A beta-glucosidase, a coding gene, a recombinant vector, an engineered bacterium and application

Abstract

The application belongs to the technical field of biology and particularly relates to a beta-glucosidase, a coding gene, a recombinant carrier, engineered bacteria and application. The beta-glucosidase is derived from Thermomyces lanuginosus DSM 10635; the amino acid sequence of the beta-glucosidase is shown in SEQ ID NO: 2. The application further provides a beta-glucosidase derived from T. lanuginosus DSM 10635 and a coding gene thereof; the beta-glucosidase coding gene can be connected with an expression carrier, transformed into a Pichia pastoris strain, and a recombinant Pichia pastoris expressing the beta-glucosidase is constructed; and the beta-glucosidase with good thermal stability expressed by the recombinant Pichia pastoris is used as a catalyst to prepare ginsenoside CK through biological transformation.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a β-glucosidase, its encoding gene, recombinant vector, engineered bacteria, and its applications. Background Technology

[0002] β-glucosidase (EC 3.2.1.21), also known as β-D-glucosidase or cellobiase, is an important component of the cellulose-degrading enzyme system. It catalyzes the hydrolysis of terminal non-reducing β-D-glycosidic bonds, producing β-D-glucose and its corresponding ligand. Furthermore, β-glucosidase can also act on glycosidic bonds between glycosyl groups and aromatic or hydrocarbon groups, degrading the corresponding glycosides to generate ligands (aglycones) and glucose. Therefore, β-glucosidase can be used in the degradation of glycosides to produce desired aglycone compounds. Currently, most industrially used β-glucosidases are derived from plants and mesophilic microorganisms. Their application is limited by poor thermal stability and a narrow range of applicable reaction conditions. β-glucosidases derived from thermophilic fungi, due to their good thermal stability, have potential applications in high-temperature industrial processes.

[0003] Ginsenosides are the main active ingredients isolated from ginseng plants. Compared to common saponins, rare saponins (CK) are difficult to isolate due to their limited natural sources, similar structures, and complex structures, making them challenging to synthesize. Ginsenoside CK is a steroid and has been proven to be the main active component of ginseng in the human body. Pharmacological studies have confirmed that ginsenoside CK exhibits high absorption rates in the blood and exerts pharmacological activity in vivo. As a drug, ginsenoside CK has attracted widespread attention due to its anti-tumor, anti-diabetic, anti-allergic, and hepatoprotective effects. However, rare ginsenoside CK is absent in ginseng and difficult to produce. Ginsenoside CK and ginsenoside Rb1 share the same core structure and belong to the PPD type of ginsenosides. Bioenzymatic conversion of derivatives with similar core structures but different degrees of glycosylation is more feasible. Compared to physical and chemical methods with harsh reaction conditions, bioenzymatic methods offer relatively mild, efficient, and specific reaction conditions, producing almost no waste and thus are pollution-free and environmentally friendly. Therefore, developing a highly efficient, specific, mild, low-cost, and environmentally friendly process for producing ginsenoside CK, converting ginsenoside Rb1 into rare ginsenoside CK, is an important approach. Using an enzymatic conversion method with low pollution and high purity and specificity to obtain rare ginsenoside CK is both an urgent practical need and has significant practical application value.

[0004] . Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a β-glucosidase, its encoding gene, a recombinant vector, an engineered bacterium, and its applications. Specifically, the following technical solution is adopted:

[0006] A β-glucosidase derived from *Thermophilus spp.* ( Thermomyces lanuginosus DSM10635; The amino acid sequence of the above β-glucosidase is shown in SEQ ID NO: 2.

[0007] SEQ ID NO: 2:

[0008] 1 MVQLDVEKTL SELELQEKVA LTAGIDNWHT VPVPRLNIPSIRVSDGPNGV RGTRFFNGVP

[0009] 61 AACLPCSTAL GSTWDKELLE RLGELLGDEA RAKGAHVLLGPTINIQRSPL GGRGFESFSE

[0010] 121 DGLLSGVLAG HLVKGLQSKG VAGTLKHFVC NDQEHERMAVDTIVTPRALR EIYLLPFQQA

[0011] 181 LKISQAACIM TSYNKVNGKH VSESREIITD ILRNEWKWNGLVMSDWFGTY STSEAVIAGL

[0012] 241 DLEMPGKTRW RGGNLIHAVW SRVVPEHVLD ERVRNVLNLINFADKSGIPE NGEEKVLDRE

[0013] 301 EDRKLLRKAA ADSVVLLKNN GNVLPFDKTK PIAVIGPNSKVASYSGGGSA SLAPYYTVTP

[0014] 361 FEGVVNNSQA DVLFSQGVYA HKSLPQFGSS IKTPDGKPGITFKVYNEPPE AENRECVDEL

[0015] 421 HLTQTFGTLT DYENPKVKSF TFYVDMEGIF TPEEDGIYDFGVMVAGTGRL YVDGELVVDN

[0016] 481 YTTQREGVSF FNTGTLEERG SKELKAGVSY KILFQFGSGPTTKLAKRNVI GENAGGFHFG

[0017] 541 VAKRLNPEES IARAVELAAK TEQVVVFAGL NGEWESEGSDREHMDLPPGT DELISRVLEA

[0018] 601 NPNAAIVIQS GTPVTMPWAD KANVLAQAWF GGNELGNGIADVLYGNVNPS GKLPLSFPVR

[0019] 661 LEDNPAYINF GSDRGRVLYG EDVYVGYRYY EKKKVKPLFPFGHGLSYTVF ERSGLTLESS

[0020] 721 PEQPTLEDGE TITATLTVTN KGSVAGAEVV QLWVRPPLTSIQRPLRELK GFTKVFLEPG

[0021] 781 ASEKVSIVVE KKLATSYWDEIRQSWASESG EYEVLITGTG DEVLRAPFTV ERTRYWRGL

[0022] This invention uses RT-PCR technology to... T. lanuginosus β-glucosidase gene isolated from DSM 10635 bacteria. Extraction was performed using the TRIzol method. T. lanuginosus Total RNA from DSM 10635 was used to synthesize the first strand of cDNA using an RT-PCR kit. The reaction system and conditions were in accordance with the kit's instructions. Then, using the designed specific primers, a 2520 bp β-glucosidase encoding gene was obtained by PCR, with the sequence shown in SEQ ID NO: 1.

[0023] SEQ ID NO: 1:

[0024] 1 ATGGTCCAGC TTGACGTTGA GAAGACTCTC TCGGAGCTTG AGCTCCAAGAGAAGGTAGCT

[0025] 61 CTCACTGCCG GTATTGATAA CTGGCACACT GTCCCTGTGCCGCGCTTGAA TATTCCGTCT

[0026] 121 ATCCGTGTCT CTGACGGGCC CAATGGAGTC CGCGGTACAAGATTCTTCAA TGGCGTCCCT

[0027] 181 GCCGCGTGCT TGCCGTGCTC TACCGCGCTC GGCTCTACGTGGGACAAGGA ACTGCTAGAG

[0028] 241 CGTCTGGGCG AGCTGCTGGG AGACGAAGCC CGTGCCAAGGGTGCTCACGT TCTCCTTGGC

[0029] 301 CCAACCATAA ACATTCAGCG ATCCCCACTG GGAGGACGCGGGTTTGAGTC GTTTTCCGAA

[0030] 361 GATGGATTGC TATCGGGTGT TCTGGCTGGT CATCTTGTTAAGGGGCTCCA GTCGAAAGGT

[0031] 421 GTCGCAGGGA CCCTGAAGCA TTTCGTGTGC AATGATCAAGAGCATGAGCG AATGGCAGTG

[0032] 481 GATACAATCG TAACTCCAAG GGCTCTGCGA GAAATCTATTTGCTTCCGTT CCAGCAAGCT

[0033] 541 CTGAAAATTT CTCAAGCAGC TTGTATCATG ACGTCCTACAACAAGGTCAA TGGGAAGCAC

[0034] 601 GTGAGCGAGT CACGGGAAAT CATAACTGAC ATTCTACGCAACGAATGGAA GTGGAATGGT

[0035] 661 CTGGTCATGA GTGATTGGTT CGGCACATAC AGTACGTCCGAGGCAGTCAT TGCTGGCCTT

[0036] 721 GACCTTGAGA TGCCCGGTAA AACCCGCTGG CGAGGCGGGAACTTGATCCA TGCTGTATGG

[0037] 781 TCGAGGGTAG TGCCTGAGCA CGTCCTCGAC GAAAGGGTGCGAAATGTCCT CAACCTGATC

[0038] 841 AATTTTGCGG ACAAATCGGG CATCCCAGAG AATGGAGAGGAGAAAGTCCT TGATAGAGAA

[0039] 901 GAAGACCGCA AACTCCTACG AAAGGCCGCT GCGGACTCTGTTGTATTGCT GAAGAACAAC

[0040] 961 GGCAATGTCC TTCCATTCGA CAAGCAAAG CCGATTGCCGTCATTGGACC AAACTCGAAG

[0041] 1021 GTGGCCTCTT ACAGTGGTGG TGGCTCTGCC TCACTGGCTCCCTACTACAC CGTGACGCCG

[0042] 1081 TTTGAGGGCG TTGTCAACAA TAGCCAGGCC GACGTGTTGTTCTCTCAGGG TGTCTACGCG

[0043] 1141 CACAAATCGC TACCGCAGTT TGGTTCGTCG ATCAAGACGCCAGATGGAAAA GCCAGGAATT

[0044] 1201 ACGTTCAAAG TGTACAACGA ACCGCCAGAG GCTGAGAACCGCGAATGCGT GGATGAGCTG

[0045] 1261 CATCTGACCC AGACGTTCGG GACTCTCACC GACTATGAAAACCCCAAGGT GAAGTCCTTT

[0046] 1321 ACCTTCTACG TCGACATGGA AGGCATCTTC ACGCCTGAAGAGGATGGAAT CTACGACTTT

[0047] 1381 GGAGTCATGG TCGCCGGCAC GGGCCGACTC TACGTCGACGGTGAACTGGT TGTCGACAAC

[0048] 1441 TACACGACGC AGCGCGAGGG CGTGTCCTTC TTCAATACAGGAACCCTTGA AGAACGGGGG

[0049] 1501 TCCAAGGAAC TGAAGGCAGG AGTTTCCTAC AAGATCCTGTTTCAATTCGG CAGCGGTCCC

[0050] 1561 ACGACAAAGT TGGCGAAACG GAACGTTATC GGCGAGAATGCCGGTGGATT CCACTTTGGG

[0051] 1621 GTGGCCAAAC GGCTTAACCC AGAGGAGTCG ATTGCGCGGGCAGTCGAGTT GGCCGCGAAA

[0052] 1681 ACGGAGCAAG TTGTCGTCTT TGCAGGCCTC AACGGCGAGTGGGAGAGCGA AGGGAGTGAC

[0053] 1741 CGGGAGCACA TGGATCTGCC GCCTGGCACC GACGAGCTCATCTCGCGGGT CCTCGAAGCG

[0054] 1801 AACCCCAACG CTGCGATCGT CATCCAGAGC GGCACACCGGTTACGATGCC GTGGGCTGAC

[0055] 1861 AAGGCCAATG TGCTGGCGCA GGCATGGTTT GGCGGTAATGAGCTGGGCAA CGGCATTGCG

[0056] 1921 GATGTTCTCT ACGGCAATGT TAACCCTTCG GGGAAGCTTCCGCTCTCCTT CCCCGTGCGG

[0057] 1981 CTGGAGGATA ATCCGGCGTA CATCAACTTC GGCTCTGACCGGGGCCGCGT CCTCTACGGT

[0058] 2041 GAGGACGTCT ACGTCGGATA TCGGTATTAC GAGAAGAAGAAGGTCAAACC CTTGTTCCCC

[0059] 2101 TTCGGCCACG GACTTTCCTA CACCGTCTTC GAGCGCTCAGGTTTGACCTT GGAATCGTCT

[0060] 2161 CCGGAACAAC CTACACTTGA AGACGGCGAG ACGATCACGGCAACGCTGAC CGTGACCAAT

[0061] 2221 AAAGGTTCCG TGGCCGGCGC GGAGGTCGTC CAACTGTGGGTGCGTCCTCC ACTGACGAGC

[0062] 2281 AGCATTCAAC GACCTCTGCG GGAGCTCAAG GGGTTCACCAAGGTGTTCCT CGAGCCTGGA

[0063] 2341 GCAAGCGAGA AAGTGTCCAT CGTCGTCGAG AAGAAGCTAGCAACTAGTTA CTGGGATGAG

[0064] 2401 ATTCGTCAAT CGTGGGCCTC GGAAAGCGGC GAGTATGAGGTTCTCATCAC TGGTACGGGT

[0065] 2461 GACGAGGTTC TGCGGGCGCCATTCACCGTG GAGAGGACCA GATACTGGCG AGGTCTGTAG

[0066] The present invention also provides a recombinant vector containing the above-mentioned coding gene.

[0067] The present invention also provides an engineered bacterium containing the above-mentioned encoding gene. The engineered bacterium is constructed by first constructing a recombinant vector containing a β-glucosidase encoding gene, and then transforming the recombinant vector into Pichia pastoris to obtain the engineered bacterium.

[0068] The specific construction steps of the above-mentioned engineered bacteria are as follows:

[0069] Based on the β-glucosidase coding sequence, primers were designed and synthesized. The full-length β-glucosidase gene sequence was cloned by PCR using cDNA as a template, and then cloned into pPICZαA using a splicing method. Eco RI and Xba Between the I sites, a Pichia pastoris secretory expression plasmid was constructed. The obtained plasmid was confirmed by sequencing and used... Sac Linearization was performed, and electroporation transformation was carried out using the Bio-Rad gene porator according to the operation manual. Pichia pastoris X33; Linearized, insert-free empty pPICZαA was used as a negative control. Transformants were screened on YPDS medium containing 200 mg / L Zeocin™ and confirmed by PCR to construct the engineered bacteria.

[0070] This invention provides a method for preparing the β-glucosidase enzyme powder catalyst of the above-mentioned engineered bacteria, comprising the following steps:

[0071] The engineered bacteria were inoculated into BMGY medium and cultured at 30 °C and 220 rpm. After 24 hours, the cells were collected by centrifugation and resuspended in BMGY medium. The cells were cultured at 30 °C and 220 rpm for 5 days. 0.5% methanol was added to the culture every 12 hours for continuous methanol induction. After the culture was completed, the culture supernatant of the recombinant Pichia pastoris was collected, centrifuged at 12000g for 15 min, and the supernatant was concentrated using an Amicon Ultra-15 ultrafiltration membrane with a molecular weight cutoff of 30 kDa. The supernatant was then freeze-dried to prepare β-glucosidase enzyme powder catalyst.

[0072] As a further preferred embodiment, the BMGY culture medium comprises the following components at the following final concentrations: 1.0% glycerol, 1.0% yeast extract, 2.0% peptone, 1.34% yeast nitrogen-free, amino acid-free ammonium sulfate, 4 × 10⁻⁶ ppm. -5 % Biotin, 100 mM potassium phosphate, pH=6.0.

[0073] As a further preferred embodiment, 1.0% methanol, 1.0% yeast extract, 2.0% peptone, 1.34% yeast nitrogen-free, amino acid-free ammonium sulfate, 4×10 -5 % Biotin, 100 mM potassium phosphate, pH=6.0.

[0074] The β-glucosidase provided by this invention can be well applied in the preparation of ginsenoside CK. The specific preparation method includes the following steps:

[0075] Using ginsenoside Rb1 as a substrate, β-glucosidase enzyme powder catalyst prepared by the aforementioned β-glucosidase was added, and the reaction was carried out at 50 °C in a reaction system consisting of a pH=7.0 buffer solution. After the reaction was completed, the resulting conversion solution contained ginsenoside CK.

[0076] As a further preferred embodiment, the amount of β-glucosidase powder used in the above reaction system is 10 g / L, and the initial concentration of the above substrate is 5 g / L.

[0077] The beneficial effects of this invention are as follows: This invention provides a β-glucosidase derived from T. lanuginosus DSM 10635 and its encoding gene; the β-glucosidase encoding gene can be ligated with an expression vector and transformed into Pichia pastoris strains to construct recombinant Pichia pastoris expressing β-glucosidase; ginsenoside CK can be prepared by biotransformation using the thermostable β-glucosidase expressed by the recombinant Pichia pastoris as a catalyst. Attached Figure Description

[0078] Figure 1 The image shown is an agarose gel electrophoresis diagram of RT-PCR for the β-glucosidase gene.

[0079] Figure 2 The diagram shown is a physical representation of the pPICZαA-TLG recombinant plasmid.

[0080] Figure 3 The image shown is an SDS-PAGE image of engineered bacteria induced expression. Lane M is the protein molecular weight marker, and lane 1 is the methanol-induced expression lane. Pichia pastoris X33 / pPICZαA-TLG fermentation broth supernatant;

[0081] Figure 4 The diagram shows the reaction process for the preparation of ginsenoside CK catalyzed by β-glucosidase. Detailed Implementation

[0082] The following will provide a clear and complete description of the concept, specific structure, and technical effects of the present invention in conjunction with embodiments and accompanying drawings, so as to fully understand the purpose, solution, and effects of the present invention.

[0083] Example 1

[0084] Obtaining the β-glucosidase gene from *Thermophilic filamentosa*

[0085] T. lanuginosusDSM 10635 (from the German Collection of Microorganisms and Cell Cultures, DSMZ) was inoculated into PDA medium and cultured at 50°C for 3 days. Mycelia were collected, ground thoroughly into powder using liquid nitrogen, and total RNA was extracted using the TRIzol method. Promega's GoScript was then used for further analysis. TM The first strand of cDNA was prepared using a reverse transcription kit, and the reaction system and conditions were in accordance with the kit's instructions. Primers P1 (ATGGTCCAGCTTGACGTTGAG) and P2 (CTACAGACCTCGCCAGTATC) were designed based on the genome sequencing results of *Thermophilus floscens*. Using cDNA as a template, the first strand of cDNA was prepared via LA-... Taq PCR amplification was performed under the action of DNA polymerase; 50 μL PCR reaction system: 10×LA- Taq DNA Polymerase Buffer 5 μL, 10 mM dNTP mixture (2.5 mM each of dATP, dCTP, dGTP, and dTTP) 0.5 μL, P1 and P2 solutions (10 μM each) 1.0 μL, cDNA 1 μL, LA- Taq DNA Polymerase 0.5 μL, nucleic acid-free water 41 μL; PCR was performed using a BioRad PCR instrument. The PCR reaction conditions were: pre-denaturation at 94 ℃ for 5 min, followed by temperature cycling at 94 ℃ for 30 s, 55 ℃ for 30 s, and 72 ℃ for 2.5 min, for a total of 30 cycles, with a final extension at 72 ℃ for 10 min, and a termination temperature of 4 ℃; PCR yielded a band of approximately 2.5 kb. Figure 1 Lane M contains the DL5000 DNA Marker (band sizes from top to bottom are 5000, 3000, 2000, 1500, 1000, 750, 500, 250, and 100 bp), and lane 1 contains the β-glucosidase gene fragment amplified by RT-PCR. The PCR product 1 is compared with pGEM... ® -T vector(Promega) concatenation, obtaining plasmid pGEM ® -TLG, sequencing identification. Analysis of the sequencing results yielded an open reading frame of 2520 bp (SEQ ID NO: 1); analysis of the gene sequence using DNAMAN software and BLAST revealed the amino acid sequence encoded by the gene as shown in SEQ ID NO: 2.

[0086] Example 2

[0087] Construction and expression of engineered Pichia pastoris strains containing β-glucosidase

[0088] Using the Pichia pastoris secretory expression vector pPICZαA in Pichia pastoris Heterologous expression of β-glucosidase in strain X33. Primer P3( AAAAGAGAGGCTGAAGCT ATGGTCCAGCTTGACGTTGAG, where the underlined part is designed to be spliced ​​with the vector pPICZαA sequence) and P4 ( TGAGATGAGTTTTTGTTC CTACAGACCTCGCCAGTATC), using the plasmid pGEM obtained in Example 1 ® Using TLG as a template, the gene fragment encoding β-glucosidase was amplified by PCR. The PCR system consisted of 50 μL of plasmid template pGEM. ® -TLG 0.5 μL, forward and reverse primers (10 mM) 1 μL each, KOD-Plus-NeoDNA polymerase 1 μL. PCR program: 94℃ pre-denaturation for 3 min, 98℃ for 10 s, 68℃ for 2.5 min, 30 cycles, 68℃ for 10 min, 16℃ for 5 min. After amplification, 1% agarose gel electrophoresis was performed, and the target gene amplification product was recovered by gel excision. Vector pPICZαA was... Eco RI and Xba I enzyme digestion, 1% agarose gel electrophoresis, and plasmid DNA fragment recovery by gel extraction. Plasmid DNA fragments and gene fragments were analyzed using ClonExpress. ® II. Use the One Step Cloning Kit for splicing, following the kit instructions. Convert the splicing solution using thermal shock. E. coli DH5α competent cells were plated on LB agar plates containing 100 μg / mL Zeocin and incubated overnight at 37°C. Positive clones from the Zeocin plates were picked and sequenced for verification. The β-glucosidase encoding gene was cloned into pPICZαA. Eco RI and Xba Between the I sites, construct the Pichia pastoris secretory expression plasmid pPICZαA-TLG (plasmid map see...). Figure 2 ).use Sac Linearize plasmid pPICZαA-TLG and transform it using electroporation with a Bio-Rad gene perforator according to the instruction manual. Pichia pastoris X33. Linearized empty pPICZαA was used as a negative control. Transformants were screened on YPDS medium containing 200 mg / L Zeocin™ and confirmed by PCR.

[0089] Example 3

[0090] Heterologous expression and preparation of β-glucosidase in engineered Pichia pastoris

[0091] The positive clone yeast colonies obtained in Example 2 were inoculated into BMGY medium (1.0% glycerol, 1.0% yeast extract, 2.0% peptone, 1.34% yeast nitrogen-free amino acid-free ammonium sulfate, 4 × 10⁻⁶). -5 Cells were cultured at 30 °C and 220 rpm using 1% biotin, 100 mM potassium phosphate, pH 6.0. After 24 hours, cells were collected by centrifugation and resuspended in BMMY medium (same composition as BMGY, but with 1% methanol instead of glycerol). The cells were cultured at 30 °C and 220 rpm for 5 days, with 0.5% (v / v) methanol added to the culture every 12 hours for continuous methanol induction. The SDS-PAGE results of heterologous expression of β-glucosidase in engineered Pichia pastoris are shown below. Figure 3 As shown, lane M is the protein molecular weight marker, and lane 1 is the supernatant of the methanol-induced Pichia pastoris X33 / pPICZαA-TLG fermentation broth. After cultivation, the culture supernatant of the recombinant Pichia pastoris was collected, centrifuged at 12000g for 15 min, the supernatant was collected, concentrated using an Amicon Ultra-15 ultrafiltration membrane with a molecular weight cutoff of 30 kDa, and then freeze-dried to prepare β-glucosidase powder, which was stored at -20 ℃ for later use.

[0092] Example 4

[0093] Using the β-glucosidase powder obtained in Example 3 as a biocatalyst, ginsenoside Rb1 was used as a substrate for biotransformation to prepare ginsenoside CK. The composition and operation of the transformation system were as follows: 5 g / L ginsenoside Rb1, 20 mM PBS (pH 7.0) buffer, and 0.1 g of enzyme powder were added to 10 mL of the transformation system. The reaction was carried out at 50℃ and 150 r / min for 12 h. Samples were taken periodically during the transformation, and HPLC was used to detect the conversion of ginsenoside Rb1 to rare ginsenoside CK. A Shimadzu HPLC system with a Wondasil C18-WR reversed-phase column (4.6 mm × 250 mm, 5 μm) was used. The detection conditions were: column temperature: 35℃; injection volume: 10 μL; detection wavelength: 210 nm; mobile phase: acetonitrile-water solution (0–27 min, 35% acetonitrile; 27–50 min, 65% acetonitrile); flow rate: 0.9 mL / min. The reaction process for the preparation of ginsenosides CK catalyzed by β-glucosidase is shown in the figure. Figure 4 .from Figure 4 It can be seen that β-glucosidase can effectively convert ginsenoside Rb1 to prepare ginsenoside CK. The conversion rate is >95% after 10 h of reaction and >99% after 12 h of reaction.

[0094] Although the description of the invention has been quite detailed and particularly of several described embodiments, it is not intended to limit it to any of these details or embodiments or any particular embodiment, but should be considered as providing a broad possible interpretation of the claims by referring to the appended claims and taking into account the prior art, thereby effectively covering the intended scope of the invention. Furthermore, the invention has been described above with respect to embodiments foreseeable by the inventors in order to provide a useful description, and non-substantial modifications to the invention that have not yet been foreseen may still represent equivalent modifications.

Claims

1. A β-glucosidase, characterized in that, Derived from the thermophilic filamentous fungus ( Thermomyces lanuginosus DSM 10635; the amino acid sequence of the β-glucosidase is shown in SEQ ID NO:

2.

2. A gene encoding the β-glucosidase as described in claim 1.

3. The encoding gene according to claim 2, characterized in that, The sequence of the encoding gene is shown in SEQ ID NO:

1.

4. A recombinant vector containing the encoding gene of claim 2 or 3.

5. An engineered bacterium containing the encoding gene of claim 2 or 3.

6. A method for constructing the engineered bacteria according to claim 5, characterized in that, Includes the following steps: A recombinant vector containing a gene encoding β-glucosidase was constructed, and then the recombinant vector was transformed into Pichia pastoris to obtain an engineered strain.

7. A method for preparing the β-glucosidase enzyme powder catalyst of the engineered bacteria according to claim 5, characterized in that, Includes the following steps: The engineered bacteria were inoculated into BMGY medium and cultured at 30 °C and 220 rpm. After 24 hours, the cells were collected by centrifugation and resuspended in BMGY medium. The cells were cultured at 30 °C and 220 rpm for 5 days. 0.5% methanol was added to the culture every 12 hours for continuous methanol induction. After the culture was completed, the culture supernatant of the recombinant Pichia pastoris was collected, centrifuged at 12000g for 15 min, and the supernatant was collected. The supernatant was concentrated using an Amicon Ultra-15 ultrafiltration membrane with a molecular weight cutoff of 30 kDa and then freeze-dried to prepare β-glucosidase enzyme powder catalyst.

8. The application of the β-glucosidase of claim 1 in the preparation of ginsenoside CK.

9. The application according to claim 8, characterized in that, The preparation method of ginsenoside CK includes the following steps: Using ginsenoside Rb1 as a substrate, β-glucosidase enzyme powder catalyst prepared by the aforementioned β-glucosidase was added, and the reaction was carried out at 50 °C in a reaction system consisting of a pH=7.0 buffer solution. After the reaction was completed, the resulting conversion solution contained ginsenoside CK.

10. The application according to claim 9, characterized in that, The amount of β-glucosidase used in the reaction system is 10 g / L, and the initial concentration of the substrate is 5 g / L.

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