CN112410321B

CN112410321B - Beta-glucosidase Ttbgl3 and application thereof

Info

Publication number: CN112410321B
Application number: CN202011343650.8A
Authority: CN
Inventors: 严金平; 张宇; 曲媛; 罗媛; 李雪杰; 杨旭磊; 伊日布斯
Original assignee: Kunming University of Science and Technology
Current assignee: Kunming University of Science and Technology
Priority date: 2020-11-26
Filing date: 2020-11-26
Publication date: 2022-01-28
Anticipated expiration: 2040-11-26
Also published as: CN112410321A

Abstract

The invention discloses a beta-glucosidase Ttbgl3, the amino acid sequence of which is shown as SEQ ID NO. 1, the nucleotide sequence of which is shown as SEQ ID NO. 2, and the beta-glucosidase Ttbgl3 is from Trametes trogii S0301, the invention is applied to the preparation of rare aglycone and monoglycoside compounds, the experimental result shows that the beta-glucosidase Ttbgl3 has the capability of hydrolyzing a plurality of glucosides, simultaneously has the hydrolytic activity of beta-glucoside bond and beta-glucuronide bond, and has stronger transformation capability to gastrodine, esculin, daidzin and baicalin; the beta-glucosidase Ttbgl3 is simple to prepare and has market popularization and application values.

Description

Beta-glucosidase Ttbgl3 and application thereof

Technical Field

The invention belongs to the fields of genetic engineering technology and biomedicine, and particularly relates to cloning, prokaryotic expression and purification of a Ttbgl3 gene of beta-glucosidase and conversion of glycoside compounds.

Background

White rot fungi such as trametes thermotolerans obtain a specific enzyme group for decomposing refractory lignocellulose in the evolution process, and different types of carbohydrate degrading enzymes including Glycoside Hydrolase (GH) exist in a CAZy database. Glycoside Hydrolases (GH) have a number of families involved in the degradation of lignocellulose. Research shows that during the response process of lignocellulose, the expression level of beta-glucosidase genes distributed in GH1 and GH3 families is obviously up-regulated by filamentous fungi, and the filamentous fungi can synthesize and secrete extracellular enzymes such as beta-glucosidase to degrade lignocellulose simultaneously and selectively. Beta-glucosidase can catalyze the hydrolysis of the glycosidic bond formed between the hemiacetal-OH group of an aldose or glucose and the-OH group of another non-sugar compound (e.g., an amino alcohol, an aryl alcohol, or a primary, secondary, tertiary alcohol); the cleavage of glycosidic bonds by this hydrolysis plays an important role in various basic biological processes and has attracted great interest; such as hydrolysis of isoflavone glycosides in the industry, converts ineffective or inefficient soy isoflavone glycosides to potent soy isoflavone aglycones, enhancing their biological activity. Beta-glucosidase also has wide applications in food bioprocessing, pharmaceuticals, and animal feed processing.

Disclosure of Invention

The invention provides a beta-glucosidase Ttbgl3, the amino acid sequence of which is shown as SEQ ID NO. 1, the nucleotide sequence of which is shown as SEQ ID NO. 2, the enzyme is from Trametes trogii S0301, the beta-glucosidase Ttbgl3 of the invention has the hydrolytic activity of beta-glucosidic bond and beta-glucuronic bond, the catalytic activity is better at pH 5-10 and 45-55 ℃, the beta-glucosidase Ttbgl3 also has better metal ion tolerance, Fe³⁺The activity of Ttbgl3 can be improved to 119.6%; the beta-glucosidase Ttbgl3 has strong conversion capability on compounds such as gastrodin, esculin, daidzin, baicalin and the like.

The full-length cDNA of Ttbgl3 is 2547bp, encodes 849 amino acid polypeptides, and the predicted protein size is 97 kDa; using NCBI database, the Ttbgl3 amino acid sequences were aligned on line by BLAST; ttbgl3 is closest to the beta-glucosidase from Polyporus arcularius (Pabgl3, ID: TFK93148.1) with 81.74% homology; in addition, the beta-glucosidase from Trametes versicolor has 80.26% homology (Tvbgl3, ID: XP-008036945.1); beta-glucosidase from Trametes coccinea with homology of 79.34% (Tcbgl3, ID: OSD 02721.1); beta-glucosidase from Gelatiopsia subvermispora with homology of 74.30% (Gsbgl3, ID: EMD34486.1)

The invention connects beta-glucosidase Ttbgl3 gene with enzyme-cut pET-28b plasmid to obtain recombinant plasmid containing beta-glucosidase Ttbgl3 gene; transforming the recombinant plasmid into an expression host bacterium Roseeta (DE3), carrying out induction culture by IPTG, centrifugally collecting thalli, crushing the thalli, and purifying by a Ni-NTA pre-loaded gravity column to obtain beta-glucosidase Ttbgl 3; the beta-glucosidase Ttbgl3 is applied to the preparation of rare aglycone and monoglycoside compounds, and experimental results show that the beta-glucosidase Ttbgl3 has the capability of hydrolyzing various glucosides and has stronger conversion capability on gastrodin, esculin, daidzin and baicalin; through homologous modeling and molecular docking, two residues of His85 and Lys467 in the beta-glucosidase Ttbgl3 are presumed to be enzyme binding sites of substrates containing beta-glucuronic bonds, and Glu377 and Thr424 are presumed to be enzyme binding sites of substrates containing beta-glucuronic bonds.

Drawings

FIG. 1 is an agarose gel map specific to Ttbgl3 gene of the invention as described in example 1;

FIG. 2 is a graph showing the results of purity identification of purified Ttbgl 3; FIG. A is an SDS-PAGE electrophoresis of Ttbgl3 protein purified by imidazole eluents of different concentration gradients, wherein lane M is protein Marker (available from New Biotechnology Co., Ltd., Beijing Ongzhico, cat # TSP021), lane 1 is protein produced by adding optimum IPTG concentration (0.05mM) to Roseteta (DE3) strain transformed with pET28b-Ttbgl3 recombinant vector; 2 is protein eluted using an eluent containing 25mM imidazole; 3 is a protein eluted using an eluent containing 50mM imidazole; 4 is protein eluted using an eluent containing 100mM imidazole; 5 is protein eluted using an eluent containing 200mM imidazole; 6 is protein eluted using an eluent containing 250mM imidazole; FIG. B is a graph showing the results of ultrafiltration and concentration of proteins in 200mM imidazole eluate, in which lane M is protein Marker (available from New Biotechnology Co., Ltd., Beijing Optimalaceae, cat # TSP021), and lane 1 is protein produced by Roseteta (DE3) strain transformed with pET 28B; lane 2 is a protein produced by the Roseeta (DE3) strain transformed with the pET28b-Ttbgl3 recombinant vector without the addition of IPTG inducer; lane 3 is a protein produced by addition of optimum IPTG concentration (0.05mM) to the Roseeta (DE3) strain transformed with pET28b-Ttbgl3 recombinant vector; lane 4 is the supernatant protein collected by ultrasonication after IPTG induction; lane 5 is the precipitated protein collected by ultrasonication after IPTG induction; lane 6 shows that the supernatant was purified by Ni-NTA pre-packed gravity column (Bio-Industrial, cat # C600793), eluted with 200mM imidazole and concentrated to 0.16mg/mL pure enzyme protein using 10kDa ultrafiltration tube (Merck, cat # UFC 9003);

FIG. 3 is a graph showing the results of pH measurement of the optimum reaction of beta-glucosidase Ttbgl3, with pH on the abscissa and relative enzyme activity on the ordinate in units%;

FIG. 4 is a graph showing the results of measurement of the optimum reaction temperature of Ttbgl3, wherein the abscissa is temperature in degrees Celsius (C) and the ordinate is relative enzyme activity in units;

FIG. 5 is a graph of pH stability measurements with pH on the abscissa and relative enzyme activity on the ordinate in units%;

FIG. 6 is a graph of the results of temperature stability measurements, with the abscissa being the incubation time in minutes (min) and the ordinate being the relative enzyme activity in units%;

FIG. 7 is a graph of the results of a sugar tolerance measurement, with the abscissa being the concentration of various monosaccharides in units (mM) and the ordinate being the relative enzyme activity in units%;

FIG. 8 shows the kinetic parameters of recombinant enzyme Ttbgl3 using p-NPG as a substrate in the qualitative determination result of Ttbgl3 of β -glucosidase of the present invention in example 3;

FIG. 9 is a high performance liquid chromatogram of Gastrodin transformed by beta-glucosidase Ttbgl3, the upper graph is a chromatogram of Gastrodin; the lower graph is a chromatogram after transformation; in the figure 1: gastrodine; 1 a: 4-hydroxybenzyl alcohol;

FIG. 10 is a high performance liquid chromatogram of the conversion of esculin by β -glucosidase Ttbgl3, the upper panel being a chromatogram of esculin; the lower graph is a chromatogram after transformation; in the figure 2: esculin; 2 a: esculetin;

FIG. 11 is a high performance liquid chromatogram of Ttbgl3 converted daidzin, the upper graph is a chromatogram of esculin; the lower graph is a chromatogram after transformation; in the figure 3: daidzin; 3 a: daidzein;

FIG. 12 is a high performance liquid chromatogram of the beta-glucosidase Ttbgl3 transformed into baicalin, and the upper graph is a chromatogram of baicalin; the lower graph is a chromatogram after transformation; in the figure 4: baicalin; 4 a: baicalein;

FIG. 13 is a graph showing the results of mass spectrometry of a product obtained by hydrolyzing gastrodin with β -glucosidase Ttbgl 3;

FIG. 14 is a graph showing the results of mass spectrometry of the conversion product of esculin hydrolysis by β -glucosidase Ttbgl 3;

FIG. 15 is a graph showing the results of mass spectrometry of the conversion product of soybean glycoside hydrolyzed by β -glucosidase Ttbgl 3;

FIG. 16 is a graph showing the results of mass spectrometry of the conversion product of baicalin hydrolysis by β -glucosidase Ttbgl 3;

FIG. 17 shows the effect of Ttbgl3 enzyme at different concentrations on the hydrolysis efficiency of gastrodine, esculin and daidzin; the bar graph shows that 500 microgram/mL substrates including gastrodine, esculin and daidzin are treated by recombinase Ttbgl3 with different concentrations, and the residual contents of the substrates in the reaction system;

FIG. 18 shows the effect of Ttbgl3 enzyme concentration on the content of converted products of gastrodine, esculin and daidzin; the bar graph shows the amount of the conversion product in the reaction system when 500. mu.g/mL substrates including gastrodine, esculin and daidzin were treated with different concentrations of recombinase Ttbgl 3;

FIG. 19 shows the effect of different reaction times on the content of gastrodine, esculin, daidzin and their conversion products; respectively treating 500 mu g/mL substrates including gastrodine, esculin and daidzin with 60U/mL, 5U/mL and Ttbgl3 at 50 deg.C for enzyme treatment time analysis; the solid line indicates the remaining amount of the substrate, and the dotted line indicates the amount of the product formed, wherein the abscissa is the reaction time (min) and the ordinate is the contents of the substrate and the product in units (. mu.g/mL);

FIG. 20 is a Ttbgl3 homology modeling 3D model;

FIG. 21 shows the molecular docking between Ttbgl3 and 4 glycosides; A. gastrodine; B. esculin; C. daidzin; D. baicalin is added.

Detailed Description

The present invention will be further described with reference to the drawings and examples so that those skilled in the art can understand the present invention, but the present invention is not limited to the following embodiments. In the present example, the method is carried out in a conventional manner unless otherwise specified, and reagents used therein are, for example, conventional reagents or reagents prepared in a conventional manner unless otherwise specified.

Example 1: cloning of beta-glucosidase Ttbgl3 gene, construction of recombinant plasmid pET28b-Ttbgl 31, obtaining of Ttbgl3 gene and cloning

Culturing heat-resistant trametes hirsuta S0301 at 28 deg.C and collecting mycelium; extracting total RNA by a Trizol method, and performing reverse transcription to obtain cDNA; obtaining a Ttbgl3 gene segment by using cDNA as a template and specific primer amplification by adopting a conventional PCR method; removing the stop codon, and recovering PCR products of 2589bp (the gene length is 2547bp + 40bp of the vector homologous sequences carried by the upstream and downstream primers) after PCR amplification;

(1) reverse transcription of RNA

Preparing a reverse transcription system shown in the table 1 in a PCR tube, uniformly mixing the reverse transcription system with a pipette, and placing the PCR tube in a PCR instrument, wherein the setting program is 50 ℃ and 15 min; 5s at 85 ℃;

TABLE 1 RNA reverse transcription reaction System

(2) The primer sequences for amplification of Ttbgl3 gene in trametes thermotolerans S0301 are as follows:

Ttbgl3-Y-F：5'-caagcttgcggccgctctagaATGTCGCGCGACTTCCTCG-3', the Xba I site is underlined; ttbgl 3-Y-R5' -gtggtggtggtggtgctcgagCACCCCGTTCCATGTGAATC-3', the Xho I site is underlined;

the amplification system was as follows:

TABLE 2 PCR amplification reaction System

The amplification conditions were as follows:

uniformly mixing the reaction system, performing pre-denaturation at 95 ℃ for 5min, performing denaturation at 95 ℃ for 30s, annealing at 62 ℃ for 30s, and extending at 72 ℃ for 3min, wherein after 35 cycles, extending at 72 ℃ for 10 min; after completion of the reaction, 5. mu.L of the product was collected and analyzed by electrophoresis on a 1% agarose gel (see FIG. 1).

2. Gel recovery purification of PCR products

(1) Preparing 1.0% agarose gel in an electrophoresis gel tank;

(2) carrying out sample application electrophoresis on the PCR product for 25min at 180V;

(3) cutting off the gel containing the target segment under an ultraviolet lamp, and transferring the gel into a 1.5mL centrifuge tube;

(4) and (3) recovering the target fragment by using a raw gelatin recovery kit, wherein the recovery method is carried out according to the operation of the instruction.

3. Construction of pET28b-Ttbgl3 recombinant expression vector

(1) The pET28b vector was digested with restriction endonucleases Xba I and Xho I, and reacted at 37 ℃ for 4 hours to recover a 5317bp cleavage product as follows:

TABLE 3 double digestion reaction System

pET28b plasmid	5μg
		10×M Buffer	5μL
Xba I	1μL
		Xho I	1μL
Double distilled water	Make up to 50. mu.L

；

(2) The double-enzyme digestion linearized pET28b vector and the PCR product were ligated using a ligase independent single-fragment rapid cloning kit (Novozam, cat # C112); the connection system and temperature are shown in table 4;

TABLE 4 ligation reaction System of linearized vector and Gene fragment

Linearized pET28b plasmid	100ng
			5×CE II Buffer	4μL
Ttbgl3 gene fragment	300ng
		Exnase II	2μL
Double distilled water	Make up to 20. mu.L

；

(3) Transformation of E.coli DH5 alpha competent cells

The ligation product is transformed into escherichia coli DH5 alpha competent cells (purchased from Beijing Ongjingkidaceae New Biotechnology Co., Ltd.), positive clones are screened, and sequence analysis is carried out to obtain a recombinant plasmid pET28b-Ttbgl 3.

Example 2: preparation of beta-glucosidase Ttbgl3

Transforming an Escherichia coli Roseeta (DE3) strain with a recombinant plasmid pET28b-Ttbgl3, culturing overnight at 37 ℃ on an LB plate medium (tryptone 10g/L, yeast extract 5g/L, NaCl5g/L and agar 15g/L) containing kanamycin (50. mu.g/mL), picking up the transformant to 200mL of LB liquid medium (50. mu.g/mL kanamycin) to culture the transformant at 37 ℃ and 200rpm with shaking until OD600 is 0.6-0.8, adding an isopropyl beta-D-thiogalactopyranoside (IPTG) inducer to the culture solution to culture the transformant at 18 ℃ for 10h, centrifuging the culture solution at 4 ℃ and 13,000rpm by a high-speed refrigerated centrifuge for 15min, and collecting the thallus; since recombinant plasmid pET28b-Ttbgl3 contains His-tag, it is purified by Ni-NTA preloaded gravity column (manufactured under the trademark C600793) to obtain purified Ttbgl 3; the specific operation process is as follows:

(1) sample processing

Resuspending the washed thallus by using 1 multiplied Binding Buffer (8 mL), and breaking the wall by ultrasonic waves (150W, 5 seconds of ultrasonic waves and 7 seconds of intermission, and crushing for 20 min);

② after wall breaking, centrifuging for 30min at 13,000g, taking the supernatant as a sample; the activity of the beta-glucosidase Ttbgl3 in the crushed supernatant is measured by using a p-NPG substrate, and the result shows that the supernatant has enzyme activity reaction, which indicates that the Ttbgl3 protein is successfully expressed and has activity, and the subsequent protein purification can be continued;

(2) treating columns

Firstly, taking 1mLNi-NTA to pre-load a gravity column;

washing the column with 5mL of PBS buffer;

③ washing the column with 5mL of 1 XCharge Buffer;

fourthly, washing the column by using 3mL of 1 XBinding Buffer.

(3) Sample loading

Adding a sample into a column, and controlling the flow rate to be about 6 drops per minute;

② washing the column with 3mL of 1 × Binding Buffer to remove unbound protein;

thirdly, eluting the column by using 10mmol/L imidazole eluent to remove foreign proteins;

fourthly, respectively washing the column by using 4mL of imidazole eluents containing different concentration gradients to elute the target protein;

fifthly, washing the column by using 5mL of 1 × Strip Buffer, and preserving the packed column by using PBS Buffer solution containing 80% absolute ethyl alcohol;

the purified beta-glucosidase Ttbgl3 was obtained by this procedure, and the purity of beta-glucosidase Ttbgl3 was identified by staining after SDS-PAGE electrophoresis, and the results are shown in FIG. 2; as can be seen from the results of fig. 2A, after the target protein is purified by the His tag, a slight band is present at the 95kDa, which is consistent with the predicted molecular weight, and the activity of β -glucosidase Ttbgl3 in imidazole eluents with different concentration gradients is determined by using a p-NPG substrate, and the results show that both enzymatic reactions are present, which indicates that the protein Ttbgl3 has been successfully purified, and the purification effect is expected; as shown in fig. 2B, imidazole was separated after concentration by the ultrafiltration tube, and Ttbgl3 protein with high concentration and single band was obtained, which was advantageous for subsequent experiments.

Example 3: enzymological properties and enzyme activity test of beta-glucosidase Ttbgl3

The enzyme activity unit (U) is defined as: under the measuring condition, the enzyme amount required for generating 1 mu moL of p-NPG per minute is 1 enzyme activity unit; performing three parallel tests on all the measurement experiments, and averaging the results;

1. method for measuring enzyme activity

The reaction system is 100 mu L, 85 mu L of 100mmol/L citric acid-disodium hydrogen phosphate buffer solution (pH6.0) is added into 5 mu L of 20mmol/L p-nitrobenzene beta-D glucoside (p-NPG), the mixture is incubated at 50 ℃ for 3min, 10 mu L of enzyme solution (diluted to a proper multiple) is added for reaction for 10min, and 600 mu L of 1mol/L sodium carbonate solution is added after color development to terminate the reaction; absorbance was measured at 405 nm.

2. Determination of optimum reaction pH

Selecting buffers of different pH values, comprising: 1mol/L disodium hydrogen citrate phosphate buffer solution (pH 2.5-7), 1mol/L Tris-HcL buffer solution (pH 7-9), 1mol/L glycine-NaoH buffer solution (pH 9-11), and enzyme activity is respectively determined at 50 ℃, and the result is shown in figure 3; as can be seen from the results in FIG. 3, the optimum reaction pH of Ttbgl3, which is a β -glucosidase of the present invention, was 6.0.

3. Determination of optimum reaction temperature

Selecting optimum pH condition, incubating the enzyme reaction system at 30 deg.C, 40 deg.C, 50 deg.C, 55 deg.C, 60 deg.C, 65 deg.C, 70 deg.C, 75 deg.C, and 80 deg.C respectively, and measuring enzyme activity, the result is shown in FIG. 4; as can be seen from the results in FIG. 4, the optimum reaction temperature of Ttbgl3, which is a β -glucosidase of the present invention, is 50 ℃.

4. Determination of pH stability

And (3) incubating the enzyme solution in buffer solutions with different pH values for 2h, detecting the residual enzyme activity, and taking the highest enzyme activity as 100%, wherein the enzyme activity can be maintained at more than 90% within the pH range of 5-10 as shown in figure 5.

5. Determination of temperature stability

Respectively incubating the enzyme solutions at different temperatures (50 deg.C, 60 deg.C) for 0-300min, and detecting the temperature stability of Ttbgl 3; as shown in FIG. 6, the enzyme activity was maintained at 85% or more after incubation at 50 ℃ for 5 hours.

6. Determination of Ttbgl3 ability to tolerate glucose

Ttbgl3 was treated with glucose solutions of different concentrations and the residual enzyme activity was measured to investigate the effect of glucose concentration on the enzyme activity of Ttbgl3, as shown in fig. 7, the enzyme activity of Ttbgl3 gradually decreased with increasing glucose concentration. When the glucose concentration was increased to 20mM, the enzyme activity decreased to 50% of the maximum enzyme activity.

7. Effect of Metal ions and organic solvents on the enzymatic Activity of Ttbgl3

The purified protein was assayed for 1mmol/L of different metal ions (Fe) at 50 ℃ and pH6.0³⁺、Sr²⁺、NH⁴⁺、Ba²⁺、K⁺、Na⁺、Co²⁺、Mg²⁺、Ni²⁺、Ca²⁺、Mn²⁺、Cu²⁺、Zn²⁺) And organic solvents (10% EDTA, 5% DMSO, 5% Tuwen-20, 1% Triton X-100, 10% ethanol, 10% SDS); the reaction system without any metal ions or organic solvent was used as a control.

The results are shown in Table 5, adding FeCl₃After that, the activity of Ttbgl3 increased to 119.6%; removing CuSO₄And ZnSO₄Besides, other metal ions have little influence on the activity of Ttbgl3 enzyme, and the maximum enzyme activity of the other metal ions is kept above 90%; even in the case of CuSO₄And ZnSO₄The activity of the enzyme (D) can be maintained above 80%; in an organic solvent, the enzyme activity is inhibited; especially in SDS containing a strong mutagen, the enzyme activity is completely inhibited. In addition, the enzyme activity is respectively reserved in EDTA, DMSO, Tuween-20, Triton-X-100 and ethanol by 96.3%, 82.6%, 77.6%, 66.4% and 65.6%; therefore, the activity of Ttbgl3 can be kept more than 50% in other organic solvents except that the activity of Ttbgl3 is completely inhibited by SDS (sodium dodecyl sulfate);

TABLE 5 Effect of Metal ions and organic solvents on the enzymatic Activity of Ttbgl3

8. Enzymatic kinetic study of Ttbgl3

The Michaelis constant of Ttbgl3 was determined by the Lineweaver-Burk double reciprocal method using p-NPG as the substrate, with 1/V as the ordinate and 1/[ S ]]Drawing a graph for the abscissa; as shown in FIG. 8, the reaction was carried out under optimum conditions of 50 ℃ and pH6.0 using p-NPG as a substrate, K of Ttbgl3_mA value of 1.04m M, V_maxIt was 263.16. mu.M/mg/min.

Example 4: beta-glucosidase Ttbgl3 transformation experiment for gastrodin, esculin, daidzin and baicalin

1. Reaction system for converting beta-glucosidase Ttbgl3 into glycoside compounds

The reaction system (400 mu L) consists of sodium phosphate buffer solution (with the final concentration of 200mmol/L), substrate (with the final concentration of 0.5mg/mL gastrodin, esculin, daidzin or baicalin) and 80U/mL pure enzyme; after incubating the reaction mixture at 50 ℃ for 12h, the reaction was stopped with 400. mu.L of methanol; adding water into the sample as a negative control;

2. high performance liquid chromatography for determining content and conversion efficiency of glycoside compounds before and after conversion

The instrument comprises the following steps: high performance liquid chromatograph (Shimadzu corporation, Japan, including on-line degasser DGU-20A3R (C), binary pump LC-20AB, autosampler SIL-20A, column oven CTO-20A, detector SPD-20A);

chromatographic conditions are as follows: a chromatographic column: a Unitry C18 column (4.6X 250mm,5 m); the mobile phase is 0.05 percent phosphoric acid water solution (A) -acetonitrile (B), and the elution procedure and the detection wavelength are shown in Table 6; the flow rate is 1mL/min, and the column temperature is 30 ℃;

TABLE 6 elution procedure and detection wavelength for determining the conversion of glycosides by high performance liquid chromatography

By high performance liquid chromatography analysis, as shown in fig. 9-12, it was observed that the reaction system of gastrodin, esculin, daidzin and baicalin all produced new component peaks after Ttbgl3 treatment;

as shown in table 7, 3 substrates including gastrodin, esculin and daidzin were completely hydrolyzed after Ttbgl3 treatment, and the conversion rate reached 100%; baicalin hydrolysis efficiency is slightly low, about 50%; at a substrate concentration of 500. mu.g/mL, the highest conversion was escin (329. mu.g/mL), followed by p-hydroxybenzyl alcohol (228.23. mu.g/mL) and daidzein (78.92. mu.g/mL). Therefore, Ttbgl3 has strong conversion capability on coumarin compounds;

TABLE 7 Ttbgl3 transformation of Gastrodin, esculin, daidzin and baicalin

3. UHPLCESI-Q-TOF-MS determination of product components after glycoside compound conversion

After the beta-glucosidase Ttbgl3 with the concentration of 80U/mL is treated for 12 hours at 50 ℃ and pH6.0, the reaction product is identified by UHPLCESI-Q-TOF-MS, and the content of the product is analyzed by High Performance Liquid Chromatography (HPLC).

As shown in FIGS. 13 to 16, by comparing the relative molecular masses of the respective components, it was found that gastrodine, esculin and daidzin all lost one glucose molecule and were converted into 4-hydroxybenzyl alcohol (4-hydroxybenzyl alcohol M/z:125.1065[ M + H ] +), esculetin (esculetin M/z:177.0145[ M-H ] -) and daidzein (daidzein M/z:253.0438[ M-H ] -). In addition, baicalin loses one glucuronic acid molecule and is converted to baicalein (baicalein M/z:269.2418[ M-H ] -). Through the chemical structure analysis of gastrodin, esculin, daidzin and baicalin, Ttbgl3 is presumed to act on the beta-glucosidic bonds of gastrodin, esculin and daidzin, and then be converted into 4-hydroxybenzyl alcohol, esculetin and daidzein respectively. In addition, the β -glucuronic acid bond in baicalin is cleaved by Ttbgl3 to produce baicalein; ttbgl3 may also act on the β -glucuronic acid bond. The Ttbgl3 is used to deglycosylate baicalin as flavonoid substrate to prepare baicalein as a new compound. However, Ttbgl3 has low hydrolysis efficiency on baicalin. When the substrate concentration is 500 mug/mL and the incubation is carried out for 12h, the hydrolysis rate of baicalin is 49.35 percent and the yield of the product baicalein is 36.22 mug/mL as determined by high performance liquid chromatography.

Example 5: optimization experiment for hydrolysis of beta-glucosyl bond-containing compound by beta-glucosidase Ttbgl3

Through LC-MS analysis, the beta-glucosidase Ttbgl3 is presumed to be capable of hydrolyzing an oxygen-glycoside bond to release a glucose molecule so as to generate a new compound, and in order to further improve the hydrolysis capacity of the Ttbgl3, three compounds (gastrodin, esculin and daidzin) containing the beta-glucoside bond are selected to optimize the conditions of the Ttbgl3 for hydrolyzing the oxygen-glycoside bond; considering the damage of enzyme reaction for a long time, the enzyme reaction temperature is controlled at 50 ℃, and the enzyme concentration and the enzyme reaction time are optimized;

further analyzing the optimal enzyme concentration and the transformation time required by the hydrolysis process by adopting a single-factor analysis method; adding 1U/mL, 5U/mL, 10U/mL, 20U/mL, 40U/mL, 60U/mL and 80U/mL of beta-glucosidase Ttbgl3 into the reaction system at 50 ℃ to determine the optimal dosage of Ttbgl 3; hydrolyzing the Ttbgl3 for 0.5h, 1h, 2h, 4h, 8h and 12h under the optimal enzyme concentration respectively, adding 400 mu L of methanol to terminate the reaction after the reaction is finished, and determining the optimal reaction time;

as shown in FIG. 17, the residual content of the substrate becomes lower as the enzyme concentration increases, esculin and daidzin were almost completely degraded when the enzyme concentration reached 5U/mL, while gastrodine was completely degraded when the enzyme concentration reached 60U/mL. Likewise, new compounds are generated while 3 substrates are hydrolyzed; as shown in FIGS. 17 and 18, after incubation at 50 ℃ for 12h, when the concentration of Ttbgl3 is 60U/mL, gastrodine is hydrolyzed to generate 269.15. mu.g/mL of 4-hydroxybenzyl alcohol with the highest content, so that the optimal enzyme concentration for converting gastrodine is 60U/mL. After 5U/mL Ttbgl3 is added into a hydrolysis system of esculin and daidzin, the generation amount of esculin tends to be stable, and the generation amount of daidzein reaches up to 206.99 mug/mL; in the process of product formation, except 4-hydroxybenzyl alcohol, the yield of the rest products of esculetin and daidzein is gradually reduced after the optimum enzyme concentration is exceeded, so the optimum enzyme concentration for converting esculetin and daidzin is 5U/mL.

Ttbgl3 with the optimal concentration of each substrate is added into the same reaction system to optimize the enzyme reaction time; wherein the optimal enzyme concentration for converting gastrodin is 60U/mL, the optimal enzyme concentration for converting esculin is 5U/mL, and the optimal enzyme concentration for converting daidzin is 5U/mL. As shown in FIG. 19, when the substrate concentration was 500. mu.g/mL, the hydrolysis of glycosides was substantially completed after 2 hours of the reaction. Meanwhile, with the prolonging of the reaction time, the yield of the 4-hydroxybenzyl alcohol is the highest after 12 hours, and reaches 254.04 mu g/mL, and the conversion efficiency is 0.17 mM/h; after 1h, the yield of the daidzein is the highest (360.01 mu g/mL), and the conversion efficiency is 1.47 mM/h; after 2h, the yield of esculetin reached the highest (342.03. mu.g/mL) with a conversion efficiency of 0.96 mM/h. The rate of hydrolysis of the substrate is inversely proportional to the yield of product, being the most efficient within 2 hours after the reaction and then tending to be flat;

the optimal transformation parameters for the three natural compounds were finally determined: 500 mu g/mL of gastrodin, 360/mL of Ttbgl 360U, and incubation for 12h at 50 ℃, wherein the conversion rate of the p-hydroxybenzyl alcohol is 0.17 mM/h; esculin 500 μ g/mL, Ttbgl 35U/mL, incubation for 2h at 50 deg.C, esculetin conversion rate is 0.96 mM/h; the daidzin is incubated for 1h at 50 ℃ with 500 mu g/mL and Ttbgl 35U/mL, and the conversion rate of daidzein is 1.47 mM/h.

Example 6: beta-glucosidase Ttbgl3 homology modeling and molecular docking

1. Molecular docking process

(1) Ligand treatment: downloading a Catalpol structure file on Pubchem, processing the Catalpol structure file by Mgltools1.5.6, calculating charges by hydrogenation, merging nonpolar hydrogen and storing the nonpolar hydrogen into a pdbqt file;

(2) receptor treatment: processing the protein structure of the modeled receptor protein by Mgltools1.5.6, storing the protein structure into a pdb file after hydrogenation, charge calculation and nonpolar hydrogen combination, and finally storing the pdb file into a pdbqt file

(3) Determination of active site: uploading the modeling structure to POCASA (http:// altair. sci. hokudai. ac. jp/g6/service/POCASA /) to predict the potential binding site, and setting the Grid Box coordinate and the Box size according to the potential binding site;

(4) plotting: docking of small molecules to proteins was performed using AutodockVina 1.1.2, yielding 9 conformations in total, selecting the best conformation for affinity as the final docking conformation. And analyzed using a Meastro 11.9 plot;

2. homologous modeling and molecular docking

The structural model is shown in FIG. 20; establishing a three-dimensional structure of Ttbgl3 by adopting Swiss-model database online homologous modeling; the quaternary structure quality evaluation (QSQSQSQSQSQSQSQSQSQS) score is 0.4, and the sequence re-matching degree is 41.73, which shows that the structure model is reliable;

analyzing the binding sites of the different substrates to Ttbgl3 by molecular docking procedures; calculating the binding energy of the protein and the small molecule by using Autodockvina 1.1.2 software, and finding that the absolute values of the binding constants of gastrodin, esculin, daidzin and baicalin are 7.9, 8.2, 9.1 and 9.6 respectively after the docking is finished as shown in table 8; the larger the absolute value of the binding constant, the less free energy is required for binding of the compound, and it is considered that when the absolute value thereof is larger than 7, the greater the possibility of binding both the small molecule compound and the protein; as can be seen in fig. 21, the small molecules and the protein surface are bound more tightly; the two have previously been in better match, providing the possibility for small molecules to bind thereto. Although the compounds vary in structure, binding to enzymes is primarily through hydrophobic and hydrogen bonding; table 8 summarizes docking conditions of 4 natural compounds, wherein 3 substrates (including gastrodin, esculin and daidzin) containing beta-glucuronide bonds all contain catalytic sites of His85 and Lys467, and baicalin containing substrates containing beta-glucuronide bonds has catalytic sites of Glu377 and Thr 424;

TABLE 8 molecular docking enzyme binding site prediction

Sequence listing

<110> university of Kunming science

<120> beta-glucosidase Ttbgl3 and application thereof

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 849

<212> PRT

<213> Thermotoga lanuginosus S0301(Trametes trogii S0301)

<400> 1

Met Ser Arg Asp Phe Leu Asp Val Asp Ile Pro Asp Leu Val Ser Lys

1 5 10 15

Leu Thr Thr Asp Glu Lys Ile Ala Leu Leu Ala Gly Pro Asn Trp Trp

20 25 30

Asn Thr Asn Ala Ile His Arg Leu Gly Val Pro Ala Val Arg Met Ser

35 40 45

Asp Gly Pro Asn Gly Val Arg Gly Ser Ser His Phe Val Ser Thr Pro

50 55 60

Ala Gln Cys Leu Pro Cys Ala Thr Ser Met Ala Ser Thr Phe Asp Ile

65 70 75 80

Asp Met Leu Tyr Glu Val Gly Thr Phe Leu Gly Glu Glu Ala Lys Ile

85 90 95

Lys Ser Ser Val Ile Leu Leu Ala Pro Thr Cys Asn Ile Gln Arg Asn

100 105 110

Pro Leu Gly Gly Arg Ala Phe Glu Ser Phe Ser Glu Asp Pro His Leu

115 120 125

Ser Gly Met Met Ala Ala Ala Tyr Val Glu Gly Leu Gln Ser Arg Gly

130 135 140

Val Ala Ser Thr Ile Lys His Phe Val Ala Asn Asp Gln Glu His Glu

145 150 155 160

Arg Thr Ala Ala Glu Ser Val Met Ser Asp Arg Ala Leu Arg Glu Val

165 170 175

Tyr Leu Tyr Pro Phe Met Leu Ala His Lys Lys Ala Asn Pro Trp Ala

180 185 190

Phe Met Thr Ser Tyr Gly Arg Ile Asn Gly Val His Cys Ala Glu Asn

195 200 205

Pro Met Leu Leu Gln Asp Ile Leu Arg Lys Glu Trp Gly Phe Asp Gly

210 215 220

Ile Val Met Ser Asp Trp Phe Gly Thr Tyr Ser Val Asp Leu Ala Ile

225 230 235 240

Asn Ala Gly Leu Asp Leu Glu Met Pro Gly Pro Pro Arg Trp Arg Thr

245 250 255

Pro Leu Leu Val Lys His Met Leu Ser Cys Gln Lys Val Thr Asp Asp

260 265 270

Thr Leu Asp Glu Arg Ala Thr His Leu Leu Arg Phe Val Gln Arg Gln

275 280 285

Ala Arg Arg Asn Pro Asp Val Val Phe Gly Asp Gly Gln Glu Arg Thr

290 295 300

Arg Asp Ser Pro Glu Gly Arg Leu Phe Cys Arg Arg Leu Ala Ala Glu

305 310 315 320

Gly Met Ile Val Leu Lys Asn Asn Gly Asp Val Leu Pro Leu Lys Ala

325 330 335

Glu Lys Val Lys Thr Ile Ala Val Ile Gly Pro Asn Ala Lys Glu Arg

340 345 350

Val Val Ser Gly Gly Gly Ser Ala Ala Leu Lys Ala Ser Tyr Ile Ile

355 360 365

Thr Pro Tyr Ala Gly Leu Val Asp Asn Ala Pro Lys Gly Val Glu Ile

370 375 380

Lys Tyr Glu Leu Gly Cys Tyr Ala Tyr Lys Tyr Thr Pro Thr Leu Glu

385 390 395 400

Thr Tyr Leu Lys Thr Pro Ser Gly Asn Ala Gly Trp Leu Cys Ser Phe

405 410 415

Tyr Asn His Asp Glu Asp Gly Asn Pro Thr Gly Glu Thr Val Gln Glu

420 425 430

Thr Val Leu Gln Asp Thr Arg Val Lys Leu Asn Asp Phe Leu Pro Ile

435 440 445

Gly Leu Thr Glu Thr Trp Thr Ile Lys Leu Arg Gly Ser Leu Thr Met

450 455 460

Glu Lys Thr Ala Glu Tyr Gln Leu Gly Leu Thr Val Ala Gly Arg Ala

465 470 475 480

Lys Leu Phe Val Asn Gly Glu Leu Ile Ile Asp Asn Trp Thr Lys Gln

485 490 495

Arg Pro Gly Asp Phe Phe Tyr Gly Gln Gly Thr Val Glu Glu Met Gly

500 505 510

Thr Val Ser Leu Thr Ala Gly Lys Pro Val Asp Ile Leu Val Glu Tyr

515 520 525

Thr Asn Thr Lys Pro Pro Gly Pro Glu Ala Asp Arg Ser Gln Pro Ala

530 535 540

Leu Met Arg Gly Val Arg Leu Gly Gly Cys Glu Lys Ile Asp Pro Glu

545 550 555 560

Glu Ala Met Val Ala Ala Glu Thr Leu Ala Ala Ala Ser Asp Ala Val

565 570 575

Val Ile Val Ala Gly Leu Ser Pro Asp Trp Glu Ser Glu Gly Phe Asp

580 585 590

Arg Pro Thr Leu His Met Pro Gly Lys Gln Asp Glu Leu Ile Ala Arg

595 600 605

Val Ala Lys Ala Asn Pro Lys Thr Val Val Cys Val Gln Ala Gly Ser

610 615 620

Ala Val Ala Met Pro Trp Val His Asp Val Ser Gly Ile Ile Gln Ala

625 630 635 640

Trp Tyr Ser Gly Asn Glu Val Gly Asn Ala Leu Ser Asp Val Val Tyr

645 650 655

Gly Ala Ile Asn Pro Ser Gly Arg Leu Pro Leu Thr Leu Pro Val Arg

660 665 670

Val Glu Asp Ile Pro Ala Tyr Pro Asn Phe Arg Ser Glu Asn Gly Gln

675 680 685

Ile His Tyr Arg Glu Asp Leu Phe Val Gly Tyr Lys Gly Tyr Ala Ala

690 695 700

Lys Gly Val Lys Pro Leu Phe Pro Phe Gly His Gly Leu Ser Tyr Thr

705 710 715 720

Thr Phe Ser Phe Ser Asn Leu Arg Val Ser Ala Ser Ser Ser Arg Lys

725 730 735

Gly Pro Asp Phe Glu Leu Asp Val Ala Val Thr Val Thr Asn Thr Gly

740 745 750

Ser Val Pro Gly Ser Glu Val Val Gln Val Tyr Val Ser Leu Pro Glu

755 760 765

Phe Gly Leu Thr Thr Pro Lys Leu Gln Leu Arg Gly Phe Ala Lys Ala

770 775 780

Arg Asp Ile Ala Pro Gly Gln Ser Lys Thr Val Ser Val Lys Leu Asp

785 790 795 800

Lys Tyr Ala Ile Ser Trp Trp Asp Val Arg Gly His Gln Trp Lys Val

805 810 815

Val Pro Gly Lys Tyr Gly Leu His Ile Gly Lys Ser Ser Ala Asp Ile

820 825 830

Val Leu Glu Gly Gly Phe Ala Leu Gln Glu Gly Phe Thr Trp Asn Gly

835 840 845

Val

<210> 2

<211> 2547

<212> DNA

<213> Thermotoga lanuginosus S0301(Trametes trogii S0301)

<400> 2

atgtcgcgcg acttcctcga tgttgacatt cccgacctcg tcagtaaact caccaccgac 60

gagaagatcg cccttcttgc aggtcccaat tggtggaata ccaacgccat tcaccggctt 120

ggcgtccctg ccgttcgtat gagcgacggc ccaaacggag ttcgcggctc ctctcacttc 180

gtttccacgc ctgcacaatg ccttccttgc gctacttcaa tggcgtccac attcgacatc 240

gacatgctct acgaagtcgg gaccttcctg ggtgaagaag ccaagatcaa gtcgtctgtg 300

atcttgctcg cacctacttg caacatacaa cgtaacccct taggagggcg ggcctttgag 360

tctttctctg aagaccccca tttatccggt atgatggctg ctgcgtatgt cgagggcctt 420

cagtcccggg gtgtagcttc gacgatcaag cactttgttg caaatgacca ggaacacgaa 480

cggacagctg cagagtctgt catgtccgat cgtgctctac gcgaggtcta cctttatccc 540

tttatgctgg cccacaagaa ggccaatcca tgggccttta tgacatccta cggaagaatc 600

aatggcgtgc attgcgcaga aaacccgatg ctgctccagg atatcttgcg gaaagaatgg 660

ggcttcgatg gcatagtcat gagcgactgg ttcggcacgt acagcgtcga tctggctata 720

aatgcaggtc tagacctgga gatgcccggc ccgcctcgtt ggcggactcc gcttcttgtg 780

aagcacatgc tgtcgtgcca gaaggtcaca gacgacacac tcgacgagcg cgcgacgcac 840

cttcttcgct tcgtgcaacg ccaggctcgc cggaatcccg atgtggtgtt cggtgacgga 900

caggagcgca cacgcgattc gccagagggc cgcctatttt gcaggagact cgctgctgag 960

gggatgatcg tgctcaagaa caatggcgac gtgctgccgt tgaaggcaga gaaggtaaag 1020

accattgcgg tgattggacc caatgccaag gagcgagtcg tttccggcgg tgggtctgcg 1080

gctttgaagg ccagctacat catcacaccg tatgcaggct tagtcgacaa cgccccgaaa 1140

ggtgtagaga taaagtacga gctgggatgt tacgcttaca agtacacacc gactttggag 1200

acctacctga agacaccttc cggaaacgcg gggtggctct gcagcttcta caaccacgac 1260

gaggacggta accccactgg cgaaacggtt caggagaccg ttctgcaaga cactcgtgtc 1320

aaactgaatg acttcttgcc cattggtctg acagagacat ggactatcaa actccgcggc 1380

agcctcacaa tggagaagac agcagagtat cagctcggac tgacagtcgc aggccgcgcg 1440

aagctgttcg tcaatggcga attgatcatc gacaactgga cgaagcagcg tccgggagac 1500

ttcttctacg ggcagggcac tgtggaagag atggggacag tctctctgac tgctggcaag 1560

ccggtcgaca ttttggtcga gtacacaaac acgaagcctc caggacccga ggcagatcga 1620

tcccagcctg cactgatgcg cggagtccgc ctcggtggct gcgagaagat cgacccagag 1680

gaagcaatgg tcgccgccga aacacttgcg gccgcgtccg acgcggtcgt catagttgct 1740

ggactgtctc cagactggga gagcgaaggt ttcgacaggc cgactctgca catgccaggg 1800

aaacaggacg agctcatcgc gcgtgttgca aaggcgaacc ccaagacggt cgtgtgcgtg 1860

caggcgggct ccgcagtcgc gatgccatgg gttcacgacg tgagcggcat catccaagcc 1920

tggtactcgg gcaacgaagt cgggaacgcg ctctccgacg tcgtctacgg cgcgatcaac 1980

cccagcggcc gccttccgct caccctccct gtgcgtgtgg aggacatccc tgcgtacccg 2040

aacttcagga gtgaaaatgg ccagattcat tatcgtgagg acctctttgt gggctacaag 2100

gggtacgccg cgaagggtgt aaagcctctc ttcccctttg gccacgggtt gtcgtacacg 2160

accttctcgt tctcgaacct gcgcgtgtcc gcctcgtcga gccggaaagg accggatttc 2220

gagctcgatg ttgcagttac cgtcacaaac actggctctg tccctggctc ggaagtcgtc 2280

caggtctacg tttcacttcc cgagttcggt ttgaccactc ctaaactaca gcttcgtgga 2340

ttcgccaagg cgcgcgacat tgccccagga cagagcaaga cggtatccgt caagttggac 2400

aagtatgcca tttcctggtg ggacgtgcgc ggtcatcagt ggaaggtcgt tcccggcaag 2460

tacggactgc acattggaaa gagcagcgca gacattgttt tagagggcgg attcgcatta 2520

caggaaggat tcacatggaa cggggtg 2547

<210> 3

<211> 40

<212> DNA

<213> Artificial sequence (Artificial)

<400> 3

caagcttgcg gccgctctag aatgtcgcgc gacttcctcg 40

<210> 4

<211> 41

<212> DNA

<213> Artificial sequence (Artificial)

<400> 4

gtggtggtgg tggtgctcga gcaccccgtt ccatgtgaat c 41

Claims

1. The amino acid sequence of the beta-glucosidase Ttbgl3 is shown in SEQ ID NO. 1.

2. The gene coding the beta-glucosidase Ttbgl3 of claim 1, the nucleotide sequence of which is shown in SEQ ID NO. 2.

3. Use of the β -glucosidase Ttbgl3 of claim 1 for the preparation of rare aglycone and monoglycoside compounds: beta-glucosidase Ttbgl3 can hydrolyze gastrodine, esculin and daidzin, and lose a glucose molecule, and respectively convert into 4-hydroxybenzyl alcohol, esculetin and daidzein; can hydrolyze baicalin to lose one glucuronic acid molecule and convert into baicalein.