WO2011160484A1 - 具有β-木糖苷酶和β-葡萄糖苷酶活性的新的糖基水解酶及其应用 - Google Patents
具有β-木糖苷酶和β-葡萄糖苷酶活性的新的糖基水解酶及其应用 Download PDFInfo
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Definitions
- Novel glycosyl hydrolase having ⁇ -xylosidase and ⁇ -glucosidase activity and application thereof
- the present invention relates to a novel glycosyl hydrolase (GH), an amino acid sequence of the enzyme, and a nucleotide sequence encoding the enzyme.
- the enzyme has ⁇ -xylosidase- ⁇ -glucosidase activity and is capable of specifically hydrolyzing a xylose group from a 7-xylostaxane compound, and the present invention relates to encoding the 7-wood
- the nucleotide sequence of the glycosidase hydrolase, the amino acid sequence of the enzyme, and the use of the enzyme or the enzyme producing bacteria are examples of the enzyme produced by the enzyme.
- Paclitaxel (Taxol®, Paclitaxel) is mainly produced by the genus Taxus, and is one of the important achievements of anticancer drugs in the 1990s. Since its inception, its unique anti-tumor mechanism and remarkable anti-tumor activity have been produced. The world is eye-catching (Kingston DGI, et al. The taxane diterpenoids. In: Herz W, et al. eds. Progress in the chemistry of organic natural products. New York: Springer-Verlag, 1993, 161-165).
- paclitaxel paclitaxel
- Paclitaxel is low in yew plants, and only about two-tenths of the bark is found in the highest content (US Patent US 6028206).
- a centuries-old yew tree can get about 3 kg of bark, from which about 300 mg of paclitaxel can be obtained (Horwitz, SB. How to make taxol from scratch. Nature 1994b, 367: 593-594), therefore, About 3,000 large trees are needed for each kilogram of paclitaxel extracted from the bark, and the dose of one patient is equivalent to destroying 3 to 4 centuries old trees.
- An alternative method is to extract a higher content of 10-deacetylbaccatin oxime (about 0.1%) from the branches of Taxus baccata L.), and use it as a raw material for semi-synthetic paclitaxel or its structure.
- the analog is docetaxel, which is slightly more active than paclitaxel and more water soluble than paclitaxel [Denis JN, et al. A highly efficient, practical approach to natural taxol. J Am Chem Soc. 1988. 1 10 (17): 5917-5919; Horwitz RI. Studies with RP 56976 (Taxotere): A semisynthet- Ic analogue of taxol. J Nat Cancer Inst. 1991 , 83(4): 288-291 ; US Patent US
- nursery cultivation of shrub-type yew hybrids is also considered to be the most simple, resource-renewable, and least costly solution to the source of paclitaxel.
- a C-7 xylose taxane compound having a paclitaxel core structure was isolated from the bark of the yew, including 7-xylose-10- Acetyl paclitaxel (7-P-xylosyl- 10-deacytyltaxol, XDT), 7-p-xylosyl- 10-deacetylcepholomanine (XDC), 7-xylose-10 - Deacetylpacillol C ( 7-P-xylosyl-l O-deacytyltaxol C, XDTC ), etc., wherein 7-P-xylosyl 10-deacytyltaxol (XDT) is the most abundant Rich (Senilh V, et al.
- Hydrolysis removes the xylose group to produce the corresponding 7-hydroxy taxane, which can be used for chemical semi-synthetic paclitaxel or docetaxel to improve the utilization of Taxus resources and alleviate the supply and demand of Taxus resources. contradiction.
- chemical methods have certain deficiencies, such as relatively low yields, relatively complex reaction processes, and environmental pollution, while biological methods are environmentally friendly.
- FEMS Microbiol Lett 2008, 284:204-212 discloses the hydrolysis of C-7 xylose taxane to C-7 hydroxy taxane using actinomycete Leifsonia shinshuensis DICP 16 (CCTCC No. M 206026) method.
- CTCC No. M 206026 actinomycete Leifsonia shinshuensis DICP 16 (CCTCC No. M 206026) method.
- 1 mg of XDT was added to 2 ml of the bacterial suspension, and the reaction was carried out at 30 ° C and 120 rpm for 21 hours, and then the reaction was terminated with 2 ml of methanol, and the reaction liquid was determined by HPLC. There is no XDT residue in it, producing 0.4 mg/ml DT.
- the wet cell concentration in 2 ml of reaction solution was 231.58 mg/ml, and 7-xylose-10-deacetylbaccatin III was at different concentrations (0.5, 0.9, 1.95, 3.1, 4.4, 5.2, and 6.75). Mg/ml) were added separately and reacted at 31 °C and 120 rpm for more than 40 h.
- the yield of 10-deacetylbaccatin III reached the highest at 1j when the substrate concentration was 1.95 mg/ml (Hao DC, Et al. Bacterial diversity of Taxus rhizosphere: culture-independent and culture-dependent approaches. FEMS Microbiol Lett 2008, 284:204-212).
- Another Chinese patent discloses the actinomycete Cellulosimicwbium cellulans, XZ-5CCTCC No. M 207130), hydrolase and its use in taxane conversion: 10 ml XDT (Concentration 5 mg/ml) was added to 90 ml of crude enzyme solution (100 ml of culture solution was added to 1 ml of 30 ⁇ C cultured for 2 days of culture of M. fibrosus, cultured at 150 rpm, shake flask for 30 days at 30 °C. The supernatant was collected by centrifugation, and the reaction was carried out at 50 rpm for 30 hours at 30 ° C to produce 40 mg of DT.
- beta-xylosidase has been isolated from fungi and other organisms [Tuohy MG, et al.
- the inventors of the present invention conducted intensive research.
- the hydrolase (GH), LC-MS/MS De novo sequencing of the purified enzyme, gave the amino acid sequence of some oligopeptides.
- a series of degenerate primers were designed based on the amino acid sequences of these oligopeptides, and the cDNA and structural genes of the enzyme were cloned by molecular biology techniques such as nested PCR, RACE and Genome Walking.
- a cDNA fragment encoding an open reading frame (ORF) of the enzyme is ligated to an appropriate expression vector to construct a recombinant plasmid, which is introduced into a corresponding host cell, such as Pichia pastoris which grows fast and can be fermented at a high density.
- the recombinant bacteria can efficiently catalyze the glycosyl hydrolysis of 7-xylose taxane to produce a 7-hydroxy taxane.
- the inventors have also discovered that the enzyme is a bifunctional enzyme that hydrolyzes glucose residues on glucosides.
- the nucleotide sequence has almost no homology with all the nucleotide sequences registered in GenBank, and the closest one is mostly the putative protein gene sequence, and the coverage ratio of both is only 3 ⁇ 7%;
- the putative amino acid sequence of the sequence is closest to the putative protein sequence of stilago maydis (GenBank accession: XP_760179), and the agreement between the two is 43%, and the similarity is 59%.
- the present invention encompasses the following inventions:
- a novel 7-xylosylosidase hydrolase codenamed Lx ⁇ -P l, is provided.
- a second object of the present invention is to provide a nucleotide sequence encoding the enzyme.
- a third object of the present invention is to provide a recombinant plasmid containing the nucleotide sequence.
- a fourth object of the present invention is to provide a host cell comprising the recombinant plasmid or the nucleotide sequence.
- a fifth object of the present invention is to provide an application of the enzyme.
- the technical solution adopted is:
- the present invention provides a 7-xylose taxane glycosyl hydrolase (a bifunctional ⁇ -xylosidase- ⁇ -glucosidase), which is encoded by LXYL-P1.
- the amino acid sequence of the 7-xylosylosidase hydrolase (LXYL-P1) comprises at least 30% of the amino acid sequence of the sequence of SEQ ID NO: 2;
- it comprises an amino acid sequence having at least 40% homogeneity of SEQ ID NO: 2;
- it comprises an amino acid sequence having at least 60% homogeneity of SEQ ID NO: 2; further preferably comprising an amino acid sequence having at least 70% homogeneity of SEQ ID NO: 2; further preferably comprising at least 80% having SEQ ID NO: a constitutive amino acid sequence; further preferably comprising an amino acid sequence having at least 90% homogeneity of SEQ ID NO: 2.
- amino acid sequence having at least 95% homogeneity to SEQ ID NO: 2.
- amino acid sequence of SEQ ID NO: 2 is substituted, deleted or added by one or several amino acid residues and has the same activity as the amino acid residue sequence of SEQ ID NO: 2, and the protein derived from SEQ ID NO: 2.
- the present invention also provides a nucleotide sequence or a coding gene encoding the 7-xylosylosidase hydrolase (LXYL-P1), coded L ⁇ /-W, the nucleotide sequence thereof has SEQ ID NO: 1 or at least 30% of the nucleotide sequence of the nucleotide sequence shown in SEQ ID NO: 3;
- nucleotide sequence comprising at least 40% of SEQ ID NO: 1 or SEQ ID NO: 3 is included;
- nucleoside having at least 50% homogeneity of SEQ ID NO: 1 or SEQ ID NO: 3.
- Acid sequence
- nucleotide sequence comprising at least 60% of SEQ ID NO: 1 or SEQ ID NO: 3 is included;
- nucleotide sequence having at least 70% homogeneity of SEQ ID NO: 1 or SEQ ID NO: 3 is included;
- nucleotide sequence having at least 80% homogeneity of SEQ ID NO: 1 or SEQ ID NO: 3 is included;
- nucleotide sequence having at least 95% of SEQ ID NO: 1 or SEQ ID NO: 3.
- the present invention also provides a recombinant plasmid comprising the nucleotide sequence encoding L ⁇ /-/ ⁇ , which can be introduced into a suitable host cell.
- the invention also provides a suitable host cell which can carry a L/-/ ⁇ gene sequence having at least 30% of the nucleotides shown in SEQ ID NO: 1 or SEQ ID NO: 3. sequence.
- the host organism may be a homologous producing strain of the peptide (LXYL-P1) having the SEQ ID NO:
- At least 30% of the amino acid sequence shown in 2 may also be a heterologous host cell.
- Suitable host organisms are selected from the group consisting of bacteria, actinomycetes, yeasts, filamentous fungi, plant cells or animal cells.
- Preferred bacteria are selected from the group consisting of Escherichia species Bacillus species;
- Preferred actinomycetes are selected from the group consisting of Streptomyces species;
- Preferred yeasts are selected from the group consisting of the yeast ( ⁇ cc/Mramyce species) and the Pichia genus Pichia. Species of Schizophyllum genus (.Schizosacchawmyce species);
- Preferred filamentous fungi are selected from the group consisting of Aspergillus species, Trichoderma species, Penicillium species, Tricholoma species, Lentinula species, Agaricus species;
- Preferred plant cells are selected from dicotyledon
- the animal cells are selected from insect cells.
- Preferred Escherichia species are preferably Escherichia coli (E. coli; preferably Bac/Zi ⁇ species is preferably Bacillus subtilis B. subtilis', preferably Streptomyces species
- S. lividans is preferred
- the preferred Saccharomyce species is preferably Saccharomyce cerevisiae
- P. pastoris preferred Pichia species P. pastoris
- preferred chizosaccharomyce species preferably Schizosacchawmyce pombe
- Preferred Aspergillus species is preferably A. niger, A. nidulans of A. oryz e;
- Preferred Trichoderma species is preferably T. ef T. viride;
- the Pem'd painting species is preferably Pem'd ⁇ chrysogenum;
- the preferred Tricholoma species is preferably Trichoderma mongolicum, preferably entirmla species. (L. edodes;
- the preferred Agaricus species is preferably Agaricus bisporus; the preferred dicotyledon is preferably Arabidopsis thaliana;
- Preferred insect cells are preferably Spodopterafmgiperda S cells.
- the present invention also provides the nucleotide sequence of the present invention, the 7-xylosylosidase hydrolase of the present invention, and the use of a host cell comprising the nucleotide sequence of the present invention.
- the application is as follows.
- the DNA is transformed into a suitable host cell using a method conventional in the art, and the transformed recombinant cell is hydrolyzed by a recombinant enzyme produced thereby to hydrolyze various substrates, particularly glycoside compounds.
- Preferred glycoside compounds as substrates are selected from compounds containing xylose residues or compounds containing glucose residues; i.e., the use of the present invention is to hydrolyze xylose and/or glucosyl groups from such glycoside compounds.
- the compound containing a xylose residue is selected from the group consisting of taxane xylosides; the substrate is preferably a taxane compound containing a 7-xylose residue, ⁇ , 7-xylose taxane, It is naturally occurring or non-naturally occurring, such as chemically or biosynthetically, or semi-synthetically.
- the use of the 7-xylosylosidase hydrolase of the present invention is preferably a biotransformation or biocatalytic preparation of a 7-hydroxy taxane for 7-xylose taxane.
- 7-xylose taxanes include, but are not limited to, the following compounds: 7-xylose-10-deacetylpaclitaxel, 7-xylose-10-deacetyl cephalosporin, 7-xylose-10- Deacetylpaclitaxel (:, 7-xylose-10-deacetylbaccatin III, 7-xylocheol, 7-xylose cephalosporin, 7-xylocheol (:, 7-xylose) Ting ⁇ ⁇ ; products obtained after hydrolysis to remove xylosyl include, but are not limited to: 10-deacetylpaclitaxel, 10-deacetyl cephalosporin, 10-deacetylpaclitaxel (:, 10-deacetylbaccatin III , paclitaxel, cephalosporin, paclitaxel (:, bacatein III.
- the substrate may also be selected from a mixture of taxane-containing compounds containing xylose residues, such mixtures being, but not limited to, Taxus (T plant tissue, preferably Taxus plants selected from the group consisting of European yew)
- Taxus T plant tissue, preferably Taxus plants selected from the group consisting of European yew
- T. baccata T. brevifolic, Himalayan yew T. wallichiana yew (7: m "), Chinese yew (7: cfe 'brain ⁇ ), Yunnan yew (7: y awake ';?), and Northeast yew (7: cuspidate, or cell culture of these plants, or a microbial cell culture capable of producing 7-xylose taxanes.
- Ph is a phenyl group
- Bz is a benzoyl group
- Ac is a solvent for the acetyl group to dissolve the substrate.
- the solvent may be selected from the group consisting of: water, methanol, ethanol, ethyl acetonate, n-hexane, chloroform, dichloromethane, hydrazine, hydrazine- Dimethylformamide (DMF), dimethyl sulfoxide (DMSO).
- the use of the present invention also includes the use of the glycosyl hydrolase of the present invention for improving bread properties, improving animal feed characteristics, producing D-xylose for the manufacture of xylitol, and deinking of recycled paper.
- Glycosyl hydrolase of the present invention can be used with cellulase (ce ll u l ases) and hemicellulases (hemicellulases) other combination, further ethanol, butanol and the like obtaining biofuels hydrolyzed lignocellulosic monosaccharide.
- the glycosyl hydrolase of the present invention can also release bioactive molecules from other glycoside compounds and is used in the field of medicine.
- the invention also provides a biotransformation preparation method of paclitaxel and the like: using 7-xylose taxane as a raw material, and using the host cell containing the gene sequence of the invention or the enzyme produced thereby to remove the wood on the raw material Glycosyl, paclitaxel or an analogue thereof is obtained.
- a preferred host cell is a fungus or a recombinant strain of the family Tricholoma, more preferably Pichia pastoris (abbreviated as Pichia).
- the bifunctional glycosyl hydrolase of the present invention whose amino acid sequence comprises at least 30% of the amino acid sequence of the sequence of SEQ ID NO: 2, can be used from 7-xylose taxane or other Xylose or glucose is removed from the glycoside compound.
- the present invention also relates to a recombinant plasmid comprising the above nucleotide sequence, and a host cell comprising the above nucleotide sequence.
- the invention further relates to the use of a 7-xylosylosidase hydrolase or a host cell comprising a 7-xylose taxane hydrolase for the hydrolysis to remove xylose and/or to hydrolyze glucose groups.
- the present invention provides a bifunctional ⁇ -xylosidase- ⁇ -glucosidase capable of clarifying its amino acid sequence, 7-xylosylosidase hydrolase, which is a fungus of Tricholomareceae.
- Lentinula edodes M95.33 is produced by recombinant cells containing the gene encoding the enzyme, may be located in cells or secreted outside the cell, and is used to transform 7-xylose taxane into paclitaxel or an analogue thereof.
- the nucleotide sequence encoding the glycosyl hydrolase of the present invention can be used to construct various types of recombinant expression plasmids which are transferred to the original fungal or other fungal host, Or is transferred to host cells such as prokaryotic cells (including Escherichia coli, actinomycetes), plant cells, and animal cells, and the expression of the glycosyl hydrolase gene allows these hosts to obtain hydrolysis of 7-xylose taxanes as 7- The ability of hydroxy taxanes.
- the recombinant host can also be used for biotransformation of other sugar-containing compounds.
- the use of the present invention also includes the use of the glycosyl hydrolase of the present invention for improving bread characteristics, improving animal feed characteristics, producing D-xylose for use in the manufacture of xylitol, and deinking of recycled paper.
- the glycosyl hydrolase of the present invention can also be used in combination with cellulases and hemicellulases to hydrolyze lignocellulosic fibers to obtain monosaccharides to prepare biofuels such as ethanol and butanol.
- the glycosyl hydrolase of the present invention can also release bioactive molecules from other glycoside compounds and is used in the field of medicine.
- the present invention firstly clones and heterologously expresses a gene capable of specifically catalyzing a glycosyl hydrolase of 7-xyose taxane to 7-hydroxy taxane, and prepares a bioengineered bacteria having the activity of the enzyme. , providing a new and effective way for large-scale preparation of 7-hydroxy taxanes.
- Term and abbreviation Term and abbreviation
- CDS refers to the sequence of a protein encoded by a gene, from the start codon to the stop codon.
- FIG. 1 Fungal M95.33 protein extract Phenyl Sepharose hydrophobic column chromatography (A) and XDT-transformed thin layer chromatography (TLC) (B) for ⁇ -xylosidase- ⁇ -glucosidase.
- the abscissa is Absorbance values for different fractions (different section collection tube numbers) with ordinate on A405 (405 nm)
- A is a mixed substrate (control); B is a recombinant yeast + mixed substrate (control) introduced into an empty vector; C is a recombinant bacteria + mixed substrate into which the Lxyl-pl gene is introduced.
- 1, 2, 3 represent 7-xylose-10-deacetyl cephalosporin (XDC), 7-xylose-10-deacetylpaclitaxel (XDT), 7-xylose-10-desacetylpacepol C, respectively (XDTC); 1', 2' and 3' are their respective 7-hydroxy taxane products 10-deacetyl cephalosporin (DC), 10-deacetylpaclitaxel (DT), 10-deacetylated Paclitaxel C (DTC).
- Figure 7 represent 7-xylose-10-deacetyl cephalosporin (XDC), 7-xylose-10-deacetylpaclitaxel (XDT), 7-xylose-10-desacetylpacepol C, respectively (
- Example 1 Purification of Lentinus edodes P-xylosidase-P-glucosidase (LXYL-P1) Culture of fungus M95.33: Pick up about 1 cm 2 square of the lawn from the cultivated strain, and inoculate 100 Ml sterile wheat bran liquid medium [wheat bran medium composition, per liter containing: wheat bran 50.00g (add appropriate amount of water, boil for 30min, filter to remove residue), peptone 20.00g, KH 2 P0 4 1.50g, MgSO 4 0.75g, natural pH ⁇ 6.3], 25 ⁇ 26. C, 160 rpm shake flask culture for 6-8d.
- glycosyl hydrolase The mycelium was collected by filtration, and after adding liquid nitrogen, 50 mM Tris-HCl (pH 8.0) protein lysate was added in a volume of 3 to 5 times, and sonicated in an ice bath for 5 min (130 W). , 10 seconds / time, interval 10 seconds). After centrifugation (12,000 rpm, lOmin), the supernatant was collected as a crude enzyme solution for further separation and purification.
- Tris-HCl pH 8.0
- the protein having ⁇ -xylosidase activity was followed by p-nitrophenyl- ⁇ -D-xyloside (PNP-Xyl) as a specific chromogenic substrate.
- PNP-Xyl p-nitrophenyl- ⁇ -D-xyloside
- One enzyme unit is defined as the amount of enzyme required to catalyze the production of 1 nmol of p-nitrophenol in 1 min at 50 °C, pH 5.0 with PNP-Xyl as the substrate.
- DEAE Sepharose FF anion exchange column (1.6 cm X 20 cm), equilibrated with Tris-HCl buffer (50 mM, pH 8.0), and the above crude enzyme solution (80-90 ml/time) was applied to the column with 0, 0.1, 0.25. And a gradient elution of 2.0 MNaCl in 50 mM Tris-HCl buffer (pH 8.0) (flow rate 2 ml/min), collecting the enzyme-active 0.1 ⁇ 0.25 M NaCl elution fraction and adding 1 M (NH 4 ) 2 S0 4 for the next chromatography.
- Phenyl Sepharose hydrophobic column (1.6 cm X 20 cm) was equilibrated with 1 M (NH 4 ) 2 S0 4 (50 mM, pH 8.0), and the previous elution fraction was applied to the column with 1.0 ⁇ 0M (NH 4 ).
- 2 SO 4 Tris-HCl buffer (50 mM, pH 8.0) linear gradient elution (flow rate 2 ml/min), collect active enzyme components, and dialyze with Tris-HCl buffer (50 mM, pH 8.0) .
- the dialyzed solution was applied to a DEAE Sepharose FF anion exchange column [1.6 cm X 20 cm, equilibrated with Tris-HCl buffer (50 mM, pH 8.0)] with Tris-HCl buffer (50 mM, pH 8. containing 0.1 ⁇ 0.25 M NaCl).
- LXYL-P 1 or P 1
- LXYL-P2 or P2
- Both P 1 and P 2 can hydrolyze 7-xylose-10-deacetylpaclitaxel (XDT) to 10-deacetylpaclitaxel (DT) (as shown in Figure 1).
- XDT 7-xylose-10-deacetylpaclitaxel
- DT 10-deacetylpaclitaxel
- A. is an active peak of the enzyme obtained by chromatography
- B. is a thin layer chromatography (TLC) for converting the substrate XDT by the P1, P2 active sample.
- TLC thin layer chromatography
- 1 in B is the XDT reference
- 2 is the DT control
- 3 is the P1 conversion XDT
- 4 is the P2 conversion XDT.
- PNP-Glc p-Nitrophenyl-beta-D-glucopyranoside
- PNP-Gal p-Nitrophenyl-PD-galactopyranoside
- PNP-Ara p-Nitrophenyl aL-arabinopyranoside
- a solution of 5 mM, pH 5.0 was prepared using 50 mM acetate buffer.
- PNP-Xyl can not hydrolyze PNP-Gal and PNP-Ara, the results are shown in Table 2.
- Example 2 After the LXYL-P1 obtained in Example 1 was subjected to SDS-PAGE electrophoresis (see FIG. 2), the sample was subjected to reduction treatment, and the electrophoresis band having an apparent molecular weight of 110 KDa was subjected to LC-MS/MS analysis, and 5 was selected. The highest peak peptide was subjected to De novo sequencing, and the amino acid residue sequences of the five peptides were obtained, respectively:
- TLTPLEALQK (where I and BL are indistinguishable, K and Q are indistinguishable) The relative position of the five peptides is evaluated by bioinformatics means, and the order before and after LXYL-P1 is determined as follows: 3 , 2, 5, 1, 4. Design the upper and lower degenerate primers according to peptides 3 and 5, respectively:
- the above degenerate primers were used for nest-PCR amplification using the fungus M95.33 total R A as a template. After the PCR product was confirmed to contain the peptide 3, 2 and 5 coding sequences, the cDNA fragment containing the above five peptide coding regions was amplified by RACE technology, and the fragment contained a 2412 bp open reading frame (Open Reading Frame). , ORF, or CDS, named Lxyl-pJ), encodes 803 amino acids.
- the cDNA sequence SEQ ID NO: 3 and its encoded amino acid sequence SEQ ID NO: 2 are shown in the Appendix.
- the PCR amplification process is shown in Figure 8:
- the ORF CLxyl-pl of the P1 coding region obtained in Example 3 was introduced into the 5'-, 3'-end of the ⁇ I and Not I restriction sites by PCR, and ⁇ VNot I was digested and ligated.
- the recombinant expression plasmid pPIC9K-Pl-2 or pPIC3.5K was formed on the Pichia expression vector pPIC9K (secreted expression vector) or pPIC3.5K (non-secretory expression vector) which was also digested with SnaB®/ ⁇ 3 ⁇ 4 I. -Pl-2.
- the recombinant plasmid was linearized by Sac l digestion and electroporated into Pichia pastoris GS115 competent cells.
- the empty vector PPIC9K or pPIC3.5K was electroporated into Pichia pastoris GS115 competent cells as a control.
- the transformed yeast cells were applied to MD plates [containing 20.00 g per glucose, 13.40 g of amino yeast nitrogen source (YNB), 0.4 mg of biotin, 15.00 g of agar), and cultured at 28 ° C for 2 to 3 days. Single colonies were picked and inoculated onto YPD-Geneticin® resistant plates (1 liter per yeast: 10.00 g of yeast extract, 20.00 g of peptone, 20.00 g of glucose, 15.00 g of agar, antibiotic G418 ⁇ 4.00 g), and continued for 2 to 3 days. Resistant colonies were screened and colony PCR was performed on resistant colonies. Take the pPIC9K and PPIC9K-P1-2 transformants as an example ( Figure 3):
- PCR primers were matched to the sequences flanking the cloning site on the PPIC9K vector, respectively: Forward: 5' GACTGGTTCCAATTGACAAGC 3';
- 1 is the recombinant yeast genome amplification result of introducing the empty vector pPIC9K; 2 is the recombinant yeast genome amplification result introduced into pPIC9K-Pl-2; 3 is the pPIC9K-Pl-2 recombinant plasmid control amplification result.
- the seed and fermentation medium used to culture the recombinant yeast were BMGY (1 liter per yeast: 10.00 g of yeast extract, 20.00 g of peptone, 100 mM potassium phosphate buffer, pH 6.0, 10 ml of glycerol) and BMMY (replaced with 10 ml of methanol). 10 ml of glycerol in BMGY as a carbon source) medium.
- BMGY 1 liter per yeast: 10.00 g of yeast extract, 20.00 g of peptone, 100 mM potassium phosphate buffer, pH 6.0, 10 ml of glycerol
- BMMY replacement with 10 ml of methanol
- the cells were transferred to 50 ml fermentation medium, cultured at 30 °C, 220 rpm, and 1% methanol was added every 24 h to induce the expression of recombinant protein.
- the enzyme activity of the recombinant strain was observed periodically.
- the cells were washed twice by centrifugation in distilled water and suspended in the same volume of distilled water.
- the 50 ⁇ suspension was added to 100 ⁇ 5 mM PNP-Xyl, and reacted at 30-55 ° C for 20 min.
- the recombinant bacteria had a hydrolyzed substrate PNP-Xyl.
- the ability of the control bacteria transferred to the empty vector was not enzymatically active (see Figure 4).
- Example 5 Recombinant yeast hydrolysis 7-xylose-10-deacetylpaclitaxel (XDT)
- the recombinant yeast GS115-9K-P1-2 (secreted expression recombinant plasmid transformant) obtained in Example 4 was induced to culture for 5 days according to the method of Example 4, and the cells were collected by centrifugation and washed, and directly or lyophilized with pH 3.5.
- - Suspension of cells 65 mg wet cells/ml, or 16 mg stem cells/ml
- 7.5 50 mM acetate buffer or phosphate buffer as a hydrolysis reaction solution.
- XDT 7-xylose-10-deacetylpaclitaxel
- the recombinant yeast GS115-3.5K-P1-2 (non-secretory expression recombinant plasmid transformant) obtained in Example 4 was subjected to the following biotransformation reaction, and the transformation substrate was a 7-xylose taxane mixture, and the main component content was : 7-xylose-10-deacetylpacepol 62.12%, 7-xylose-10-deacetyl cephalosporin 12.75%, 7-xylose-10-desacetylpacepol C 17.04%, other components accounted for 8.09 %.
- the recombinant culture method was the same as in Example 5. Add 16 ml of the concentration to the 200 ml recombinant reaction solution.
- the conversion rates are: 7-xylose-10-deacetylpaclitaxel (XDT) 92.45%, 7-xylose-10-deacetyl cephalosporin (XDC) 93.60%, 7- Xylose-10-deacetylpacepol C (XDTC) 92.00%
- the yields of the three main products were: 10-deacetylpaclitaxel (DT) 3.27 mg/ml, 10-deacetylated cephalosporin (DC) 0.74 Mg/ml, 10-deacetylpacillin C (DTC) 0.92 mg/ml, the sum of the three was 4.93 mg/ml; whereas the control did not have the above activity (Fig. 6).
- A is a mixed substrate (control); B is a recombinant yeast + mixed substrate (control) into which an empty vector is introduced; and C is a recombinant bacteria + mixed substrate into which the Lxyl-pl gene is introduced.
- 1 is 7-xylose-10-deacetyl cephalosporin; 2 is 7-xylose-10-deacetylpaclitaxel; 3 is 7-xylose-10-desacetylpacepol C; 1', 2' and 3' is the corresponding 7-hydroxy taxane product, respectively.
- Example 7 Recombinant Yeast Hydrolysis 7-Xylose Baccarin III (XDB)
- the strain was the same as in Example 6, and the substrate was 7-xylose-10-deacetylbaccatin I I I (XDB).
- XDB 7-xylose-10-deacetylbaccatin I I I
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WO2014201595A1 (zh) * | 2013-06-21 | 2014-12-24 | 中国科学院大连化学物理研究所 | 一种木糖苷酶Xy1_S及其编码基因与应用 |
CN105331596A (zh) * | 2014-08-04 | 2016-02-17 | 本田技研工业株式会社 | 属于GH3家族的耐热性β-木糖苷酶 |
CN105420216A (zh) * | 2014-09-17 | 2016-03-23 | 本田技研工业株式会社 | 属于GH3家族的耐热性β-木糖苷酶 |
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CN104232605B (zh) * | 2013-06-21 | 2016-07-06 | 中国科学院大连化学物理研究所 | 一种木糖苷酶Xyl_S及其编码基因与应用 |
CN103864729A (zh) * | 2014-03-03 | 2014-06-18 | 无锡紫熙生物科技有限公司 | 一种10-去乙酰基巴卡亭iii的制备方法 |
CN105331596A (zh) * | 2014-08-04 | 2016-02-17 | 本田技研工业株式会社 | 属于GH3家族的耐热性β-木糖苷酶 |
CN105331596B (zh) * | 2014-08-04 | 2018-11-23 | 本田技研工业株式会社 | 属于GH3家族的耐热性β-木糖苷酶 |
CN105420216A (zh) * | 2014-09-17 | 2016-03-23 | 本田技研工业株式会社 | 属于GH3家族的耐热性β-木糖苷酶 |
CN105420216B (zh) * | 2014-09-17 | 2018-11-30 | 本田技研工业株式会社 | 属于GH3家族的耐热性β-木糖苷酶 |
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