Truncation enzyme GlSK-20a of glucosidase as well as preparation method and application of truncation enzyme GlSK-20a
By designing a glucosidase GlSK-20a with improved heat resistance and byproduct tolerance, the problems of heat stability and byproducts of existing natural glycosidases in the preparation of ginsenosides were solved, and efficient industrial production was achieved.
Patent Information
- Application Number
- CN202510814513.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-18
- Publication Date
- 2025-09-09
AI Technical Summary
Existing natural glycosidases have insufficient thermal stability and by-product tolerance during the preparation of ginsenosides, resulting in low reaction efficiency and serious by-products, making it difficult to meet industrial needs.
A truncated glucosidase, GlSK-20a, was designed. Through 200ns kinetic simulation based on the three-dimensional structure of wild glucosidase, 20 amino acids in the unstable N-terminal region were truncated to obtain an enzyme with significantly improved heat resistance and byproduct tolerance, which was used in the preparation of ginsenoside F1.
The thermal stability of glucosidase GlSK-20a was increased by 9.17 times, and the by-product tolerance was increased by 5.2 times, which significantly improved the preparation efficiency and yield of ginsenoside F1, making it suitable for large-scale industrial production.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of enzyme catalysis, and in particular relates to a truncated enzyme of glucosidase GlSK-20a, a preparation method and an application thereof. Background Art
[0002] Ginsenosides are the active ingredients in the traditional Chinese medicinal plant Panax ginseng, exhibiting a wide range of pharmacological effects. However, due to their high polarity and molecular weight, ginsenosides have low absorption and bioavailability in the body. Their deglycosylated secondary metabolites, known as "rare ginsenosides," are more readily absorbed into the bloodstream and exert their active effects. Rare saponins possess promising pharmacological effects, including anti-cancer and protective effects on the immune, nervous, and cardiovascular systems, and possess enormous potential for pharmaceutical development and market application. Traditionally, rare saponins have been primarily prepared using acid-base hydrolysis. Compared to chemical methods, which are characterized by harsh reaction conditions, numerous byproducts, and severe environmental pollution, biotransformation, with its milder reaction conditions, higher efficiency, and pollution-free nature, has become a more widely used preparation method.
[0003] Currently, β-glucosidase, β-xylosidase, and α-rhamnosidase have been successfully used to prepare rare ginsenosides. However, due to the poor solubility of ginsenosides, modern industrial production often requires high temperatures to promote dissolution to improve reaction efficiency and reduce the risk of microbial contamination during long fermentation processes. At the same time, the byproduct sugars produced during the preparation of ginsenosides inevitably severely reduce their catalytic activity. Existing natural glycosidases generally exhibit limited thermal stability and sugar tolerance. Therefore, the development of industrial biocatalysts with strong thermal stability and byproduct tolerance is of great significance. Summary of the Invention
[0004] In view of this, the object of the present invention is to provide a truncated enzyme GlSK-20a of glucosidase and its preparation method and application; the truncated enzyme GlSK-20a has good heat resistance, high tolerance to by-products, simple preparation process, wide range of applications, and has the potential to be applied to large-scale industrial production; the application of the truncated enzyme to the preparation of the high-value compound ginsenoside F1 can significantly increase the yield.
[0005] The present invention provides a truncated glucosidase GlSK-20a, the amino acid sequence of which is shown in SEQ ID NO.1.
[0006] The present invention provides a gene encoding the truncation enzyme GlSK-20a, the nucleotide sequence of which is shown in SEQ ID NO.3.
[0007] The present invention provides a method for preparing the truncated enzyme GlSK-20a, comprising the following steps:
[0008] 1) Recombining the gene into an expression vector to obtain a recombinant vector;
[0009] 2) The recombinant vector obtained in step 1) is transferred into a host bacterium to induce expression to obtain the truncated glucosidase.
[0010] Preferably, in step 1), the expression vector is pETduet1 or pET28a, and the host bacteria is Escherichia coli BL21 (DE3).
[0011] Preferably, after the induction expression in step 2) is completed, the bacteria are collected and crushed to obtain a crude enzyme solution.
[0012] The present invention provides the use of the truncated enzyme GlSK-20a in catalyzing the synthesis of ginsenoside F1.
[0013] Preferably, the substrate for catalytic synthesis of ginsenoside F1 is ginsenoside Rg1, and the substrate concentration is 0.5-5 mg / ml.
[0014] Preferably, the temperature for catalytic synthesis of ginsenoside F1 is 30-50°C.
[0015] Preferably, the pH of the catalytic synthesis of ginsenoside F1 is 5.5 to 8, and the time is 3 to 24 hours.
[0016] Compared with the prior art, the present invention has the following beneficial effects: the present invention is based on the three-dimensional structure of wild glucosidase GlSK, uses gromacs to perform 200ns kinetic simulation, analyzes the RMSF in its trajectory file, and obtains the first 35 amino acids of N-terminus as an unstable region; the truncated enzyme GlSK-20a of glucosidase is obtained by designing 20 amino acids of N-terminal truncation, and the truncated enzyme GlSK-20a has good heat resistance, high by-product tolerance, and thermal stability and sugar tolerance are respectively increased by 9.17 times and 5.2 times than the wild enzyme. The present invention applies the glucosidase GlSK-20a to the preparation of catalytic synthesis of ginsenoside F1, and the target substance yield is greatly improved compared with the wild enzyme. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Figure 1 Schematic diagram of the truncated enzyme RMSF and its design.
[0018] Figure 2 Comparison of the thermal stability and sugar tolerance of wild-type enzyme GlSK and truncated enzyme GlSK-20a;
[0019] Figure 3 The yield diagram of the high-value compound ginsenoside F1 produced by wild enzyme GlSK and truncated enzyme GlSK-20a;
[0020] Figure 4 Comparison results of the enzymatic activities of wild-type enzyme GlSK and different truncated enzymes are shown. DETAILED DESCRIPTION
[0021] The present invention provides a truncated glucosidase enzyme GlSK-20a, the amino acid sequence of which is shown in SEQ ID NO.1, specifically as follows:
[0022] .
[0023] Based on the three-dimensional structure of wild-type glucosidase GlSK, this study used Gromacs to perform a 200 ns kinetic simulation. RMSF analysis of the trajectory file revealed that the first 35 amino acids at the N-terminus represent an unstable region. A 20-amino acid truncation of the N-terminus was designed to generate glucosidase GlSK-20a. The amino acid sequence of the wild-type enzyme is shown in SEQ ID NO. 2 and is as follows:
[0024] M GSSHHHHHHSQDPPTPLTTL.
[0025] The bold and underlined part is the truncated 20 amino acids.
[0026] The present invention provides a gene encoding the truncation enzyme GlSK-20a, the nucleotide sequence of which is shown in SEQ ID NO.3, as follows:
[0027]
[0028] The present invention also provides a method for preparing the truncated enzyme GlSK-20a, comprising the following steps: 1) recombining the gene into an expression vector to obtain a recombinant vector; 2) transferring the obtained recombinant vector into a host bacterium to induce expression to obtain the truncated enzyme GlSK-20a.
[0029] In the present invention, the gene is recombined into an expression vector to obtain a recombinant vector, preferably pETduet1 or pET28a. The gene is preferably recombined between the NcoI and XhoI restriction sites of the expression vector. The present invention does not particularly limit the specific steps and parameters of the recombinant vector, and conventional gene cloning methods in the art can be used.
[0030] After obtaining the recombinant vector, the present invention transfers the obtained recombinant vector into a host bacterium to induce expression and obtain the truncated glucosidase GlSK-20a. In the present invention, the host bacterium is preferably Escherichia coli BL21 (DE3). The present invention does not specifically limit the specific method for transferring the recombinant vector into the host bacterium, and the conventional chemical transformation method in the field can be used. The present invention does not specifically limit the specific parameters of the chemical transformation method. In the present invention, the induced expression specifically includes the following steps: S1) culturing the host bacterium into which the recombinant vector has been transferred at 36-38°C for 10-14h; S2) inducing the cultured host bacterium at 15-17°C for 20-24h.
[0031] After the induced expression is completed, the cells are collected and disrupted to obtain a crude enzyme solution. Preferably, the collected cells are resuspended in a phosphate buffer and disrupted using a high-pressure homogenization method to obtain a crude enzyme solution. In the present invention, the concentration of the phosphate buffer is preferably 15 to 25 mM, more preferably 18 to 22 mM, and most preferably 20 mM; the pH of the phosphate buffer is preferably 7.8 to 8.2, more preferably 8.0.
[0032] The present invention also provides the use of the truncated enzyme GlSK-20a in catalyzing the synthesis of ginsenoside F1.
[0033] In the present invention, the substrate for the catalytic synthesis of ginsenoside F1 is preferably ginsenoside Rg1, and the substrate concentration is preferably 0.5-5 mg / ml. In the present invention, the temperature for the catalytic synthesis of ginsenoside F1 is 30-50°C; the pH for the catalytic synthesis of ginsenoside F1 is preferably 5.5-8, and the time for the catalytic synthesis of ginsenoside F1 is preferably 3-24 h.
[0034] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.
[0035] Example 1
[0036] This example is based on the three-dimensional structure of wild-type glucosidase GlSK (SEQ ID NO. 2). A 200 ns kinetic simulation was performed using gromacs. The RMSF analysis in the trajectory file revealed that the first 35 amino acids at the N-terminus are unstable. A sequence with a 20-amino acid truncation of the N-terminus was designed, and the truncated sequence is shown in SEQ ID NO. 1.
[0037] The restriction sites were selected as NcoI and XhoI, and the gene fragment of the truncated enzyme GlSK-20a was obtained by PCR using the wild-type GlSK as a template. At the same time, the pETduet1 empty plasmid was double-digested with NcoI and XhoI to obtain a plasmid fragment with sticky ends. After the plasmid and gene fragment were reacted at 50°C for 30 minutes using homologous recombinase, the ligated plasmid was transformed into Escherichia coli trans5α. The correctly sequenced plasmid GlSK-20aa was introduced into Escherichia coli BL21 (DE3) by chemical transformation to obtain a recombinant bacterium that can express the truncated enzyme.
[0038] In this example, a control bacterium expressing a wild enzyme was also set up. The gene encoding the wild enzyme was constructed into the expression vector pETduet1, and the restriction sites were NcoI and XhoI. The constructed plasmid GlSK was introduced into Escherichia coli BL21 (DE3) by chemical transformation to obtain a control bacterium that can express the wild enzyme.
[0039] 20 μL of recombinant bacteria expressing the truncated enzyme and control bacteria expressing the wild-type enzyme were inoculated into 4 mL of LB liquid medium. After overnight culture at 37°C, 2 mL of the culture was transferred to 250 mL of LB liquid medium and induced for protein expression at 16°C for 22 hours. After expression, the cells were collected and resuspended in 20 mM pH 8.0 phosphate buffer and disrupted using high-pressure homogenization to obtain crude enzyme solutions of the truncated enzyme and the wild-type enzyme, respectively.
[0040] Weigh 5 g of p-nitrophenyl-β-D-pyranoglucopyranoside (pNPG) and dissolve it in 5 mL of deionized water to obtain a pNPG solution. Take 0.3 mL of the pNPG solution, add GlSK-20aa and GlSK crude enzyme solution, which contains 0.005 mg of glucosidase (truncated enzyme or wild enzyme), and react at 30°C for 1 min. Add 0.2 mL of 1 M Na2CO3, and the absorbances are measured at 405 nM to be 2.244 and 2.174, respectively. Substituting the p-nitrophenol standard curve Y = (A-0.02593) / 0.00545, the final reaction rates are calculated to be 407.06 μmol / L / min for GlSK-20aa and 394.18 μmol / L / min for GlSK.
[0041] Example 2
[0042] The wild enzyme GLSK and the truncated enzyme GlSK-20aa were incubated at 30 and 40°C for 20-140 min, respectively, and the activity changes of each group of enzymes were measured. The activity at time 0 was set as 100%. The results of the thermal stability test are shown in Figure 2. Figure 2 As shown in A in FIG, it can be seen that after incubation under different temperature conditions, the enzymatic activity of the truncated enzyme GlSK-20a is significantly higher than that of the wild enzyme, indicating that the truncated enzyme prepared by the present invention has better thermal stability than the wild enzyme.
[0043] Example 3
[0044] The wild enzyme GLSK and the truncated enzyme GlSK-20aa were reacted in a system with a final concentration of 0-0.4M glucose. The reaction system contained 0.005mg enzyme and 0.3g substrate pNPG. The reaction system was reacted at 30℃ for 1min, and the absorbance of each group was measured. The activity of the 0M glucose group was set as 100%. The glucose tolerance test results are shown in Figure 2. Figure 2 As shown in B, it can be seen that the sugar tolerance of the truncated enzyme is significantly better than that of the wild enzyme, especially when the sugar concentration is high.
[0045] Example 4
[0046] The wild enzyme GLSK and the truncated enzyme GlSK-20aa were used.
[0047] Weigh ginsenoside Rg1 and dissolve it in water to prepare a 0.5-5 mg / ml substrate solution. Add 0.5 mL of the control bacteria expressing the wild enzyme GLSK and the recombinant bacteria expressing the truncated enzyme GlSK-20aa constructed in Example 1 with an OD of 1.8, respectively. React at 30°C for 12 hours, and detect the product ginsenoside F1 by liquid chromatography. The results are as follows. Figure 3 As shown in Figure 2, the yields of wild enzyme GLSK and truncated enzyme GlSK-20aa for hydrolyzing ginsenoside Rg1 were 0.204 mg / mL and 0.205 mg / mL at low substrate concentration (0.5 mg / mL), and 0.4056 mg / mL and 1.636 mg / mL at high substrate concentration ( Figure 3 ).
[0048] Comparative Example 1
[0049] According to the method of Example 1, based on the three-dimensional structure of wild-type glucosidase GlSK SEQ ID NO.2, 200 ns kinetic simulation was performed using gromacs. The RMSF in the trajectory file was analyzed and the first 35 amino acids of the N-terminus were found to be unstable regions. Sequences of 30 and 35 amino acids of N-terminal truncation were designed and the enzyme activity was measured. The results are as follows: Figure 4 As shown, the enzymatic activity of the sequences with N-terminal truncations of 30 and 35 amino acids was significantly lower than that of the wild-type enzyme and the truncated enzyme GlSK-20aa.
[0050] As can be seen from the above examples, the truncated enzyme GlSK-20a of the glucosidase provided by the present invention is 9.17 times more thermostable than the wild-type enzyme, and the byproduct glucose tolerance is increased by 5.2 times. The truncated enzyme GlSK-20a has good heat resistance, high byproduct tolerance, simple preparation process, wide application range, and has the potential for application in large-scale industrial production.
[0051] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.
Claims
1. A truncated enzyme of glucosidase GlSK-20a, characterized in that The amino acid sequence is shown in SEQ ID NO.
1.
2. The gene encoding the truncation enzyme GlSK-20a according to claim 1, characterized in that The nucleotide sequence is shown in SEQ ID NO.
3.
3. The method for preparing the truncated enzyme GlSK-20a according to claim 1, wherein The following steps are involved: 1) Recombining the gene according to claim 2 into an expression vector to obtain a recombinant vector; 2) The recombinant vector obtained in step 1) is transferred into a host bacterium to induce expression to obtain the truncated enzyme GlSK-20a.
4. The preparation method according to claim 3, characterized in that In step 1), the expression vector is pETduet1 or pET28a, and the host bacteria is Escherichia coli BL21 (DE3).
5. The preparation method according to claim 3, characterized in that Step 2) After the induction expression is completed, the bacteria are collected and crushed to obtain a crude enzyme solution.
6. Use of the truncated enzyme GlSK-20a according to claim 1 in catalyzing the synthesis of ginsenoside F1.
7. The use according to claim 6, characterized in that The substrate for the catalytic synthesis of ginsenoside F1 is ginsenoside Rg1, and the substrate concentration is 0.5-5 mg / ml.
8. The use according to claim 7, characterized in that The temperature for catalytic synthesis of ginsenoside F1 is 30-50°C.
9. The use according to claim 8, characterized in that The pH value for catalytic synthesis of ginsenoside F1 is 5.5 to 8, and the time is 3 to 24 hours.