CN103805581B - Beta-glycosidase mutant and coding gene thereof, and application thereof in producing ginsenoside CK - Google Patents

Abstract

The invention discloses a beta-glycosidase mutant and a coding gene thereof, and an application thereof in producing ginsenoside CK. The protein disclosed by the invention is named as lacS-mut, and is one of defined as the following items: 1) the protein is composed of amino acid residue sequence of a sequence 4 in the sequence table; 2) the protein is derived from the protein as defined in the item 1) by substituting and/or deleting and/or adding of one or several amino acid residues in the amino acid residue sequence of the sequence 4 in the sequence table, and has beta-glycosidase activity. Experiments prove that the beta-glycosidase mutant is obtained by mutating a wild type beta-glycosidase gene; compared with the wild type, the beta-glycosidase mutant has the advantage that the activity of the beta-glycosidase mutant in synthesizing the ginsenoside CK is obviously improved, and therefore the beta-glycosidase mutant has better industrial application prospect.

Description

β-glucosidase mutant and its coding gene and its application in the production of ginsenoside CK

technical field

The invention relates to the field of biotechnology, in particular to a beta-glucosidase mutant and its coding gene and its application in the production of ginsenoside CK.

Background technique

Ginseng is a traditional Chinese medicine. According to modern pharmacological research reports, ginseng has the effects of enhancing human immunity, improving nutrition, anti-aging and relieving fatigue. As one of the main effective active ingredients of ginseng, ginsenoside CK is more easily absorbed by the human body, and has medical effects such as promoting the death of malignant tumor cells and curbing the spread or deterioration of tumors, so it has extremely high production value and application prospects. However, ginsenoside CK does not exist in natural ginseng extracts, but is produced by other ginsenoside components (such as ginsenosides Rb ₁ , Rb ₂ , Rd, Re and Rg ₁ ) through the metabolic transformation of intestinal bacteria, so The content in nature is very low. The conversion process of ginsenoside mainly includes the hydrolysis of glucose and other monosaccharide groups from the main structure. The hydrolysis methods include chemical hydrolysis and biocatalytic reaction methods. Although the chemical method has high conversion efficiency, the lack of specificity in catalysis limits its wide application. The biocatalytic reaction method, the use of enzymatic methods to realize the synthesis of ginsenoside CK is less, mainly the purification and property research of some enzyme proteins derived from fungi, such as the glucosidase purified from Paecilomyces Bainier and the glucosidase purified from Thermuscaldophilus etc. have been found to have the activity of synthesizing ginsenoside CK, but on the one hand, the activity is low and intermediate products accumulate during the transformation process; on the other hand, the gene sequences of these enzymes have not been reported, and recombinant expression cannot be achieved. In the study of enzymatic synthesis of ginsenoside CK, not only the conversion efficiency needs to be improved, but also the accumulation of intermediate products should be minimized or even eliminated, so as to maximize the yield of ginsenoside CK and reduce the difficulty of downstream separation and purification.

β-Glycosidase (LacS, β-Glycosidase) from Sulfolobus solfataricus has been reported to have the ability to synthesize ginsenoside CK, but the conversion efficiency is limited and cannot meet the needs of large-scale production. The advantages of using this enzyme to synthesize CK are: 1. The enzyme has been expressed recombinantly, which is suitable for further improving its catalytic efficiency by means of protein engineering; 2. The reaction temperature of the enzyme is high and the thermal stability is very good, which is helpful for The product synthesis is completed under high temperature production conditions, the conversion efficiency is improved, and the possibility of pollution in the production process is reduced; 3. The intermediate process of the enzyme catalyzed synthesis of ginsenoside CK has been clearly explained; 4. The crystal structure of the enzyme has been reported, for its Further modification and analysis of enzyme mutants provided important reference information.

Contents of the invention

One object of the present invention is to provide a β-glucosidase mutant and its coding gene.

The protein provided by the present invention, named lacS-mut, is a protein of the following 1):

1) A protein composed of the amino acid residue sequence of sequence 4 in the sequence listing;

2) A protein derived from 1) that undergoes substitution and/or deletion and/or addition of one or several amino acid residues in the amino acid residue sequence of Sequence 4 in the sequence listing and has β-glucosidase activity.

Sequence 4 in the above sequence listing consists of 489 amino acids, and the substitution and/or deletion and/or addition of one or several amino acid residues is a substitution and/or deletion and/or addition of no more than 10 amino acid residues .

Genes encoding the above proteins are also within the protection scope of the present invention.

The above-mentioned gene is a DNA molecule as follows (1) or (2) or (3):

(1) The DNA molecule shown in sequence 3 in the sequence listing;

(2) A DNA molecule that hybridizes to the DNA sequence defined in (1) and encodes a protein with β-glucosidase activity under stringent conditions;

(3) At least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97% of the DNA sequence defined in (1) , a DNA molecule having at least 98% or at least 99% homology and encoding a protein having β-glucosidase activity.

The above-mentioned stringent conditions were hybridization at 65°C in a solution of 6×SSC, 0.5% SDS, and then the membrane was washed once with 2×SSC, 0.1% SDS and 1×SSC, 0.1% SDS.

The above sequence 3 is composed of 1470 nucleotides, and the coding region of the sequence 3 is 1-1470 nucleotides from the 5' end.

Recombinant vectors, expression cassettes, transgenic cell lines or recombinant bacteria containing the above genes are also within the protection scope of the present invention.

The above-mentioned recombinant vector is a recombinant vector obtained by inserting the coding gene of the above-mentioned protein into the expression vector to obtain the expression of the above-mentioned protein, specifically, it is obtained by inserting the DNA molecule shown in Sequence 3 in the sequence listing between the NheI and XhoI recognition sites of the vector pET28a recombinant vector.

The application of the above-mentioned protein, above-mentioned gene or above-mentioned recombinant vector, expression cassette, transgenic cell line or recombinant bacteria as β-glucosidase is also within the protection scope of the present invention.

The application of the above protein in the production of ginsenoside CK is also within the protection scope of the present invention.

Another object of the present invention is to provide a method for producing ginsenoside CK.

The method provided by the invention comprises the steps of:

1) Ferment the above-mentioned recombinant bacteria, collect the fermentation broth, collect the cells by centrifugation, and collect the supernatant by centrifugation after crushing the cells;

2) Reacting the supernatant with ginsenoside substrate Rb ₁ in MC buffer to obtain ginsenoside CK.

In the above method, 1) the fermentation is carried out under the induction of IPTG;

2) The MC buffer solution is an MC buffer solution with a pH value of 5.5; the MC buffer solution with a pH value of 5.5 is specifically composed of 200 mM disodium hydrogen phosphate, 100 mM citric acid and water at a final concentration.

Experiments of the present invention prove that the wild-type β-glucosidase gene of the present invention is mutated to obtain a β-glucosidase mutant. Compared with the wild-type, it has a significant improvement in the activity of synthesizing ginsenoside CK, so it has better industrial application prospect.

Detailed ways

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Embodiment 1, the acquisition of beta-glucosidase mutant and its coding gene

1. Acquisition of β-glucosidase and its coding gene

Genomic DNA of Sulfolobus solfataricus (ATCC 35091, DSM 1616) was extracted and used as a template, and 5'-atggctagcatgtactcatttccaaatagc-3'(forward)and 5'-gtgctcgagttagtgccttaatggctttac-3'(reverse) were used as primers Perform PCR amplification. The PCR amplification conditions are as follows: first, 95°C pre-denaturation for 4min, then 95°C for 45s, 55°C for 30s, 72°C for 1min30s, a total of 30 cycles; finally, 72°C for 10min.

The above PCR reaction product was recovered and detected by agarose gel electrophoresis. As a result, a fragment with a size of about 1470 bp was obtained.

The PCR product obtained above was double-digested with NheI and XhoI, and the digested product was ligated with the same digested pET28a vector (Novagen, No. 69864-3) with T4 ligase (purchased from Treasure Biotechnology) overnight at 16°C. The ligation product was transformed into Escherichia coli MC1061 competent cells to obtain recombinant bacteria.

The plasmid sequencing verification and sequencing analysis of the recombinant bacteria were extracted. The gene of the PCR product was named lacS, and its nucleotide sequence was sequence 1 in the sequence table. Through comparison, it was the β-glucosidase lacS of Sulfolobus solfataricus The gene encoded by the gene is named LacS, and the amino acid sequence of the protein is sequence 2 in the sequence listing. The plasmid containing the PCR product was named pET28a-lacS, which is a vector obtained by inserting the sequence 1 in the sequence listing between the NheI and XhoI double restriction sites of the pET28a vector.

2. Mutations to obtain β-glucosidase mutants

On the basis of the β-glucosidase lacS gene, a random mutation library was constructed by using the method of error-prone PCR. Mainly by changing the concentration of manganese ions in the PCR amplification system and increasing the mismatch rate of Taq DNA polymerase, mutations are introduced into the gene sequence. In this study, a round of mutant library construction was performed to obtain mutated PCR product 1 and mutated PCR product 2.

The above mutated PCR product 1 and mutated PCR product 2 were double digested with NheI and XhoI, respectively, and the digested products were respectively ligated with the pET28a vector that had been digested by the same enzyme, and the ligated products were respectively transformed into BL21 (DE3) competent cells to obtain Mutation library. To screen recombinant bacteria, the screening method is to use a sterile toothpick to pick single colony clones containing mutants into 800 ul LB medium (tryptone 10 g/L, NaCl 10 g/L, Yeast extract 5g/L, add antibiotic kanamycin to 50μg/mL) After 2h of shaker culture at 37°C, add inducer IPTG to a final concentration of 0.4mM, and induce for about 16h at 30°C shaker. Centrifuge, discard the supernatant, collect the bacteria, freeze-thaw the cell wall, add lysozyme (10mg/ml, Tris-HCl buffer, pH8.0) to react at 37°C for 1h, centrifuge, and the supernatant is Crude enzyme solution after wall breaking. React the crude enzyme solution with the ginsenoside substrate Rb ₁ (at 85°C, pH 5.5), and detect the content of the reducing sugar in the by-product of the reaction by the dinitrosalicylic acid method (DNS method), which indirectly reflects the conversion into ginsenoside CK Ability.

For the recombinant bacteria that produce more reducing sugars, extract the plasmid and send it to sequencing. As a result, the gene of the PCR product in the plasmid has the nucleotide shown in sequence 3 in the sequence table, and the gene is named as lacS-mut. The protein is named lacS-mut, and the amino acid sequence of the protein is sequence 4 in the sequence listing.

Compare the amino acid and nucleotide sequence of the above-mentioned lacS-mut with the amino acid and nucleotide sequence of wild-type β-glucosidase lacS:

The amino acid sequence of lacS-mut (sequence 4) is the amino acid sequence of the wild-type β-glucosidase lacS (sequence 2 in the sequence listing) from the N' end of the 218th valine Val to glycine Gly mutation, lacS-mut The nucleotide sequence of the gene (sequence 3) is that the nucleotide sequence of the wild-type β-glucosidase lacS gene (sequence 1 in the sequence table) is mutated from the 5' end of the 652-654th GTT to GGT. It shows that lacS-mut is a mutant of lacS.

The plasmid containing lacS-mut is named pET28a-lacS-mut, which is a vector obtained by inserting sequence 3 in the sequence table between the NheI and XhoI double restriction sites of pET28a, and the recombinant bacteria containing the plasmid is named BL21(DE3)/pET28a-lacS-mut.

The above BL21(DE3)/pET28a-lacS-mut can also be prepared according to the following method: artificially synthesize sequence 3 in the sequence listing, and insert the DNA molecule shown in sequence 3 in the sequence listing between the NheI and XhoI double restriction sites of pET28a to obtain Then pET28a-lacS-mut was introduced into BL21(DE3) competent cells to obtain BL21(DE3)/pET28a-lacS-mut.

Using the same method, the plasmid pET28a-lacS was transferred into the competent cells of BL21(DE3), and the recombinant strain BL21(DE3)/pET28a-lacS was obtained.

Example 2, lacS-mut has β-glucosidase activity

1. Obtaining of β-glucosidase mutants

1. Induced expression

Inoculate the single colony of BL21(DE3)/pET28a-lacS-mut obtained above into LB liquid medium containing kanamycin (final concentration: 50 μg/ml), culture at 37°C for 12 hours, collect the fermentation broth, and ferment The solution was transferred to 100ml of fresh LB liquid medium according to the inoculation amount of 1% (volume percentage content), cultured at 37°C until the OD ₆₀₀ reached 0.6, and then sterile IPTG was added to the medium to make IPTG in the medium The final concentration was 0.4mM and the induction fermentation was carried out at 30°C. After 16 hours, the fermentation was finished, and the fermentation liquid was centrifuged at 4000 g for 10 minutes to collect the bacteria.

Resuspend the bacteria in 50ml of 50mM binding buffer (Tris-hydrochloric acid, pH 8, containing 300mM NaCl and 10mM imidazole), ultrasonically break the wall (200W, work for 3s, pause for 3s, work 100 times), centrifuge (15,700 g, 4°C, 30min) to remove cell debris and collect the supernatant.

2. Purification

After the above supernatant is filtered through a 0.22 μm filter membrane, the purification conditions are as follows:

Wash buffer: 50 mM pH8 Tris-HCl, 300mM NaCl, 20 mM imidazole;

Elution buffer: 50 mM pH8 Tris-HCl, 300 mM NaCl, 250 mM imidazole.

The effluent is collected and dialyzed (the dialysate is MC buffer solution with a pH value of 5.5, which consists of a final concentration of 200mM disodium hydrogen phosphate, a final concentration of 100mM citric acid and water; the molecular weight cut-off of the dialysis bag is 8000-14000) After that, a purified enzyme solution (mutation) is obtained.

Use methods such as enzyme activity test and SDS-PAGE to verify the purity of the purified components; use Bradford protein concentration assay to test the concentration of purified protein.

Using the same method, the recombinant strain BL21(DE3)/pET28a-lacS was induced, expressed and purified to obtain purified enzyme solution (wild).

2. Detection of β-glucosidase activity and its application in the production of ginsenoside CK

Mix 10 μl of the purified enzyme solution (mutated) obtained above, 10 μl of ginsenoside substrate Rb ₁ (MC buffer solution with a pH value of 5.5, which consists of a final concentration of 200 mM disodium hydrogen phosphate, a final concentration of 100 mM citric acid and water) , reacted at 85°C for 1 hour, dried the sample in vacuo after the reaction and redissolved it in methanol, and detected it by high-pressure liquid chromatography. The detection conditions of high-pressure liquid chromatography are C18 column (Waters Symmetry, 250 mm×4.6 mm, 5 μm), the column temperature is 35° C., and the mobile phase is acetonitrile. 0-20min, 70-60% water, 30-40% acetonitrile; 2-35min, 60-0% water, 40-100% acetonitrile; 35-40min, 100% acetonitrile; 40-45min, 70% water, 30% Acetonitrile. The flow rate is 1.0ml/min, and the detection wavelength is 203nm. The purified enzyme solution (wild) was used as the control. Standard products ginsenoside Rb ₁ (Beijing Bailingwei Chemical Technology Co., Ltd., product number ASB-00007190-010) and ginsenoside CK (Chengdu Purifa Technology Development Co., Ltd., product number G4038) refer to.

Results The retention time of the standard ginsenoside CK was 32.402 min, and the retention times of the purified enzyme solution (wild) reaction product and the purified enzyme solution (mutated) reaction product were 32.397 min and 32.400 min, respectively; indicating that ginsenoside CK was obtained.

The amount of ginsenoside CK in the reaction product of purified enzyme solution (wild) is 0.09g/L/h;

The amount of ginsenoside CK in the reaction product of purified enzyme solution (mutation) was 0.24g/L/h.

The above results indicated that the ability of the mutant to produce CK was 2-5 times that of the wild type.

Claims (11)

1. an albumen, the protein be made up of the aminoacid sequence shown in sequence in sequence table 4.

2. the gene of albumen described in coding claim 1.

3. gene as claimed in claim 2, is characterized in that: described gene is the DNA molecular as shown in sequence in sequence table 3.

4. the recombinant vectors containing gene described in Claims 2 or 3.

5. recombinant vectors as claimed in claim 4, is characterized in that:

Described recombinant vectors, for being inserted in expression vector by the encoding gene of albumen described in claim 1, obtains the recombinant vectors of expressing albumen described in claim 1.

6. the expression cassette containing gene described in Claims 2 or 3.

7. the transgenic cell line containing gene described in Claims 2 or 3.

8. the recombinant bacterium containing gene described in Claims 2 or 3.

9. albumen described in claim 1 is as the application in beta-glycosidase.

10. recombinant bacterium described in transgenic cell line described in expression cassette, claim 7 described in recombinant vectors, claim 6 described in gene, claim 4 described in albumen, Claims 2 or 3 described in claim 1 or claim 8 is producing the application in Ginsenoside compound K.

11. 1 kinds of methods of producing Ginsenoside compound K, comprise the steps:

1) ferment recombinant bacterium described in claim 8, collected by centrifugation supernatant liquor after collection fermented liquid, collected by centrifugation thalline, broken thalline; Described fermentation is carried out under IPTG induction;

2) by described supernatant liquor and ginsenoside substrate Rb ₁react in MC damping fluid, obtain Ginsenoside compound K; Described MC damping fluid to be pH value be 5.5 MC damping fluid; Described pH value be 5.5 MC damping fluid specifically by final concentration be 200mM Sodium phosphate dibasic, final concentration is that 100mM citric acid and water form.