A beta-glucosidase mutant, related biological material and preparation method and application thereof

Abstract

The application discloses a beta-glucosidase mutant, related biological materials and a preparation method and application thereof, relates to the field of genetic engineering, enzyme engineering and biological conversion technology. The amino acid sequence of the mutant is shown as SEQ ID NO:1, the encoding nucleic acid molecule is shown as SEQ ID NO:2, high-efficiency soluble expression can be realized in escherichia coli, and the target protein accounts for about 35% of total soluble protein of the bacterial body. The application further provides a recombinant expression vector and a recombinant microorganism containing the nucleic acid molecule, and a method for preparing high-activity tag-removed enzyme by removing a 6*His tag through a TEV protease, the specific activity of the tag-removed enzyme reaches 152 U / mg, and is increased by about 40% compared with the tagged enzyme. The beta-glucosidase mutant can efficiently catalyze high-concentration ginsenoside Rb1 to generate rare ginsenoside CK, the molar conversion rate reaches 85.1%, the purity of the product is more than 92%, and the mutant has a good industrial application prospect.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering, enzyme engineering and biotransformation technology, and in particular to a β-glucosidase mutant, related biomaterials, their preparation methods and applications. Background Technology

[0002] Rare ginsenoside CK is a key active product resulting from the metabolism of protopanaxadiol-type saponins (such as Rb1) in ginseng by intestinal microorganisms. Compared to Rb1, CK has a lower content in natural ginseng materials, making direct extraction difficult to meet the needs of large-scale preparation.

[0003] The deglycosylation of ginsenoside Rb1 to CK using β-glucosidase has advantages such as relatively mild reaction conditions, good selectivity, and low environmental burden, making it one of the important technical routes for CK preparation. However, existing β-glucosidases for ginsenoside conversion still have shortcomings in terms of engineered expression and scale-up preparation, mainly including: Some natural β-glucosidase encoding genes do not match the codon preference of commonly used expression hosts such as Escherichia coli, resulting in low heterologous expression levels, insufficient soluble expression ratios, or the formation of inclusion bodies. In existing expression construction, if the connection method between the affinity purification tag and the target protein is unreasonable, it may affect the enzyme protein conformation or catalytic activity. Some enzyme preparation methods lack expression and purification processes that match high-density fermentation, limiting their stable supply as industrial enzyme preparations. Existing technologies mostly focus on β-glucosidase itself or a single conversion reaction, and there is insufficient disclosure of integrated engineering solutions for "coding sequence optimization - expression vector construction - detagged active enzyme preparation - Rb1 directed conversion of CK".

[0004] Therefore, it is necessary to provide a β-glucosidase-encoded nucleic acid molecule that is suitable for efficient soluble expression in common hosts, and to construct a recombinant expression system that can balance expression level, purification convenience and enzyme activity, so as to improve the engineering applicability of ginsenoside Rb1 biotransformation to prepare CK. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a β-glucosidase mutant, related biomaterials, preparation methods and applications thereof, in order to solve the problems existing in the prior art.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows: In a first aspect, the present invention provides a biomaterial, said biomaterial being at least one of the following: A1) Nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO: 2; A2) An expression cassette containing the nucleic acid molecules described in A1); A3) A recombinant expression vector containing the nucleic acid molecule described in A1) or a recombinant expression vector containing the expression cassette described in A2); A4) Recombinant microorganisms containing the nucleic acid molecules described in A1), recombinant microorganisms containing the expression cassette described in A2), or recombinant microorganisms containing the recombinant expression vector described in A3).

[0007] In this invention, the nucleic acid molecule undergoes codon optimization for the Escherichia coli expression system, and the codon optimization includes at least one of the following: (1) Replace low-frequency or rare codons with high-frequency codons of E. coli; (2) Adjust the GC content to 50%–55%; (3) Optimize the mRNA secondary structure of the 5' translation initiation region.

[0008] The examples show that the original sequence has a rare codon ratio of about 12% and a GC content of about 42%; the optimized sequence has a GC content of 52%, which reduces the stability of the secondary structure.

[0009] In this invention, under the same expression conditions, the target protein in the SEQ ID NO: 2 expression strain accounts for approximately 35% of the total soluble protein in the bacterial cells, while the target protein in the unoptimized original gene expression strain accounts for approximately 8%. The soluble expression level of the target protein is increased by approximately 3.4 times after optimization.

[0010] After codon optimization, this nucleic acid molecule is suitable for achieving high levels of soluble expression in host cells such as Escherichia coli.

[0011] Preferably, the sequence of the nucleic acid molecule is as shown in SEQ ID NO:2 or has at least 90% nucleotide sequence identity with SEQ ID NO:2.

[0012] Preferably, in the biological material, the recombinant expression vector further comprises a 6×His tag coding sequence, a TEV protease recognition site coding sequence, and a flexible linker peptide coding sequence; and the TEV protease recognition site coding sequence and the flexible linker peptide coding sequence are located between the 6×His tag coding sequence and the nucleic acid molecule.

[0013] In this invention, the 6×His tag encoding sequence is: caccaccaccaccaccac.

[0014] Preferably, the amino acid sequence of the TEV protease recognition site is shown in SEQ ID NO:3, and the amino acid sequence of the flexible linker peptide is shown in SEQ ID NO:4.

[0015] Preferably, the recombinant expression vector is a pET-28a(+) modified vector, and the nucleic acid molecule is inserted into the vector through Nde I and Xho I sites, so that it is in the same open reading frame as the N-terminal 6×His tag, the TEV protease recognition site and the coding sequence of the flexible linker peptide.

[0016] In this invention, the recombinant expression vector is equipped with an affinity purification tag and a protease recognition site that can be cleaved, which makes the fusion protein obtained by expression easy to purify, and the tag can be removed after purification to obtain β-glucosidase with enhanced activity.

[0017] Preferably, the recombinant expression vector comprises: The T7 promoter, the 6×His affinity purification tag coding sequence, the TEV protease recognition site coding sequence, the flexible linker peptide coding sequence, and the multiple cloning site for insertion into SEQ ID NO: 2.

[0018] The 6×His tag is used for nickel ion affinity chromatography purification; the TEV protease recognition site is used for tag removal after purification; and the flexible linker peptide is used to reduce the tag's influence on the spatial conformation and catalytic activity of β-glucosidase.

[0019] In this invention, after removing the His tag, the specific activity of β-glucosidase increased from 110 U / mg to 152 U / mg, an increase of approximately 40%.

[0020] Preferably, the recombinant microorganism is selected from Escherichia coli BL21 (DE3), Rosetta (DE3), or Origami (DE3).

[0021] In a second aspect, the present invention provides a β-glucosidase mutant, wherein the β-glucosidase mutant is expressed from any of the biological materials described above.

[0022] Preferably, the amino acid sequence of the β-glucosidase mutant is shown in SEQ ID NO:1.

[0023] In a third aspect, the present invention provides a method for preparing the β-glucosidase mutant described above, which is obtained by culturing and purifying recombinant microorganisms from any of the above-described biological materials.

[0024] Specifically, it includes the following steps: (1) The nucleic acid molecule is cloned into a recombinant expression vector to construct a recombinant plasmid; (2) The recombinant plasmid was transformed into Escherichia coli BL21(DE3) competent cells to obtain recombinant microorganisms; (3) The recombinant microorganisms are inoculated into TB or 2×YT medium containing antibiotics and cultured. (4) When the OD of the culture medium 600 When the concentration reaches 2.0–4.0, add IPTG to a final concentration of 0.1–1.0 mM, and lower the culture temperature to 16–20°C to induce expression for 16–24 hours; (5) Collect bacterial cells by centrifugation, and then take the supernatant after high-pressure homogenization; (6) The fusion protein was purified by Ni-NTA or Ni-IDA nickel ion affinity chromatography; (7) The 6×His tag was removed by TEV protease and the flow-through was collected again by nickel ion affinity chromatography to obtain the detagged β-glucosidase mutant.

[0025] Preferably, the high-pressure homogenization crushing condition is 950 bar, with 2–3 cycles.

[0026] Preferably, in nickel ion affinity chromatography, after sample loading, the sample is washed with a buffer containing 20 mM imidazole, and then the target protein is eluted with a buffer containing 200–300 mM imidazole.

[0027] Preferably, in the TEV enzymatic digestion reaction, the His-tagged fusion protein and the His-labeled TEV protease are mixed at a mass ratio of 50:1 and dialyzed overnight at 4°C in dialysate. The dialysate consists of 50 mM Tris-HCl, 0.5 mM EDTA, 1 mM DTT, and pH 8.0.

[0028] In a fourth aspect, the present invention provides the application of the β-glucosidase mutant described above, or any of the biomaterials described above, in the preparation of rare ginsenoside CK.

[0029] In a fifth aspect, the present invention provides a method for producing rare ginsenoside CK, comprising reacting the aforementioned β-glucosidase mutant with ginsenoside Rb1.

[0030] Preferably, in the catalytic reaction: the pH is 6.0~7.0, the reaction temperature is 70~90℃, the concentration of ginsenoside Rb1 is 15~25 g / L, the concentration of MgCl2 is 2~4mM, and the reaction time is 24~30h.

[0031] Specifically, the reaction system is illustrated using 1 mL as an example, including: 50 mM sodium phosphate buffer, pH 6.5; 3 mM MgCl2; 50 g / L ginsenoside Rb1; 5 U of unlabeled purified enzyme.

[0032] The reaction system was subjected to an HPLC reaction at 80°C for 24 hours. After the reaction, the products were analyzed by HPLC at a detection wavelength of 203 nm. The results showed that the peak of ginsenoside Rb1 basically disappeared, and a main peak appeared at the retention time position of the CK standard, accompanied by trace amounts of intermediate products Rd and F2. The molar conversion rate of CK was 85.1%, and the product purity exceeded 92%.

[0033] The beneficial effects of this invention include at least the following: (1) Increase the soluble expression level of β-glucosidase The nucleic acid molecule SEQ ID NO: 2 provided by this invention has undergone codon optimization for the Escherichia coli expression system. Results from examples show that, under the same expression conditions, the target protein accounts for approximately 35% of the total soluble protein in the SEQ ID NO: 2 expression strain, while it accounts for approximately 8% in the unoptimized original gene expression strain. The optimized soluble expression level is increased by approximately 3.4 times.

[0034] (2) Balancing purification efficiency and enzyme activity through resectable tag design This invention incorporates a TEV protease recognition site and a Gly-Ser-Gly flexible linker peptide between the 6×His tag and the target β-glucosidase, enabling rapid purification of the fusion protein via nickel ion affinity chromatography and subsequent tag removal. Results from examples show that the specific activity of the untagged enzyme is 152 U / mg, while the specific activity of the tagged enzyme is 110 U / mg, representing an approximately 40% increase in specific activity after tag removal.

[0035] (3) Suitable for high-density fermentation preparation of recombinant β-glucosidase This invention establishes high-density fermentation and low-temperature induction expression conditions matched to the recombinant expression strain. In a 5 L fermenter, the OD at the end of fermentation... 600 The concentration reached 45, and the wet bacterial weight was approximately 120 g per liter of fermentation broth, indicating that this expression system has the potential for further scale-up preparation of β-glucosidase.

[0036] (4) It can efficiently catalyze the conversion of ginsenoside Rb1 to CK. HPLC analysis showed that the molar conversion rate of CK was 85.1% and the purity of the product exceeded 92%, indicating that the enzyme has good application value for the conversion of Rb1 to prepare CK. Attached Figure Description

[0037] Figure 1 This is an SDS-PAGE gel image of the recombinant protein. The second channel shows the optimized protein, and the third channel shows the unoptimized protein.

[0038] Figure 2 The plasmid map of the recombinant expression vector pET28a-SEQ2.

[0039] Figure 3 The results are the plasmid sequencing results for the recombinant expression vector pET28a-SEQ2.

[0040] Figure 4 This is a comparison of the specific activities of tagged and untagged enzymes.

[0041] Figure 5 Protein expression was cultured in a 5 L fermenter.

[0042] Figure 6 This is an HPLC chromatogram of the biotransformation product of protopanaxadione Rb1.

[0043] Figure 7 MS analysis of the biotransformation product of protopanaxadione Rb1. Detailed Implementation

[0044] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0045] Furthermore, the technical solutions of the various embodiments of the present invention can be combined with each other, but only if they are feasible for those skilled in the art. If the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention.

[0046] The following specific embodiments illustrate the solution proposed in this invention: Example 1: Codon Optimization and Gene Synthesis 1.1 Sequence Analysis Using the amino acid sequence shown in SEQ ID NO: 1 as the target protein sequence, the corresponding original coding sequence was analyzed. The results showed that the original coding sequence contained a certain proportion of low-frequency or rare codons that are unfavorable to E. coli expression, such as AGG, AGA, and CUA, with rare codons accounting for approximately 12%; the GC content of the original sequence was approximately 42%; and the 5' mRNA secondary structure free energy was -8.5 kcal / mol.

[0047] 1.2 Codon Optimization and Gene Synthesis The original coding sequence was optimized based on the codon preference of Escherichia coli BL21(DE3).

[0048] Table 1. Conversion of rare codons into high-frequency codons

[0049] Optimize the mRNA secondary structure of the 5' translation initiation region. Specific optimization methods include: (1) Avoid forming consecutive GC pairings near the start codon ATG; (2) Reduce the local GC content at the 5' end; (3) Avoid ribosome binding sites, start codons and their downstream early coding regions from participating in the stable stem-loop structure; (4) Without changing the amino acid sequence, perform synonymous substitutions on the first 20 or so codons at the 5' end; (5) By predicting the secondary structure of mRNA, the free energy of the 5' translation initiation region was adjusted from about -8.5 kcal / mol in the original sequence to about -4.2 kcal / mol, thus reducing structural stability.

[0050] After optimization, the secondary structure stability of the mRNA initiation region at the 5' end is reduced, and the start codon ATG and its surrounding sequences are in a relatively open state, which is conducive to ribosome binding and translation initiation. This optimization does not change the amino acid sequence of the β-glucosidase mutant; it only improves the local structure of the mRNA through synonymous codon adjustments.

[0051] Right now: Replace the rare codons in the original sequence with codons that are frequently used by E. coli; The overall GC content was adjusted to 52%; The 5' end sequence was optimized to adjust the secondary structure free energy of the mRNA initiation region to -4.2 kcal / mol.

[0052] The optimized nucleotide sequence SEQ ID NO: 2 was obtained, and the full-length nucleic acid molecule was synthesized by a gene synthesis company. The results are shown in [see attached image]. Figure 1 .

[0053] 1.3 Comparison of Expression Levels The unoptimized original gene (SEQ ID NO: 8) and the optimized SEQ ID NO: 2 were cloned into the same pET-28a(+) vector, and transformed into Escherichia coli BL21(DE3) under the same conditions and induced to express.

[0054] After successful expression of the recombinant β-glucosidase expression strain in a small-scale experiment, the preserved glycerol bacteria were inoculated at a ratio of 1:1000 into small test tubes containing 5 mL of LB liquid medium containing kanamycin at a final concentration of 100 μg / mL. After inoculation, the culture was incubated overnight at 37℃ and 220 r / min using a shaker to obtain the primary seed culture.

[0055] The above-mentioned primary seed culture was transferred to 800 mL of 2×YT liquid medium containing 100 μg / mL kanamycin, and cultured on a shaker at 37°C and 220 r / min. When the OD of the culture medium... 600 When the concentration reaches approximately 0.6, the culture time is typically 3–4 hours, and the temperature of the shaker culture is lowered to 16–28°C. After the bacterial culture temperature has decreased, IPTG is added to induce expression, with a final IPTG concentration of 0.5–1.0 mM, and the induction time is 16 hours.

[0056] After the induction of expression is complete, the culture medium is transferred to a collection bottle and centrifuged at 5500 r / min for 15 min. The supernatant is discarded, and the bacterial pellet is collected. The bacterial pellet is then resuspended in 25 mL of protein buffer. The resuspended bacterial suspension is transferred to a 50 mL centrifuge tube and can be used directly for subsequent disruption or stored at -80℃ for later use.

[0057] For protein extraction, the bacterial suspension described above was used for cell disruption using a high-pressure cryogenic disruptor. The disruption conditions were: pressure 900-1200 bar, temperature 4°C, and 2-3 cycles to ensure complete lysis of the recombinant cells and release of the target protein into the protein buffer. During disruption, the sample was kept at a low temperature to minimize degradation or inactivation of the target protein.

[0058] After disruption, the lysate was placed in a high-speed refrigerated centrifuge and centrifuged at 4°C and 6000-8000 r / min for 30 min to separate cell debris, undisturbed bacterial cells, and insoluble components. After centrifugation, the supernatant and precipitate were collected separately, and SDS-PAGE analysis was performed to determine whether the target protein existed primarily in the form of soluble protein or inclusion bodies.

[0059] When the SDS-PAGE results showed that the target protein was mainly present in the lysed supernatant, the lysed supernatant was collected and filtered through a 0.45 μm microporous membrane to remove residual particulate matter, resulting in a crude extract of soluble protein that could be used for subsequent Ni affinity chromatography purification.

[0060] Since the target protein is constructed on the pET-28a(+) expression vector, which has a 6×His histidine tag at its N-terminus, it can specifically bind to the Ni-NTA nickel ion affinity chromatography column. Therefore, the filtered bacterial supernatant was loaded onto the equilibrated Ni affinity chromatography column, and non-specifically bound contaminating proteins were removed by imidazole gradient elution, and the target protein was obtained.

[0061] The specific purification steps are as follows: Before loading the sample, rinse the Ni affinity chromatography column with two column volumes of deionized water, and then equilibrate the Ni affinity chromatography column with two column volumes of protein buffer containing 10 mM imidazole. Then, slowly pass the lysed supernatant through the Ni affinity chromatography column at an appropriate flow rate to ensure that the 6×His-tagged fusion protein is fully bound to the packing material. After loading, the flow-through buffer can be passed through the column again to improve the binding efficiency of the target protein, and the flow-through sample is used for SDS-PAGE detection.

[0062] After sample loading, two column volumes were washed with protein buffer to remove unbound protein. Subsequently, gradient elution was performed using imidazole protein buffers of varying concentrations: 30 mL of 50 mM imidazole protein buffer, 30 mL of 100 mM imidazole protein buffer, 30 mL of 150 mM imidazole protein buffer, 30 mL of 200 mM imidazole protein buffer, and 300 mM imidazole protein buffer. During each imidazole gradient elution, the first few drops of flow-through sample and the eluted medium were collected. After sample preparation, 12% SDS-PAGE analysis was performed to determine the elution concentration, purification efficiency, and protein purity of the target protein.

[0063] After SDS-PAGE analysis, the elution fractions containing the target protein and with fewer contaminating proteins were combined to obtain the purified β-glucosidase fusion protein with a 6×His tag. This purified protein can be further used for TEV protease tag removal, enzyme activity assays, and CK preparation experiments using ginsenoside Rb1.

[0064] SDS-PAGE analysis showed that in the SEQ ID NO: 2 expression strain, the target protein had a molecular weight of approximately 57 kDa, accounting for about 35% of the total soluble protein in the bacterial cell; in the original gene expression strain, the target protein accounted for about 8% of the total soluble protein in the bacterial cell. Therefore, the optimized soluble expression level of the target protein increased by approximately 3.4 times.

[0065] Example 2: Construction and tag removal of the recombinant expression vector pET28a-SEQ2-TEV 2.1 Carrier Construction A double-stranded DNA fragment with Nde I and Xho I restriction sites was synthesized. The fragment was then double-digested with Nde I and Xho I and ligated into a pET-28a(+) modified vector that had been double-digested with the same restriction enzymes.

[0066] The pET-28a(+) modified vector pre-inserted nucleotide sequences encoding the TEV protease recognition site ENLYFQG and the flexible linker peptide Gly-Ser-Gly after the 6×His tag coding sequence and before the multiple cloning site. The resulting recombinant plasmid was named pET28a-SEQ2-TEV, and its nucleotide sequence is shown in SEQ ID NO: 9. It was then transformed into *E. coli* BL21(DE3). The plasmid map is shown below. Figure 2 .

[0067] The nucleotide sequence of Gly-Ser-Gly is: GGTAGTGGT (SEQ ID NO: 5) The nucleotide sequence of ENLYFQG is: GAA AAC CTG TAT TTT CAG GGT (SEQ ID NO: 6) 6×His tag encoding sequence: caccaccaccaccaccac (SEQ ID NO: 7).

[0068] 2.2 Expression and purification of tagged fusion proteins The recombinant strain was cultured under the induction expression conditions of Example 1, and the cells were collected and lysed. After centrifugation and collection of the supernatant, the fusion protein with a 6×His tag was purified by Ni-NTA nickel ion affinity chromatography.

[0069] The specific purification steps are as follows: Before loading the sample, rinse the Ni affinity chromatography column with two column volumes of deionized water, and then equilibrate the Ni affinity chromatography column with two column volumes of protein buffer containing 10 mM imidazole. Then, slowly pass the lysed supernatant through the Ni affinity chromatography column at an appropriate flow rate to ensure that the 6×His-tagged fusion protein is fully bound to the packing material. After loading, the flow-through buffer can be passed through the column again to improve the binding efficiency of the target protein, and the flow-through sample is used for SDS-PAGE detection.

[0070] 2.3 Preparation of detagged enzyme The tagged fusion protein was mixed with His-labeled TEV protease at a mass ratio of 50:1 and dialyzed overnight at 4°C in dialysate. The dialysate consisted of 50 mM Tris-HCl, 0.5 mM EDTA, 1 mM DTT, and pH 8.0.

[0071] After dialysis, the enzyme digestion mixture was re-loaded onto a Ni-NTA column, and the flow-through was collected to obtain β-glucosidase with the His tag removed.

[0072] 2.4 Specific activity determination Using 50 g / L ginsenoside Rb1 as a substrate, the reaction was carried out at 80℃ and pH 6.5 for 30 minutes, and the specific activities of the tagged and untagged enzymes were determined.

[0073] The results showed that the specific activity of the tagged enzyme was 110 U / mg, and the specific activity of the untagged enzyme was 152 U / mg. After removing the tag, the specific activity of β-glucosidase increased by approximately 40%, as shown in the results below. Figure 4 .

[0074] Example 3: High-density fermentation preparation of recombinant β-glucosidase 3.1 Seed Culture The recombinant strain of Escherichia coli BL21(DE3) containing pET28a-SEQ2-TEV was inoculated into LB medium containing 50 μg / mL kanamycin and cultured overnight at 37°C and 220 rpm to obtain seed culture.

[0075] 3.2 5 L fermenter culture The seed culture was inoculated into a 5 L fermenter at a 2% inoculation rate. The fermenter contained 3 L of TB medium with 50 μg / mL kanamycin. See fermenter details below. Figure 5 . Fermentation parameters are as follows: Incubation temperature: 37℃; pH: Controlled at 7.0±0.1 using ammonia water; Dissolved oxygen (DO): maintained above 30%; Aeration and agitation: Dissolved oxygen is maintained through correlated control; Feeding strategy: Exponential flow feeding is adopted; Feeding solution: 50% glycerin.

[0076] 3.3 Induced Expression and Results After approximately 8 hours of incubation, OD 600 Reach 3.5. Add IPTG to a final concentration of 0.5 mM, and simultaneously lower the culture temperature to 18°C, continuing induction culture for another 20 hours.

[0077] At the end of fermentation, the OD of the culture medium 600 The temperature reached 45°C. The cells were collected by centrifugation, yielding approximately 120g of wet cells per liter of fermentation broth.

[0078] After purification, the total yield of the target protein was approximately 5.2 times that of the shake-flask level.

[0079] Example 4: Recombinant β-glucosidase catalyzes the conversion of high-concentration ginsenoside Rb1 into CK. 4.1 Biotransformation reaction Add the following to a 1 mL reaction system: 1) 50 mM sodium phosphate buffer, pH 6.5; 2) 3 mM MgCl2; 3) 50 g / L ginsenoside Rb1; 4) 5 U of delabeled purified β-glucosidase prepared in Example 2.

[0080] The reaction system was placed in an 80°C water bath and reacted for 24 hours.

[0081] Table 2 Comparison of the effects of different methods

[0082] Compared with conventional acid hydrolysis, microbial fermentation, and ordinary β-glucosidase conversion methods in the field, the present invention can still achieve a CK molar conversion rate of 85.1% under high concentration of Rb1 (50 g / L) and the product purity exceeds 92%. These results indicate that the recombinant β-glucosidase of the present invention has good thermostability, substrate tolerance, and targeted desugaring ability, and can reduce the accumulation of intermediate products Rd and F2, making it suitable for large-scale bioprocessing of CK.

[0083] 4.2 HPLC detection After the reaction was complete, an equal volume of DMSO was added to treat the sample, which was then analyzed by HPLC at a detection wavelength of 203 nm.

[0084] HPLC results are shown in Figure 6 The results showed that the ginsenoside Rb1 peak basically disappeared, and the main peak appeared at the corresponding retention time position of the CK standard, accompanied only by trace amounts of intermediate products Rd and F2 peaks. The calculated molar conversion rate of CK was 85.1%, and the product purity exceeded 92%.

[0085] 4.3 Mass spectrometry confirmation The main peak obtained by HPLC separation was further analyzed by LC-MS. Results ( Figure 7 The data shows that an ion signal of m / z 667.44 was detected in this peak, which can be attributed to [M+COOH]. + Its theoretical m / z value is 667.44, which is consistent with the molecular weight of Compound K (CK).

[0086] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0087] The sequence numbers of the above embodiments of the present invention are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.

[0088] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A biomaterial, characterized in that, The biomaterial is at least one of the following: A1) Nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO: 2; A2) An expression cassette containing the nucleic acid molecules described in A1); A3) A recombinant expression vector containing the nucleic acid molecule described in A1) or a recombinant expression vector containing the expression cassette described in A2); A4) Recombinant microorganisms containing the nucleic acid molecules described in A1), recombinant microorganisms containing the expression cassette described in A2), or recombinant microorganisms containing the recombinant expression vector described in A3).

2. The biomaterial according to claim 1, characterized in that, The recombinant expression vector further comprises a 6×His tag coding sequence, a TEV protease recognition site coding sequence, and a flexible linker peptide coding sequence; and the TEV protease recognition site coding sequence and the flexible linker peptide coding sequence are located between the 6×His tag coding sequence and the nucleic acid molecule.

3. The biomaterial according to claim 2, characterized in that, The amino acid sequence of the TEV protease recognition site is shown in SEQ ID NO:3, and the amino acid sequence of the flexible linker peptide is shown in SEQ ID NO:

4.

4. The biomaterial according to claim 1, characterized in that, The recombinant microorganisms are selected from Escherichia coli BL21 (DE3), Rosetta (DE3), or Origami (DE3).

5. A β-glucosidase mutant, characterized in that, The β-glucosidase mutant is obtained by expression of the biological material described in any one of claims 1 to 4.

6. The mutant according to claim 5, characterized in that, The amino acid sequence of the β-glucosidase mutant is shown in SEQ ID NO:

1.

7. A method for preparing the β-glucosidase mutant of claim 5 or 6, characterized in that, Obtained by culturing and purifying the recombinant microorganisms in the biological material according to any one of claims 1 to 4.

8. The use of the biomaterials described in any one of claims 1 to 4, or the β-glucosidase mutant described in any one of claims 5 to 6, in the preparation of rare ginsenoside CK.

9. A method for producing rare ginsenoside CK, characterized in that, This includes catalyzing the reaction of the β-glucosidase mutant of claim 5 or 6 with ginsenoside Rb1.

10. The method according to claim 9, characterized in that, In the catalytic reaction: pH is 6.0~7.0, reaction temperature is 70~90℃, ginsenoside Rb1 concentration is 15~25 g / L, MgCl2 concentration is 2~4mM, and reaction time is 24~30h.

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