction system containing astragaloside IV to react to obtain a reaction solution, and the reaction solution is separated to obtain the prepared ginsenoside CK;

[0047] The volume of the mutant enzyme solution added to the reaction system is 30-50% of the volume of the reaction system, the concentration of the substrate ginsenoside Rb1 in the reaction system is 5-10 g / L, the reaction pH is 6.5-7.5, and the temperature is 40-50°C.

[0048] A method for preparing resveratrol using the mutant as described above, the method comprising adding the Microbacterium dextranolyticum β-glucosidase mutant to a reaction system containing ginsenoside Rb1 to react to obtain a reaction solution, and the reaction solution can be separated to obtain the prepared polydatin;

[0049] The volume of the mutant enzyme solution added to the reaction system is 30-50% of the volume of the reaction system, the concentration of the substrate ginsenoside Rb1 in the reaction system is 5-10 g / L, the reaction pH is 6.5-7.5, and the temperature is 40-50°C.

[0050] Use of the mutant as described above in the preparation of ginsenoside CK and / or ginsenoside 75 and / or resveratrol and / or cycloastragenol-β-D-xyloside.

[0051] A method for converting polydatin into resveratrol using the mutant as described above, wherein the obtained β-glucosidase mutant is added to a reaction system containing polydatin for reaction, and the reaction solution obtained after the reaction is completed is extracted to obtain resveratrol;

[0052] Wherein, in the reaction system, the addition amount of the β-glucosidase mutant is 10-100 U / mL;

[0053] Alternatively, the reaction system contains a β-glucosidase mutant, polydatin and an appropriate buffer system;

[0054] Alternatively, in the reaction system, the buffer used is a phosphate buffer with a concentration of 0.02-0.1 M;

[0055] Alternatively, the reaction is carried out at 30-40°C and pH 6-7.

[0056] Specifically, the relevant preparation and detection are as follows:

[0057] The E. coli BL21 (DE3) involved in the following examples was purchased from Qingke Biotechnology; the pETDuet-1 plasmid involved in the following examples was purchased from GenScript Corporation; and the natural products involved in the following examples were all purchased from Shanghai Yuanye Company.

[0058] The culture medium involved in the following examples is as follows:

[0059] LB medium: 5.0 g / L yeast extract, 10 g / L tryptone, 10 g / L sodium chloride; add 15-20 g / L agar to solid culture medium.

[0060] The enzyme activity and liquid phase detection methods in the following examples are as follows:

[0061] The β-glucosidase and mutant enzyme activity detection method used in the present invention is mainly based on the determination of the concentration of the substrate generated by enzymatic hydrolysis. The synthetic substrate p-nitrophenyl-β-D-pyranoglucoside (pNPG) is used as the substrate, and the product p-nitrophenol (pNP) produced by hydrolysis is colored under alkaline conditions, and the content of the substance at 405nm is determined. The specific detection method is as follows: take 10ul of appropriately diluted enzyme solution and add it to 90ul of the reaction system preheated at 37°C, so that the system contains 2mM pNPG, 100mM phosphate buffer, pH 7.0. After mixing, react for 1min, and add 100ul 2M sodium carbonate solution to terminate the reaction. Using the boiled inactivated enzyme solution as a control, measure the absorbance at OD405nm, and substitute it into the standard curve to calculate the pNP concentration in the system.

[0062] Drawing of pNP standard curve: Prepare 8-10 pNP systems containing 2-10 μM, use sterile water as blank, measure the absorbance at OD405nm, draw a linear regression curve of the relationship between absorbance and concentration, and calculate R 2 Value, up to R 2 Values ​​are between 0.99 and 1.

[0063] Calculation formula for β-glucosidase and mutant enzyme activity: Enzyme activity S = C × V1 × n / 1000 × t × V2

[0064] Wherein, C is the concentration of pNP (μM); V1 is the volume of the reaction solution (mL); V2 is the volume of the enzyme solution (mL); n is the dilution factor of the enzyme solution; and t is the reaction time (min).

[0065] Definition of enzyme activity: The amount of enzyme required to hydrolyze 1 μmol of p-nitrobenzene (pNP) per minute at 37°C is defined as 1 enzyme activity unit (U).

[0066] Liquid chromatography detection conditions of polydatin and its hydrolysis products: Shimadzu LC40 high performance liquid chromatograph was used for analysis, the chromatographic column was Agilent C18 column (4.6×250mm, 5μm), the detector was PDA detector, the detection wavelength was 306nm, the column temperature was set to 40℃, the flow rate was 1mL / min, and the gradient elution program of mobile phase A (water) and B (acetonitrile) was as follows: 0min, 72:28; 7min, 28:72; 9min, 28:72; 20min, 72:28.

[0067] Liquid chromatography detection conditions of ginsenoside Rb1 and its hydrolyzate: Shimadzu LC40 high performance liquid chromatograph was used for analysis, the chromatographic column was Agilent C18 column (4.6×250mm, 5μm), the detector was PDA detector, the detection wavelength was 203nm, the column temperature was set to 40℃, the flow rate was 0.8mL / min, and the gradient elution program of mobile phase A (water) and B (acetonitrile) was as follows: 0min, 70:30; 5min, 70:30; 15min, 43:57; 25min, 30:70; 30min, 30:70; 45min; 70:30. Liquid chromatography detection conditions of astragaloside IV (ASI) and its hydrolysis product: Shimadzu LC40 high performance liquid chromatograph was used for analysis, the chromatographic column was an Agilent C18 chromatographic column (4.6×250 mm, 5 μm), the detector was an ELSD detector, the column temperature was set to 40°C, the flow rate was 1 mL / min, and the mobile phase was 75% methanol.

[0068] The conversion rate is calculated as follows: (C1 / C1+C2+…+Cn)×100%, where C1 is the product concentration (mM), and C2,…,Cn are the concentrations of the remaining components (mM).

[0069] Example 1: Expression of β-glucosidase BgMd from Microbacterium dextranolyticum

[0070] The sequence Genebank accession number WP_204962958.1 is shown in the sequence SEQ ID NO.1. After optimizing the codons of Escherichia coli, it was synthesized by GenScript and constructed between the BamH I and Kpn I restriction sites of the expression vector pETDuet-1 to obtain the recombinant plasmid pETDuet-BgMd containing β-glucosidase, as shown in SEQ ID NO.2.

[0071] The obtained recombinant plasmid pETDuet-BgMd containing β-glucosidase was transformed into BL21 (DE3). The specific steps were as follows: the recombinant plasmid Escherichia coli pETDuet-BgMd was mixed with competent cells of Escherichia coli BL21 (DE3) on ice, and after an ice bath for 30 minutes, heat-shocked in a 42°C water bath for 1 minute, followed by an ice bath for 2-3 minutes, and 1 mL of LB medium was added to recover at 37°C for 30 minutes to 1 hour. The cells were then collected by centrifugation, and the excess supernatant was removed and spread on an LB solid culture medium plate containing 100 μg / mL ampicillin, and placed in a 37°C incubator for inversion culture for 12 hours. A single colony was obtained and verified to be recombinant Escherichia coli.

[0072] The obtained recombinant E. coli was inoculated into LB liquid medium containing 100 μg / mL ampicillin and cultured overnight at 37°C and 220 rpm in a shaker for strain preservation, and then transferred to fresh LB medium containing 100 μg / mL ampicillin at 1% (v / v), cultured to OD600 of 0.6-0.8, and isopropyl-β-D-thiogalactoside was added to a final concentration of 1 mmol / L, and placed in a shaker at 16°C and 160 rpm for low-temperature induction. After the induction, the cells were collected by centrifugation, resuspended with an appropriate amount of phosphate buffer, and centrifuged after ultrasonic disruption to obtain the supernatant, which was the crude enzyme solution of the wild type.

[0073] Example 2: Construction of mutants

[0074] According to the gene sequence of β-glucosidase shown in nucleotide sequence SEQ ID NO.2, mutation primers of E329K, E329G, E329N and R72N were designed respectively, and site-directed mutagenesis of β-glucosidase BgMd was performed. The specific steps are as follows:

[0075] Using the whole plasmid PCR technology, the above-mentioned recombinant plasmid pETDuet-BgMd carrying the encoding β-glucosidase BgMd was used as a template, and the following mutant primers were added and the corresponding PCR system was configured for amplification.

[0076] The site-directed mutagenesis primers for introducing the E329K mutation are:

[0077] Forward primer: 5'-CCGACTGG AAA TTGGTCAACGATAATCATGTTGGTGATC-3'(SEQ ID NO.3)

[0078] Reverse primer: 5'-TTGACCAA TTT CCAGTCGGGTGACGACCACG-3'(SEQ ID NO.4)

[0079] The site-directed mutagenesis primers for introducing the E329G mutation were:

[0080] Forward primer: 5'-CCGACTGG GGT TTGGTCAACGATAATCATGTTGGTGATC-3'(SEQ ID NO.5)

[0081] Reverse primer: 5'-TTGACCAA ACC CCAGTCGGGTGACGACCACG-3'(SEQ ID NO.6)

[0082] The site-directed mutagenesis primers for introducing the E329N mutation are:

[0083] Forward primer: 5'-CCGACTGG AAT TTGGTCAACGATAATCATGTTGGTGATC-3'(SEQ ID NO.7)

[0084] Reverse primer: 5'-TTGACCAA ATT CCAGTCGGGTGACGACCACG-3'(SEQ ID NO.8)

[0085] The configured PCR system is: 2×PhantaFlash Master Mix 15ul, forward and reverse primers (10mM) 1.2ul each, recombinant plasmid template (10ng / ul) 1ul, and dd water to 30ul.

[0086] The PCR amplification program is as follows: the first step is pre-denaturation at 98°C for 30s; then enter 28-35 cycles: denaturation at 98°C for 10s, annealing at 60°C for 5s, extension at 72°C for 1min 50s; and finally extension at 72°C for 1min.

[0087] Take 2ul of the amplified product and perform electrophoresis detection. After detecting the correct PCR product, add 1ul of DpnI enzyme and digest the template in a 37°C water bath for 2-4h. After digestion, the product is recovered by a product recovery kit. The recovered product is transformed into Escherichia coli BL21 (DE3), and the positive colonies on the plate are picked for sequencing verification. The verified correct colonies are inoculated into LB liquid culture medium containing 100μg / mL ampicillin and placed at 37°C, 220rpm constant temperature shaker for 12h and cultured for strain preservation, and induced expression as in Example 1 above.

[0088] The double-point mutation is based on the correct single-point mutation. The plasmid is extracted using a plasmid extraction kit, the mutant plasmid is used as a template, the following mutation primers are added and the corresponding PCR system is configured for amplification, and the subsequent steps are carried out as described above.

[0089] The site-directed mutagenesis primers for introducing the R72N mutation are:

[0090] Forward primer: 5'-TGCTGGAAGCGCCAGGT AAT ATCAGCAA-3' (SEQ ID NO.9)

[0091] Reverse primer: 5'-GTGGTCGGGCTTTTGCTGAT ATT ACCTGGCG-3' (SEQ ID NO. 10)

[0092] The amino acid sequences of the obtained mutants E329K / R72N, E329G and E329N are shown in the sequence listing SEQ ID NO.11, SEQ ID NO.12 and SEQ ID NO.13.

[0093] like Figure 1 As shown, the amplified product was detected by electrophoresis, and the band was single and bright, with a size between 5000bp and 8000bp, which was basically consistent with the theoretical size of the recombinant plasmid 7042bp. After subsequent verification, it can be said that the mutant was successfully constructed.

[0094] Example 3: Purification of wild-type and mutant β-glucosidase

[0095] The obtained crude enzyme solution was purified using a Ni ion affinity column. The purification process was as follows: first, 6 column volumes of buffer A (50mM phosphate buffer, pH7.4, 500mM NaCl, 20mM imidazole) were used to balance the column, and then the crude enzyme solution was filtered through a microporous filter, and 1-2 column volumes of crude enzyme solution were added for adsorption. 10-20 column volumes of buffer A were used for washing, and then 5 column volumes of buffer B (50mM phosphate buffer, pH7.4, 500mM NaCl, 500mM imidazole) were used for elution and the eluate was collected to obtain the purified wild-type and mutant β-glucosidase. 10ul of the purified enzyme was added to 2ul of 6× protein loading buffer, boiled at 100°C for 3-5min, and analyzed by SDS-PAGE electrophoresis.

[0096] like Figure 2 As shown, the pure enzyme solution obtained after purification has a single, bright band with a size between 50 kDa and 70 kDa, which is basically consistent with the molecular weight of 67.55 kDa calculated based on the amino acid sequence, indicating that the wild-type and mutant β-glucosidase are well expressed in Escherichia coli.

[0097] The enzyme activities of the wild type and mutants were measured and the results are shown in the following table.

[0098]

[0099] Compared with the mutant enzyme reported in the authorized patent CN 115232802 B, which has a specific activity significantly increased by 1.5 times or more than that of the wild enzyme, the mutant obtained in this example has an activity increased by more than 3 times compared with the wild type, and the activity of the double-point mutant is increased by more than 15 times, with significant effects.

[0100] Example 4: Application of wild-type and mutant β-glucosidase to catalyze the transformation of natural products

[0101] Transformation of Polydatin:

[0102] The wild-type and mutant enzyme solutions obtained in Examples 1 and 2 were added to the polydatin conversion system, which contained 2 g / L polydatin, 0.1 M phosphate buffer pH 7.0, and 40% (v%, volume concentration) enzyme solution. After mixing, the mixture was converted in a constant temperature shaker at 37°C and 200 rpm for 12 h. After the reaction was completed, an equal volume of methanol was added to inactivate the enzyme. The supernatant was centrifuged and passed through an organic microporous filter membrane for detection by liquid chromatography.

[0103] like Figure 3 As shown, the conversion rate of the wild type to 2g / L polydatin in 12h is 45.3%, while the conversion rate of the mutant E329K / R72N is the highest, with a conversion rate of 99.7% for 2g / L polydatin in 12h, which is about 25% higher than that of the wild type. Compared with the patent authorization number CN 115011582 B reporting that the conversion rate of polydatin increased from about 15% to 99.7%, it has obvious advantages.

[0104] Transformation of ginsenoside Rb1:

[0105] The crude enzyme solutions of the wild type and mutant obtained in Examples 1 and 2 were added to the conversion system of ginsenoside Rb1, which contained ginsenoside Rb1 2g / L, 0.1M phosphate buffer pH 7.0, and 40% (v%, volume concentration) of crude enzyme solution. After mixing, the mixture was converted in a constant temperature shaker at 37°C and 200rpm for 12h. After the reaction was completed, an equal volume of methanol was added to inactivate the enzyme. The supernatant was centrifuged and passed through an organic microporous filter membrane for detection by liquid chromatography.

[0106] like Figure 4 As shown, the conversion rate of the wild type to CK was 36.6% for 2g / L ginsenoside Rb1 in 12h, and 14.2% for ginsenoside 75 (Gyp75), while the mutant E329N had the highest conversion rate, with a conversion rate of 40.8% for 2g / L ginsenoside Rb1 in 12h, and a conversion rate of 59.2% for CK, which were increased by about 11% and 317% respectively compared with the wild type. Compared with the results reported in Patent Publication No. CN 117210441 A, this example can obtain more rare and valuable ginsenoside CK.

[0107] Conversion of Astragaloside IV:

[0108] The crude enzyme solutions of the wild type and mutant obtained in Examples 1 and 2 were added to the astragaloside IV conversion system, which contained 2 g / L astragaloside IV, 0.1 M phosphate buffer pH 7.0, and 40% (v%, volume concentration) of crude enzyme solution. After mixing, the mixture was converted in a constant temperature shaker at 37°C and 200 rpm for 12 h. After the reaction, an equal volume of water-saturated n-butanol was added to inactivate the enzyme and extract it. The supernatant was taken by centrifugation and the extraction was repeated once. After the collected organic phase was dried, a certain volume of methanol was added to dissolve it, the supernatant was taken and dd water was added to a volume ratio of methanol to 75%, and the mixture was passed through an organic microporous filter membrane and detected by liquid chromatography.

[0109] like Figure 5 As shown, the conversion rate of the wild type to 2 g / L astragaloside IV in 12 h was 83.1%, while the mutant E329G had the highest conversion rate, with the conversion rate of 2 g / L astragaloside IV to cycloastragenol-β-D-xyloside (CMX) being 99.8%, which was about 20% higher than that of the wild type.

[0110] The sequences used in the present invention are as follows:

[0111] SEQ ID NO.1 Protein sequence BgMd amino acid sequence

[0112] MTPHLSTAADGTSYRDLNGNGVMDPYEDPRLSPEERADDLVSRMSLEEKCGLMFQTVIEVGDDGELLEAPGRISKSPTTEVVVGKNLSHFNVHAIRTARQAAVWNNNLQALAERTPHGVPVTISTDPRHAFVENTGVGFAAGPFSQWPEGLGLAAIDDVETVREFADVARREYRAVGIRAALHPQIDLATEPRWGRQAQTLGQDAARVAEFTAAYLQGFQGDELGPDSVACTTKHFPGGGPQKDGEDSHFPYGREQVYPGGMFEYHLEPFREAIRRGTAAMMPYYGMPVGLERGGEKIEEVGFGYNRQIVTDLLRGELGYDGVVVTDWELVNDNHVGDQVLPARAWGVEELTPAERMEKILDAGADQFGGEECVDLLLDLVRSGRIGEERIDASARRLLLVKFRLGLFDDPYVDPDEAERIVGNADFRAQGERAQARALTVLVNREDASGAPTLPLRPVGRVYVEGFRADEAAELGVVVDDPADADLALVRLGAPFDPRDDLFLEAWFHQGSLEFPPGRVYRMRRIAEQCPLVLVANLDRAAVLTPFAEFAAAIAADFGSSGAAVLDALTGRIAPEGRLPVELPRSMDAVRASREDVPSDTEDPLFPVHFGLSLTTARA

[0113] Nucleotide sequence of recombinant plasmid pETDuet - BgMd, SEQ ID NO.2

[0114]

[0115] SEQ ID NO.3BgMd nucleotide sequence

[0116]

[0117] SEQ ID NO.4E329K-F Primer sequence

[0118] CCGACTGGAAATTGGTCAACGATAATCATGTTGGTGATC

[0119] SEQ ID NO.5E329K-R Primer sequence

[0120] TTGACCAATTTCCAGTCGGTGACGACCACG

[0121] SEQ ID NO.6E329G-F Primer sequence

[0122] CCGACTGGGGTTTGGTCAACGATAATCATGTTGGTGATC

[0123] SEQ ID NO.7E329G-R Primer sequence

[0124] TGACCAAACCCCAGTCGGTGACGACCACG

[0125] SEQ ID NO.8E329N-F Primer sequence

[0126] CCGACTGGAATTTGGTCAACGATAATCATGTTGGTGATC

[0127] SEQ ID NO.9E329N-R Primer sequence

[0128] TTGACCAAATTCCAGTCGGTGACGACCACG

[0129] SEQ ID NO.10R72N-F primer sequence

[0130] TGCTGGAAGCGCCAGGTAATATCAGCAA

[0131] SEQ ID NO.11R72N-R primer sequence

[0132] GTGGTCGGGCTTTTGCTGATATTACCTGGCG

[0133] SEQ ID NO.12 Mutant E329K / R72N amino acid sequence

[0134] MTPHLSTAADGTSYRDLNGNGVMDPYEDPRLSPEERADDLVSRMSLEEKCGLMFQTVIEVGDDGELLEAPGNISKSPTTEVVVGKNLSHFNVHAIRTARQAAVWNNNLQALAERTPHGVPVTISTDPRHAFVENTGVGFAAGPFSQWPEGLGLAAIDDVETVREFADVARREYRAVGIRAALHPQIDLATEPRWGRQAQTLGQDAARVAEFTAAYLQGFQGDELGPDSVACTTKHFPGGGPQKDGEDSHFPYGREQVYPGGMFEYHLEPFREAIRRGTAAMMPYYGMPVGLERGGEKIEEVGFGYNRQIVTDLLRGELGYDGVVVTDWKLVNDNHVGDQVLPARAWGVEELTPAERMEKILDAGADQFGGEECVDLLLDLVRSGRIGEERIDASARRLLLVKFRLGLFDDPYVDPDEAERIVGNADFRAQGERAQARALTVLVNREDASGAPTLPLRPVGRVYVEGFRADEAAELGVVVDDPADADLALVRLGAPFDPRDDLFLEAWFHQGSLEFPPGRVYRMRRIAEQCPLVLVANLDRAAVLTPFAEFAAAIAADFGSSGAAVLDALTGRIAPEGRLPVELPRSMDAVRASREDVPSDTEDPLFPVHFGLSLTTARA

[0135] Amino acid sequence of SEQ ID NO.13 mutant E329G

[0136] MTPHLSTAADGTSYRDLNGNGVMDPYEDPRLSPEERADDLVSRMSLEEKCGLMFQTVIEVGDDGELLEAPGRISKSPTTEVVVGKNLSHFNVHAIRTARQAAVWNNNLQALAERTPHGVPVTISTDPRHAFVENTGVGFAAGPFSQWPEGLGLAAIDDVETVREFADVARREYRAVGIRAALHPQIDLATEPRWGRQAQTLGQDAARVAEFTAAYLQGFQGDELGPDSVACTTKHFPGGGPQKDGEDSHFPYGREQVYPGGMFEYHLEPFREAIRRGTAAMMPYYGMPVGLERGGEKIEEVGFGYNRQIVTDLLRGELGYDGVVVTDWGLVNDNHVGDQVLPARAWGVEELTPAERMEKILDAGADQFGGEECVDLLLDLVRSGRIGEERIDASARRLLLVKFRLGLFDDPYVDPDEAERIVGNADFRAQGERAQARALTVLVNREDASGAPTLPLRPVGRVYVEGFRADEAAELGVVVDDPADADLALVRLGAPFDPRDDLFLEAWFHQGSLEFPPGRVYRMRRIAEQCPLVLVANLDRAAVLTPFAEFAAAIAADFGSSGAAVLDALTGRIAPEGRLPVELPRSMDAVRASREDVPSDTEDPLFPVHFGLSLTTARA

[0137] Amino acid sequence of SEQ ID NO.14 mutant E329N

[0138] MTPHLSTAADGTSYRDLNGNGVMDPYEDPRLSPEERADDLVSRMSLEEKCGLMFQTVIEVGDDGELLEAPGRISKSPTTEVVVGKNLSHFNVHAIRTARQAAVWNNNLQALAERTPHGVPVTISTDPRHAFVENTGVGFAAGPFSQWPEGLGLA AIDDVETVREFADVARREYRAVGIRAALHPQIDLATEPRWGRQAQTLGQDAARVAEFTAAYLQGFQGDELGPDSVACTTKHFPGGGPQKDGEDSHFPYGREQVYPGGMFEYHLEPFREAAIRRGTAAMMPYYGMPVGLERGGEKIEEVGFGYNRQI VTDLLRGELGYDGVVVTDWNLVNDNHVGDQVLPARAWGVEELTPAERMEKILDAGADQFGGEECVDLLLDLVRSGRIGEERIDASARRLLLVKFRLGLFDDPYVDPDEAERIVGNADFRAQGERAQARALTVLVNREDASGAPTLPLRPVGRVYV EGFRADEAAELGVVVDDPADADLALVRLGAPFDPRDDLFLEAWFHQGSLEFPPGRVYRMRRIAEQCPLVLVANLDRAAVLTPFAEFAAAIAADFGSSGAAVLDALTGRIAPEGRLPVELPRSMDAVRASREDVPSDTEDPLFPVHFGLSLTTARA

[0139] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will appreciate that various substitutions, changes and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the scope of the present invention is not limited to the contents disclosed in the embodiments.

Claims

1. A Microbacterium dextranolyticum β-glucosidase mutant, characterized in that: The β-glucosidase mutant is obtained by mutating the 329th position of the β-glucosidase with the amino acid sequence as shown in SEQ ID NO.1 from glutamic acid to glycine.

2. A gene encoding the Microbacterium dextranolyticum β-glucosidase mutant according to claim 1.

3. A recombinant plasmid carrying the gene as claimed in claim 2.

4. A microbial cell carrying the gene according to claim 2 or the recombinant plasmid according to claim 3.

5. A method for preparing a Microbacterium dextranolyticum β-glucosidase mutant enzyme solution using the microbial cell according to claim 4, characterized in that: The method inoculates the recombinant cells into a culture medium for culturing, adds an inducer and continues culturing to obtain a bacterial solution containing a β-glucosidase mutant; centrifuges the bacterial solution to collect bacterial cells, and after ultrasonic disruption, centrifuges the solution obtained by disruption to obtain a supernatant to obtain the Microbacterium dextranolyticum β-glucosidase mutant enzyme solution.

6. A method for preparing cycloastragenol-β-D-xyloside using the mutant according to claim 1, characterized in that: The method comprises adding a Microbacterium dextranolyticum β-glucosidase mutant to a reaction system containing astragaloside IV to carry out a reaction to obtain a reaction solution, and the reaction solution is separated to obtain the prepared cycloastragenol-β-D-xyloside; The volume of the mutant enzyme solution added to the reaction system is 30-50% of the volume of the reaction system, the concentration of the substrate astragaloside IV in the reaction system is 5-10 g / L, the reaction pH is 6.5-7.5, and the temperature is 40-50°C.

7. Use of the mutant according to claim 1 in the preparation of cycloastragenol-β-D-xyloside.

Images