Beta-glucuronidase as well as nucleotide sequence and application of gene of beta-glucuronidase

Abstract

The invention belongs to the technical field of biology, and discloses a gene of beta-glucuronidase, an amino acid sequence, an expression vector and a host cell of the beta-glucuronidase and application of the beta-glucuronidase in preparation of glycyrrhetinic acid monoglucuronide. The gene of the beta-glucuronidase is connected with an expression vector and is transformed into a saccharomyces cerevisiae cell, so that the saccharomyces cerevisiae engineering strain with high yield of the beta-glucuronidase is successfully constructed. The glycyrrhizic acid can generate a single product glycyrrhetinic acid monoglucuronide under the catalytic action of the enzyme, and no by-product is generated, so that the aim of efficiently, specifically and safely preparing glycyrrhetinic acid monoglucuronide is fulfilled, and the application and development prospect is wide.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a β-glucuronidase, a nucleotide sequence encoding the enzyme, and their applications. Background Art

[0002] Glycyrrhizin (GL) is the most important triterpenoid saponin in Glycyrrhiza uralensis. It is extremely sweet, with a sweetness 200-250 times that of sucrose. It is a natural product with high sweetness and low calories, and has stable physical and chemical properties. Therefore, it is widely used in food additives and daily cosmetics. At the same time, glycyrrhizin has various pharmacological activities, such as anti-inflammatory, antioxidant, anti-tumor, liver protection, and enhancement of cell immune regulation, etc., and has been widely studied. However, due to the strong polarity of glycyrrhizin, it is not easy to penetrate the cell membrane and enter the cell, which limits the exertion of its pharmacological effects.

[0003] Glycyrrhizin is linked with two glucuronic acid molecules at the C3 position. After hydrolyzing and removing one molecule of glucuronic acid group, the product is glycyrrhetic acid monoglucuronide (GAMG); after removing another molecule of glucuronic acid group, glycyrrhetinic acid (GA) is formed. The sweetness of GAMG is 5 times that of GL and 1000 times that of sucrose. It is a functional sweetener with high sweetness and low calories. In terms of the application in food and cosmetics, GAMG has stable properties. It is not only resistant to high temperature, acid, and pressure, but also has good emulsifying and foaming properties. It can significantly enhance the fragrance and cover the sour and astringent taste. It is a good food sweetener. GAMG also has good pharmacological activities such as anti-tumor, anti-inflammatory, and anti-hepatic fibrosis. Compared with GL and GA, GAMG contains only one molecule of glucuronic acid group, and its polarity is between GL and GA. It is more likely to penetrate the cell membrane, and its solubility and transmembrane transport ability in the body are better than those of GL. Therefore, it shows better physiological activity. Research shows that the median lethal dose LD 50 of GAMG is 5000 mg / kg, which is 6 times that of glycyrrhetinic acid, and it has no teratogenic effect and has relatively high safety. The pharmacokinetic study of GAMG in rats shows that oral administration of GAMG can be rapidly and effectively absorbed and widely distributed in tissues such as kidney, spleen, liver, lung, and brain, exerting potent and multiple pharmacological activities. Therefore, the production and application of GAMG have important research value.

[0004] When producing GAMG by chemical methods, there are disadvantages such as poor selectivity, high energy consumption, and serious pollution. Compared with chemical methods, biotransformation methods have advantages such as high reaction efficiency, mild reaction conditions, high substrate specificity, and strong product specificity, and thus have received extensive attention. Research has found that β-glucuronidase is a type of glycosidase that can catalyze the hydrolysis of β-glucuronide glycosides and is widely present in humans, animals, plants, and microorganisms. This enzyme can catalyze the formation of GA or GAMG from GL, but there are certain differences in the catalytic performance of enzymes from different sources. The substrate specificity of β-glucuronidase from animal sources is relatively poor. For example, after GL is hydrolyzed to GAMG, GAMG sometimes serves as a substrate and is further hydrolyzed to produce GA, so the product is a mixture of GAMG and GA. In addition, the preparation process of enzymes from animal tissues is complex and costly, and it is not suitable for large-scale production.

[0005] Microbial transformation is a reaction in which enzymes produced during the metabolic process of microorganisms catalyze substrates. A large number of studies have shown that when using β-glucuronidase from certain microbial sources to hydrolyze GL, the substrate is more specific and can specifically produce GAMG without the generation of by-product GA. For example, Li Chun et al. isolated and screened a Penicillium fungus, Penicillium purpurogenum, which uses GL as the sole carbon source, from the soil in the licorice-producing area of Xinjiang. The β-glucuronidase expressed by this fungus has high substrate specificity and can directionally hydrolyze GL to produce GAMG. Wang Yun et al. used a Trichoderma viride isolated from the soil for the biotransformation of glycyrrhizic acid to produce GAMG. Wu Shaojie et al. inoculated Aspergillus oryzae 39 and Aspergillus niger UV-48 on a solid medium for cultivation to obtain an enzyme solution. The enzyme from Aspergillus niger UV-48 can produce GAMG, but it further hydrolyzes GAMG to GA. Therefore, if the β-glucuronidase from Aspergillus niger is used for directional catalysis to obtain the product GAMG, the reaction conditions must be optimized to reduce the occurrence of tandem side reactions. Zhu et al. first screened a novel β-glucuronidase (cg-GUS) from the plant endophytic fungus Chaetomium globosum DX-THS3. This β-glucuronidase has excellent thermal stability and pH stability. However, whether from the perspective of safety or technical conditions, it is still necessary to modify the discovered β-glucuronidase and its source strains to obtain better GAMG conversion conditions.

[0006] In summary, searching for new β-glucuronidase genes, constructing and optimizing genetically engineered bacteria can obtain recombinant β-glucuronidase with high expression levels, strong substrate specificity, and high enzyme activity, which is an effective way to achieve the directional conversion of GL into a single product, GAMG. Using the method of microbial transformation to prepare GAMG has high economic benefits, is suitable for industrial production, and has broad application prospects.

[0007] The present invention adopts the technology of homologous recombination to clone the β-glucuronidase of Talaromyces verruculosus into a yeast expression vector, construct a Saccharomyces cerevisiae engineering strain with high-yield β-glucuronidase, and achieve an efficient reaction for the directional catalysis of GL to obtain a single product GAMG. The above invention has not been reported. Summary of the Invention

[0008] In view of the above technical limitations, it is impossible to meet the industrial-level preparation of monoglucuronide glycyrrhetinic acid. The purpose of the present invention is to provide an efficient β-glucuronidase, construct a Saccharomyces cerevisiae engineering strain with high-yield β-glucuronidase, significantly improve the fermentation level of β-glucuronidase, and under the catalysis of this enzyme, it is possible to prepare monoglucuronide glycyrrhetinic acid from glycyrrhizic acid, realizing the safe, specific and efficient preparation of this product.

[0009] In the first aspect, the present invention provides a β-glucuronidase, characterized in that the β-glucuronidase has the amino acid sequence shown in SEQ ID NO.3.

[0010] In the second aspect, the present invention provides a gene fragment encoding β-glucuronidase, and its nucleotide sequence is as shown in SEQ ID NO.1 or the optimized gene fragment, and the nucleotide sequence is as shown in SEQ ID NO.2.

[0011] In the third aspect, the present invention provides the recombinant expression vector containing the nucleotide sequence described in the second aspect. The recombinant vector described in the present invention should be understood as the recombinant vector of any gene in the prior art, such as various plasmids, that is, introducing β-glucuronidase into a DNA vector plasmid capable of stably expressing this enzyme.

[0012] In the fourth aspect, the present invention provides the host cell containing the expression vector described in the third aspect. Further, the provided host cell is characterized in that the host cell is selected from bacteria, actinomycetes, filamentous fungi, yeasts, plant cells, animal cells, wherein the bacteria are selected from Escherichia coli, the actinomycetes are selected from Streptomyces, the filamentous fungi are selected from Basidiomycetes, filamentous ascomycetes, the yeasts are selected from Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and the animal cells are selected from insect cells. The selected cells include but are not limited to the host cells listed above.

[0013] In the fifth aspect, the present invention provides a recombinant protein with the function of β-glucuronidase, characterized in that the amino acids in the functional region of the recombinant protein are encoded by the nucleotide sequence shown in SEQ ID NO.1 or SEQ ID NO.2.

[0014] In a sixth aspect, the present invention provides the use of the β-glucuronidase, or a recombinant protein of this enzyme, or a host cell containing this enzyme, in hydrolyzing and removing glucuronic acid glycosyl groups.

[0015] Furthermore, the present invention provides the above-mentioned use, characterized in that the β-glucuronidase is used in the preparation of mono-glucuronyl glycyrrhetinic acid using glycyrrhizic acid as a raw material.

[0016] Furthermore, for a product used in the preparation of mono-glucuronyl glycyrrhetinic acid, according to its characteristics, the present invention provides the above-mentioned β-glucuronidase, and / or the above-mentioned gene fragment, and / or the above-mentioned expression vector, and / or the above-mentioned host cell, and / or the above-mentioned recombinant protein.

[0017] Furthermore, for a product used in the preparation of mono-glucuronyl glycyrrhetinic acid, according to its characteristics, the present invention provides that the above-mentioned product further includes conventional components for the preparation of mono-glucuronyl glycyrrhetinic acid, and / or conventional reagents for ligating and transforming the above-mentioned gene fragment, and / or conventional reagents for constructing the above-mentioned expression vector, and / or conventional culture components for propagating the above-mentioned host cell, and / or conventional reagents for transforming and expressing the above-mentioned recombinant protein.

[0018] In a seventh aspect, the present invention provides a method for preparing mono-glucuronyl glycyrrhetinic acid, characterized in that the above-mentioned β-glucuronidase, and / or the gene fragment of this enzyme, and / or an expression vector containing the gene fragment of this enzyme, and / or a host cell containing the expression vector of this enzyme, and / or the recombinant protein of this enzyme are added and / or used during the preparation of mono-glucuronyl glycyrrhetinic acid.

[0019] In an eighth aspect, the present invention provides a preparation method for a product used in the preparation of mono-glucuronyl glycyrrhetinic acid, characterized in that the above-mentioned β-glucuronidase, and / or the gene fragment of this enzyme, and / or an expression vector containing the gene fragment of this enzyme, and / or a host cell containing the expression vector of this enzyme, and / or the recombinant protein of this enzyme are added and / or used during the preparation of mono-glucuronyl glycyrrhetinic acid.

[0020] In a ninth aspect, the present invention provides an expression vector related to the third aspect, characterized in that the gene fragment described in SEQ ID NO.1 or SEQ ID NO.2 is inserted into the pESC-Leu vector.

[0021] In a tenth aspect, the present invention provides a host cell, characterized in that the host cell is a Saccharomyces cerevisiae engineering bacterium containing the expression vector described in the ninth aspect and capable of expressing β-glucuronidase.

[0022] The technical solution of the present invention will be further described below:

[0023] The present invention provides a glycosyl hydrolase whose amino acid sequence can be determined, specifically β-glucuronidase, which is produced by a Talaromyces verruculosus, or produced by a recombinant cell or transformant containing the enzyme-encoding gene, and can be located intracellularly or secreted extracellularly, and is used for converting glycyrrhizic acid into monoglucuronyl glycyrrhetinic acid.

[0024] The amino acid sequence of the β-glucuronidase of the present invention has the amino acid sequence shown in SEQ ID NO: 3, and the gene encoding the β-glucuronidase has the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0025] Beneficial technical effects

[0026] For the first time, the present invention clones the β-glucuronidase gene in Talaromyces verruculosus into a yeast expression vector to construct an engineering strain of Saccharomyces cerevisiae with high production of β-glucuronidase, realizing an efficient reaction for specifically catalyzing the conversion of glycyrrhizic acid into the product monoglucuronyl glycyrrhetinic acid, and the Saccharomyces cerevisiae has high safety. In summary, the present invention provides a novel and effective approach for the industrial large-scale preparation of monoglucuronyl glycyrrhetinic acid. Brief description of the drawings

[0027] Figure 1 It is a map of the recombinant expression vector of β-glucuronidase KUL85516.1. In the map, GUS represents the KUL85516.1 protein sequence, which is correctly constructed into the PESC-LEU yeast expression plasmid.

[0028] Figure 2 It is an agarose gel electrophoresis map of the PCR product of the β-glucuronidase gene, where M is the Marker, i.e., the DNA molecular weight standard; 1 is the PCR amplification product of the β-glucuronidase gene.

[0029] Figure 3 It is a reaction schematic diagram of β-glucuronidase catalyzing glycyrrhizic acid (GL) to generate monoglucuronyl glycyrrhetinic acid (GAMG) and glucuronic acid (GlcA).

[0030] Figure 4 It is an HPLC analysis map of recombinant Saccharomyces cerevisiae hydrolyzing glycyrrhizic acid into monoglucuronyl glycyrrhetinic acid. Among them, A is the product after the reaction of recombinant Saccharomyces cerevisiae introduced with the β-glucuronidase gene and the substrate glycyrrhizic acid; B is the glycyrrhizic acid standard; C is the monoglucuronyl glycyrrhetinic acid standard. Detailed implementation manners

[0031] The present invention will be further described below in conjunction with specific embodiments. It should be understood that the following embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods used in the following embodiments are all conventional methods unless otherwise specified. The materials, reagents, etc. used in the following embodiments can be obtained from commercial sources unless otherwise specified.

[0032] Example 1: Construction of the recombinant plasmid pESC-Leu-gus

[0033] Search for the β-glucuronidase gene of Penicillium verruculosum in the NCBI database. Its gene sequence number is LHCL01000038.1. The nucleotide sequence encoding this protein is shown in SEQ NO.1, with a length of 1554 bp; the protein sequence number is KUL85516.1, and the sequence is shown in SEQ NO.3, with a length of 517 aa. It was synthesized by Nanjing GenScript Co., Ltd. The β-glucuronidase gene was ligated to the yeast expression vector pESC-Leu to construct the recombinant plasmid pESC-Leu-gus, ( Figure 1 ). This plasmid was transformed into Escherichia coli DH5α and spread on an LB plate containing the antibiotic Amp, and cultured overnight at 37 °C. Single colonies were picked and inoculated into an LB liquid medium containing Amp, and cultured overnight at 200 rpm at 37 °C. Then the plasmid was extracted and stored at -20 °C, and sequenced, and the result was completely matched with the target sequence.

[0034] Example 2: Codon optimization of the nucleotide sequence of the β-glucuronidase gene of Penicillium verruculosum

[0035] In order to obtain better protein expression efficiency in the model strain of Saccharomyces cerevisiae, the nucleotide sequence SEQ ID NO.1 in Example 1 was codon-optimized, and the optimized sequence is shown in SEQ NO.2. It was synthesized by Nanjing GenScript Co., Ltd. The optimized recombinant plasmid was named pESC-Leu-gus1. The amplification and extraction of the plasmid containing the SEQ NO.2 sequence were the same as in Example 1. Primers for the target sequence SEQ NO.2 were designed, and PCR verification was carried out using the recombinant plasmid, and then the PCR product was identified by agarose gel electrophoresis. The results are as Figure 2 shown. The fragment size is 1554 bp, and at the same time, sequencing verification was carried out, and the result was completely matched with the target sequence.

[0036] Example 3: Construction of recombinant yeast engineering strains

[0037] The preparation of competent Saccharomyces cerevisiae cells and the transformation of competent cells were completed using the Super Yeast Transformation Kit. Pipette 5 μL of the pESC-Leu-gus1 plasmid and transfer it into 50 μL of competent cells of the Saccharomyces cerevisiae BY4742 strain, and then add 350 μL of Y3 solution. Mix well with a pipette. Use the heat shock method for transformation. The heat shock temperature is 30 °C and the time is 1 h. During this period, shake once every 10 min to ensure that the solution remains evenly suspended. After the transformation is completed, centrifuge at 4000 rmp for 5 min, discard the supernatant, add 200 μL of sterile water to resuspend the cells, and spread them on a plate lacking SD-Leu. Place the plate in an incubator at 30 °C and culture for 2 - 4 days. Pick single colonies from the plate and inoculate them into a 10 mL test tube containing 5 mL of SD-Leu-deficient liquid medium. Place the test tube on a shaker at 220 rpm and culture with shaking at 30 °C for 24 hours. Take out 500 μL of the bacterial solution from the test tube and transfer it to an eppendorf tube. Then add 500 μL of 50% glycerol, mix well with a pipette, and store it in a -80 °C freezer.

[0038] Example 4: Preparation of SD Medium

[0039] Weigh 6.7 g of YNB and place it in a beaker. Add 10 mL of the amino acid auxotrophic stock solution (100×), add deionized water to make the volume up to 900 mL, and dispense it into 20 250 ml conical flasks. Sterilize at 121 °C for 20 min. Weigh a total of 20 g of glucose and galactose, with glucose:galactose = 1:9, place it in a beaker, add 100 mL of deionized water, and sterilize at 121 °C for 20 min. Mix the above sterilized components before use. The final concentration of the glucose-galactose mixture in the SD-deficient medium is 20 g / L. To prepare the solid medium of the deficient medium, 20 g / L of agar powder needs to be added. The amino acid auxotrophic stock solution is shown in the following table (100×).

[0040]

[0041]

[0042] Example 5: Hydrolysis of Glycyrrhizic Acid by Recombinant Yeast Engineering Strains

[0043] Under the catalytic action of β-glucuronidase, glycyrrhizic acid (GL) is hydrolyzed to generate monoglucuronyl glycyrrhetinic acid (GAMG) and glucuronic acid (GlcA) as Figure 3As shown. The constructed recombinant yeast engineering strain was inoculated into 5mL SD-Leu defective liquid culture medium, and cultured at 220rpm and 30℃ for 24h to obtain the primary seed solution. 1ml of the primary seed solution was inoculated into 50mL SD-Leu defective liquid culture medium and cultured for another 24h. Then 30mg of glycyrrhizic acid was added, and the culture was continued for 5d under the same conditions, followed by centrifugation at 4000rpm for 5min. The supernatant was transferred to a new 50ml centrifuge tube, 15ml of methanol was added to the bacteria, and ultrasonicated for 20min. The reaction solution was filtered at a pore size of 0.22μm and analyzed by HPLC. A binary elution system was used, with mobile phase A being water and B being acetonitrile, and the chromatographic column being Thermo Acclaim TM 120C18 (5μm, 4.6×250mm), gradient elution, 0-30min 5%-95% B, 30-40min 100% B, mobile phase flow rate of 1ml / min, column temperature of 25℃, detection wavelength of 254nm. The test results are as follows Figure 4 As shown in the figure, A is the product of the reaction between the recombinant Saccharomyces cerevisiae introduced with the β-glucuronidase gene and the substrate glycyrrhizic acid; B is the glycyrrhizic acid standard; C is the monoglucuronic acid glycyrrhetinic acid standard, and the glycyrrhizic acid conversion rate using the recombinant yeast engineering strain is calculated to be 92%.

Claims

1. A β-glucuronidase, characterized in that, The β-glucuronidase has the amino acid sequence shown in SEQ ID NO.

3.

2. A gene fragment encoding the β-glucuronidase according to claim 1.

3. The gene fragment according to claim 2, wherein the nucleotide sequence is as shown in SEQ ID NO.1 or SEQ ID NO.

2.

4. An expression vector containing the nucleotide sequence according to claim 2 or 3.

5. A host cell containing the expression vector according to claim 4.

6. The host cell according to claim 5, wherein, The host cell is selected from bacteria, actinomycetes, filamentous fungi, yeasts, plant cells, and animal cells. Among them, the bacteria are selected from Escherichia coli, the actinomycetes are selected from Streptomyces, the filamentous fungi are selected from Basidiomycetes and filamentous ascomycetes, the yeasts are selected from Pichia pastoris, Saccharomyces cerevisiae, and Schizosaccharomyces pombe, the animal cells are selected from insect cells, and the selected cells include but are not limited to the host cells listed above.

7. Recombinant protein with β-glucuronidase function, characterized in that The amino acid sequence of the functional region of the recombinant protein is encoded by the nucleotide sequence shown in SEQ ID NO.1 or SEQ ID NO.

2.

8. Use of the β-glucuronidase according to claim 1 or the recombinant protein of the β-glucuronidase according to claim 7 or a host cell containing β-glucuronidase in hydrolyzing and removing glucuronic acid groups.

9. The application according to claim 8, wherein Use of the β-glucuronidase in the preparation of mono-glucuronyl glycyrrhetinic acid using glycyrrhizic acid as a raw material.

10. A product for preparing mono-glucuronide glycyrrhetinic acid, characterized in that, Its active ingredient includes the β-glucuronidase according to claim 1, and / or the gene fragment according to claim 2 or 3, and / or the expression vector according to claim 4, and / or the host cell according to claim 5, and / or the recombinant protein according to claim 7.

11. The article according to claim 10, characterized in that, The product further includes conventional components for the preparation of mono-glucuronyl glycyrrhetinic acid, and / or conventional reagents for ligating and transforming the gene fragment according to claim 2 or 3, and / or conventional reagents for constructing the expression vector according to claim 4, and / or conventional culture components for propagating the host cell according to claim 5, and / or conventional reagents for transforming and expressing the recombinant protein according to claim 7.

12. A preparation method for a product for preparing mono-glucuronide glycyrrhetinic acid, characterized in that, Using the β-glucuronidase according to claim 1, and / or the gene fragment according to claim 2 or 3, and / or the expression vector according to claim 4, and / or the host cell according to claim 5, and / or the recombinant protein according to claim 7 as one of the active ingredients for preparing the product; and / or placing the β-glucuronidase according to claim 1, and / or the gene fragment according to claim 2 or 3, and / or the expression vector according to claim 4, and / or the host cell according to claim 5, and / or the recombinant protein according to claim 7 in a packaging box marked with the use for preparing mono-glucuronyl glycyrrhetinic acid.

13. A method for preparing mono-glucuronide glycyrrhetinic acid, characterized in that, Adding and / or using the β-glucuronidase according to claim 1, and / or the gene fragment according to claim 2 or 3, and / or the expression vector according to claim 4, and / or the host cell according to claim 5, and / or the recombinant protein according to claim 7 during the preparation of mono-glucuronyl glycyrrhetinic acid.

14. The expression vector according to claim 4, wherein To insert the gene fragment described in claim 2 or 3 into the pESC-Leu vector.

15. The host cell according to claim 5, characterized in that, It is a genetically engineered Saccharomyces cerevisiae bacterium containing the expression vector described in claim 14.

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