A beta-glucosidase Bgl-Gs2, a coding gene and application thereof

Abstract

The application discloses beta-glucosidase Bgl-Gs2, a coding gene and application thereof, and belongs to the technical field of genetic engineering and enzyme engineering. The application first mines and discloses beta-glucosidase Bgl-Gs2 derived from Ganoderma applanatum, and the amino acid sequence of the enzyme is shown as SEQ ID NO:1. The enzyme can specifically break the terminal glucose glycosidic bond of mogroside V, and directionally catalyze the generation of mogroside IV and siamenoside I, and the conversion rate can reach more than 55.39%, and the comprehensive yield of the target product is considerable. The enzyme catalysis reaction system condition is mild, the production energy consumption is low, no pollutants are generated, the reaction product is convenient for subsequent separation and purification, and the development concept of green processing is met. The enzyme can efficiently prepare high-value mogroside IV and siamenoside I at a low cost, effectively improves the economic benefits of deep processing products of Siraitia grosvenorii, and has a good popularization and application prospect in the food, health food and pharmaceutical industries.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and enzyme engineering technology, and relates to a β-glucosidase Bgl-Gs2 and its encoding gene and applications. In particular, it relates to a β-glucosidase encoding gene, recombinant expression vector, and recombinant strain derived from Ganoderma lucidum sessile, as well as the application of the β-glucosidase encoded by this gene in the catalytic directed hydrolysis of mogroside V to prepare mogroside IV and symmenidine I. Background Technology

[0002] Mogrosides are the main flavor and functional active components of monk fruit, classified as cucurbitane-type triterpenoid saponins. Among them, mogroside V has the highest abundance among natural components, with a sweetness up to 300 times that of sucrose, and is now widely used in the food, health food, and pharmaceutical industries. Secondary glycosides such as mogroside IV and symmenidine I possess superior sensory characteristics, with a refreshing taste and a slight bitter aftertaste, showing outstanding application potential in high-end sweeteners, functional food development, and new drug research; however, these substances are scarce in natural monk fruit raw materials, and the separation and purification process is cumbersome and the yield is low, making large-scale industrial production impossible.

[0003] Currently, the preparation routes of secondary glycosides from monk fruit are mainly divided into two categories: chemical hydrolysis and enzymatic hydrolysis. Chemical hydrolysis has harsh reaction conditions, is prone to inducing various side reactions, produces complex products, and is difficult to separate and purify, while also posing environmental challenges. Enzymatic hydrolysis, with its advantages of mild reaction conditions, excellent catalytic selectivity, high purity of the target product, and green and low-carbon process, is gradually becoming the preferred technology route for development.

[0004] Current β-glucosidases used for the conversion of mogrosides generally have shortcomings: poor substrate recognition specificity, limited directional catalytic conversion efficiency, difficulty in precisely controlling the product component ratio, and narrow thermal stability and acid-base tolerance ranges, making them unsuitable for the industrial-scale production of mogroside IV and sympathomimetic I. While existing literature and patents document research on the conversion of mogroside V to mogroside IV using β-glucosidases, most utilize commercially available compound enzymes or known enzyme resources already reported in studies. Currently, there is a lack of novel β-glucosidase genes capable of specifically catalyzing mogroside V, achieving efficient directional conversion, and simultaneously enriching mogroside IV and sympathomimetic I. Furthermore, there are no reports of related new genes derived from *Ganoderma lucidum*. Summary of the Invention

[0005] To overcome the shortcomings and deficiencies of existing technologies, such as poor selectivity of β-glucosidase for the hydrolysis of mogroside V, low efficiency in the targeted preparation of mogroside IV and symmenidine I, and lack of novel gene resources, the present invention aims to provide a β-glucosidase Bgl-Gs2, its encoding gene, and its application in the targeted transformation of mogroside V. This enzyme exhibits high substrate specificity and high catalytic efficiency for mogroside V.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] This invention provides a β-glucosidase Bgl-Gs2, which was cloned from sessile Ganoderma lucidum, and its amino acid sequence is shown in SEQ ID NO: 1.

[0008] The present invention also provides a gene encoding the above-mentioned β-glucosidase Bgl-Gs2, the nucleotide sequence of which is shown in SEQ ID NO: 2 or SEQ ID NO: 3.

[0009] The present invention also provides a biomaterial related to the above-mentioned β-glucosidase Bgl-Gs2, which is any one or more combinations of the following biomaterials:

[0010] (a) An expression cassette containing the above-mentioned encoded genes;

[0011] (b) Recombinant expression vectors containing the above-mentioned coding genes;

[0012] (c) A recombinant expression vector containing the expression cassette described in (a);

[0013] (d) Recombinant strains containing the above-mentioned encoding genes;

[0014] (e) A recombinant strain containing the expression cassette described in (a);

[0015] (f) A recombinant strain containing the recombinant expression vector described in (b) or (c).

[0016] Furthermore, the starting vector of the recombinant expression vector described in (b) and (c) is a vector suitable for prokaryotic or eukaryotic expression;

[0017] Furthermore, the vector suitable for prokaryotic expression is a vector suitable for Escherichia coli expression, preferably a pET series vector, etc.; more preferably a pET-28a(+) vector.

[0018] Furthermore, the vector suitable for eukaryotic expression is a vector suitable for yeast expression; preferably a vector suitable for Pichia pastoris expression; more preferably a pPIC series vector, and even more preferably a pPIC9K vector.

[0019] Furthermore, the host bacteria corresponding to the recombinant strains described in (d), (e), and (f) are selected from prokaryotes, yeasts, or higher eukaryotic cells; the prokaryotes include bacteria such as *Escherichia* and *Bacillus*; the yeasts include yeasts such as *Komagataella phaffii* and *Saccharomyces cerevisiae*. More specifically, the prokaryote is *Escherichia coli* (E. coli), specifically *Escherichia coli* BL21(DE3) or *Escherichia coli* DH5α; the yeast is *Pichia pastoris* GS115.

[0020] This invention also provides an application of the above-mentioned β-glucosidase Bgl-Gs2, encoding gene, and biological material, which is one of the following applications:

[0021] (I) Application in the preparation of β-glucosidase Bgl-Gs2;

[0022] (II) Application in the directed catalysis of mogroside V.

[0023] Furthermore, application (II) is used in the directed catalytic preparation of mogroside V into mogroside IV and / or symmenoside I.

[0024] The present invention also provides a method for preparing mogroside IV and / or symbioside I, comprising the step of using mogroside V as a substrate and adding the above-mentioned β-glucosidase Bgl-Gs2 for catalytic reaction.

[0025] Specifically, the steps include the following:

[0026] The above recombinant strains were cultured, and induction reagents were added to induce expression. The bacterial cells were then collected.

[0027] The bacterial cells were broken down and purified to obtain recombinant β-glucosidase Bgl-Gs2;

[0028] Using mogroside V as a substrate, mogroside IV and / or symbioside I were prepared by directional catalysis of the substrate with the above-mentioned recombinant β-glucosidase Bgl-Gs2.

[0029] Preferably, the culture conditions are 35–37°C, 200–250 rpm shaking culture until OD. 600 =0.6-0.8, the inducing reagent is IPTG, the final concentration is 0.2-1.0 mM, induce at 16-25℃ for 12-20 h, and collect the bacterial cells.

[0030] More preferably, the culture conditions are 37°C and 220 rpm shaking culture until OD. 600=0.6-0.8, the inducing reagent was IPTG, the final concentration was 0.2 mM, the induction was carried out at 16℃ for 12 h, and the bacterial cells were collected.

[0031] Preferably, the purification of the bacterial cells after disruption is carried out by Ni-NTA affinity chromatography.

[0032] Preferably, the concentration of mogroside V is 1.0–10.0 mg / mL, and the amount of recombinant β-glucosidase Bgl-Gs2 added is 200–3000 U / g substrate.

[0033] Furthermore, the concentration of mogroside V was 5.0–6.0 mg / mL, and the amount of recombinant β-glucosidase Bgl-Gs2 added was 1500–2000 U / g substrate.

[0034] Preferably, the catalytic conditions are a reaction at 40–60°C for 2–12 h; more preferably, a reaction at 40–50°C for 2–12 h.

[0035] Preferably, the catalytic pH is 3–8; further, it is 4–6; and even further, it is 5.

[0036] The present invention has the following advantages and effects compared with the prior art:

[0037] (1) Novel gene resources: This study is the first to discover and disclose the β-glucosidase encoding gene Bgl-Gs2 from Ganoderma lucidum, filling the gap in specific catalytic gene resources in this direction; at the same time, the efficient soluble expression of this gene in the expression host was completed, and a recombinant β-glucosidase with industrial development potential was obtained.

[0038] (2) Excellent substrate selectivity: This recombinase can specifically break the terminal glucose glycosidic bond of mogroside V and directionally catalyze the generation of mogroside IV and symmenoside I. The amount of by-products generated is low, and the product component ratio can be controlled.

[0039] (3) Excellent conversion efficiency: After optimization of the reaction system conditions, the conversion efficiency of substrate mogroside V can reach more than 55.39%, and the overall yield of the target product is considerable, with potential for process scale-up.

[0040] (4) Green process characteristics: The enzyme catalytic reaction system has mild conditions, with a suitable reaction range of 40-60 ℃ and pH 5.0. The production energy consumption is low, no pollutants are generated, and the reaction products are easy to separate and purify, which is in line with the concept of green processing development.

[0041] (5) Outstanding industrialization potential: It can efficiently prepare high-value-added mogroside IV and symmenoside I at a low cost, effectively improving the economic benefits of deep-processed mogroside products, and has good prospects for promotion and application in the food, health food and pharmaceutical industries. Attached Figure Description

[0042] Figure 1 This is an electrophoresis diagram of the PCR amplification of the β-glucosidase gene Bgl-Gs2 of this invention.

[0043] Figure 2 This is an SDS-PAGE electrophoresis image of recombinant β-glucosidase Bgl-Gs2; lane M: protein marker 0-180 kDa; lane 1: induced and purified protein.

[0044] Figure 3 The graph shows the results of the optimal pH measurement.

[0045] Figure 4 The graph shows the results of the measurement at the optimal temperature.

[0046] Figure 5 The HPLC chromatograms are for the hydrolysis of mogroside V by recombinant β-glucosidase Bgl-Gs2 to generate mogroside IV and sympathomimetic acid I; where MG-SI standard refers to sympathomimetic acid I standard, MG-IV standard refers to mogroside IV standard, and MG-V standard refers to mogroside V standard. Detailed Implementation

[0047] The present invention will be further described in detail below with reference to embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto. Test methods in the following embodiments that do not specify specific experimental conditions are generally performed under conventional experimental conditions or according to the manufacturer's recommended experimental conditions. Unless otherwise specified, the materials and reagents used are commercially available.

[0048] The sessile Ganoderma involved in the examples is Ganoderma sessile, which is a conventional commercially available product.

[0049] Example 1: Cloning of the β-glucosidase gene Bgl-Gs2 from Ganoderma lucidum

[0050] (1) Select sessile Ganoderma lucidum strains with mogroside V hydrolysis activity after screening, inoculate them into PDA liquid medium, and culture at 28℃ and 180 rpm for 4-6 days.

[0051] (2) Collect mycelium, extract total RNA using the Trizol method, and after testing its purity and integrity, reverse transcribe to synthesize cDNA.

[0052] (3) Based on the transcriptome sequencing results, specific primers were designed, and PCR amplification was performed using cDNA as a template to obtain the target gene fragment. The electrophoresis results are as follows: Figure 1 As shown.

[0053] (4) The PCR product was recovered by gel, ligated into a cloning vector, transformed into Escherichia coli DH5α, and the positive clone was sequenced and verified to obtain the β-glucosidase gene Bgl-Gs2 with the nucleotide sequence shown in SEQ ID NO: 2.

[0054] The β-glucosidase Bgl-Gs2 has a length of 954 amino acids, and its amino acid sequence is shown in SEQ ID NO: 1.

[0055] 。

[0056] The gene encoding β-glucosidase Bgl-Gs2 is 2865 bp in length, and its nucleotide sequence is shown in SEQ ID NO: 2:

[0057]

[0058] Example 2: Construction of recombinant expression vector and recombinant strain

[0059] (1) The β-glucosidase Bgl-Gs2 obtained in Example 1 was optimized by E. coli codon optimization to obtain the optimized gene sequence, the nucleotide sequence of which is shown in SEQ ID NO: 3; then BamH1 and XhoI restriction sites were introduced at both ends of the gene sequence, and the Bgl-Gs2 gene was double-digested with restriction endonucleases (BamH1 / XhoI) and ligated with expression vector pET-28a(+) and T4 ligase to construct recombinant plasmid pET28a-Bgl-Gs2.

[0060] The nucleotide sequence optimized using E. coli codons is shown in SEQ ID NO: 3:

[0061]

[0062] (2) The recombinant plasmid was chemically transformed into Escherichia coli BL21(DE3) competent cells, plated on LB agar plates containing 25 μg / mL kanamycin, and incubated overnight at 37°C.

[0063] (3) Colony PCR and sequencing verification yielded a positive recombinant Escherichia coli expression strain, denoted as BL21(DE3) / pET28a-Bgl-Gs2.

[0064] Example 3: Induction and preparation of recombinant β-glucosidase

[0065] (1) Pick a single positive colony and inoculate it into LB liquid medium, and culture at 37°C and 220 rpm with shaking until OD. 600 ≈0.6-0.8.

[0066] (2) Add IPTG to a final concentration of 0.2 mM and induce recombinase expression at 16℃ for 12 h.

[0067] (3) Collect the bacterial cells by centrifugation, resuspend them in buffer solution, sonicate to disrupt them, centrifuge to collect the supernatant, and obtain the crude enzyme solution of recombinant β-glucosidase.

[0068] (4) The recombinant β-glucosidase was purified by Ni-NTA affinity chromatography and subjected to SDS-PAGE protein electrophoresis. The results are as follows: Figure 2 As shown, the protein size is consistent with the theoretical value (104.94 kDa).

[0069] Example 4: Determination of optimal pH and optimal temperature for recombinant β-glucosidase

[0070] 1. Experimental materials

[0071] Enzyme solution: Recombinant β-glucosidase Bgl-Gs2 from Escherichia coli purified in Example 3;

[0072] Reaction substrate: p-nitrophenyl-β-D-glucoside (pNPG);

[0073] Gradient buffers: pH 3.0–5.0: 50 mM citrate-sodium citrate buffer, pH 5.0–7.0: 50 mM acetate-sodium acetate buffer, pH 7.0–9.0: 50 mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer.

[0074] Termination solution: 1 mol / L Na2CO3 solution;

[0075] Testing instruments: constant temperature water bath, ultraviolet spectrophotometer (detection wavelength 405 nm).

[0076] 2. Optimal pH Measurement Procedure

[0077] Prepare gradient pH buffer solutions: 3.0, 4.0, 5.0, 6.0, 7.0, 8.0;

[0078] Total reaction system 1 mL: 800 μL of corresponding pH buffer + 100 μL of 5 mM pNPG substrate solution, 3 replicates per group, and the blank control group was replaced with buffer solution instead of enzyme solution;

[0079] Add 100 μL of purified recombinant enzyme solution to each group, mix well, and incubate in a 50 ℃ water bath for 120 min.

[0080] Immediately after the incubation period, add 500 μL of 1 mol / L Na2CO3 to terminate the enzymatic reaction;

[0081] The absorbance (OD) values ​​of each group were measured at a wavelength of 405 nm, and the relative enzyme activity under different pH conditions was calculated.

[0082] Enzyme activity is defined as: the amount of enzyme required to catalyze the production of 1 μmol of product per minute under optimal reaction conditions = 1 U.

[0083] The highest measured enzyme activity was set as 100%, and the enzyme activities at other pH values ​​were converted to relative activities. A pH-relative enzyme activity curve was then plotted. Figure 3 As shown, the results indicate that the optimal reaction pH for β-glucosidase Bgl-Gs2 is 5, the relative enzyme activity at pH 3-8 can reach over 50%, and the relative enzyme activity at pH 4-6 can reach over 60%.

[0084] 3. Procedure for determining the optimal temperature

[0085] The reaction system was constructed using a uniform 50 mM, pH 5.0 citrate-disodium hydrogen phosphate buffer solution;

[0086] Set temperature gradients: 30 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 70 ℃, 80 ℃, with 3 parallel groups for each group;

[0087] 1 mL reaction system: 800 μL buffer + 100 μL 5 mM pNPG + 100 μL recombinant enzyme solution, incubated in a water bath at the corresponding temperature for 120 min;

[0088] The reaction was terminated by adding Na2CO3, the OD value was measured at 405 nm, and the relative enzyme activity at each temperature was calculated.

[0089] Plot a temperature-relative enzyme activity curve with the maximum enzyme activity value as 100%, as follows: Figure 4As shown, the results indicate that the optimal reaction temperature for β-glucosidase Bgl-Gs2 is 45 ℃, the relative enzyme activity can reach over 70% at 40–60 ℃, and the relative enzyme activity can reach over 90% at 40–50 ℃.

[0090] Example 5: Preparation of mogroside IV and symbioside I by recombinase catalysis of mogroside V

[0091] (1) Prepare a 50 mM citrate-disodium hydrogen phosphate buffer (pH 5.0) and add mogroside V to a final concentration of 6.0 mg / mL.

[0092] (2) The recombinant β-glucosidase Bgl-Gs2 prepared in Example 3 was added at a concentration of 1740 U / g substrate. The treatment without enzyme addition served as the control group.

[0093] (3) Place in a 45℃ water bath for 2 h and shake gently during the reaction.

[0094] (4) The reaction was terminated by boiling in a water bath for 10 min, the supernatant was collected by centrifugation and analyzed by HPLC. Each group had 3 parallel experiments.

[0095] The HPLC detection conditions were as follows: Column: Agilent Eclipse XDB C18 column (4.6 mm × 250 mm, 5 μm); water as phase A, methanol as phase B, gradient elution (0 → 5 min, methanol ratio increased from 5% to 15%; 5 → 10 min, increased to 20%; 10 → 15 min, increased to 25%; 15 → 20 min, increased to 30%; 20 → 25 min, increased to 35%; 25 → 30 min, decreased to 30%; 30 → 35 min, decreased to 25%; 35 → 40 min, decreased to 20%; 40 → 45 min, decreased to 15%; 45 → 50 min, decreased to 5%), flow rate: 0.500 mL / min; column temperature limit: 35℃; detection wavelength: 203 nm; injection volume: 10 μL; system pressure limit: 200 bar.

[0096] The results are as follows Figure 5 As shown, mogroside V was successfully converted, with mogroside IV and symbioside I as the main products, very few byproducts, high total purity of the target product, and a conversion rate of 55.39%.

[0097] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A β-glucosidase Bgl-Gs2, characterized in that: The amino acid sequence of the β-glucosidase Bgl-Gs2 is shown in SEQ ID NO:

1.

2. A gene encoding the β-glucosidase Bgl-Gs2 of claim 1.

3. The gene according to claim 2, characterized in that: The nucleotide sequence of the gene encoding β-glucosidase Bgl-Gs2 is shown in SEQ ID NO: 2 or SEQ ID NO:

3.

4. The biomaterial related to β-glucosidase Bgl-Gs2 as described in claim 1, characterized in that, It can be any one or more combinations of the following biological materials: (a) An expression cassette containing the gene of claim 2 or 3; (b) A recombinant expression vector containing the gene of claim 2 or 3; (c) A recombinant expression vector containing the expression cassette described in (a); (d) A recombinant strain containing the gene described in claim 2 or 3; (e) A recombinant strain containing the expression cassette described in (a); (f) A recombinant strain containing the recombinant expression vector described in (b) or (c).

5. The biomaterial according to claim 4, characterized in that: The starting vector for the recombinant expression vectors described in (b) and (c) is a vector suitable for expression in prokaryotes or eukaryotes; And / or, the host bacteria corresponding to the recombinant strains described in (d), (e), and (f) are selected from prokaryotes, yeast, or higher eukaryotic cells.

6. The use of the gene according to any one of claims 2 to 3 or the biomaterial according to any one of claims 4 to 5 in the preparation of β-glucosidase Bgl-Gs2.

7. The application of the β-glucosidase Bgl-Gs2 of claim 1, the gene of any one of claims 2 to 3, or the biomaterial of any one of claims 4 to 5 in the directed catalysis of mogroside V.

8. The application according to claim 7, characterized in that: The application is in the directed catalytic preparation of mogroside V into mogroside IV and / or symmenidine I.

9. A method for preparing mogroside IV and / or symbioside I, characterized in that: The method includes the step of using mogroside V as a substrate and adding the β-glucosidase Bgl-Gs2 described in claim 1 to catalyze the reaction.

10. The method according to claim 9, characterized in that, Specifically, the steps include the following: The recombinant strain according to any one of claims 4 to 5 is cultured, and an inducing reagent is added to induce expression, and the bacterial cells are collected. The bacterial cells were broken down and purified to obtain recombinant β-glucosidase Bgl-Gs2; Using mogroside V as a substrate, recombinant β-glucosidase Bgl-Gs2 was added to catalyze the substrate to prepare mogroside IV and / or symbioside I.

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