Glucoside hydrolase ScBGLU as well as coding gene and application thereof
By cloning and expressing the glycoside hydrolase ScBGLU in horse blue leaves, the problem of insufficient catalytic indigo yield and efficiency in the prior art is solved, and efficient and stable indigo catalysis is achieved, which is suitable for industrial applications.
Patent Information
- Application Number
- CN202411276171.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-12
- Publication Date
- 2025-02-14
AI Technical Summary
The existing glycoside hydrolase has shortcomings in catalyzing the yield and efficiency of indigo, which is difficult to meet the needs of modern industry.
ScBGLU, a glycoside hydrolase from marblu leaves, was cloned and expressed. The protein encoded by its genes had high thermal stability and pH stability, and was able to selectively recognize β-glycosidic bonds. The enzyme was expressed in E. coli Rosetta (DE3) by the recombinant expression vector pET-28a(+) and obtained by HIS-TAG purification.
The glycoside hydrolase ScBGLU significantly improves the catalytic efficiency and yield of indigo, has high thermal stability and pH stability, and is suitable for applications in the food industry and agriculture.
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Abstract
Description
Technical Field
[0001] The invention relates to the field of genetic engineering, in particular to a glycoside hydrolase ScBGLU and a coding gene and application thereof. Background Art
[0002] Indigo, as an ancient and important industrial pigment, has a wide range of applications in textiles, food, cosmetics, medicine and other fields. The production of indigo mainly relies on the conversion of indole glycosides extracted from plants such as Indigofera odorifera through enzymatic catalysis or chemical oxidation. However, traditional extraction and conversion methods are inefficient and cannot meet the demand for indigo production in modern industry. Therefore, research and development of efficient indigo production methods have become a hot topic and difficulty at present.
[0003] Indigofera serrata, as a plant rich in glycoside compounds such as indole glycosides, is an important raw material for indigo production. Indole glycosides can be converted into indigo under the catalysis of specific enzymes. Therefore, the research and development of efficient hydrolases for indigofera serrata glycosides is of great significance for improving the yield and efficiency of indigo.
[0004] Glycoside hydrolases are a class of enzymes that can specifically hydrolyze glycosidic bonds and play an important role in the development and utilization of plant resources. In recent years, with the rapid development of biotechnology, the research and application of glycoside hydrolases have made significant progress. However, there are relatively few studies on efficient hydrolases for indigo glycosides, and the existing hydrolases have deficiencies in catalytic efficiency and stability, which makes it difficult to meet the needs of indigo production. Summary of the invention
[0005] The purpose of the present invention is to provide a glycoside hydrolase ScBGLU and the cloning, expression and application of its encoding gene in order to solve the problems of low catalytic efficiency and high use cost of existing glycoside hydrolases. The present invention comprises amplifying the full-length gene sequence of the glycoside hydrolase ScBGLU, a recombinant expression vector pET-28a(+)-ScBGLU, and recombinant bacteria, and obtaining the glycoside hydrolase ScBGLU by protein induction expression, purification and other methods.
[0006] In a first aspect of the present invention, a gene encoding glycoside hydrolase ScBGLU is provided, and the nucleotide sequence thereof is shown as SEQ ID No.1.
[0007] In a second aspect of the present invention, a glycoside hydrolase ScBGLU is provided, and its amino acid sequence is shown in SEQ ID No.2.
[0008] Using cDNA obtained from fresh indigofera leaves as a template, specific primers BGLU-F and BGLU-R were designed to construct plasmid Bluntzero-ScBGLU, which was amplified in TOP10 competent state and sequenced to obtain the glycoside hydrolase ScBGLU of the present invention.
[0009] The third aspect of the present invention provides a recombinant expression vector, wherein the recombinant expression vector contains the gene encoding glycoside hydrolase ScBGLU (SEQ ID No. 1).
[0010] The fourth aspect of the present invention provides a recombinant bacterium, wherein the recombinant bacterium contains the gene encoding the glycoside hydrolase ScBGLU, SEQ ID No. 1.
[0011] Furthermore, the recombinant bacteria are formed by transforming the recombinant expression vector as described above into a host cell.
[0012] Furthermore, when preparing the recombinant expression vector or recombinant bacteria, the primers used to amplify the coding gene of glycoside hydrolase ScBGLU are shown in SEQ ID No.3 and SEQ ID No.4:
[0013] BGLU-F: 5'-ATGGCGATGACCACGAGC-3' (SEQ ID No. 3)
[0014] BGLU-R: 5'-TCATGTTGCTCTTGATCTTTTCCC-3' (SEQ ID No. 4).
[0015] The fifth aspect of the present invention provides a method for preparing glycoside hydrolase ScBGLU, comprising the recombinant expression vector pET-28a(+)-ScBGLU and recombinant bacteria as described above, and the steps of obtaining glycoside hydrolase ScBGLU by protein induction expression, purification, etc.
[0016] Furthermore, the method specifically includes the following steps:
[0017] (A) amplifying the nucleotide sequence shown in SEQ ID No. 1 by PCR;
[0018] (B) Construction of recombinant plasmid pET-28a(+)-ScBGLU;
[0019] (C) The recombinant plasmid pET-28a(+)-ScBGLU was introduced into Escherichia coli Rosetta (DE3) and named as recombinant bacteria;
[0020] (D) selecting a single clone of the recombinant bacteria for culture, then expanding the culture, and after overnight culture on a shaking table, adding an inducer for induction culture, collecting the bacterial liquid, and collecting the precipitate after centrifugation;
[0021] (E) Buffer was added to the precipitate to resuspend the strain, and then ultrasonically lysed and centrifuged. The supernatant was collected and frozen for later use.
[0022] (F) The supernatant in step E is graded and purified using a nickel column, and eluted with a 50 mM-300 mM imidazole / Tri-HCl solution to finally obtain the purified protein.
[0023] In a sixth aspect, the present invention provides a use of the above-mentioned glycoside hydrolase ScBGLU, its encoding gene, recombinant expression vector, and recombinant bacteria in indigo production.
[0024] In a seventh aspect, the present invention provides a method for preparing indigo, which uses the glycoside hydrolase ScBGLU as described above to catalyze indole glycoside to produce indigo.
[0025] The present invention has the advantages that:
[0026] 1. The gene of the glycoside hydrolase ScBGLU of the present invention is from the leaves of Indigofera scabra, the nucleotide sequence is shown in SEQ ID No. 1, and the amino acid sequence is shown in SEQ ID No. 2. The gene of the enzyme is cloned, and pET-28a (+) is used as an expression plasmid, Escherichia coli Rosetta (DE3) is used as an expression host, a recombinant strain is constructed, and the protein of the glycoside hydrolase ScBGLU gene is expressed. After HIS-TAG purification, it is identified that the recombinant protein has the properties of glycoside hydrolase.
[0027] 2. The glycoside hydrolase ScBGLU of the present invention has high thermal stability and pH stability, with an optimum temperature of 55°C and an optimum pH of 8.0. It has certain selectivity for reaction substrates, can recognize β-glycosidic bonds, but cannot recognize α-glycosidic bonds.
[0028] 3. The present invention lays a foundation for the efficient biosynthesis of indigo pigment and has great application potential in food industry and agriculture. BRIEF DESCRIPTION OF THE DRAWINGS
[0029] Figure 1 : SDS-PAGE image of glycoside hydrolase ScBGLU;
[0030] Figure 2 : Optimum temperature of glycoside hydrolase ScBGLU;
[0031] Figure 3 : Optimal pH diagram of glycoside hydrolase ScBGLU;
[0032] Figure 4 :Glycoside hydrolase ScBGLU catalyzes the conversion of indole glycoside indican to indigo. DETAILED DESCRIPTION
[0033] The specific implementation methods provided by the present invention are described in detail below in conjunction with examples.
[0034] Example 1: Cloning of the ScBGLU glycoside hydrolase gene
[0035] (1) Glycoside hydrolase ScBGLU, whose amino acid sequence is shown in SEQ ID No. 2 and nucleotide sequence is shown in SEQ ID No. 1.
[0036] (2) Primers BGLUF and BGLUR were designed based on the gene sequence and amplified using the cDNA of S. indigofera as a template.
[0037] Primer BGLU-F: 5'-ATGGCGATGACCACGAGC-3' (SEQ ID No. 3)
[0038] Primer BGLU-R: 5'-TCATGTTGCTCTTGATCTTTTCCC-3' (SEQ ID No. 4)
[0039] (3) PCR reaction conditions were as follows: pre-denaturation at 95°C for 5 min; 34 cycles of 94°C for 30 sec, 55°C for 30 sec, and 72°C for 40 sec; and finally extension at 72°C for 10 min.
[0040] (4) The PCR product was recovered after agarose gel electrophoresis, connected to Bluntzero and transformed into E. coli DH5α, and positive clones were screened on LB plates containing kanamycin resistance (100 mg / L). The selected positive clones were cultured in LB liquid medium at 37°C for 12-16 hours, and the plasmid was extracted and named Bluntzero-ScBGLU, which was sent to the company for sequencing.
[0041] Embodiment 2:
[0042] (1) Based on the gene sequence, primers 28a-ScBGLUF and 28a-ScBGLUR were designed with EcoRI and HindⅢ restriction sites, respectively, and PCR was performed using the correctly sequenced Bluntzero-ScBGLU as a template.
[0043] Primer 28a-ScBGLUF:
[0044] 5'-GTCGCGGATCCGAATTCATGGCGATGACCACGAGC-3'(SEQ ID No.5)
[0045] Primer 28a-ScBGLUR:
[0046] 5'-CTCGAGTGCGGCCGCAAGCTTTCATGTTGCTCTTGATCTTTTCCC-3' (SEQ ID No. 6).
[0047] The reaction conditions for PCR amplification were as follows: pre-denaturation at 95°C for 5 min; 34 cycles of 94°C for 30 sec, 68°C for 30 sec, and 72°C for 40 sec; and finally extension at 72°C for 10 min.
[0048] (2) The expression vector pET-28a(+) was double-digested with restriction endonucleases EcoRI and HindⅢ.
[0049] (3) The PCR amplification products and double enzyme digestion products were subjected to agarose gel electrophoresis, and then the two target bands were recovered from the gel.
[0050] (4) The two target bands recovered in the above step were connected and transformed into E. coli DH5α competent cells, and positive transformants were screened on LB plates containing kanamycin. After selective cultivation, the recombinant plasmid pET-28a(+)-ScBGLU was extracted and then sequenced.
[0051] Example 3: Protein expression and purification of glycoside hydrolase ScBGLU gene
[0052] (1) The correctly sequenced recombinant plasmid pET-28a(+)-ScBGLU was introduced into E. coli Rosetta (DE3) competent cells to obtain recombinant bacteria.
[0053] (2) Select a single clone of the recombinant bacteria and inoculate it into 1 mL LB medium containing 100 μg / mL kanamycin and culture it in a shaking incubator at 37°C and 200 rpm for about three hours and observe the OD 600 In the range of 0.6-0.8, transfer the bacterial solution to 100 mL LB medium containing 100 μg / mL kanamycin and continue to culture in a shaking incubator at 28°C and 180 rpm until the OD 600 In the range of 0.6-0.8.
[0054] (3) IPTG (inducer) was then added for induction, and the cells were cultured at 25°C and 180 rpm with shaking for 20 h.
[0055] (4) Collect the bacterial solution, centrifuge at 8000 rpm at 4°C for 10 min, remove the supernatant, and collect the precipitate.
[0056] (5) Take 2 mL of the bacterial solution and add the precipitate into a 2 mL centrifuge tube. Add 1 mL of pH 7.0 HEPES buffer to the bacterial precipitate and resuspend the bacteria.
[0057] (6) Ultrasonic lysis of bacteria in an ice bath.
[0058] (7) Centrifuge at 4°C, 6000 rpm / min for 10 min, collect the supernatant of the bacterial lysate, and place on ice.
[0059] (8) The collected supernatant was purified by HIS-TAG column recombinant protein. The purified recombinant protein solution was subjected to SDS-PAGE, and a 65.3 kDa protein band was obtained (the result is shown in Figure 1 shown).
[0060] Example 4: Identification of ScBGLU glycoside hydrolase gene activity
[0061] The international unit of β-glucosidase activity is defined as the amount of enzyme required to release 1 μmol of p-nitrophenol (pNP) per minute using p-nitrophenol-β-D-glucoside (pNPG) as a substrate under certain conditions.
[0062] Under certain conditions, glycoside hydrolase can hydrolyze the glycosidic bonds in pNPG to generate pNP. pNP appears yellow in alkaline solution and has an obvious absorption peak at a wavelength of 410 nm. Therefore, the absorbance at 410 nm was determined by spectrophotometry, and a standard curve was drawn for the concentration of the p-nitrophenol standard solution. The activity of glycoside hydrolase was then calculated based on the standard curve.
[0063] Example 5: Determination of the Optimal Reaction Temperature of Glycoside Hydrolase ScBGLU
[0064] When p-nitrophenol-β-D-glucoside (pNPG) is used as substrate, the reaction is carried out at a temperature of 25-65°C, and the enzyme activity is calculated by the amount of p-nitrophenol released. 3 μL of glycoside hydrolase ScBGLU is added to 300 μL of pH 8.0 solution containing 0.25 mM pNPG, reacted at 55°C for 30 minutes, and 1 mL of DMSO was added to terminate the reaction. Enzyme activity is defined as the amount of enzyme that generates 1 μmol pNP per minute at 55°C.
[0065] The protein content was determined by Coomassie Brilliant Blue method, and the protein concentration was 7 g / L.
[0066] The enzyme reaction rate of ScBGLU was measured at reaction temperatures of 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, and 65°C. The results were averaged from 3 replicates.
[0067] The results are as follows Figure 2 As shown in the figure, the highest enzyme activity was 12.3 U / μmol when the reaction temperature was 55°C. The highest enzyme reaction rate was taken as 100%, and the remaining enzyme activities were calculated as Figure 2 .
[0068] Example 6: Determination of the Optimal pH of Glycoside Hydrolase ScBGLU
[0069] With p-nitrophenol-β-D-glucoside (pNPG) as substrate, glycoside hydrolase ScBGLU was placed at different pH values (pH=3.0-10.0) to react to determine the optimal pH value. The buffers used were: when pH=3.0-7.0, the buffer was citric acid-phosphate buffer, and when pH=8.0-10.0, the buffer was glycine-sodium hydroxide buffer.
[0070] The protein content was determined by referring to the Coomassie Brilliant Blue method, and the protein concentration was 8 g / L;
[0071] The enzyme activity was 11.3 U / μmol at pH 8.0. The highest enzyme reaction rate was taken as 100%, and the reactions at other pH values were divided by the highest reaction rate to obtain the corresponding relative reaction rates. Figure 3 shown.
[0072] Example 7: Substrate selectivity of glycoside hydrolase ScBGLU
[0073]
[0074] This example shows that the glycoside hydrolase ScBGLU can recognize β-glycosidic bonds and can recognize a variety of different glycosides, including galactoside, glucoside, etc., but cannot recognize α-glycosidic bonds, indicating that the glycoside hydrolase ScBGLU is selective for substrates.
[0075] Example 8: Glycoside hydrolase ScBGLU catalyzes the production of indole glycoside indican
[0076] According to the optimal reaction temperature of 55°C, the optimal pH value of 8.0, and the glycine-sodium hydroxide buffer solution in the above embodiment, as the reaction conditions of this embodiment, the indole glycoside indican is used as the substrate to carry out the reaction. The final reaction solution changes from colorless and transparent to blue, proving that indigo is generated therein. The results and reaction principle are shown in FIG. Figure 4 .
[0077] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the embodiments. Those skilled in the art may make various equivalent modifications or substitutions without violating the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.
Claims
1. A gene encoding a glycoside hydrolase ScBGLU, characterized in that: Its nucleotide sequence is shown in SEQ ID No.
1.
2. A glycoside hydrolase ScBGLU, characterized in that Its amino acid sequence is shown in SEQ ID No.
2.
3. A recombinant expression vector, characterized in that: The recombinant expression vector contains the coding gene SEQ ID No. 1 of glycoside hydrolase ScBGLU.
4. A recombinant bacterium, characterized in that: The recombinant bacteria contains the coding gene SEQID No.1 of glycoside hydrolase ScBGLU.
5. The recombinant bacterium according to claim 4, characterized in that The recombinant bacteria are formed by transforming the recombinant expression vector described in claim 3 into host cells.
6. Primers for amplifying the gene encoding the glycoside hydrolase ScBGLU, characterized in that: The nucleotide sequences of the primers are shown in SEQ ID No.3 and SEQ ID No.
4.
7. A method for preparing glycoside hydrolase ScBGLU, characterized in that: The preparation method comprises the steps of obtaining the glycoside hydrolase ScBGLU by the recombinant expression vector according to claim 3, the recombinant bacteria according to claim 4, and inducing protein expression and purifying the protein.
8. The preparation method according to claim 7, characterized in that: The following steps are involved: (A) amplifying the nucleotide sequence shown in SEQ ID No. 1 by PCR; (B) Construction of recombinant plasmid pET-28a(+)-ScBGLU; (C) The recombinant plasmid pET-28a(+)-ScBGLU was introduced into Escherichia coli Rosetta (DE3) and named as recombinant bacteria; (D) selecting a single clone of the recombinant bacteria for culture, then expanding the culture, and after overnight culture on a shaking table, adding an inducer for induction culture, collecting the bacterial liquid, and collecting the precipitate after centrifugation; (E) Buffer was added to the precipitate to resuspend the strain, and then ultrasonically lysed and centrifuged. The supernatant was collected and frozen for later use. (F) The supernatant in step E is graded and purified using a nickel column, and eluted with a 50 mM-300 mM imidazole / Tri-HCl solution to finally obtain the purified protein.
9. Use of the gene encoding the glycoside hydrolase ScBGLU according to claim 1, the glycoside hydrolase ScBGLU according to claim 2, the recombinant expression vector according to claim 3, and the recombinant bacteria according to claim 4 in indigo production.
10. A method for preparing indigo, characterized in that: The method comprises using the glycoside hydrolase ScBGLU as claimed in claim 2 to catalyze indole glycoside to produce indigo.