Glycoside hydrolase mutants and uses thereof

Abstract

The application belongs to the technical field of enzyme engineering, and relates to a glycoside hydrolase mutant and application thereof. The application successfully obtains a high-temperature stable glycoside hydrolase mutant Bgl8-4X (M332S / H177A / N367P / L254K) through a semi-rational modification strategy, and significantly improves the conversion efficiency of CK. In a high-temperature condition of 70 DEG C, the CK yield of Bgl8-4X reaches 1311.02% of that of the wild-type Bgl8 when a crude ginsenoside extract is used as a substrate, and the application provides a potential candidate enzyme for solving the poor thermal stability difference in multi-enzyme combination and the industrial high-temperature environment adaptation problem.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering technology and relates to a glycoside hydrolase mutant and its application. Background Technology

[0002] Ginseng is a precious Chinese medicinal herb, and ginsenosides are its main active ingredients. Rare ginsenosides, due to their lower glycosyl content, exhibit significant advantages in terms of absorption and activity in vivo. Rare ginsenoside Compound K (CK) possesses various potential medicinal values. Currently, the main components of ginsenosides, such as Rb1, Rb2, and Rc, can be converted into CK through multiple single-enzyme catalysis or multi-enzyme combination. The preparation of CK requires hydrolysis of glycosyl groups at different positions. For the multi-enzyme combination process of CK preparation, different glycoside hydrolases exhibit significant differences in thermal stability. Furthermore, in industrial production, high temperatures reduce microbial contamination and lower solution viscosity, making thermosensitive enzymes indispensable. Therefore, this invention aims to modify glycoside hydrolases to obtain a glycoside hydrolase that retains activity at high temperatures, providing a candidate thermostable enzyme for the preparation of CK using single-enzyme or multi-enzyme combination methods at high temperatures. Summary of the Invention

[0003] To address the shortcomings of existing technologies, the present invention adopts the following technical solution:

[0004] The first aspect of this invention discloses a glycoside hydrolase mutant, the amino acid sequence of which is shown in SEQ ID NO: 1;

[0005] MSLPKGFLWGAATASYQIEGAWNEDGKGESIWDRFTHQKGNILYGHNGDVACDHYHRFEEDVSLMKELGLKAYRFSIAWARIFPDGFGTVNQKGLEFYDRLINKLVENGIEPVV TIYHWDLPQKLQDIGGWANPEIVNYYFEYAMLIVNRYKDKVKKWITFNEPYCIAFLGHFYGVAAPGIKDFKVAMDVVHNIMLSHFKVVKAVKENNIDVEVGITLNLTPVYFQTE RLGYKVSEIEREMVNLSSQLDNELFKDPVLKGSYPQKLFDYLVQKDLLETQKVLSMQQEVKENFVFPDFLGINYYTRAVRLYDENSNWIFPIRWEHPAGEYTESGWEVFPQGLY DLLIWIKESYPQIPIYITENGAAYPDKVEDGRVHDQKRVEYLKQHFEAARKAIENGVDLRGYFVWSLLDNLEWAMGYTKRFGVIYVDYETQKRIKKDSFYFYQQYIKENS (SEQ ID NO: 1).

[0006] The glycoside hydrolase mutant Bgl8-4X, compared with wild-type Bgl8, includes the following mutations: M332S, H177A, N367P, and L254K.

[0007] The amino acid sequence of Bgl8 is shown in SEQ ID NO: 2.

[0008] MSLPKGFLWGAATASYQIEGAWNEDGKGESIWDRFTHQKGNILYGHNGDVACDHYHRFEEDVSLMKELGLKAYRFSIAWARIFPDGFGTVNQKGLEFYDRLINKLVENGIEPVV TIYHWDLPQKLQDIGGWANPEIVNYYFEYAMLIVNRYKDKVKKWITFNEPYCIAFLGHFYGVHAPGIKDFKVAMDVVHNIMLSHFKVVKAVKENNIDVEVGITLNLTPVYFQTE RLGYKVSEIEREMVNLSSQLDNELFLDPVLKGSYPQKLFDYLVQKDLLETQKVLSMQQEVKENFVFPDFLGINYYTRAVRLYDENSNWIFPIRWEHPAGEYTEMGWEVFPQGLY DLLIWIKESYPQIPIYITENGAAYNDKVEDGRVHDQKRVEYLKQHFEAARKAIENGVDLRGYFVWSLLDNLEWAMGYTKRFGVIYVDYETQKRIKKDSFYFYQQYIKENS (SEQ ID NO:2)

[0009] In one embodiment of the present invention, the nucleotide sequence encoding Bgl8 is shown in SEQ ID NO: 4.

[0010]

[0011] Wherein, M332S refers to replacing methionine at position 332 in the Bgl8 amino acid sequence with serine;

[0012] H177A refers to replacing the histidine at position 177 in the Bgl8 amino acid sequence with alanine;

[0013] The N367P refers to replacing the asparagine at position 367 in the Bgl8 amino acid sequence with proline;

[0014] The L254K refers to replacing the leucine at position 254 in the Bgl8 amino acid sequence with lysine.

[0015] A second aspect of the present invention discloses a polynucleotide encoding the above-mentioned glycoside hydrolase mutant, the sequence of which is shown in SEQ ID NO: 3.

[0016]

[0017] A third aspect of the present invention discloses a recombinant expression vector comprising the aforementioned polynucleotides.

[0018] A fourth aspect of this invention discloses a host cell comprising the aforementioned recombinant expression vector, wherein the host cell is a prokaryotic cell or a eukaryotic cell. The host cell is *Escherichia coli*.

[0019] The fifth aspect of this invention discloses a method for preparing the above-mentioned glycoside hydrolase mutant, which uses a stepwise directed evolution strategy to modify Bgl8 glycoside hydrolase: firstly, multiple alanine mutants with enhanced catalytic activity are obtained by screening using alanine scanning technology; then, a saturated mutant library with 7 sites is constructed based on these beneficial mutation sites, from which 4 optimal mutants are identified: M332S / H177A / N367P / L254K; finally, the mutant Bgl8-4X is obtained through combined saturated mutagenesis technology.

[0020] The sixth aspect of this invention discloses a method for preparing rare ginsenosides, comprising the following steps:

[0021] In the catalytic reaction, the glycoside hydrolase mutant as described in any one of claims 1-3 is contacted with the ginsenoside substrate, thereby catalyzing the conversion of the substrate into CK.

[0022] The ginsenoside substrate is selected from one or more combinations of Rb1, Rb2, Rc, Rd, and F2.

[0023] Preferably, the catalytic reaction is carried out in a temperature range of 30°C to 70°C, and more preferably at 70°C;

[0024] The pH of the catalytic reaction is 5.0 to 7.0, preferably 6.0.

[0025] This invention compares the CK production of Bgl8 wild-type, single-site mutant (Bgl8-1X), and combined saturated mutant (Bgl8-4X) at different temperatures. At 70℃, the CK production of both the combined saturated mutant and the single-site mutant was higher than that of the Bgl8 wild-type. The CK production of the combined saturated mutant was higher than that of the Bgl8 wild-type at all temperatures from 40℃ to 70℃, being 1.16 times higher at 40℃ and 13 times higher at 70℃. The combined saturated mutant significantly enhanced the CK conversion ability of glycoside hydrolases under high-temperature conditions, improving catalytic efficiency and stability. The optimal pH for the combined saturated mutant was tested and found to be 6.0, consistent with the optimal pH for the Bgl8 wild-type.

[0026] Compared with the prior art, the present invention has the following advantages:

[0027] 1. This invention successfully constructed a quadruple mutant Bgl8-4X (M332S / H177A / N367P / L254K) through multiple rounds of combined mutagenesis. Under standard reaction conditions of 70℃, using total ginsenosides as raw material, the CK yield of this mutant Bgl8-4X reached 1311.02% of that of wild-type Bgl8, with significantly improved catalytic performance. The synergistic effect of the four sites significantly enhanced the enzyme's substrate binding capacity and thermal stability.

[0028] 2. The glycoside hydrolase mutant Bgl8-4X prepared by this invention exhibits catalytic efficiency and temperature stability for the conversion of Rd to CK, thereby obtaining an engineered glycoside hydrolase with both high temperature stability and high catalytic efficiency. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention or existing methods and experiments, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained from these drawings without creative effort.

[0030] Figure 1 Protein validation for Bgl1-12.

[0031] Figure 2 In this context, 'a' represents the conversion rate of Bgl 1-12 at 70℃. Figure 2 In the figure, b represents the conversion rate of Bgl 1-12 at 30℃.

[0032] Figure 3 Homologous structure model and catalytic pocket analysis for Bgl8.

[0033] Figure 4 This is a comparison of relative yields for the first round of alanine scanning screening.

[0034] Figure 5 For the second round of alanine scanning screening, a relative yield comparison was conducted.

[0035] Figure 6 Displays the results of saturation mutation screening.

[0036] Figure 7 A comparison of the yields of four combined mutants and wild-type CK.

[0037] Figure 8 The CK yield of the three enzymes at different temperatures.

[0038] Figure 9 The CK yield of Bgl8-4X at different pH values.

[0039] Figure 10 The CK production of Bgl8-4X at different time periods.

[0040] Figure 11 The liquid phase spectrum of Bgl8 reaction at 30℃.

[0041] Figure 12 The liquid phase spectrum of Bgl8 reaction at 70℃.

[0042] Figure 13 The liquid phase spectrum of Bgl8-H177A at 70℃.

[0043] Figure 14 The liquid phase spectrum of Bgl8-1X reaction at 70℃ is shown.

[0044] Figure 15 The liquid phase spectrum of Bgl8-2X reaction at 70℃ is shown.

[0045] Figure 16 The liquid phase spectrum of Bgl8-3X reaction at 70℃.

[0046] Figure 17 The liquid phase spectrum of Bgl8-4X reaction at 30℃ is shown.

[0047] Figure 18 The liquid phase spectrum of Bgl8-4X reaction at 70℃ is shown. Detailed Implementation

[0048] To make the technical problems, technical solutions, and beneficial effects to be solved by this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of this application.

[0049] Example 1

[0050] 1. Recombinant expression of glycoside hydrolases

[0051] Twelve glycoside hydrolases from different sources were expressed in *E. coli* using the pET-28a vector (Table 1). Among them, Bgl8, a glycoside hydrolase derived from *Caldicellulosiruptor bescii*, can hydrolyze the glycosyl groups at positions 3 and 20 of PPD-type ginsenosides, hydrolyzing various PPD-type ginsenosides into CK. This enzyme can simultaneously synthesize the rare ginsenoside CK from Rb1, Rb2, and Rc, which are more abundant in ginseng extracts.

[0052] Table 1

[0053] Serial Number source Gene number Bgl1 WP_025332314.1 Bgl2 KAB7237138.1 Bgl3 KAA8825186.1 Bgl4 WP_163229936.1 Bgl5 WP_046725991.1 Bgl6 MCI1641647.1 Bgl7 TDD71648.1 Bgl8 WP_015907053.1 Bgl9 WP_045173846.1 Bgl10 WP_045170481.1 Bgl11 MBO2493568.1 Bgl12 WP_111572365.1

[0054] 2. Screening results of glycoside hydrolase activity

[0055] Overexpression of Bgl1-12 resulted in a protein band between 25-35 kDa, and the protein size was as expected. Figure 1 In experiments using Rd and total ginsenosides as substrates, this invention investigated the biotransformation capacity of Bgl1-12 for CK. At 30℃ and 70℃, Bgl8 exhibited the strongest biotransformation capacity for CK. Figure 2 As shown in a and b), this invention selected Bgl8 as the parent material for subsequent experiments.

[0056] Example 2 Construction of Bgl8 alanine scanning mutant

[0057] The Bgl8 protein sequence was used to construct a homology model in SWISS-MODEL. This model was constructed using the crystal structure of GH1 as a template. Figure 3 In (a), the sequence homology was 88.32%.

[0058] To determine the catalytic residues of Bgl8, CcBglA, which also belongs to the β-glycosyl hydrolase family, was used. Figure 3 The active centers in b) are E166 and E352, and the catalytic residues of Bgl8 are inferred through structural overlap.

[0059] The catalytic residues of Bgl8 are E163 and E361. Figure 3 (d) , centered on the active site, select an 8 Å range ( Figure 3 Mutations were performed at sites c), and a total of 21 sites were selected, namely 74R, 115T, 118H, 119W, 159I, 160T, 217T, 218L, 219N, 299G, 301N, 303Y, 334W, 358W, 359I, 360T, 362N, 405F, 407W, 408S, and 414E.

[0060] For the first batch of alanine mutant strains constructed, this study used IPTG to induce protein expression and compared it with the wild-type Bgl8. Twenty-one loci (74R, 115T, 118H, 119W, 159I, 160N, 217T, 218L, 219N, 299G, 301N, 303Y, 334W, 358W, 359I, 360T, 362N, 405F, 407W, 408S, 414E) were screened for alanine content and subjected to mutant PCR.

[0061] After the first alanine scan mutation site screening, a second alanine scan mutation site screening was performed, selecting sites adjacent to the sites screened in the first screening: a total of 15 sites were constructed in the first batch of alanine scans (see...). Figure 4(Among them, alanine mutations at L218A, N219A, G299A, and S408A sites resulted in significantly higher CK yields than Bgl8. The first site screening was based on...) Figure 4 The activity levels were reflected, with L218A, N219A, G299A, N362A, S408A, and E414A alanine mutants showing increased activity, W119A, I159A, I359A, and T360A showing decreased activity, and alanine mutants T115A, H118A, T160A, T217A, N301A, and W407A showing loss of activity. Sites with increased activity were selected for subsequent saturation mutations, and sites near these sites were screened.

[0062] After the first batch of alanine mutations, the G299A alanine mutant had the highest CK yield, which was 228.9% of the Bgl8 wild type, and was the highest CK yield among the alanine mutants in the first batch of alanine scans. Figure 4 ).

[0063] The second batch of alanine scans constructed a total of 13 sites (see...). Figure 5 Comparing the results, the P164A mutant lost its activity; after alanine mutations at F253A, L254A, H170A, H177A, M332A, S76A, and N367A, the CK yield was higher than that of Bgl8; the alanine mutants at L410A, M411A, G419A, T421A, and M418A showed decreased activity. Among them, the CK yield after an alanine mutation at H177 was 454.4% of that of Bgl8, the highest yield among the same mutants.

[0064] Example 3 Construction of Bgl8 saturated mutant

[0065] Saturation mutagenesis: Site-directed saturation mutagenesis can mutate an amino acid at a specific position into 19 other amino acids, greatly enriching the amino acid mutation library. The goal is to find the most dominant amino acid at that position by mutating a single amino acid into 19 other amino acids.

[0066] Beneficial mutants obtained in Example 2 were screened: S76, L170, H177, F253, L254, M332, and N367. This study first constructed a saturation mutant for H177, which had the highest alanine yield in the scan. Next, saturation mutants M332 and N367, which are closer to the active site, were induced. Finally, saturation mutants S76, L170, F253, and L254, which are farther from the catalytic site, were induced. Primers for the saturation mutants are shown in Table 2.

[0067] Table 2

[0068]

[0069] First, this study constructed a saturation mutant library at the H177 position. By comparing the first batch of saturation mutants with H177A obtained from the alanine mutation, this study found that among the saturation mutations at the H177 position, other mutants had lower CK conversion efficiency than the H177A alanine mutation, while H177A exhibited higher CK conversion ability. Figure 6 ).

[0070] Then, this study performed saturation mutations on two sites, M332 and N367, which are close to the active site. At the M332 site, three beneficial mutations were obtained: M332S, M332Y, and M332L. Among them, the M332S mutant showed the highest activity. Figure 6 The percentage of the M332A alanine mutant (f) was 132%. At the N367 site, this study obtained three beneficial mutants: N367D, N367L, and N367P, with the N367L mutant exhibiting the highest activity (f). Figure 6 (g), which is 134% of the N367A alanine mutant.

[0071] This study performed saturation mutation analysis on the remaining four alanine scanning sites (S76, L170, F253, and L254). The results showed that at the S76 site, the CK transformation efficiency of all saturation mutants was lower than that of the S76A alanine mutant. Figure 6 (a); No beneficial mutations were found at the L170 site either, and the activity of its saturated mutants was lower than that of L170A (a). Figure 6 (b). The adjacent F253 and L254 sites were screened to yield F253N (CK yield was 110% of F253A) and L254K (CK yield was 112% of L254A), respectively. Figure 6 (d, e), and finally L254K was selected as the combined mutation site.

[0072] This study constructed saturation mutant libraries for seven key sites (S76, L170, H177, F253, L254, M332, and N367) of the Bgl8 enzyme using saturation mutagenesis. By comparing the CK conversion efficiency of saturation mutants and alanine scanning mutants, M332S, H177A, N367P, and L254K were selected as the optimal mutant combinations, providing experimental basis for subsequent multi-site combination mutations.

[0073] like Figure 7As shown, this study constructed combinatorial mutants using a multi-round directed evolution strategy and evaluated their impact on CK synthesis efficiency. Experimental data showed that, after enzymatic catalysis, the yield of 5 mg / mL total ginsenosides from ginseng was significantly increased by the single-site mutant M332S ((Bgl8-1X)) compared to the wild-type Bgl8 (WT) baseline of 0.035 mg / mL, with a relative yield of 729.57% ± 5.20 and an absolute yield increase to 0.261 ± 0.003 mg / mL, representing a 7.30-fold increase in catalytic efficiency. Introducing the H177A mutation to form a double mutant (Bgl8-2X, M332S / H177A) further increased the yield to 787.15% ± 4.80, with a 7.87-fold increase in catalytic efficiency, indicating a synergistic effect between the two sites. Subsequently, the triple mutant (Bgl8-2X, M332S / H177A) integrating the N367P mutation was further improved. The gl8-3X mutant (M332S / H177A / N367P) showed a more significant synergistic effect (relative yield 952.46% ± 6.10; absolute yield 0.341 ± 0.004 mg / mL; catalytic efficiency 9.52 times). Finally, the quadruple mutant (Bgl8-4X, i.e., M332S / H177A / N367P / L254K) achieved the highest catalytic activity, with a relative yield of 1311.02% ± 8.50 (0.469 ± 0.005 mg / mL) and a 13-fold increase in catalytic efficiency. This demonstrates that the efficiency bottleneck of single-point mutation can be overcome through progressive site combination, providing an important example for industrial enzyme modification.

[0074] Example 4: Optimal Catalytic Efficiency Study of Bgl-4X

[0075] 1. Optimal Temperature Study of Bgl8

[0076] Plasmids Bgl8, Bgl8-1X, and Bgl8-4X were transformed into E. coli BL21(DE3) competent cells via heat shock and plated on LB agar plates containing kanamycin (50 μg / mL) for single-clone selection. Typical single colonies were picked and inoculated into 400 μL of LB liquid medium containing kanamycin (50 μg / mL) and cultured at 37°C with shaking at 200 rpm for 14 h for amplification. 30 μL of the primary culture was transferred to 3 mL of LB liquid medium containing the same antibiotic and cultured until the logarithmic growth phase (OD600 = 0.6-0.8). Then, IPTG was added to a final concentration of 0.1 mM and the cells were induced at 16°C with 200 rpm for 16 h for low-temperature expression induction. When collecting the induced bacterial cells, wet cells were first obtained by centrifugation (8000 × g, 4°C, 15 min), then washed once with 30 mL of sterile physiological saline and centrifuged to remove water. Accurately weigh 0.5g of bacterial cell precipitate, resuspend in 10mL of PBS buffer (pH 6.0), and then sonicate. The disruption parameters were set as follows: 6mm amplitude transformer, 20% amplitude, cyclic mode (2s operation / 3s interval), total processing time 20min. The disrupted solution was centrifuged (8000×g, 4℃, 15min), and the supernatant was collected to obtain the crude enzyme solution. The crude enzyme solution was used for the reaction at temperatures of 30, 40, 50, 60, and 70℃ for 24h. After the reaction, 1mL of methanol was added to the reaction system to stop the reaction, mixed well, and then centrifuged at 12000rpm, 20℃ for 5min. The supernatant was collected. 100μg of the supernatant was added to a liquid chromatography vial for HPLC analysis (results...). Figure 11-18 (As shown).

[0077] HPLC operating conditions and analytical methods: Column: C18 column; Detection wavelength: 203 nm; Mobile phase: Ultrapure water treated with an ultrasonic vibrator for 30 min; Solvent ratio: Acetonitrile:Water = 60:40; Peristaltic pump total flow rate: 1 mL / min; Column oven: 25℃; Detector: VWD detector; Injection volume: 10 μL, isocratic elution; Processing time for each sample: 15 min (since the CK peak time is 11.67 min, the analysis time for each sample is determined to be 20 min). CK yield was calculated using the CK standard curve.

[0078] Determining the CK standard curve using the external standard method: Accurately weigh 3 mg of CK standard and place it in a 1 mL volumetric flask. Dissolve and dilute to volume with methanol, then shake well to obtain a 3 mg / mL CK standard stock solution. Take 1.5 mL of the CK standard stock solution into another clean 1 mL brown volumetric flask, dilute to volume with methanol, and shake well to obtain a 0.75 mg / mL CK standard stock solution. Accurately pipette 250 and 500 μL CK standard solutions, dilute to 1 mL with methanol, and mix thoroughly to obtain CK standard solutions with mass concentrations of 0.375 and 0.75 mg / mL. After removing impurities and particles through a 0.45 μm microporous membrane, inject the solutions into 1.5 mL sample vials, number them sequentially, and perform HPLC analysis.

[0079] The conversion efficiency of Bgl8 to CK at high temperature (70℃) is relatively low. Through directed evolution, this study increased the yield of Bgl8-4X to 12 times that of Bgl8 at 70℃. This study compared Bgl8, Bgl8-1X, and Bgl8-4X, and compared their CK conversion efficiencies at various temperatures.

[0080] The results showed that the CK yield of wild-type Bgl8 reached 1.252 mg / mL at 30℃, but dropped sharply to 0.035 mg / mL at 70℃ (only 2.8% of that at 30℃), indicating that its high-temperature stability was significantly insufficient.

[0081] The optimal single-point mutant Bgl8-1X was obtained through alanine scanning and saturation mutation screening. Figure 8 (b): The mutant produced 0.963 mg / mL of CK at 30℃ (slightly lower than the wild type), but increased to 0.271 mg / mL at 70℃ (7.7 times that of the wild type), and its high-temperature yield (28.1%) was significantly better than that of the wild type.

[0082] Further optimization through saturation mutations ultimately yielded the Bgl8-4X mutant. Figure 8 The concentration of C at 70℃ reached 0.469 mg / mL, which is 13 times that of the wild type at 70℃. The high temperature stability was significantly improved, and the conversion efficiency at 40℃ exceeded that of the wild type (1.16 times).

[0083] 2. Optimal pH determination of Bgl8-4X

[0084] Plasmids Bgl8, Bgl8-1X, and Bgl8-4X were transformed into E. coli BL21(DE3) competent cells via heat shock and plated on LB agar plates containing kanamycin (50 μg / mL) for single-clone selection. Typical single colonies were picked and inoculated into 400 μL of LB liquid medium containing kanamycin (50 μg / mL) and cultured at 37°C with shaking at 200 rpm for 14 h for amplification. 30 μL of the primary culture was transferred to 3 mL of LB liquid medium containing the same antibiotic and cultured until the logarithmic growth phase (OD600 = 0.6-0.8). Then, IPTG was added to a final concentration of 0.1 mM and the cells were induced at 16°C with 200 rpm for 16 h for low-temperature expression induction. When collecting the induced bacterial cells, wet cells were first obtained by centrifugation (8000 × g, 4°C, 15 min), then washed once with 30 mL of sterile physiological saline and centrifuged to remove water. Accurately weigh 0.5g of bacterial cell precipitate, resuspend in 10mL of PBS buffer (pH 6.0), and then sonicate. The disruption parameters were set as follows: 6mm amplitude transformer, 20% amplitude, cyclic mode (2s operation / 3s interval), total processing time 20min. After centrifugation (8000×g, 4℃, 15min), the supernatant was collected to obtain the crude enzyme solution. The crude enzyme solution was used for reactions at pH 5.0, 5.5, 6.0, 6.5, and 7.0 for 24h. After the reaction, 1mL of methanol was added to the reaction system to stop the reaction, and the mixture was stirred. Then, the mixture was centrifuged at 12000rpm and 20℃ for 5min, and the supernatant was collected. 100μg of the supernatant was added to a liquid chromatography vial for HPLC analysis. The HPLC analysis method was the same as above, and the CK yield was calculated using the CK standard curve.

[0085] The CK yield of Bgl8-4X was verified at various pH values ​​around 6.0. Consistent with Bgl8, the highest CK yield was observed at pH 6.0, which is the optimal pH value for Bgl8-4X. Figure 9 At pH=5.0, 5.5, 6.5, and 7.0, the yields of CK were 78.98%, 89.38%, 78.20%, and 72.23% of that at pH=6.0, respectively.

[0086] 3. Production observation of Bgl8-4X over 36 hours

[0087] Glycoside hydrolases hydrolyze Rb1, Rb2, and Rc in crude ginsenosides. Rd is obtained by hydrolyzing the outer glycosyl group at position 20, and CK is obtained by continuous hydrolysis of the glycosyl group at position 3. Figure 10 As shown. When crude ginsenosides are added, this study hopes that glycoside hydrolases can convert all Rb1, Rb2, and Rc into CK, but this process is slow, and this study requires long-term hydrolysis research.

[0088] Plasmids Bgl8 and Bgl8-4X were transformed into E. coli BL21(DE3) competent cells via heat shock and plated on LB agar plates containing kanamycin (50 μg / mL) for single-clone selection. Typical single colonies were picked and inoculated into 400 μL of LB liquid medium containing kanamycin (50 μg / mL) and cultured at 37°C with shaking at 200 rpm for 14 h for amplification. 30 μL of the primary culture was transferred to 3 mL of LB liquid medium containing the same antibiotic and cultured until the logarithmic growth phase (OD600 = 0.6-0.8). Then, IPTG was added to a final concentration of 0.1 mM and the cells were induced at 16°C with 200 rpm for 16 h for low-temperature expression induction. When collecting the induced bacterial cells, wet cells were first obtained by centrifugation (8000 × g, 4°C, 15 min), then washed once with 30 mL of sterile physiological saline and centrifuged to remove water. Accurately weigh 0.5g of bacterial cell precipitate, resuspend in 10mL of PBS buffer (pH 6.0), and then sonicate. The disruption parameters were set as follows: 6mm amplitude transformer, 20% amplitude, cyclic mode (2s operation / 3s interval), total processing time 20min. After centrifugation (8000×g, 4℃, 15min), the supernatant was collected to obtain the crude enzyme solution. The crude enzyme solution was used for the reaction at 70℃, pH 6.0, and for 36h. After the reaction, 1mL of methanol was added to the reaction system to stop the reaction, mixed well, and then centrifuged at 12000rpm, 20℃ for 5min. The supernatant was collected. 100μg of the supernatant was added to a liquid chromatography vial for HPLC analysis. The HPLC detection method was as described above, and the CK yield was calculated using the CK standard curve.

[0089] The reaction yield of Bgl8-4X at 70℃ for 36 hours was compared at different time points to analyze the efficiency of CK production at each time point. This study sampled samples every 6 hours, measuring the reaction yield at 6h, 12h, 18h, 24h, 30h, and 36h.

[0090] According to the biotransformation process of ginsenosides, in the step of biotransformation into CK, Rb1, Rb2, and Rc in ginsenosides must be converted into Rd, Rd into Rf2, and Rf2 is biotransformed into CK. Crude ginsenosides contain Rb1, Rb2, Rc, and Rd, but not CK. In the initial stage of the reaction, Rb1, Rb2, and Rc in the crude ginsenosides are continuously converted into Rd, while Rd in the crude product also begins to convert into Rf2, and Rf2 is simultaneously converted into CK.

[0091] The biotransformation process of ginsenoside CK exhibits significant stage-specific characteristics, and the variation of its yield with reaction time can be summarized as follows based on experimental data: Figure 10The initial conversion stage is the substrate pretreatment period. Glycoside hydrolases preferentially bind to the initial substrates Rb1, Rb2, and Rc, generating the intermediate product Rd through stepwise deglycosylation, accumulating the key intermediate product Rd for subsequent conversions. The efficient synthesis stage is the critical period for CK yield growth. With the large accumulation of Rd and Rf2, the enzymatic reaction enters the efficient conversion stage, and CK yield increases significantly. Experimental data show that the increase in CK yield within 16-24 hours accounts for 39.80% of the total yield in 24 hours, and the yield in this stage is 3.01 times that of 0-6 hours, indicating that this 8-hour period is the main contributing period for CK synthesis, and the binding of substrate and enzyme in the reaction system is dominated by the Rd→Rf2→CK pathway. The reaction decay and termination stage occurs after 24 hours, with a significant decrease in CK production rate, mainly due to the gradual depletion of substrates (Rd, Rf2) and weakened enzymatic reaction kinetics. After 30-36 hours, the yield of CK basically stabilized, and the slope of the curve approached zero, indicating that the reaction had reached its endpoint and the convertible substrate in the system had been completely consumed.

[0092] The catalytic properties of Bgl8-4X were evaluated. Results showed that during the 0-30 hour reaction period, the CK yield increased slowly from 0-6 hours, presumably indicating continuous production of the intermediate product Rd during this stage. The CK yield increased steadily from 6-12 hours, reaching its fastest growth rate from 12-18 hours. The growth rate of CK concentration began to decline from 18-24 hours, and the reaction essentially ceased from 24-30 hours. Notably, at 70°C, the yield of Bgl8-4X (0.469 mg / mL) was 13 times that of the wild type, demonstrating a significant improvement in catalytic efficiency at high temperatures. These data confirm that this mutant possesses both high-temperature stability and improved catalytic efficiency, laying an important foundation for further applications.

[0093] It should be noted that this invention is not limited to the embodiments described in the specific examples above. Those skilled in the art should understand that these embodiments are only used to illustrate the core principles of this invention. Without departing from the design concept and scope of protection of this invention, those skilled in the art can make appropriate adjustments and improvements to the model construction method, evaluation indicators, and application scenarios according to actual needs. The scope of protection of this invention is defined by the appended claims. All equivalent substitutions or reasonable improvements based on the technical solutions of this invention should be included within the scope of protection of this invention.

Claims

1. A glycoside hydrolase mutant Bgl8-4X, characterized in that, The amino acid sequence of the glycoside hydrolase mutant Bgl8-4X is shown in SEQ ID NO: 1; compared with wild-type Bgl8, the glycoside hydrolase mutant Bgl8-4X includes the following mutations: M332S, H177A, N367P and L254K.

2. A polynucleotide, characterized in that, Its encoding is the glycoside hydrolase mutant Bgl8-4X as described in claim 1.

3. A recombinant expression vector, characterized in that, It contains the polynucleotide as described in claim 2.

4. A host cell, characterized in that, It comprises the recombinant expression vector of claim 3, wherein the host cell is a prokaryotic cell or a eukaryotic cell.

5. The host cell according to claim 4, characterized in that, The host cell is Escherichia coli.

6. A method for preparing rare ginsenosides, characterized in that, Includes the following steps: In the catalytic reaction, the glycoside hydrolase mutant Bgl8-4X as described in claim 1 is contacted with the ginsenoside substrate to catalyze the conversion of the substrate into CK. The ginsenoside substrate is selected from one or more combinations of Rb1, Rb2, Rc, Rd, and F2.

7. The method according to claim 6, characterized in that, The catalytic reaction is carried out in a temperature range of 30°C to 70°C; the pH of the catalytic reaction is 5.0 to 7.

0.

8. The method according to claim 7, characterized in that, The catalytic reaction is carried out at a temperature of 70°C; the pH of the catalytic reaction is 6.0.

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