Beta-glucosidase bgl-gs1 from ganoderma boninense and application thereof

Abstract

The application discloses a beta-glucosidase Bgl-Gs1 from Ganoderma applanatum and an application thereof, and belongs to the technical field of genetic engineering and enzyme engineering. The application obtains a beta-glucosidase Bgl-Gs1 from Ganoderma applanatum, and the amino acid sequence of the beta-glucosidase Bgl-Gs1 is shown as SEQ ID NO: 2. The enzyme can efficiently and specifically catalyze the directional hydrolysis of mogroside V to generate mogroside IV and siamenoside I, the conversion rate can be higher than 34.58 %, the total yield of target products is high, and the enzyme is suitable for scale-up production. The enzymatic reaction condition is mild, energy consumption is low, there is no pollution, the product is easy to separate and purify, and the green manufacturing requirement is met. In conclusion, the enzyme can efficiently, mildly and at low cost prepare high-value mogroside IV and siamenoside I, significantly improves the added value of deep processing of momordica grosvenori, and has a wide industrialization prospect in the fields of food, health products and medicine.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and enzyme engineering technology, and relates to a β-glucosidase Bgl-Gs1 derived from sessile Ganoderma lucidum and its application. In particular, it relates to a β-glucosidase encoding gene, recombinant expression vector, recombinant strain derived from sessile Ganoderma lucidum, and the application of the β-glucosidase encoded by the gene in the catalytic directional hydrolysis of mogroside V to prepare mogroside IV and symmenidine I. Background Technology

[0002] Monk fruit glycosides are the core functional and sweetening substances of monk fruit, belonging to the cucurbitane-type triterpenoid saponins. Among them, mogroside V has the highest content and a sweetness approximately 300 times that of sucrose, and is widely used in the food, health product, and pharmaceutical industries. Secondary glycosides such as mogroside IV and symmenidine I have a more refreshing taste and a weaker aftertaste, making them more valuable in the development of high-end sweeteners, functional foods, and drugs. However, their natural content is extremely low, and the cost of separation and purification is high, resulting in low yields and making large-scale acquisition difficult.

[0003] Currently, the main methods for preparing secondary glycosides from monk fruit are chemical hydrolysis and enzymatic hydrolysis. Chemical hydrolysis involves harsh conditions, numerous side reactions, complex products, difficult separation, and is environmentally unfriendly. Enzymatic hydrolysis, on the other hand, has become the mainstream approach due to its milder conditions, high selectivity, high product purity, and environmentally friendly process.

[0004] Existing β-glucosidases for the hydrolysis of mogrosides generally suffer from problems such as poor substrate specificity, low directional hydrolysis efficiency, difficulty in controlling product ratios, and insufficient thermal stability and pH adaptability, making it difficult to meet the needs of industrial-scale, high-efficiency preparation of mogroside IV and sympathomimetic I. While there are reports in existing public technologies on the hydrolysis of mogroside V with β-glucosidase to prepare mogroside IV, these are mostly commercially available mixed enzymes or enzymes from known sources. There is a lack of novel β-glucosidase gene resources with high selectivity, high conversion rate, and directional production of mogroside IV and sympathomimetic I from mogroside V, and no new genes from sessile Ganoderma lucidum have been reported. Summary of the Invention

[0005] In order to overcome the shortcomings and deficiencies of existing technologies, such as poor selectivity of β-glucosidase for the hydrolysis of mogroside V, low efficiency of directional preparation of mogroside IV and symmenidine I, and lack of novel gene resources, the present invention aims to provide a β-glucosidase Bgl-Gs1 derived from sessile Ganoderma lucidum, which has high substrate specificity and high catalytic efficiency for mogroside V.

[0006] Another object of the present invention is to provide the encoding gene for the above-mentioned β-glucosidase Bgl-Gs1.

[0007] Another objective of this invention is to provide the application of the above-mentioned β-glucosidase Bgl-Gs1 and its encoding gene in the targeted transformation of mogroside V.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] This invention provides a β-glucosidase Bgl-Gs1, which was cloned from sessile Ganoderma lucidum, and its amino acid sequence is shown in SEQ ID NO: 2.

[0010] The present invention also provides a gene encoding the above-mentioned β-glucosidase Bgl-Gs1, the nucleotide sequence of which is shown in SEQ ID NO: 1.

[0011] The nucleotide sequence of the gene encoding the mature peptide of the β-glucosidase Bgl-Gs1 is shown in SEQ ID NO: 3.

[0012] The present invention also provides an expression cassette, recombinant expression vector, or recombinant strain containing the encoding gene of the above-mentioned β-glucosidase Bgl-Gs1.

[0013] The starting vector of the recombinant expression vector is a vector suitable for prokaryotic or eukaryotic expression;

[0014] 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.

[0015] 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.

[0016] The host bacteria corresponding to the recombinant strain are selected from prokaryotes or yeasts, etc.; the prokaryotes include bacteria such as Escherichia; the yeasts include yeasts such as Pichia pastoris (Komagataella phaffii). More specifically, the prokaryote is Escherichia coli (E. coli), specifically Escherichia coli BL21(DE3) or Escherichia coli DH5α; the yeast is Pichia pastoris GS115.

[0017] The present invention also provides an application of the above-mentioned β-glucosidase Bgl-Gs1, its encoding gene, an expression cassette containing the encoding gene of β-glucosidase Bgl-Gs1, a recombinant expression vector, or a recombinant strain, which is one of the following applications:

[0018] (a) Application in the preparation of β-glucosidase Bgl-Gs1;

[0019] (b) Application in the directed catalysis of mogroside V.

[0020] Furthermore, application (b) is used in the directed catalytic preparation of mogroside V into mogroside IV and / or symmenidine I.

[0021] 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-Gs1 to carry out a catalytic reaction.

[0022] Specifically, the steps include the following:

[0023] The above recombinant strains were cultured, and induction reagents were added to induce expression. The bacterial cells were then collected.

[0024] The bacterial cells were broken down and purified to obtain recombinant β-glucosidase Bgl-Gs1;

[0025] Using mogroside V as a substrate, mogroside IV and / or symbioside I were prepared by directionally catalyzing the substrate with the above-mentioned recombinant β-glucosidase Bgl-Gs1.

[0026] 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.

[0027] 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.

[0028] Preferably, the purification of the bacterial cells after disruption is carried out by Ni-NTA affinity chromatography.

[0029] Preferably, the concentration of mogroside V is 1.0–10.0 mg / mL, and the amount of recombinant β-glucosidase Bgl-Gs1 added is 200–3000 U / g substrate.

[0030] Further preferred, the concentration of mogroside V is 5.0–6.0 mg / mL, and the amount of recombinant β-glucosidase Bgl-Gs1 added is 2000–3000 U / g substrate.

[0031] Preferably, the catalytic conditions are 40–60°C for 2–12 h; more preferably 40–50°C for 2–12 h; and even more preferably 45°C for 2 h.

[0032] Preferably, the catalytic pH is 5-6; more preferably 5.

[0033] The present invention has the following advantages and effects compared with the prior art:

[0034] (1) New gene resources: This invention discloses for the first time a β-glucosidase gene Bgl-Gs1 derived from sessile Ganoderma lucidum. The sequence is novel, filling the gap in related gene resources. It also achieves efficient soluble expression of the gene in the host, and obtains recombinant β-glucosidase that can be used in industrial applications.

[0035] (2) High selectivity: This enzyme specifically hydrolyzes the terminal glucose of mogroside V to generate mogroside IV and symmenoside I in a directed manner, with very few byproducts and controllable product ratio.

[0036] (3) High conversion efficiency: Under optimized conditions, the conversion rate of mogroside V can reach more than 34.58%, and the total yield of the target product is high, which is suitable for large-scale production.

[0037] (4) Process advantages: The enzyme-catalyzed reaction conditions are mild (temperature 40-50℃, pH 5.0), with low energy consumption, no pollution, and easy separation and purification of products, which meets the requirements of green manufacturing.

[0038] (5) High application value: It can efficiently and cost-effectively prepare high-value mogroside IV and symmenoside I, significantly improving the added value of deep processing of mogroside, and has broad industrialization prospects in the fields of food, health products and medicine. Attached Figure Description

[0039] Figure 1 This is an electrophoresis diagram of the PCR amplification of the β-glucosidase gene Bgl-Gs1 of this invention.

[0040] Figure 2 SDS-PAGE electrophoresis image of recombinant β-glucosidase Bgl-Gs1; where lane M: protein marker 0-180 kDa; lanes 1 and 2: uninduced cell supernatant; lane 3: induced and purified protein.

[0041] Figure 3 The graph shows the detection results for the optimal pH.

[0042] Figure 4 The graph shows the test results at the optimal temperature.

[0043] Figure 5The HPLC chromatograms are for the hydrolysis of mogroside V by recombinant β-glucosidase Bgl-Gs1 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

[0044] 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.

[0045] The sessile Ganoderma involved in the examples is Ganoderma sessile, which is a conventional commercially available product.

[0046] Example 1: Cloning of the β-glucosidase gene Bgl-Gs1 from Ganoderma lucidum.

[0047] (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.

[0048] (2) Collect mycelium, extract total RNA using the Trizol method, and after testing its purity and integrity, reverse transcribe to synthesize cDNA.

[0049] (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.

[0050] (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-Gs1 with the nucleotide sequence shown in SEQ ID NO: 1.

[0051] The gene encoding β-glucosidase Bgl-Gs1 is 2724 bp in length, and its nucleotide sequence is shown in SEQ ID NO: 1, where 1-78 bp is the signal peptide coding sequence; and 79-2724 bp is the mature peptide coding sequence.

[0052]

[0053] β-glucosidase Bgl-Gs1 is 907 amino acids long, and its amino acid sequence is shown in SEQ ID NO: 2, where 1-26 amino acids are the signal peptide and 27-907 amino acids are the mature peptide.

[0054] MLNYRLSALCALVLPVISTNASIVNAESSVKGSTIQDFPSGTESGPPSTTSQPGFSSVLSTSTNGVSVQSTTIPVSTELSATPDSTSIVSLTAATETVTTTSGGPSMETTLITLPISSYSFSSFPVPSASPIPGIFPSTSPSSPPPPGSRLIPDFGPAWAAARAKAKNLVSSWTLEQKVNTSTGIGAIGSRCSGNIAAISDDFPGLCFQDGPLGVRGTDFVSVFPAGINAAATWNRTLIRARGKAIGQEFKGKGANVELGPMMNIMRVPQAGRNWEGFGADPFLSGEAAYETVLGWQEGGAQACAKHYINNDQEALRFQVVERRRQDRARDLLHPFMRAVQASLASVMCSYNLINEAFACENDRTLNQILKGEVGFQGYVVSDWAAQRSTLAAVAGLDMSMPGDIIPGSGTSFFGANLTAFVQNGTISEARLDDMVERIVAGWYFLGRTTAFRKVRRVAIYSPSLFLMHGPVKYNTQMPDDPATNEHVDVQGDHFKIAREVGAASVVLLKNVAGALPLKQPRSLAVIGNDSGPSLRGPNGYPGNAGDDGTLAIGWGSGSGTFPYLITPLEAIQARAREDHSSVSWFLSNWDLAGAAATARGQDVALVFANADSGEGAIVVGDSSPLAQGIILVDGMAGDRTNLTLWGNADALVNAVAAVNPNTIVVVHAAGLPGQESGNALVDVLYGAVNPSARLPYTIARDPADYPARLVTAPANTTGKTGGSPLPVFIIDYTEGLHIDYRHFDANGIEPRFPFGCGMSYTTFAYSDLNVQVIHQGDADSAALETAWARGEASPNVEGGSTAIRLHRPAFEVSFSVRNTGKVKDGEIPQLYLHFPSGAGEPPNVLRRFSDVLLEPGESKVVALTLSRYDLNIWDTGAQGWRKPEGKFSFSVGASSRDFRLNGTLPL。

[0055] Example 2: Construction of recombinant expression vector and recombinant strain

[0056] (1) The mature peptide sequence of β-glucosidase Bgl-Gs1 obtained in Example 1 (serial numbers 27-907aa in SEQ ID NO: 2) was optimized for E. coli codons to obtain the optimized nucleotide sequence as shown in SEQ ID NO: 3. The target gene fragment with restriction sites was amplified using specific primers (Bgl-Gs1-F / Bgl-Gs1-R) with restriction sites. Then, the Bgl-Gs1 gene and the expression vector pET-28a(+) were double-digested with restriction endonucleases (BamH1 / XhoI) respectively, and ligated with T4 ligase to construct the recombinant plasmid pET28a-Bgl-Gs1.

[0057] The optimized nucleotide sequence is shown in SEQ ID NO: 3:

[0058]

[0059] Bgl-Gs1-F: 5'-GCATGACTGGTGGACAGCAAATGGGTCGC GGATCC ATGGAGTCTAG-3', SEQ ID NO: 4, wherein, " GGATCC "This is the sequence of the restriction enzyme site BamH1;

[0060] Bgl-Gs1-R: 5'-GGATCTCAGTGGTGGTGGTGGTGGTG CTCGAG TTACAG-3', SEQ ID NO: 5, wherein, " CTCGAG " is the sequence of the restriction enzyme site XhoI.

[0061] (2) The recombinant plasmid was chemically transformed into Escherichia coli BL21(DE3) competent cells, plated on LB plates containing 25 μg / mL kanamycin, and incubated overnight at 37°C.

[0062] (3) Colony PCR and sequencing verification yielded a positive recombinant Escherichia coli expression strain, denoted as BL21(DE3) / pET28a-Bgl-Gs1.

[0063] Example 3: Induction and preparation of recombinant β-glucosidase

[0064] (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.

[0065] (2) Add IPTG to a final concentration of 0.2 mM and induce recombinase expression at 16℃ for 12 h.

[0066] (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.

[0067] (4) The recombinant β-glucosidase was purified by Ni-NTA affinity chromatography and subjected to SDS-PAGE electrophoresis. The results are as follows: Figure 2 As shown, the protein size is consistent with the theoretical value (97.02 kDa).

[0068] Example 4: Determination of optimal pH and optimal temperature for recombinant β-glucosidase

[0069] 1. Experimental materials

[0070] Enzyme solution: Recombinant β-glucosidase Bgl-Gs1 from Escherichia coli purified in Example 3;

[0071] Reaction substrate: p-nitrophenyl-β-D-glucoside (pNPG);

[0072] 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.

[0073] Termination solution: 1 mol / L Na2CO3 solution;

[0074] Testing instruments: constant temperature water bath, ultraviolet spectrophotometer (detection wavelength 405 nm).

[0075] 2. Optimal pH Measurement Procedure

[0076] Prepare gradient pH buffer solutions: 3.0, 4.0, 5.0, 6.0, 7.0, 8.0;

[0077] 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;

[0078] Add 100 μL of purified recombinant enzyme solution to each group, mix well, and incubate in a 50 ℃ water bath for 120 min.

[0079] Immediately after the incubation period, add 500 μL of 1 mol / L Na2CO3 to terminate the enzymatic reaction;

[0080] 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.

[0081] 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.

[0082] 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-Gs1 is 5, and the relative enzyme activity at pH 5-6 can reach over 50%.

[0083] 3. Procedure for determining the optimal temperature

[0084] The reaction system was constructed using a uniform 50 mM, pH 5.0 citrate-disodium hydrogen phosphate buffer solution;

[0085] Set temperature gradients: 30 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 70 ℃, 80 ℃, with 3 parallel groups for each group;

[0086] 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;

[0087] 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.

[0088] Plot a temperature-relative enzyme activity curve with the maximum enzyme activity value as 100%, as follows: Figure 4 As shown, the results indicate that the optimal reaction temperature for β-glucosidase Bgl-Gs1 is 45 ℃, the relative enzyme activity can reach more than 70% at 40–60 ℃, and the relative enzyme activity can reach more than 80% at 40–55 ℃.

[0089] Example 5: Preparation of mogroside IV and symbioside I by recombinase catalysis of mogroside V

[0090] (1) Prepare 50 mM citrate-disodium hydrogen phosphate buffer (pH 5.0) and add mogroside V to a final concentration of 6.0 mg / mL.

[0091] (2) Add the recombinant β-glucosidase Bgl-Gs1 prepared in Example 3 at an enzyme concentration of 2550 U / g substrate. Set up a control without adding enzyme.

[0092] (3) Place in a 45℃ water bath for 2 h and shake gently during the reaction.

[0093] (4) Terminate the reaction by boiling in a water bath for 10 min, centrifuge and collect the supernatant for HPLC analysis. Set up 3 replicates for each group.

[0094] The HPLC detection conditions were as follows: an Agilent Eclipse XDB C18 column (4.6 mm × 250 mm, 5 μm) was used; water was used as phase A and methanol as phase B, with 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 was 0.500 mL / min; column temperature limit was 35℃; detection wavelength was 203 nm; injection volume was 10 μL; system pressure limit was 200 bar.

[0095] result( Figure 5 The results showed that mogroside V was successfully converted, with mogroside IV and cimanin I as the main products, very few byproducts, high total purity of the target product, and a conversion rate of 34.58%.

[0096] 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-Gs1, characterized in that: The amino acid sequence of the β-glucosidase Bgl-Gs1 is shown in SEQ ID NO:

2.

2. A gene encoding the β-glucosidase Bgl-Gs1 as described in claim 1.

3. The gene according to claim 2, characterized in that: The nucleotide sequence of the gene encoding β-glucosidase Bgl-Gs1 is shown in SEQ ID NO: 1, or the nucleotide sequence of the gene encoding the mature peptide of β-glucosidase Bgl-Gs1 is shown in SEQ ID NO:

3.

4. The biomaterial related to β-glucosidase Bgl-Gs1 as described in claim 1, characterized in that, It can be any one or more combinations of the following biological materials: (1) An expression cassette containing the gene of claim 2 or 3; (2) A recombinant expression vector containing the gene described in claim 2 or 3; (3) A recombinant expression vector containing the expression cassette described in (1); (4) A recombinant strain containing the gene described in claim 2 or 3; (5) A recombinant strain containing the expression cassette described in (1); (6) A recombinant strain containing the recombinant expression vector described in (2) or (3).

5. The biomaterial according to claim 4, characterized in that: The starting vector for the recombinant expression vectors mentioned in (2) and (3) is a vector suitable for prokaryotic or eukaryotic expression; And / or, the host bacteria corresponding to the recombinant strains described in (4), (5), and (6) are selected from prokaryotes or yeast.

6. The use of the gene according to any one of claims 2 to 3 or the biological material according to any one of claims 4 to 5 in the preparation of β-glucosidase Bgl-Gs1.

7. The application of the β-glucosidase Bgl-Gs1 according to claim 1, 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 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-Gs1 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-Gs1; Using mogroside V as a substrate, recombinant β-glucosidase Bgl-Gs1 was added to catalyze the substrate to prepare mogroside IV and / or symbioside I.

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