A high-performance β-glucuronidase modified by mutation and its applications

Abstract

This invention discloses a high-performance β-glucuronidase modified by mutation and its applications, belonging to the field of enzyme engineering. The substrate-specific β-glucuronidase can efficiently and specifically convert glycyrrhizic acid into glycyrrhetinic acid monoglucuronide (GAMG) without the formation of the byproduct glycyrrhetinic acid (GA). The gene was ligated into an expression vector and transformed into *E. coli*. After induction of expression, a whole-cell catalyst was prepared for the production of GAMG, achieving a yield of 30.5 g / L and a yield of 97%. The β-glucuronidase mutant and its whole-cell catalytic method provided by this invention have the advantages of high yield and conversion efficiency, simple enzyme preparation and reaction system, mild reaction conditions, and low cost, showing great application potential.

Description

Technical Field

[0001] This invention relates to a high-performance β-glucuronidase modified by mutation and its applications, belonging to the field of enzyme engineering. Background Technology

[0002] Licorice is a commonly used bulk traditional Chinese medicine, and its main triterpenoid active ingredient is glycyrrhizic acid (GL). GL is composed of pentacyclic triterpenoid saponins and two molecules of glucuronic acid, and has anti-tumor, antiviral, anti-inflammatory, antihypertensive, anti-hyperlipidemic, and adrenocortical hormone-promoting effects, making it widely used in the food industry and cosmetics. Monoglucuronido-glycyrrhizic acid (GAMG) is a product obtained by hydrolyzing one molecule of glucuronic acid through the β-1,2 glycosidic bond on the outer side of glycyrrhizic acid. It has better water solubility and its sweetness is 5 times that of GL (more than 900 times that of sucrose). It has better bioavailability, biosafety, and medicinal value, and its structure itself has more active functional groups, making it a promising functional sweetener for the food industry.

[0003] There are two main methods for producing GAMG: chemical and biological. Chemical methods cannot specifically hydrolyze the target glycosidic bonds and have drawbacks such as producing numerous byproducts, being environmentally unfriendly, and incurring high separation and purification costs. Biological methods utilize β-glycosides produced by microorganisms. Glucuronidase, prepared by hydrolyzing glycyrrhizic acid, possesses advantages such as high substrate specificity, high yield, and mild reaction conditions, making it a popular research direction. Currently, wild-type β-glucuronidases used to catalyze the synthesis of GAMG are mainly produced by fungal fermentation. However, this method cannot meet the demands of industrial production in practical applications for two main reasons: First, fungal enzyme production of GAMG involves long fermentation cycles, low enzyme yields, and is difficult to modify. Second, most wild-type β-glucuronidases continuously hydrolyze glucuronic acid (β-1,3 glycosidic bonds) to generate the byproduct GA, while very few β-glucuronidases can specifically hydrolyze one molecule of distal glucuronic acid to generate GAMG. The hydrolysis of glycyrrhizic acid by β-glucuronidase is as follows... Figure 1 As shown.

[0004] In response to the current technical difficulties and bottlenecks in the industrial production of GAMG, some research is currently exploring the use of Chaetomium globulus ( Chaetomium globosum β-glucuronidase (cg-GUS) of DX-THS3 was heterologously expressed in Escherichia coli BL21(DE3) and then mutated to improve its substrate specificity and catalytic activity, thereby shortening the fermentation cycle, significantly increasing GAMG yield and conversion rate, and reducing production costs. However, there are currently few reports in the literature on the modification of fungal cg-GUS using site-directed mutagenesis technology. Therefore, research on site-directed mutagenesis of fungal cg-GUS has excellent theoretical and applied value. Summary of the Invention

[0005] To address the shortcomings of the existing technology, this invention provides a high-performance β-glucuronidase modified by mutation and its application, aiming to solve the problem of β-glucuronidase derived from Chaetomium globulus ( Chaetomium globosum The insufficient substrate specificity of β-glucuronidase (DX-THS3) hinders the efficient synthesis of GAMG.

[0006] The first technical solution provided by this invention is a β-glucuronidase mutant, wherein the mutant is formed by mutating at least one site of the β-glucuronidase parent amino acid sequence as shown in SEQ ID NO.1: (1) Valine at position 79 is mutated to lysine; (2) The 101st tryptophan is mutated to lysine; (3) Glutamine at position 278 is mutated to lysine; (4) The alanine at position 389 is mutated to glutamine, phenylalanine or glutamic acid; (5) Alanine at position 391 is mutated to arginine; (6) Glycine at position 392 is mutated to glutamine; (7) Valine at position 394 is mutated to arginine; (8) The aspartic acid at position 440 is mutated to lysine; (9) Asparagine at position 474 is mutated to arginine; (10) Valine at position 501 is mutated to lysine; (11) Glutamic acid at position 532 is mutated to tryptophan; (12) The aspartic acid at position 536 is mutated to tryptophan or glutamine; (13) Serine at position 589 is mutated to lysine; (14) The alanine at position 591 is mutated to arginine, histidine, or lysine; (15) Arginine at position 592 is mutated to glutamic acid, tryptophan or glutamine; (16) Asparagine at position 596 is mutated to glutamine.

[0007] The second technical solution provided by the present invention is a gene encoding the mutant described in the first technical solution.

[0008] The third technical solution provided by the present invention is a recombinant vector carrying the gene described in the second technical solution.

[0009] In some embodiments, the recombinant vector uses plasmid pET28a as the expression vector.

[0010] The fourth technical solution provided by the present invention is to express the mutant described in the first technical solution, or to contain the gene described in the second technical solution, or to transform a host cell with the recombinant vector described in the third technical solution.

[0011] In some embodiments, the host cell is Escherichia coli.

[0012] The fifth technical solution provided by this invention is a method for improving the specificity of β-glucuronidase for the substrate β-1,2 glycosidic bond, wherein the method involves performing at least one of the following mutations on the β-glucuronidase parent with the amino acid sequence shown in SEQ ID NO.1: (1) Valine at position 79 is mutated to lysine; (2) The 101st tryptophan is mutated to lysine; (3) Glutamine at position 278 is mutated to lysine; (4) The alanine at position 389 is mutated to glutamine, phenylalanine or glutamic acid; (5) Alanine at position 391 is mutated to arginine; (6) Glycine at position 392 is mutated to glutamine; (7) Valine at position 394 is mutated to arginine; (8) The aspartic acid at position 440 is mutated to lysine; (9) Asparagine at position 474 is mutated to arginine; (10) Valine at position 501 is mutated to lysine; (11) Glutamic acid at position 532 is mutated to tryptophan; (12) The aspartic acid at position 536 is mutated to tryptophan or glutamine; (13) Serine at position 589 is mutated to lysine; (14) The alanine at position 591 is mutated to arginine, histidine, or lysine; (15) Arginine at position 592 is mutated to glutamic acid, tryptophan or glutamine; (16) Asparagine at position 596 is mutated to glutamine.

[0013] The sixth technical solution provided by the present invention is a catalyst, wherein the catalyst is the mutant described in the first technical solution, or a whole-cell catalyst prepared from host cells as described in the fifth technical solution.

[0014] The present invention provides a seventh technical solution, which is a method for biosynthesizing glycyrrhetinic acid monoglucuronide (GAMG), wherein the method utilizes the catalyst described in the sixth technical solution to hydrolyze glycyrrhizic acid to synthesize GAMG.

[0015] The eighth technical solution provided by the present invention is the application of the mutant described in the first technical solution, or the gene described in the second technical solution, or the recombinant vector described in the third technical solution, or the host cell described in the fourth technical solution, or the method described in the fifth technical solution, or the catalyst described in the sixth technical solution in the hydrolysis of glycoside compounds containing β-1,2-glycosidic bonds.

[0016] In some embodiments, the application involves hydrolyzing glycoside compounds containing β-1,2-glycosidic bonds to generate corresponding aglycone compounds or corresponding deglycosylated compounds.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: Compared to wild-type β-glucuronidase, this invention improves the substrate specificity of the wild-type enzyme, enabling it to specifically hydrolyze glycyrrhizic acid into GAMG. The yield of GAMG is 30.5 g / L, with a conversion rate of up to 97%. The specific activity of the mutant enzyme is 1565 U / mg. The improved substrate specificity and enzyme activity can effectively reduce the economic cost of industrial applications. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the process of β-glucuronidase hydrolyzing glycyrrhizic acid.

[0019] Figure 2 The results show the PCR amplification and verification of the pET28a-cgGUS plasmid vector, where A: PCR fragment amplification; B: PCR verification of transformant colonies; lanes A1-A3: pET28a vector fragment; lanes A4-A6: cgGUS gene fragment; lanes B1-B10: PCR amplification fragments of positive recombinant clone colonies.

[0020] Figure 3 HPLC chromatograms for substrate-specific screening of wild-type and mutant strains.

[0021] Figure 4 Gel electrophoresis image of purified mutant β-glucuronidase protein.

[0022] Figure 5 HPLC chromatogram for detecting GAMG content in whole-cell catalytic production. Detailed Implementation

[0023] refer to Figures 1-5The preferred embodiments of the present invention will be described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0024] Test method: Determination of β-glucuronidase activity: Enzyme activity assay conditions: 100 μL of enzyme solution was added to 900 μL of 50 mM sodium acetate buffer solution (pH 6.0) containing 40 g / L GL. After mixing, the mixture was reacted at 45℃ for 30 min. After the reaction was completed, the reaction was terminated by boiling in a water bath for 10 min. 200 μL of the reaction solution was mixed with 800 μL of methanol and filtered through a 0.22 μm filter membrane to prepare the HPLC detection sample.

[0025] HPLC detection conditions: mobile phase 70% methanol and 30% water (containing 0.5% acetic acid), flow rate 1 mL / min, detection time 40 min.

[0026] Enzyme activity unit (U) definition: The amount of enzyme required to produce 1 nmol of glycyrrhetinic acid monoglucuronide per minute under the above conditions.

[0027] Enzyme activity (U / mg) = Enzyme activity / Protein mass.

[0028] GAMG substrate specificity S cb (%): The specific calculation formula is as follows: S cb (%)=GAMG mol / (GAMG) mol +GA mol ) × 100%. Among them, GAMG mol This indicates the molar concentration of GAMG. mol This indicates the molar concentration of GA.

[0029] Materials used in the examples: Derived from Chaetomium globosum ( Chaetomium globosum The coding sequence of DX-THS3 (GenBank: MN207130.1) was optimized for codon preference in E. coli, and then Yixin Biotechnology (Shanghai) Co., Ltd. was commissioned to conduct codon optimization synthesis.

[0030] Commonly used Escherichia coli recipient bacteria E. coli BL21(DE3) and the commonly used expression vector pET 28a, purchased from Yixin Biotechnology (Shanghai) Co., Ltd.

[0031] Peptone and yeast extract were purchased from Oxoid for LB liquid medium (10.0 g / L peptone, 5.0 g / L yeast extract, 5.0 g / L NaCl) and TB fermentation medium (12.0 g / L peptone, 24.0 g / L yeast extract, 4.0 g / L glycerol, 17 mmol / L KH2PO4, 72 mmol / L K2HPO4). Other biochemical reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. Plasmid extraction and product purification were performed using kits from Vazyme.

[0032] Protein Marker, DNA Marker, High-Fidelity DNA Polymerase, and Recombinase 2×CE Mix were purchased from Vazyme.

[0033] The substrate glycyrrhizic acid was provided by Beijing Jinke Biotechnology Co., Ltd., while sodium acetate and sodium hydroxide were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0034] Example 1: Construction of recombinant plasmid pET28a-cgGUS The recombinant plasmid pET28a-cgGUS is a recombinant vector encoding the wild-type β-glucuronidase gene. First, based on the original gene sequence (GenBank: MN207130.1), the gene was synthesized using *E. coli* as the expression host through codon optimization. The optimized β-glucuronidase gene was then seamlessly inserted between the Nco I and Xho I restriction sites of the pET-28a expression vector to obtain the recombinant vector pET28a-cgGUS. The recombinant vector pET28a-cgGUS was then transformed into the host cell. E. coli BL21 (DE3) was plated onto LB resistant plates containing 50 μg / mL kanamycin and incubated overnight at 37°C. Transformants were selected for PCR verification, and positive transformants were sequenced. After successful sequencing, single bacteria were picked for culture and the recombinant vector pET28a-cgGUS was extracted.

[0035] Example 2: Construction of substrate-specific mutant strains (1) Construction of substrate-specific mutant libraries First, the structure of the cg-GUS protein was predicted using Alphafold 2. Then, it was molecularly docked with the 3D structure of the substrate glycyrrhizic acid molecule using Autodock Vina. The conformation with the lowest binding energy was selected. Amino acid residues within a 6A radius centered on the substrate were defined as the active pocket components. The substrate binding pocket was determined to consist of 27 amino acids: V79, W101, R184, H185, D186, Q278, A389, I390, G392, V394, N440, E441, E474, Q476, R481, N494, Y496, Y550, E532, D536, W578, F583, S589, A591, R592, N596, and K598. Mutation screening was then performed on the amino acids in the substrate binding pocket. Using the constructed pET28a-cgGUS as a template, site-directed mutagenesis primers were designed. Primer sequences are shown in Table 1. PCR reaction conditions were: 95℃ for 3 min, 30 cycles (95℃ for 15 s, 60℃ for 15 s, 72℃ for 4 min), 72℃ for 5 min. PCR amplification system: 1 μL template, 2 μL each of forward and reverse primers, 25 μL 2× Phanta Max Master Mix, 20 μL ddH2O. After PCR amplification, the original template was digested with Dpn I, and 10 μL of the PCR amplification product was transformed into... E. coli BL21(DE3) competent cells were spread onto LB resistant plates containing 50 μg / mL kanamycin and cultured overnight at 37°C. Transformants were selected for sequencing to obtain recombinant strains that successfully underwent site-directed saturation mutagenesis.

[0036] Table 1. Site-directed mutagenesis primers

[0037] (2) Screening of substrate-specific mutants Single colonies of these mutant strains were picked and cultured in LB medium containing kanamycin resistance at 37°C for 12 h. 500 μL of each colony was then transferred to 50 mL of TB medium and cultured at 37°C and 220 rpm until OD (dose elapsed). 600=0.8, add 0.05 mM IPTG inducer, and induce at 25℃ and 220 rpm for 20 h. Centrifuge at 8000 rpm for 10 min to collect bacterial cells, discard the supernatant, wash and resuspend the bacterial cells with sodium acetate buffer, centrifuge again at 8000 rpm for 10 min, discard the supernatant, and finally resuspend the bacterial cells with 25 mL sodium acetate buffer and sonicate. Take 100 μL of the lysate and add it to 900 μL of 50 mM sodium acetate buffer solution (pH 6.0) containing 2 g / L GL, mix well, and react at 45℃ for 12 h. After the reaction is completed, stop the reaction by boiling water bath for 10 min. Take 200 μL of the reaction solution and mix it with 800 μL of methanol, filter through a 0.22 μm filter membrane, and detect the content of glycyrrhetinic acid monoglucuronide by HPLC to screen for mutant strains with high substrate specificity.

[0038] In all mutants, the following mutations were made: valine at position 79 of β-glucuronidase was replaced with lysine; tryptophan at position 101 was replaced with lysine; glutamine at position 278 was replaced with lysine; alanine at position 389 was replaced with glutamine, phenylalanine, or glutamic acid; alanine at position 391 was replaced with arginine; glycine at position 392 was replaced with glutamine; valine at position 394 was replaced with arginine; aspartic acid at position 440 was replaced with lysine; asparagine at position 474 was replaced with arginine; valine at position 501 was replaced with lysine; glutamic acid at position 532 was replaced with tryptophan; aspartic acid at position 536 was replaced with tryptophan or glutamine; serine at position 589 was replaced with lysine; alanine at position 591 was replaced with arginine, histidine, or lysine; arginine at position 592 was replaced with glutamic acid, tryptophan, or glutamine; and asparagine at position 596 was replaced with glutamine. When performing single-point mutations, the substrate specificity of mutant β-glucuronidase was improved to varying degrees compared with the wild type (as shown in Table 2). Among them, the substrate specificity of mutants 592W, A389Q, A391R, D536W, S589K, A591R, A591K, and R592Q was significantly improved, at 58%, 54%, 71%, 66%, 63%, 52%, 65%, and 65%, respectively.

[0039] Table 2. Substrate specificity of wild-type and single-point mutant strains

[0040] Example 3: Substrate specificity and enzyme activity detection of combined mutants To further improve the substrate specificity of the enzyme, the dominant unit point mutants obtained in Example 2 were selected for combined mutation, and their substrate specificity and enzyme activity were tested. The substrate specificity detection method was the same as in Example 2, and the enzyme activity detection method is as follows: Single colonies of the combined mutant strain were picked and placed into 10 mL LB medium containing 50 μg / mL kanamycin. The culture was activated overnight at 37°C and 220 rpm using a shaker. The colonies were then transferred to 100 mL TB medium at a 1% inoculum and cultured until OD500. 600 The concentration was set to 0.8, 0.05 mM IPTG was added, and the cells were induced at 25 °C for 20 h. The cells were then centrifuged at 8000 rpm for 10 min to collect the bacteria. After resuspending the cells twice in 20 mM Tris buffer (pH 7.4), the cells were washed and then resuspended in 50 mL of buffer. The cells were sonicated for 20 min, and the supernatant was collected by centrifugation. The supernatant was filtered through a 0.22 μm filter membrane and loaded onto an AKTA protein purifier equipped with a HISTRAP HP 1×5 mL pre-packed column. The recombinant protein was eluted with a gradient of imidazole solutions (50, 100, 200, 300, 500 mM), and the eluent was collected. The protein was concentrated by centrifugation at 4 °C and 4500×g using a 30 kDa ultrafiltration tube. The final concentration of the purified protein was determined using the Bradford method. Figure 5 As shown. 0.5 mg of pure enzyme was added to 1 mL of 50 mM sodium acetate buffer solution (pH 6.0) containing 40 g / L GL. After mixing, the mixture was reacted at 45 °C for 30 min. After the reaction was completed, the reaction was terminated by boiling in a water bath for 10 min. 200 μL of the reaction solution was mixed with 800 μL of methanol, filtered through a 0.22 μm filter membrane, and the content of glycyrrhetinic acid monoglucuronide was detected by HPLC. The specific enzyme activity of the mutant strain was calculated.

[0041] The substrate specificity and enzyme activity of the combined mutants are shown in Table 3. The substrate specificity of A389Q / A391R, A591R / R592W, A389Q / A591R, A391R / D536W, A391R / R592Q, W101K / A389Q, A391R / R592Q, A391R / S589K, and A389Q / A591K were increased to 77%, 84%, 82%, 85%, 80%, 74%, 83%, 95%, and 97%, respectively. Under the premise of high substrate specificity, the specific enzyme activity of the combined mutants with high substrate specificity was compared. Finally, the optimal mutant was determined to be the A389Q / A591K combined mutant. This mutant catalyzes only one product, glycyrrhetinic acid monoglucuronide, with a substrate specificity of up to 97% for the target product GAMG and a specific enzyme activity of 1565 U / mg.

[0042] Table 3. Substrate specificity and enzyme activity of the combined mutant strains

[0043] Example 4: E. coliWhole-cell catalytic production of GAMG using BL21-pET28a-A389Q / A591K Preparation of whole-cell catalyst: A single colony of mutant strain A389Q / A591K was inoculated into LB medium containing 50 μg / mL kanamycin and activated overnight at 37°C and 220 rpm. The activated culture was then transferred to TB fermentation medium at a 1% inoculum and cultured at 37°C and 220 rpm until OD200. 600 The concentration was 0.8, 0.05 mM IPTG was added, and the cells were induced at 25 ℃ for 20 h. The cells were collected by centrifugation at 8000 rpm for 10 min, resuspended and washed twice with 50 mM sodium acetate buffer solution (pH 6.0), centrifuged at 8000 rpm for 10 min, and the weight of the wet cells was measured.

[0044] Whole-cell catalytic reaction system: 60 g of wet bacterial cells were weighed and mixed with 200 mL of 40 g / L glycyrrhizic acid substrate solution (pH 6.0). The mixture was reacted at 45℃ and 220 rpm for 12 h on a shaker. After the reaction was complete, 200 μL of the reaction solution was mixed with 800 μL of methanol, filtered through a 0.22 μm filter membrane, and analyzed by HPLC. Figure 5 The yield of glycyrrhetinic acid monoglucuronide was 30.5 g / L, and the GAMG conversion rate was 97%.

[0045] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A β-glucuronidase mutant, characterized in that, The mutant is a β-glucuronidase parent with the amino acid sequence shown in SEQ ID NO. 1, with mutations at at least one of the following sites: (1) valine at position 79 is mutated to lysine; (2) tryptophan at position 101 is mutated to lysine; (3) glutamine at position 278 is mutated to lysine; (4) alanine at position 389 is mutated to glutamine, phenylalanine, or glutamic acid; (5) alanine at position 391 is mutated to arginine; (6) glycine at position 392 is mutated to glutamine; (7) valine at position 394 is mutated to arginine; (8) aspartic acid at position 440 is mutated to lysine; (9) asparagine at position 474 is mutated to arginine; (10) valine at position 501 is mutated to lysine; (11) glutamic acid at position 532 is mutated to tryptophan; (12) aspartic acid at position 536 is mutated to tryptophan or glutamine; (13) alanine at position 589 is mutated to lysine; (4) alanine at position 389 is mutated to arginine, phenylalanine, or glutamic acid; (5) alanine at position 391 is mutated to arginine; (6) glycine at position 392 is mutated to glutamine; (7) valine at position 394 is mutated to arginine; (8) aspartic acid at position 440 is mutated to lysine; (9) asparagine at position 474 is mutated to arginine; (10) valine at position 50 (14) The 591st alanine is mutated to arginine, histidine or lysine; (15) The 592nd arginine is mutated to glutamic acid, tryptophan or glutamine; (16) The 596th asparagine is mutated to glutamine.

2. The gene encoding the mutant of claim 1.

3. A recombinant vector carrying the gene of claim 2.

4. The recombinant vector according to claim 3, characterized in that, The recombinant vector uses plasmid pET28a as the expression vector.

5. A host cell expressing the mutant of claim 1, or containing the gene of claim 2, or transformed with the recombinant vector of any one of claims 3 to 4.

6. The host cell according to claim 5, characterized in that, The host cell is Escherichia coli.

7. A method for improving the specificity of β-glucuronidase for substrate β-1,2 glycosidic bonds, characterized in that, The method involves performing at least one of the following mutations on the β-glucuronidase parent with the amino acid sequence shown in SEQ ID NO.1: (1) Valine at position 79 is mutated to lysine; (2) The 101st tryptophan is mutated to lysine; (3) Glutamine at position 278 is mutated to lysine; (4) The alanine at position 389 is mutated to glutamine, phenylalanine or glutamic acid; (5) Alanine at position 391 is mutated to arginine; (6) Glycine at position 392 is mutated to glutamine; (7) Valine at position 394 is mutated to arginine; (8) The aspartic acid at position 440 is mutated to lysine; (9) Asparagine at position 474 is mutated to arginine; (10) Valine at position 501 is mutated to lysine; (11) Glutamic acid at position 532 is mutated to tryptophan; (12) The aspartic acid at position 536 is mutated to tryptophan or glutamine; (13) Serine at position 589 is mutated to lysine; (14) The alanine at position 591 is mutated to arginine, histidine, or lysine; (15) Arginine at position 592 is mutated to glutamic acid, tryptophan or glutamine; (16) Asparagine at position 596 is mutated to glutamine.

8. A catalyst, characterized in that, The catalyst is the mutant described in claim 1, or a whole-cell catalyst prepared from the host cell as described in any one of claims 5 to 6.

9. A method for biosynthesizing glycyrrhetinic acid monoglucuronide, characterized in that, The method involves using the catalyst described in claim 8 to hydrolyze glycyrrhizic acid to synthesize GAMG.

10. The use of the mutant of claim 1, or the gene of claim 2, or the recombinant vector of any one of claims 3-4, or the host cell of any one of claims 5-6, or the method of claim 7, or the catalyst of claim 8 in the hydrolysis of glycoside compounds containing β-1,2-glycosidic bonds.

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