Astragaloside beta-xylosidase mutant as well as coding gene, recombinant vector, recombinant strain, enzyme preparation and application thereof

Abstract

The invention belongs to the technical field of enzyme engineering and biology, and discloses an astragaloside beta-xylosidase mutant as well as a coding gene, a recombinant vector, a recombinant strain, an enzyme preparation and application thereof. The amino acid sequence of the mutant is mutated from at least one of the 23rd site, the 699th site and the 712th site of the amino acid sequence as shown in SEQ ID NO.1. The astragaloside beta-xylosidase mutant has the advantages that the enzyme catalytic activity and the hydrolysis conversion rate are obviously improved, so that the production efficiency of preparing cycloastragenol-6-O-glucoside from astragaloside through enzyme catalysis is obviously improved; good industrialization potential is realized.

Description

Technical Field

[0001] The present invention relates to the field of enzyme engineering and biotechnology, and in particular to an astragaloside β-xylosidase mutant and its encoding gene, a recombinant vector, a recombinant strain, an enzyme preparation and their use in hydrolyzing astragaloside Ⅳ to cycloastragenol-6-O-glucoside. Background Art

[0002] β-Xylosidase is a hydrolase from the GH3 family that can hydrolyze the β-1,4-xylosidic bond of the non-reducing xylose residue at the end of β-D-xyloside and release xylose. It is mainly found in bacteria and fungi, and is common in filamentous fungi. β-Xylosidase not only has the function of transglycosidation, but also has the ability to catalyze the cleavage of glycosidic bonds and efficiently hydrolyze glycosyl. Based on its diversity of functions, β-Xylosidase has been widely used in food, agriculture, industry, medicine and environmental protection. For example, in the energy field, it can be used in combination with cellulase and pectinase to produce bioethanol from xylose extracted from plant hemicellulose. In the food field, β-Xylosidase helps beer production and reduces the turbidity of beer. β-Xylosidase can also be used to convert many biologically active compounds to obtain more valuable and more easily utilized active substances, such as ginsenoside Rb3, notoginseng saponins, epimedin B and anthocyanins.

[0003] The prokaryotic expression system often produces inactive proteins due to the lack of post-translational modification function. Pichia pastoris is currently the most commonly used eukaryotic expression system after Escherichia coli. It has a clear genetic background, a post-translational modification system, and no toxins are generated during the cultivation process. It is suitable for the recombinant expression of enzymes in the food industry.

[0004] Cycloastragenol belongs to the triterpenoid saponin class of compounds. It has the effects of inhibiting brain cell apoptosis and neuroinflammation during cerebral ischemia and maintaining the blood-brain barrier, and is a popular molecule in the development of anti-aging drugs. At the same time, cycloastragenol is the only telomerase activator discovered so far. It is mainly obtained by hydrolysis of astragaloside IV. Among the current traditional synthesis methods, the plant extraction method is easily affected by the external environment, the content of the extracted effective ingredients is low, and it is easily contaminated during the culture process; and cycloastragenol has a certain acid instability and is easily converted into astragenol under acidic conditions. Therefore, the acid hydrolysis method has a low yield, which is not conducive to industrial production. The enzymatic synthesis of cycloastragenol has the characteristics of mild conditions, specific reaction and high efficiency, which can better solve the problems existing in plant extraction and chemical synthesis methods. However, the existing astragaloside IV xylosidic bond hydrolase activity is limited. How to dig out a hydrolase with high catalytic activity to improve the production efficiency of cycloastragenol has become an urgent problem to be solved. Summary of the invention

[0005] The purpose of the present invention is to overcome the problems of limited astragaloside xylosidic bond hydrolase activity and low catalytic efficiency in the prior art, and to provide an astragaloside β-xylosidase mutant and its encoding gene, recombinant vector, recombinant strain and enzyme preparation and their application. The astragaloside β-xylosidase mutant has high catalytic efficiency.

[0006] In order to achieve the above object, the first aspect of the present invention provides an astragaloside β-xylosidase mutant, wherein at least one of the 23rd, 699th and 712th positions of the amino acid sequence shown in SEQ ID NO.1 is mutated.

[0007] Preferably, the astragaloside β-xylosidase mutant is an enzyme having an amino acid sequence as shown in SEQ ID NO.1 subjected to at least one of the mutations described in (1) to (3);

[0008] (1) Cysteine ​​C at position 23 mutated to threonine T, represented by C23T;

[0009] (2) Threonine T at position 699 mutated to tyrosine Y, represented by T699Y;

[0010] (3) Aspartic acid D at position 712 mutates to leucine L, represented by D712L.

[0011] Preferably, the astragaloside β-xylosidase mutant is obtained by subjecting the enzyme having the amino acid sequence shown in SEQ ID NO. 1 to the mutation described in (1).

[0012] The second aspect of the present invention provides a gene encoding an astragaloside β-glucosidase mutant, wherein the gene has the nucleotide sequence of the astragaloside β-xylosidase mutant as described above.

[0013] Preferably, the nucleotide sequence of the gene encoding the astragaloside β-xylosidase mutant is shown as SEQ ID NO.23.

[0014] The third aspect of the present invention provides a recombinant vector, which contains the gene as described above.

[0015] Preferably, the expression vector is a pPIC9k plasmid.

[0016] The fourth aspect of the present invention provides a recombinant strain, which contains the gene or the recombinant vector as described above.

[0017] The fifth aspect of the present invention provides a method for preparing an astragaloside β-xylosidase mutant, the method comprising: inoculating the above-mentioned recombinant strain into a culture medium for fermentation culture.

[0018] The sixth aspect of the present invention provides an enzyme preparation, which comprises the astragaloside β-xylosidase mutant prepared by the method described above.

[0019] The seventh aspect of the present invention provides the use of at least one of the astragaloside β-xylosidase mutant, the gene, the recombinant vector, the recombinant strain, the astragaloside β-xylosidase mutant prepared by the method described above, and the enzyme preparation described above in hydrolyzing astragaloside Ⅳ to cycloastragenol-6-O-glucoside.

[0020] In an eighth aspect, the present invention provides a method for hydrolyzing astragaloside Ⅳ to cycloastragenol-6-O-glucoside using at least one of the astragaloside β-xylosidase mutant, the gene, the recombinant vector, the recombinant strain, the astragaloside β-xylosidase mutant prepared by the method described above, and the enzyme preparation described above.

[0021] Preferably, the astragaloside IV is mixed in the form of a solution with a concentration of 1-5 g / L.

[0022] Preferably, the dosage of the astragaloside β-xylosidase mutant is 0.3U-0.5U relative to 1g of astragaloside Ⅳ.

[0023] Preferably, the reaction conditions include: pH 4-5, rotation speed 150-250 rpm, and temperature 30-50°C.

[0024] Through the above technical scheme, the present invention obtains the astragaloside β-xylosidase sequence from Aspergillusaculeatus through gene hunting method, and successfully realizes heterologous expression in yeast, constructs and obtains an astragaloside β-xylosidase mutant with improved hydrolysis activity. Compared with the wild type, the astragaloside β-xylosidase mutant has higher catalytic activity and higher conversion rate of hydrolysis of astragaloside IV, so that the efficiency of enzyme-catalyzed astragaloside to prepare cycloastragenol-6-O-glucoside is significantly improved.

[0025] The astragaloside β-xylosidase mutant provided by the invention has broad industrial application prospects in the preparation of cycloastragenol-6-O-glucoside. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a relative activity test diagram of the wild-type astragaloside β-xylosidase and 9 mutant enzymes of Example 2;

[0027] Figure 2 This is a diagram showing the results of SDS-PAGE electrophoresis analysis of the astragaloside β-xyloside mutant enzyme of Example 3;

[0028] Figure 3 The result diagram of the hydrolysis of astragaloside Ⅳ by the wild-type astragaloside β-xylosidase and the C23T mutant enzyme in Example 4 to produce cycloastragenol-6-O-glucoside;

[0029] Figure 4 This is a graph showing the results of liquid chromatography detection of the wild-type astragaloside β-xylosidase and the C23T mutant enzyme in Example 4 in converting astragaloside Ⅳ to cycloastragenol-6-O-glucoside for 48 hours. DETAILED DESCRIPTION

[0030] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0031] The first aspect of the present invention provides an astragaloside β-xylosidase mutant, wherein the enzyme having the amino acid sequence shown in SEQ ID NO.1 is mutated at least one of the 23rd, 699th and 712th positions.

[0032] During the research process, the inventors of the present invention obtained the nucleotide sequence of astragaloside β-xylosidase from Aspergillusaculeatus by genome hunting method, and the amino acid sequence of the astragaloside β-xylosidase is shown in SEQ ID NO.1. At least one of the 23rd, 699th and 712th positions thereof was mutated by site-directed mutagenesis technology, which can effectively improve the activity of astragaloside β-xylosidase and improve the catalytic hydrolysis efficiency of astragaloside Ⅳ.

[0033] Preferably, the astragaloside β-xylosidase mutant is an enzyme having an amino acid sequence as shown in SEQ ID NO.1 subjected to at least one of the mutations described in (1) to (3);

[0034] (1) Cysteine ​​C at position 23 mutated to threonine T, represented by C23T;

[0035] (2) Threonine T at position 699 mutated to tyrosine Y, represented by T699Y;

[0036] (3) Aspartic acid D at position 712 mutates to leucine L, represented by D712L.

[0037] Further preferably, the mutation mode of the astragaloside β-xylosidase mutant is C23T. The inventors found that under this preferred embodiment, the mutant enzyme has higher activity, thereby increasing the catalytic hydrolysis efficiency of astragaloside IV and the yield of cycloastragenol-6-O-glucoside.

[0038] The method for determining the activity of astragaloside β-xylosidase and mutants used in the present invention is mainly based on determining the concentration of products generated by enzymatic hydrolysis of substrates for enzyme activity conversion. Enzyme activity is determined using p-nitrophenyl-β-D-pyranoxyloside (pNPX) as a substrate, and the product p-nitrophenol (pNP) generated by hydrolysis is yellow under alkaline conditions. The content of pNP is calculated by measuring the absorbance at 405nm, and a boiled inactivated enzyme solution is used as a control. The absorbance at OD405 is measured and substituted into the standard curve to calculate the pNP concentration in the system, thereby converting the enzyme activity.

[0039] The second aspect of the present invention provides a gene encoding an astragaloside β-xylosidase mutant, wherein the gene has a nucleotide sequence encoding the astragaloside β-xylosidase mutant as described above.

[0040] Preferably, the nucleotide sequence of the mutant is shown in SEQ ID NO.23.

[0041] It is well known in the art that among the 20 different amino acids that make up proteins, except for Met (ATG) or Trp (TGG) which are encoded by a single codon, the other 18 amino acids are encoded by 2-6 codons respectively (Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, second edition, 1989, see Appendix D on page 950). That is, due to the degeneracy of the genetic code, there are usually more than one codon to determine an amino acid, and the substitution of the third nucleotide in the triplet codon often does not change the amino acid composition, so the nucleotide sequence of the gene encoding the same protein can be different.

[0042] The nucleotide sequences provided by the present invention can usually be obtained by polymerase chain reaction (PCR) amplification, recombination, or artificial synthesis. Once the relevant nucleotide sequence is obtained, the relevant amino acid sequence can be obtained in large quantities by recombination. The obtained nucleotide sequence is usually cloned into a vector, then transferred into genetically engineered bacteria, and then the relevant nucleotide sequence is isolated from the propagated host cells by conventional methods.

[0043] In addition, the relevant nucleotide sequences can also be synthesized by known artificial chemical synthesis methods.

[0044] The third aspect of the present invention provides a recombinant vector, which contains the gene as described above.

[0045] In the present invention, the "vector" used in the recombinant vector can be selected from various vectors known in the art, such as various commercially available plasmids, cosmids, phages and retroviruses, etc. The preferred expression vector of the present invention is the pPIC9k plasmid.

[0046] The fourth aspect of the present invention provides a recombinant strain, which contains the gene or the recombinant vector as described above.

[0047] In the present invention, the recombinant vector can be transformed, transduced or transfected into a host cell (strain) by conventional methods in the art, such as chemical transformation by calcium chloride method or high voltage electroporation transformation. The host cell can be a prokaryotic cell or a eukaryotic cell, preferably Pichia pastoris KM71.

[0048] The fifth aspect of the present invention provides a method for preparing an astragaloside β-xylosidase mutant, the preparation method comprising: inoculating the above-mentioned recombinant strain into a fermentation medium for fermentation.

[0049] In the present invention, there is no particular limitation on the conditions for fermentation of the recombinant strain, as long as the recombinant strain can be proliferated in large quantities through the fermentation process. Preferably, the fermentation process comprises: inoculating the above-mentioned recombinant cells into YPD medium for activation, then inoculating the activated bacterial solution into BMGY fermentation medium at 1% and culturing until OD600 is above 5, collecting the bacterial cells, washing with sterile water, and resuspending in BMMY medium for continued cultivation, and centrifuging and ultrafiltration after the cultivation to obtain an enzyme solution containing astragaloside β-xylosidase mutant.

[0050] The present invention has no particular limitation on the seed culture method, as long as the recombinant strain can be activated and proliferated by the method. Preferably, the parameters such as temperature, pH, rotation speed, time, etc. used in the seed culture can be conventional settings in the art.

[0051] Preferably, the astragaloside IV is mixed in the form of a solution with a concentration of 1-5 g / L;

[0052] Preferably, the dosage of the astragaloside β-xylosidase mutant is 0.3U-0.5U relative to 1g of astragaloside Ⅳ.

[0053] Preferably, the seed culture conditions include: temperature of 30-45°C, rotation speed of 150-250rpm, and time of 8-12h.

[0054] In the present invention, there is no particular limitation on the seed culture medium and the fermentation culture medium, and they can be culture mediums conventionally used in the art. Preferably, the activation culture medium adopts YPD culture medium (tryptone 15-25 g / L, yeast extract 8-12 g / L, glucose 15-25 g / L), the primary seed culture medium adopts BMGY culture medium (tryptone 15-25 g / L, yeast extract 8-12 g / L, glycerol 0.8-1.2%, YNB 10-15 g / L), and the secondary seed culture medium adopts BMMY culture medium (tryptone 15-25 g / L, yeast extract 8-12 g / L, YNB 10-15 g / L).

[0055] The sixth aspect of the present invention provides an enzyme preparation, which comprises the astragaloside β-xylosidase mutant prepared by the method described above.

[0056] In the present invention, the astragaloside β-xylosidase mutant can be prepared into a corresponding enzyme preparation. Specifically, the enzyme preparation is in a liquid form.

[0057] In the present invention, the fermentation broth obtained by the above-mentioned preparation method can be directly used as an enzyme preparation, or the fermentation broth can be separated and purified to obtain the astragaloside β-xylosidase mutant as an enzyme preparation, and the separation and purification can adopt the conventional enzyme separation method of Ni+ column purification in the art. The astragaloside β-xylosidase mutant can be used in the form of whole cells of a recombinant strain, or in the form of a crude enzyme separated from the cells of a recombinant strain and not purified, or a purified enzyme.

[0058] The astragaloside β-xylosidase mutant provided by the present invention has high enzyme activity and significantly improves the hydrolysis effect of astragaloside Ⅳ. The seventh aspect of the present invention provides the use of at least one of the astragaloside β-xylosidase mutant, the gene, the recombinant vector, the recombinant strain, the astragaloside β-xylosidase mutant prepared by the method described above, and the enzyme preparation described above in the hydrolysis of astragaloside Ⅳ.

[0059] In the present invention, the method for hydrolyzing astragaloside IV may include: treating the astragaloside IV β-xylosidase mutant, the gene, the recombinant vector, the recombinant bacteria,

[0060] The strain, the astragaloside β-xylosidase mutant prepared by the above method and at least one of the enzyme preparations described above are mixed with astragaloside IV to react.

[0061] The reaction conditions include: pH 4-5, rotation speed 150-250 rpm, and temperature 30-50° C. The reaction process can be carried out under the condition of PB buffer with pH 4-5 as solvent.

[0062] In the present invention, preferably, the astragaloside IV is mixed in the form of a solution with a concentration of 1-5 g / L.

[0063] In the present invention, preferably, the amount of the astragaloside β-xylosidase mutant is 0.3U-0.5U relative to 1g of astragaloside Ⅳ.

[0064] The astragaloside β-xylosidase mutant has high enzyme activity and can significantly improve the ability to hydrolyze astragaloside Ⅳ. The astragaloside β-xylosidase mutant will have great potential in hydrolyzing astragaloside Ⅳ to cycloastragenol-6-O-glucoside and has broad application prospects in biochemical engineering.

[0065] The present invention will be described in detail below through examples.

[0066] In the following examples, Pichia pastoris KM71 and PNGase F deglycosylation kit were purchased from Beyotime Biotechnology Co., Ltd., pPIC9k plasmid was purchased from GenScript Corporation, and p-nitrophenyl-β-D-xylopyranoside was purchased from Shanghai MacLean Biochemical Technology Co., Ltd. The natural products involved in the following examples were all purchased from Shanghai Yuanye Company.

[0067] The culture medium involved in the following examples is as follows:

[0068] YPD medium: tryptone 20 g / L, yeast extract 10 g / L, glucose 20 g / L; solid medium contains agar powder 20 g / L, sterilized by high pressure steam at 115°C for 30 min.

[0069] The first BMGY medium: 20 g / L tryptone, 10 g / L yeast extract, 1% glycerol, 13.4 g / L YNB (containing 10 g / L ammonium sulfate), sterilized by high pressure steam at 115°C for 30 min, and added with the prepared 500× biotin aqueous solution and 10× phosphate buffer (pH 6.0) when used.

[0070] The second BMMY medium: 20 g / L tryptone, 10 g / L yeast extract, 13.4 g / L YNB (containing 10 g / L ammonium sulfate), sterilized by high-pressure steam at 115°C for 30 min, and added with the prepared 500× biotin aqueous solution, 10× phosphate buffer (pH 6.0), and 0.5% methanol when used.

[0071] The enzyme activity and liquid phase detection methods in the following examples are as follows:

[0072] The method for determining the activity of β-xylosidase and mutants used in the present invention is mainly based on determining the concentration of products generated by enzymatic hydrolysis of substrates for enzyme activity conversion. The enzyme activity is determined using p-nitrophenyl-β-D-pyranoxyloside (pNPX) as a substrate, and the product p-nitrophenol (pNP) generated by hydrolysis is yellow under alkaline conditions, and the content of pNP is calculated by measuring the absorbance at 405 nm.

[0073] The specific detection method is as follows: Take 10 μL of appropriately diluted enzyme solution and add it to 90 μL of a reaction system preheated at 37°C, wherein the system contains 2 mM pNPX and 100 mM pH 4-5 PB buffer. After mixing evenly, react for 1 min, and add 100 μL of 2M sodium carbonate solution to terminate the reaction. Use the enzyme solution inactivated by boiling as a control, and measure the OD 405 The absorbance at 400 nm was substituted into the standard curve to calculate the pNP concentration in the system, thereby converting the enzyme activity.

[0074] Drawing of pNP standard curve: Prepare 6-10 pNP systems containing 2-10 μM respectively, use sterile water as blank control, and measure OD 405 Absorbance, 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.

[0075] Calculation formula for β-xylosidase and mutant enzyme activity: Enzyme activity S = C × V1 × n / 1000 × t × V2

[0076] 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).

[0077] Definition of enzyme activity: The amount of enzyme required to hydrolyze 1 μmol of p-nitrophenol (pNP) per minute at 37°C is defined as 1 enzyme activity unit (U).

[0078] 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 column (4.6, 5 μm), the detector was an ELSD detector, the column temperature was set to 40°C, the mobile phase was 75% methanol, and the flow rate was 1 mL / min.

[0079] The structural formula of astragaloside IV (ASI) is shown in formula (I),

[0080]

[0081] 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).

[0082] Example 1

[0083] This example illustrates the acquisition of astragaloside β-xylosidase (XylO2) from Aspergillus aculeatus.

[0084] The relevant sequence information of β-xylosidase in Aspergillus aculeatus was retrieved in the NCBI database, and the sequence was compared with the sequence Xy1-T reported in the literature to obtain β-xylosidase XylO2 with high homology. The sequence Genebank accession number XP_025507064.1 was optimized by yeast codons to synthesize XylO2 (the amino acid sequence is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2).

[0085] Primers were designed to use the synthesized gene as a template, and the target gene fragment XylO2 containing the homology arms of the EcoR I and Not I sites of the yeast expression vector pETDuet-1 was amplified by high-fidelity PCR. The amplified target gene was connected to the linearized expression vector pPIC9k by a one-step cloning enzyme, and the connected product was transformed into E. coli DH5α. The correct recombinant plasmid was screened and transformed into P. pastoris KM71. The transformants grown on the MD solid medium were selected and cultured on the YPD solid medium containing different concentrations of G418 antibiotics. The colonies grown on the YPD solid medium with a high concentration of G418 antibiotics were verified by colony PCR. The correct colonies were verified to be the construction of the correct expression strain.

[0086] The obtained recombinant plasmid pPic9kα-XylO2 of Astragalus β-xylosidase was transformed into P. pastoris KM71, and the specific steps were as follows:

[0087] Preparation of competent cells of Pichia pastoris: A single colony of Pichia pastoris was inoculated into 5 ml of YPD medium and cultured overnight at 28°C and 220 rpm; 1 mL was inoculated into 100 mL of YPD medium and cultured at 28°C and 220 rpm until OD 600 =1.3-1.5, and centrifuge at 5000g, 4℃ for 5min, discard the supernatant; add 100mL pre-cooled sterile water to resuspend the cells, centrifuge as above and discard the supernatant; resuspend the cells in 4mL pre-cooled 1M sorbitol solution, centrifuge as above and discard the supernatant; resuspend the cells in 300μL pre-cooled 1M sorbitol solution, and dispense into 80μL per tube to complete the preparation.

[0088] Gently mix the recombinant plasmid Pichia pastoris and the competent Pichia pastoris, transfer to a pre-cooled electroporation cup, let stand on ice for 5 minutes, and set the electroporation program Sc2 for electroporation. Quickly add 900μL of pre-cooled 1M sorbitol solution and transfer to a centrifuge tube. Let stand at 30℃ for 1h, 3000g, 4℃ refrigerated centrifugation for 5min, spread on MD medium, and culture at 30℃ for 3 days. Transfer the colonies grown on MD medium to medium with different G418 concentrations and culture at 30℃ for 2 days. Pick the colonies grown on the high-concentration medium and perform colony PCR verification after thermal lysis. The correct verification is the recombinant Pichia pastoris.

[0089] The specific steps for obtaining astragaloside β-xylosidase are as follows:

[0090] The obtained recombinant Pichia pastoris was inoculated into YPD and cultured at 30°C, 220 rpm for 12 h. It was then inoculated into BMGY medium at 1% and cultured at 30°C, 220 rpm until OD 600 5, collect the cells, wash with sterile water, resuspend in BMMY medium and continue to culture for 72 hours, add 0.5% methanol every day. The fermentation broth is centrifuged and then ultrafiltered to obtain crude astragaloside β-xylosidase.

[0091] Gently mix the recombinant plasmid and the competent cells of Pichia pastoris, transfer to a pre-cooled electroporation cup, let stand on ice for 5 minutes, and set the electroporation program Sc2 for electroporation. Quickly add 900 μL of pre-cooled 1M sorbitol solution and transfer to a centrifuge tube. Let stand at 30℃ for 1 hour, 3000g, 4℃ refrigerated centrifugation for 5 minutes, spread on MD medium, and culture at 30℃ for 3 days. Transfer the colonies grown on MD medium to medium with different G418 concentrations and culture at 30℃ for 2 days. Pick the colonies grown on the high-concentration medium and perform colony PCR verification after thermal lysis. The correct verification is the recombinant Pichia pastoris.

[0092] The specific steps for obtaining astragaloside β-xylosidase are as follows:

[0093] The obtained recombinant Pichia pastoris was inoculated into YPD and cultured at 30°C, 220 rpm for 12 h. It was then inoculated into BMGY medium at 1% and cultured at 30°C, 220 rpm until OD 600 When the pH value is above 5, the cells are collected, washed with sterile water, resuspended in BMMY medium and cultured for 72 hours, and 0.5% methanol is added every day. The fermentation broth is centrifuged and then ultrafiltered to obtain crude astragaloside β-xylosidase solution.

[0094] Example 2

[0095] This example illustrates the site-directed mutagenesis of astragaloside β-xylosidase and the construction of recombinant plasmid

[0096] The software Autodock4 was used to dock XylO2 with the model substrates p-nitrophenyl-β-D-pyranose (pNPX) and astragaloside (ASI). The protein and ligand were hydrogenated and the charge was calculated. The docking box of the protein was set near the key catalytic residues, and the docking algorithm selected the genetic algorithm. After the docking was completed, based on the complex structure obtained by docking, the distance around the substrate was selected. The residues in the range were subjected to virtual saturation mutation, and then the mutation binding free energy of the selected amino acid mutation to the remaining 19 amino acids was calculated in the software Discoverystudio. The mutation sites with the most reduced or increased virtual saturation mutation binding free energy were selected for construction, and XylO2 and pNPX were uploaded to the HotspotWizzard server for mutation hotspot prediction. Finally, F20Y, C23T, E186F, D290H, C291H, C291P, T327Q, T669Y and D712L were selected for construction.

[0097] According to the gene sequence of astragaloside β-xylosidase shown in nucleotide sequence SEQ ID NO.2, F20Y, C23T, E186F, D290H, C291H, C291P, T327Q, T669Y and D712L mutation primers were designed to perform site-directed mutagenesis. The specific steps are as follows:

[0098] Using the whole plasmid PCR technique, the recombinant plasmid pPic9kα-XylO2 carrying the XylO2 encoding gene was used as a template, and the following mutation primers (primers are shown in Table 1) were added and the corresponding PCR system was configured for amplification. The primer sequences are shown in Table 1:

[0099] Table 1

[0100]

[0101]

[0102] The configured PCR system is shown in Table 2.

[0103] Table 2

[0104] Element Volume (μL) Final concentration template 1 10ng / μL Upstream primer 1.2 10mM / μL Downstream primer 1.2 10mM / μL Phanta Flash Master Mi 15 2× <![CDATA[ddH2O]]> 11.6 -

[0105] The configured PCR reaction program is shown in Table 3.

[0106] Table 3

[0107]

[0108] Take 2 μL of the amplified product and perform electrophoresis detection. The correct PCR product is detected and recovered by a product recovery kit. The recovered product is transferred to the competent state of Pichia pastoris, and 6 positive bacteria on the plate are selected for sequencing verification. The correct colonies are verified for fermentation verification, and fermentation culture and strain preservation are performed as in the above Example 1. The activity of the mutant verified by the shake flask is measured. The results show that the relative activities of the mutants with F20Y, C23T, E186F, D290H, C291H, C291P, T327Q, T669Y and D712L mutations are as follows: Figure 1 As shown, the mutations with improved activity include C23T, T669Y and D712L, and the C23T mutation has the most significant improvement in enzyme activity, with an activity of 1.5 times that of the wild type. The amino acid sequence of the best mutation C23T obtained is shown in the sequence table SEQ ID NO.23. Therefore, the present invention uses a mutant enzyme prepared by the mutation of C23T (represented by mutant C23T, whose amino acid sequence is shown in SEQ ID NO.24 and nucleotide sequence is shown in SEQ ID NO.23) for subsequent example studies.

[0109] Example 3

[0110] This example illustrates the purification of wild-type astragaloside β-xylosidase and C23T mutant enzyme

[0111] The crude enzyme solution of the wild-type and C23T mutant enzymes obtained in Example 2 was purified using a Ni ion affinity column, and the purification process was as follows: 6 column volumes of buffer A (50mM pH 7.4 phosphate buffer, 500mM NaCl, 20mM imidazole) were first used to balance the column. The crude enzyme solution was filtered using a microporous filter, and then 2 column volumes of crude enzyme solution were added for adsorption. 15 column volumes of buffer A were used for washing, and then 5 column volumes of buffer B (50mM pH 7.4 phosphate buffer, 500mM NaCl, 500mM imidazole) were used for elution and the eluate was collected to obtain the purified wild-type astragaloside β-xylosidase and C23T mutant enzyme.

[0112] Deglycosylation of Pichia pastoris expressed protein: Refer to the instructions of the Biyuntian PNGase F deglycosylation kit, take 9μL of purified protein sample, add 1μL of denaturation buffer, heat at 100℃ for 10min, then add 2μL of reaction buffer, 2μL of re-denaturation buffer, 6μL of deionized water and 1μL of PNGase F, mix well and digest at 37℃ overnight.

[0113] The collected target protein was treated with protein loading buffer, mixed and heated at 100°C for 10 min to fully denature it, and then subjected to SDS-PAGE protein electrophoresis. The SDS-PAGE electrophoresis results of the protein β-xylosidase XlyO2 from Aspergillus aculeatus are as follows: Figure 2 As shown, where: M is DNA Marker, 1 is the analysis result of crude enzyme solution, 2 is the analysis result of pure enzyme solution, and 3 is the analysis result after deglycosylation enzyme digestion.

[0114] Depend on Figure 2 It can be seen that after purification, the mutant astragaloside β-xylosidase obtained a diffuse band near 150 kDa, which is quite different from the 84.82 kDa calculated based on the amino acid sequence. The deglycosylated mutant astragaloside β-xylosidase solution obtained an obvious band slightly less than 100 kDa, which is basically consistent with the calculated 84.82 kDa. Therefore, β-xylosidase was successfully heterologously expressed.

[0115] Example 4

[0116] This example illustrates the application of wild-type astragaloside β-xylosidase and C23T mutant enzyme in catalyzing astragaloside Ⅳ

[0117] The wild-type astragaloside β-xylosidase and C23T mutant enzyme obtained in Example 3 were added to two groups of the same astragaloside conversion system, which contained 2 g / L astragaloside, 0.1 M PB buffer (pH = 4), and 40% (v / v) enzyme preparation. After mixing, the mixture was converted in a constant temperature shaker at 37°C and 200 rpm for 48 h. The reaction results are shown in FIG. Figure 3 As shown, wherein: WT is the wild type XylO2 of the present invention, C23T is the mutant of the present invention, Figure 3 It can be seen that the amount of cycloastragenol-6-O-glucoside produced by enzymatic hydrolysis of the C23T mutant was significantly higher than that of the wild-type astragaloside β-xylosidase.

[0118] After the reaction, an equal volume of water-saturated n-butanol was added to inactivate the enzyme and extract it. The supernatant was centrifuged and extracted again. The collected organic phase was dried and then re-dissolved in a certain volume of methanol. The supernatant was taken and sterile water was added to make the methanol ratio 75%. The supernatant was passed through an organic microporous filter membrane and tested by liquid chromatography. The liquid chromatography was as follows: Figure 4 As shown by Figure 4 It can be seen that the amount of cycloastragenol-6-O-glucoside produced by the C23T mutant enzyme hydrolysis was significantly higher than that of the wild-type astragaloside β-xylosidase.

[0119] The conversion rate was calculated according to the conversion rate calculation formula, and the result was: the conversion rate of the wild-type enzyme to convert 2 g / L astragaloside Ⅳ to cycloastragenol-6-O-glucoside (CMG) in 48 hours was 36.56%, while the conversion rate of the C23T mutant enzyme to convert 2 g / L astragaloside Ⅳ to cycloastragenol-6-O-glucoside (CMG) in 48 hours was 65.81%, which was about 30% higher than that of the wild-type. It can be concluded that the C23T mutant enzyme has a higher hydrolysis conversion rate than the wild-type astragaloside β-xylosidase.

[0120] The structural formula of the cycloastragenol-6-O-glucoside (CMG) is shown in formula (II).

[0121]

[0122] Example 5

[0123] This example illustrates the effect of changes in reaction conditions (pH, rotation speed, temperature) on the hydrolysis conversion rate.

[0124] 1. This example was operated according to the method described in Example 4, except that the pH conditions were set to 3, 4, 5, and 6, respectively, and HPLC was used for detection. The results are shown in Table 4:

[0125] Table 4:

[0126]

[0127]

[0128] The results in Table 4 show that the hydrolysis conversion rate of the reaction under pH 4 and pH 5 conditions is higher than that under pH 3 and pH 6 conditions. Therefore, astragaloside β-xylosidase and C23T mutant enzyme have higher hydrolysis conversion rates under reaction conditions of pH 4-5.

[0129] 2. This embodiment is operated according to the method described in Example 4, except that the rotation speed conditions are set to 100, 150, 250, and 300 rpm respectively, and HPLC is used for detection. The results are shown in Table 5:

[0130] Table 5

[0131]

[0132] The results in Table 5 show that the hydrolysis conversion rate of the reaction under the rotation speed of 150 rpm and 250 rpm is higher than that of the reaction under the rotation speed of 100 rpm and 300 rpm. Therefore, astragaloside β-xylosidase and C23T mutant enzyme have a higher hydrolysis conversion rate under the reaction condition of a rotation speed of 150-250 rpm.

[0133] 3. This example was operated according to the method described in Example 4, except that the temperature conditions were set to 20, 30, 50, and 55° C., respectively, and HPLC was used for detection. The results are shown in Table 6:

[0134] Table 6

[0135]

[0136] The results in Table 6 show that the hydrolysis conversion rate of the reaction at 30°C and 50°C is higher than that of the reaction at 20°C and 55°C. Therefore, astragaloside β-xylosidase and C23T mutant enzyme have higher hydrolysis conversion rate under the reaction condition of temperature of 30-50°C.

[0137] The above results show that astragaloside β-xylosidase and C23T mutant enzyme have a higher hydrolysis conversion rate under the reaction conditions of pH 4-5, rotation speed 150-250rpm and temperature 30-50℃.

[0138] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. An astragaloside β-xylosidase mutant, characterized in that: The astragaloside β-xylosidase mutant is prepared by mutating at least one of the 23rd, 699th and 712th positions of the amino acid sequence shown in SEQ ID NO.

1.

2. The astragaloside β-xylosidase mutant according to claim 1, characterized in that: The astragaloside β-xylosidase mutant is obtained by subjecting the enzyme having the amino acid sequence shown in SEQ ID NO.1 to at least one of the mutations (1) to (3); (1) Cysteine ​​C at position 23 mutated to threonine T, represented by C23T; (2) Threonine T at position 699 mutated to tyrosine Y, represented by T699Y; (3) Aspartic acid D at position 712 mutated to leucine L, represented by D712L; Preferably, the astragaloside β-xylosidase mutant is obtained by subjecting the enzyme having the amino acid sequence shown in SEQ ID NO. 1 to the mutation described in (1).

3. A gene encoding an astragaloside β-xylosidase mutant, characterized in that: The gene has a nucleotide sequence encoding the astragaloside β-xylosidase mutant according to claim 1 or 2; Preferably, the nucleotide sequence of the mutant is shown in SEQ ID NO.

23.

4. A recombinant vector, characterized in that: The recombinant vector contains the gene according to claim 3; Preferably, the expression vector of the recombinant vector is a pPIC9k plasmid.

5. A recombinant strain, characterized in that: The recombinant strain contains the gene according to claim 3 or the recombinant vector according to claim 4; Preferably, the expression strain of the recombinant strain is Pichia pastoris KM71.

6. A method for preparing an astragaloside β-xylosidase mutant, characterized in that: The preparation method comprises: inoculating the recombinant strain according to claim 5 into a culture medium for fermentation culture.

7. An enzyme preparation, characterized in that The enzyme preparation comprises the astragaloside β-xylosidase mutant prepared by the method according to claim 6.

8. Use of at least one of the astragaloside β-xylosidase mutant according to claim 1 or 2, the gene according to claim 3, the recombinant vector according to claim 4, the recombinant strain according to claim 5, the astragaloside β-xylosidase mutant obtained by the method according to claim 6, and the enzyme preparation according to claim 7 in hydrolyzing astragaloside Ⅳ to cycloastragenol-6-O-glucoside.

9. A method for hydrolyzing astragaloside IV, characterized in that: The method comprises mixing at least one of the astragaloside β-xylosidase mutant described in claim 1 or 2, the gene described in claim 3, the recombinant vector described in claim 4, the recombinant strain described in claim 5, the astragaloside β-xylosidase mutant obtained by the method of claim 6 and the enzyme preparation described in claim 7 with astragaloside IV for reaction.

10. The method for hydrolyzing astragaloside IV according to claim 9, characterized in that: The product of the reaction is cycloastragenol-6-O-glucoside; Preferably, the astragaloside IV is mixed in the form of a solution with a concentration of 1-5 g / L; Preferably, relative to 1g of astragaloside IV, the dosage of astragaloside IV β-xylosidase mutant is 0.3U-0.5U; Preferably, the reaction conditions include: pH 4-5, rotation speed 150-250 rpm, and temperature 30-50°C.

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