Streptomyces rochei wa11-1-1 and application thereof

Abstract

The present application belongs to the field of microbial technology, and particularly relates to Streptomyces louchei WA11-1-1 and application thereof. The Streptomyces louchei WA11-1-1 provided by the present application is isolated from Periplaneta americana, and the strain is currently preserved in the Guangdong Microbial Culture Collection Center with a preservation number of GDMCC NO.61629 and a preservation date of May 19, 2021. The Streptomyces louchei WA11-1-1 provided by the present application can convert 7-xylose-10-deacetyl paclitaxel into 10-deacetyl paclitaxel in fermentation, and under the conditions of a xylan concentration of 0.8%, a fermentation time of 4d, a fermentation temperature of 28℃ and a liquid loading of 250mL, the conversion rate can reach 40.72±1.08%, which is 1.85 times higher than that before optimization, and the Streptomyces louchei WA11-1-1 can be used for preparation of anticancer drugs and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Streptomyces WA11-1-1 and its applications. Background Technology

[0002] Taxol is a diterpenoid anticancer compound widely used clinically to treat advanced ovarian cancer, breast cancer, and non-small cell lung cancer. Taxol is derived from the bark of endangered species of the yew genus, and its extremely limited production results in a high price.

[0003] 7-xylosyl-10-deacetylpaclitaxel (7-XDT) and other 7-xylosyltaxane compounds are analogues of the parent structure of paclitaxel. They are mainly found in the roots, stems and leaves of yew trees, and their content is at least 5 times that of paclitaxel, but they have not been fully utilized.

[0004] Although 10-deacetylpaclitaxel (10-DAT) cannot currently be used for the semi-synthesis of paclitaxel, it can be directly converted into paclitaxel via 10-deacetylbaccatin III-10-β-O-acetyltransferase (DBAT).

[0005] Hao et al. isolated a new actinomycete, DICP16, from the soil surrounding yew trees in temperate regions through enrichment culture. They then performed taxonomic identification and determined it to be... Leifsonia shinshuensis sp. The 7-β-xyl in strain DICP16 can hydrolyze the C-7 xylose residue of 7-XDT to yield 10-DAT and 10-DAB. Wang et al. screened four strains from Kunming, Yunnan Province that could produce 7-β-xyl and convert 7-XDT to 10-DAT. Morphological, physiological, and genetic identification revealed that one of these strains was *Streptomyces mattii*. Streptomyces matensi Liu Yu et al. screened 52 tested microorganisms on agar plates using xylan as the sole carbon source and obtained a strain of Streptomyces cerevisiae (sky blue). Streptomycesc coelicolor This bacterium also has the ability to transform 7-XDT into 10-DAT. Dou et al. isolated a fibrotic microbacterium (Fibrous microbacterium) from the rhizosphere soil of *Taxus yunnanensis*. Cellulosimicrobium cellulans strain F16The biotransformation of 7-XDT to 10-DAT was achieved. Wang et al. discovered that nine actinomycetes produced 7-β-xyl, which was active in the transformation of 7-XDT. High-performance liquid chromatography (HPLC) confirmed that the transformation product was 10-DAT. These actinomycetes belonged to five genera, namely the genus Micrococcus (…). Cellulosimicrobium ), Fibromospora ( Cellulomonas ), Ehrlich ( Oerskovia ), Dequinone bacteria ( Demequina ) and Actinobiliformes ( Actinotalea ). Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides *Streptomyces loucherei* WA11-1-1 and its applications. This invention is the first to use *Streptomyces loucherei* WA11-1-1 for the production of 7-β-xyl enzymes, and under the conditions of this invention, the conversion rate of 10-DAT can reach 40.72±1.08%, which is 1.85 times higher than before optimization.

[0007] To achieve the above objectives, the technical effects of this invention are as follows:

[0008] A strain of *Streptomyces loucherii* WA11-1-1, wherein *Streptomyces loucherii* WA11-1-1 is... Streptomyces rochei It is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO. 61629, and the deposit date is April 26, 2021.

[0009] The present invention also provides the application of the aforementioned Streptomyces WA11-1-1 in the production of 7-β-xyl enzyme.

[0010] Preferably, the reaction temperature of the 7-β-xyl enzyme is 35°C, the reaction pH is 6, and the storage temperature is 20°C~30°C, the pH is 7.

[0011] The present invention also provides the application of the aforementioned Streptomyces WA11-1-1 in the preparation of 10-deacetylated paclitaxel.

[0012] Preferably, the 10-deacetylated paclitaxel is prepared by converting 7-xylose-10-deacetylated paclitaxel during fermentation by Streptomyces WA11-1-1.

[0013] Preferably, the fermentation conditions are: xylan concentration 0.8%, fermentation time 4 days, fermentation temperature 28℃, and liquid volume 250mL.

[0014] The present invention also provides the use of the aforementioned Streptomyces WA11-1-1 in the preparation of a tumor-treating pharmaceutical composition.

[0015] Preferably, the tumor includes ovarian cancer, breast cancer, and non-small cell lung cancer.

[0016] Preferably, the pharmaceutical composition further includes physiological saline and other pharmaceutically acceptable carriers.

[0017] Only two reports have been published on the production of 7-β-xyl by Streptomyces: *Streptomyces mater* and *Streptomyces cerevisiae*. There are also no reports on the production of 7-β-xyl by *Copper-loving Bacteria*. This invention has screened eight *Streptomyces* strains and two *Copper-loving Bacteria* strains from cockroach enterobacteria, all of which possess the ability to produce 7-β-xyl. Among them, strain WA11-1-1 showed good transformation efficiency. Morphological identification, physiological and biochemical identification, and 16S rRNA gene sequence analysis of the 7-β-xyl-producing strain WA11-1-1 were performed to determine its taxonomic position at the species level, enriching the sources of 7-β-xyl-producing strains and providing guidance for subsequent whole-genome sequencing. Under the action of 7-β-xyl, 7-XDT may produce only 10-DAT or 10-DAB, or both, resulting in diverse enzymatic digests. This invention measures the efficiency of 7-β-xyl from strain WA11-1-1 in synthesizing the paclitaxel intermediate 10-DAT. In order to gain a preliminary understanding of the physicochemical properties of 7-β-xyl and to speculate on its application direction and potential, the enzymatic properties of 7-β-xyl are investigated from the aspects of temperature, pH and metal ions.

[0018] This invention identified the 7-β-xyl-producing strain WA11-1-1 through morphological characteristics, physiological and biochemical properties, and 16S rRNA gene sequence analysis, ultimately classifying it as *Streptomyces rochei*. WA11-1-1 can convert 7-XDT to 10-DAT and 10-DAB, with a conversion rate of 23.48 ± 0.89% for the major product, 10-DAT. The optimal temperature for the crude 7-β-xyl enzyme solution is 35℃, and the optimal pH is 6; better stability is observed at 20℃~30℃ and a pH close to 7. Na... + K + Mg 2+ It promotes the activity of this enzyme, of which Na + The effect is most significant, while Ca 2+ Fe 3+ Zn 2+ It has an inhibitory effect.

[0019] Factors influencing enzyme production during microbial fermentation mainly include the composition of the culture medium required for microbial growth and enzyme production, such as the selection of carbon sources, nitrogen sources, inorganic salts, and trace elements, as well as fermentation conditions such as fermentation time, fermentation temperature, liquid volume, inoculum size, and pH value. Single-factor experiments studying the effects of different factors on experimental indicators are a commonly used optimization strategy in experimental research; however, due to their limitations, they are often combined with orthogonal experiments. Orthogonal experiments comprehensively examine multiple factors and levels, ultimately selecting the optimal combination of factor levels. Compared with single-factor experiments, they have advantages such as fewer experiments, simpler methods, and more reliable results, and have been applied in many fields. Therefore, combining single-factor experiments with orthogonal experiments to optimize various factors can yield the optimal combination for enzyme production. Currently, there are no reports on 7-β-xyl from *Streptomyces rochei*, both domestically and internationally, and research on optimizing fermentation conditions for 7-β-xyl production is even more lacking. This study used single-factor experiments and orthogonal experiments to optimize four fermentation conditions (xylan (the only carbon source) concentration, fermentation time, fermentation temperature, and liquid volume) for the production of 7-β-xyl by strain WA11-1-1, ultimately improving the efficiency of strain WA11-1-1 in catalyzing the synthesis of paclitaxel intermediate 10-DAT.

[0020] This invention optimized the fermentation conditions for 7-β-xyl production by strain WA11-1-1 using single-factor experiments and orthogonal experiments. The optimal combination of fermentation conditions was obtained as follows: xylan concentration 0.8%, fermentation time 4 days, fermentation temperature 28℃, and liquid volume 250mL. Under these conditions, the 10-DAT conversion rate of strain WA11-1-1 increased by 1.85 times compared with that before optimization, with a conversion rate of 40.72 ± 1.08%.

[0021] Compared with the prior art, the present invention has the following advantages: The present invention successfully increased the 10-DAT conversion rate of strain WA11-1-1 to 40.72 ±1.08%; it is the first time that this strain has been used to produce 7-β-xyl enzyme, enriching the source of 7-β-xyl-producing strains, and providing guidance for subsequent whole genome sequencing. Attached Figure Description

[0022] Figure 1 Morphological characteristics of strain WA11-1-1;

[0023] Figure 2 The image shows the molecular biological identification results of strain WA11-1-1.

[0024] Figure 3 The HPLC chromatogram of the transformation product of strain WA11-1-1 is shown in the figure.

[0025] Figure 4The standard curve of 10-DAT;

[0026] Figure 5 The graph shows the effect of temperature on enzyme activity.

[0027] Figure 6 The graph shows the effect of pH on enzyme activity.

[0028] Figure 7 The graph shows the effect of temperature on enzyme activity and stability.

[0029] Figure 8 The graph shows the effect of pH on enzyme activity and stability.

[0030] Figure 9 The graph shows the effect of metal ions on enzyme activity and stability.

[0031] Figure 10 The figure shows the effect of xylan concentration, fermentation time, fermentation temperature and liquid volume on the production of 7-β-xyl by strain WA11-1-1;

[0032] Figure 11 To optimize fermentation conditions for orthogonal experiments;

[0033] Figure 12 For the effect curve;

[0034] Figure 13 HPLC chromatograms of 10-DAT produced by strain WA11-1-1 before and after optimization of fermentation conditions. Detailed Implementation

[0035] The present invention will be further explained below with reference to specific embodiments. However, it should be noted that the following embodiments are only used to explain the present invention and cannot be used to limit the present invention. All technical solutions that are the same as or similar to the present invention are within the protection scope of the present invention. Where specific techniques or conditions are not specified in this embodiment, they shall be operated in accordance with conventional technical methods and instrument manuals in the art; where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be obtained commercially.

[0036] In the experiment of this invention, the selected strain, Enterobacter bladderwrack WA11-1-1, was previously isolated by the inventors and stored in the laboratory at -80°C. It was identified as Streptomyces louchei.

[0037] The main solution preparation processes involved are as follows:

[0038] (1) 1.5% agarose gel: Accurately weigh 0.375g agarose into an Erlenmeyer flask, add 25mL of 1×TAE electrophoresis buffer (small gel), heat in a microwave oven on medium heat for 1min to completely dissolve the agarose, cool with cold water to about 55℃, add 2μL of 10mg / mL ethidium bromide (EB) substitute to the Erlenmeyer flask, shake well and pour into the gel tank, insert the comb, wait for about 30min for the gel to solidify and then remove the comb.

[0039] (2) 50×TAE buffer: Weigh 242.0 g of Tris base and 37.2 g of EDTA disodium dihydrate into a 1 L measuring cup, pour in 800 mL of double-distilled water, mix well, pour 57.1 mL of glacial acetic acid (CH3COOH) into the measuring cup, slowly add 10% sodium hydroxide (NaOH) to adjust the pH to 8.0, and then make up to 1 L with double-distilled water.

[0040] (3) 1 mol / L Na2CO3 solution: Weigh 10.6 g of sodium carbonate (Na2CO3) solid powder, dissolve it in a small amount of double-distilled water, and make up to 100 mL.

[0041] (4) 5 mmol / L p-nitrophenyl-β-D-xylopyranoside (pNPX) solution: Weigh 0.135 g of pNPX powder and dissolve it in a small amount of phosphate buffered saline (PBS) solution (pH=7), make up to 100 mL, and store at -20 °C in the dark for later use.

[0042] (5) 2.5% glutaraldehyde solution: Measure 10 mL of 25% glutaraldehyde aqueous solution and 50 mL of 0.2 M, pH 7.0 PBS, and make up to 100 mL with double distilled water. Store in a refrigerator at 4°C in the dark for later use.

[0043] (6) Seed culture medium: glucose 10.0 g / L, tryptone 12.0 g / L, yeast extract 2.0 g / L, dissolved in double-distilled water and brought to a final volume of 1 L, pH adjusted to 7.2±0.2, autoclaved at 115℃ for 20 min, used for enrichment culture of actinomycetes.

[0044] (7) Xylan liquid culture medium: xylan 2.0 g / L, ammonium sulfate ((NH4)2SO4) 1.0 g / L, magnesium sulfate (MgSO4·7H2O) 0.1 g / L, potassium dihydrogen phosphate (KH2PO4) 3.0 g / L, dipotassium hydrogen phosphate (K2HPO4) 7.0 g / L, sodium chloride (NaCl) 0.5 g / L, dissolved in double-distilled water and brought to a final volume of 1L. The pH was adjusted to 7.2±0.2, and the mixture was autoclaved at 121℃ for 15 min. This medium was used for the liquid fermentation culture of xylanase-producing strains.

[0045] The main instruments and reagents used in the experiment are shown in Table 1 below.

[0046] Table 1. Sources of instruments and reagents used in this invention

[0047]

[0048] Experimental Example 1: Identification of strain WA11-1-1

[0049] I. Experimental Procedure:

[0050] 1. Morphological identification: Strain strain WA11-1-1, preserved in a -80℃ storage chamber, was inoculated into Gao's Synthetic I solid medium and incubated upside down at 28℃ for 5-7 days. After single colonies grew, their morphological characteristics were observed. A small amount of bacterial cells was smeared onto a glass slide with an appropriate amount of sterile water added, and Gram staining was performed using a Gram staining kit. The basic morphology and staining were observed under an optical microscope.

[0051] Activated strain WA11-1-1 was inoculated at the contact point between the substrate and the culture medium. The substrate plates were inverted and cultured for 3-5 days. The growth and color of the mycelia and aerial hyphae were observed, as well as the production of soluble pigments. Soybean-sized cells of strain WA11-1-1 were collected and placed in 1.5 ml centrifuge tubes. Pre-cooled 2.5% glutaraldehyde solution was added and the cells were fixed overnight at 4°C. The fixative was discarded, and the samples were washed three times with pH 7.0 PBS. The samples were then dehydrated with ethanol solutions of different concentrations (30%, 50%, 70%, 80%, 90%, 95%, and 100%). They were then treated with pure isoamyl acetate for 1 hour, followed by critical point drying, coating, and finally, the three-dimensional morphology of the strain surface was observed using a scanning electron microscope.

[0052] 2. Physiological and Biochemical Identification: The principles and methods of the physiological and biochemical identification experiments were based on the "Technical Manual of Actinomycete Systematic Classification" (ISBN: 9787122249517, authors Guan Tongwei and Zhang Xiaoping, published January 2016). Specific experiments were conducted using reagent kits from Guangdong Huankai Biotechnology Co., Ltd.

[0053] 3. Molecular biological identification

[0054] A single colony of strain WA11-1-1 was inoculated into 5 mL of seed culture medium and incubated overnight at 28 °C and 160 rpm. DNA was then extracted from strain WA11-1-1 according to the instructions of the bacterial genomic DNA extraction kit. The 16S rRNA gene of strain WA11-1-1 was amplified using universal primers (27F: 5'-AGAGTTTGATCCTGG CTCAG-3'; 1492R: 5'-TACGGCTACCTTGTTACGACTT-3'). The PCR reaction system and conditions are shown in Tables 2 and 3. The PCR amplification products were analyzed by 1.5% agarose gel electrophoresis and sent to Qingke Biotechnology Co., Ltd. for sequencing. The 16S rRNA gene sequence of strain WA11-1-1 was compared with known sequences from NCBI using Blast sequencing. The alignment results were used to construct a phylogenetic tree using Mega 7.0 software. At the same time, the strain sequence was uploaded to the GenBank database on the NCBI website through the Submission Portal, and the accession number (GenBank Accession) of strain WA11-1-1 was obtained.

[0055] Table 2 PCR reaction system

[0056]

[0057] Table 3 PCR amplification program

[0058]

[0059] II. Experimental Results:

[0060] 1. Morphological identification: Specific results are as follows Figure 1 As shown, Figure 1 In the figures, A shows the colony morphology of strain WA11-1-1; B shows the Gram staining image of strain WA11-1-1 (100×); C shows the observation results of strain WA11-1-1 under a scanning electron microscope at 2000×; and D shows the observation results of strain WA11-1-1 under a scanning electron microscope at 10000×. It can be seen that the colonies of strain WA11-1-1 on Gao's Synthetic I medium are round, grayish-white, dry, dull, opaque, and slightly raised. The mycelia within the substrate are well-developed and slightly brownish, while the aerial mycelia are vigorous, gray, and contain no soluble pigment. Gram staining is positive, and the bacterial cells are short rod-shaped. The scanning electron microscopy results show that the spore hyphae are straight and flexible, and the spores are oblong with a smooth surface. Based on the characteristics indicated in the figures, this fungus basically conforms to the morphological characteristics of Streptomyces.

[0061] 2. Physiological and biochemical identification: The results of some physiological and biochemical tests of strain WA11-1-1 are shown in Table 4. Compared with the book "Systematic Classification of Actinomycetes", the results are basically consistent with the physiological and biochemical characteristics of Streptomyces rochei.

[0062] Table 4. Partial physiological and biochemical characteristics of strain WA11-1-1

[0063]

[0064] 3. Molecular biological identification: Results are as follows Figure 2 As shown, A is an electrophoresis diagram of the 16S rRNA gene amplification product of strain WA11-1-1; B is a phylogenetic tree of the 16S rRNA gene sequence of strain WA11-1-1. From the electrophoresis diagram of the 16S rRNA gene amplification product of strain WA11-1-1, the target band size can be seen to be between 1000-2000 bp. Figure 2 A). After sequencing and Blast homology comparison, this strain is found to be in the same clade as *Streptomyces rochei* strain NRRL B-1559, with a similarity of up to 100%. Figure 2 B). Based on morphological, physiological and biochemical characteristics and molecular biological features, strain WA11-1-1 was identified as *Streptomyces rochei*. The 16S rRNA gene sequence of strain WA11-1-1 was obtained from GenBank through the Submission Portal, with accession number OK047707.

[0065] Experimental Example 2: Determination of the efficiency of 7-β-xyl-catalyzed synthesis of 10-DAT by strain WA11-1-1

[0066] I. Experimental Procedure:

[0067] 1. Determination of the 7-β-xyl conversion product of 7-XDT: Three standards, 7-XDT, 10-DAT, and 10-DAB, were dissolved in chromatographic-grade methanol and filtered through a 0.45 μm filter membrane to prepare a 1 mL solution with a concentration of 0.5 mg / mL. Simultaneously, strain WA11-1-1 was used to convert the product. The concentrated sample was filtered. The specific conversion and concentration process was as follows: a single colony was picked and inoculated into a 300 mL shake flask containing 100 mL of xylan liquid medium and cultured at 28℃ and 180 rpm for 3 days for enrichment culture. Then, 5 mL of the seed culture suspension was inoculated into 50 mL of xylan liquid medium, and fermentation was continued in shake flasks for 2 days. 4 mL of 0.5 mg / mL 7-XDT methanol solution was added to the fermentation broth, and the culture was continued for 4 days. The fermentation broth was centrifuged at 4℃ and 8,000 rpm for 15 min, and the supernatant was collected. The supernatant was extracted three times with the same volume of ethyl acetate. The extract was concentrated under reduced pressure by rotary evaporation to obtain the concentrated sample.

[0068] The filtrate was used as the HPLC test solution. The chromatographic conditions were as follows: mobile phase: acetonitrile:water (45:55, V / V); injection volume: 10 μL; flow rate: 0.8 mL / min; detection wavelength: 230 nm; column temperature: room temperature; detector: PDA (photodiode array); column: YMC-Pack ODS-AQ C18 column (250 mm × 4.6 mm ODS-5 μm, 12 nm). The HPLC chromatogram of the transformed product sample was compared with the chromatograms of three standards: 7-XDT, 10-DAT, and 10-DAB, to determine the major product of 7-XDT transformation by strain WA11-1-1. The experiment was repeated three times.

[0069] 2. Preparation of the 10-DAT standard curve: Six different concentrations of 10-DAT solutions (0.05, 0.10, 0.15, 0.20, 0.30, and 0.40 mg / mL) were prepared. The filtrates filtered through a 0.45 μm filter membrane were used as the HPLC test solution. Chromatographic conditions were the same as in step 1. The injection was repeated three times, and the peak areas were measured to plot a 10-DAT concentration-peak area standard curve.

[0070] 3. Determination of the efficiency of 7-β-xyl-catalyzed synthesis of 10-DAT: After confirming that the main product is 10-DAT through the previous operations, the peak area of ​​10-DAT can be obtained by HPLC analysis of the conversion product sample. The peak area of ​​10-DAT is substituted into the linear regression equation of the 10-DAT standard curve to calculate the concentration of the conversion product 10-DAT.

[0071] Sample conversion rate (%) = (number of moles of 10-DAT generated / number of moles of 7-XDT before conversion) × 100%.

[0072] II. Experimental Results:

[0073] 1. Determination of the product of 7-XDT conversion from 1,7-β-xyl: Experimental results are as follows Figure 3 As shown, Figure 3 In the table, A is 7-XDT standard; B is 10-DAT standard; C is 10-DAB standard; D is the transformation product of strain WA11-1-1; and E is the control group of strain WA11-1-1 (without 7-XDT). Figure 3 It was found that the retention times of the three standards, 7-XDT, 10-DAT, and 10-DAB, were 10.595 min, 17.873 min, and 5.776 min, respectively, with specific peak shapes (UV spectra) at specific absorption wavelengths of 229.7 nm, 228.6 nm, and 230.9 nm. The substrate and product transformed by strain WA11-1-1 showed absorption peaks at 10.654 min, 17.829 min, and 5.756 min, respectively, and the UV spectra of these three substances were consistent with those of the three standards. Therefore, it can be concluded that strain WA11-1-1 can convert 7-XDT to 10-DAT and 10-DAB, but the transformation product is predominantly 10-DAT.

[0074] 2.10-DAT Standard Curve Construction: The constructed standard curve is as follows... Figure 4 As shown, the horizontal axis represents the 10-DAT concentration, and the vertical axis represents the peak area. The obtained linear regression equation is: y = 2587x + 45.60, R0 2 = 0.999, indicating a good linear fit.

[0075] 3.7 Determination of the efficiency of 7-β-xyl-catalyzed synthesis of 10-DAT: The peak area of ​​10-DAT, the product of the three transformations of strain WA11-1-1, was measured and substituted into the above linear regression equation to obtain the concentration of 10-DAT. The conversion rate of 7-XDT to 10-DAT by strain WA11-1-1 was calculated to be 23.48±0.89% (n=3).

[0076] Preliminary study on the 7-β-xyl enzymatic properties of strain WA11-1-1 (Experimental Example 3)

[0077] 1. Principle of β-xylosidase activity assay: Under certain conditions, pNPX hydrolyzes β-xylosidase to generate p-nitrophenol (pNP). pNP is yellow under alkaline conditions and has a characteristic absorption peak at a wavelength of 405 nm. The absorbance value obtained by the enzyme-linked immunosorbent assay (ELISA) reader or spectrophotometer corresponds to the corresponding pNP concentration, and the β-xylosidase activity can be calculated from this.

[0078] 2. Preparation of 7-β-xyl crude enzyme solution: A single colony of strain WA11-1-1 was inoculated into a 300 mL shake flask containing 100 mL of xylan liquid medium and cultured at 28℃ and 180 r / min for 3 days for strain enrichment. Then, 5 mL of the seed culture suspension was inoculated into 50 mL of 0.2% xylan liquid medium at a 10% inoculation rate and cultured at 28℃ and 180 r / min for another 2 days. The fermentation broth was centrifuged at 4℃ and 8000 r / min for 15 min. The supernatant was collected and filtered through gauze; the filtered result is the crude enzyme solution.

[0079] 3. Method for β-xylosidase activity assay: Following the method of Tang Yong et al., 70 µL of crude enzyme solution was added to a 2 mL EP tube, followed by 30 µL of 5 mmol / L pNPX substrate. The mixture was then placed in a water bath at 40 °C for 30 min. Finally, 1 mL of 1 mol / L Na₂CO₃ solution was added to stop the reaction. The OD was measured using a microplate reader. 405 Blank control: An enzyme solution that has been inactive after being heated to boiling at high temperature is used as a control. The reaction system and reaction method for enzyme activity determination are the same as above.

[0080] 4. Definition and Calculation of Enzyme Activity Units: An enzyme activity unit is defined as the amount of enzyme required to release 1 µmol of pNP per minute using pNPX as a substrate under certain conditions. The formula for calculating enzyme activity (U) is: U / mL = C × V1 / t × V2. Where C is the concentration of pNP (µmol / mL), V1 is the total reaction volume (mL), V2 is the volume of the enzyme solution (mL), and t is the reaction time (min).

[0081] 5. Preliminary study on the enzymatic properties of 7-β-xyl:

[0082] (1) Determination of optimal reaction temperature

[0083] The crude enzyme solution was placed in a water bath at seven different temperatures (5°C intervals) from 20°C to 50°C for reaction. Other reaction systems were the same as in step 3. The enzyme solution that was inactivated at 100°C under the same conditions was used as a blank control. The activity of β-xylosidase was measured. The highest enzyme activity was taken as 100%. The relative enzyme activity was calculated and a line graph of the optimal reaction temperature was plotted, where the horizontal axis represents temperature and the vertical axis represents relative enzyme activity.

[0084] (2) Determination of the optimal reaction pH

[0085] The crude enzyme solution was reacted under eight different pH conditions ranging from pH 3.0 to 10.0. Other reaction systems were reacted in the same way as in step 3. The enzyme solution inactivated at 100℃ under the same conditions was used as a blank control. The activity of β-xylosidase was measured. The highest enzyme activity was taken as 100%. The relative enzyme activity was calculated and a line graph of the optimal reaction pH was plotted, where the horizontal axis represents pH and the vertical axis represents relative enzyme activity.

[0086] (3) Temperature stability determination

[0087] The crude enzyme solution was incubated for 1 hour at seven different temperatures (5°C intervals) from 20°C to 50°C, and then rapidly cooled to 4°C. Other reaction systems were treated in step 3. An enzyme solution inactivated at 100°C under the same conditions was used as a blank control. The β-xylosidase activity was measured. The enzyme activity of the untreated enzyme solution was taken as 100%, and its relative enzyme activity was calculated. A temperature stability line graph was plotted, where the horizontal axis represents temperature and the vertical axis represents relative enzyme activity.

[0088] (4) Determination of acid-base stability

[0089] The crude enzyme solution was diluted 10-fold with eight different pH buffers ranging from pH 3.0 to 10.0 and incubated at 4°C for 1 h. Other reaction systems were performed as in step 3. An enzyme solution inactivated at 100°C under the same conditions was used as a blank control. The β-xylosidase activity was measured. The enzyme activity of the untreated enzyme solution was taken as 100%, and its relative enzyme activity was calculated. An acid-base stability line graph was plotted, where the horizontal axis represents pH and the vertical axis represents relative enzyme activity.

[0090] (5) Determination of metal ion stability

[0091] Preparation of 0.1 mol / L solutions of different metal ions (Mg 2+ Ca 2+ Fe 3+ Zn 2+ Na + and K + Add the enzyme solution at a volume ratio of 1:500 to the crude enzyme solution and incubate at 4℃ for 1 hour. Repeat the same steps for other reaction systems. Use the enzyme solution inactivated at 100℃ under the same conditions as a blank control to determine the β-xylosidase activity. Using the enzyme activity of the enzyme solution without the above metal ion solution treatment as 100%, calculate its relative enzyme activity and plot a metal ion stability bar chart, where the horizontal axis represents the type of metal ion and the vertical axis represents the relative enzyme activity.

[0092] II. Experimental Results:

[0093] 1. Determination of optimal reaction temperature: Experimental results are as follows Figure 5 As shown, by Figure 5It can be seen that as the temperature increases, the activity of 7-β-xyl enzyme gradually increases, reaching its peak at 35℃. After that, as the temperature continues to rise, the enzyme activity gradually decreases. Therefore, the optimal temperature for 7-β-xyl is 35℃.

[0094] 2. Determination of optimal reaction pH: The experimental results are as follows Figure 6 As shown, by Figure 6 It can be seen that the enzyme activity of 7-β-xyl enzyme reaction increases with increasing pH value. The enzyme activity is the highest at pH 6. When the pH is greater than 6, the enzyme activity continues to decrease. Therefore, the optimal pH for 7-β-xyl reaction is 6.

[0095] 3. Temperature stability determination: The test results are as follows Figure 7 As shown, by Figure 7 It can be seen that the relative enzyme activity of 7-β-xyl is greater than 80% at temperatures between 20℃ and 30℃, indicating that the enzyme has good stability and maintains high catalytic ability within this temperature range, and is most stable at 25℃. However, when the temperature exceeds 30℃, the enzyme activity decreases rapidly.

[0096] 4. Acid-base stability test: The test results are as follows Figure 8 As shown, by Figure 8 It can be seen that 7-β-xyl exhibits the best acid-base stability at pH 7. However, when the pH is less than or greater than 7, the relative enzyme activity is below 60%, and the acid-base stability of 7-β-xyl also deteriorates. This indicates that 7-β-xyl is adapted to a neutral environment but not to acidic or alkaline environments.

[0097] 5. Determination of metal ion stability: The stability results of metal ions are as follows... Figure 9 As shown, Na + K + Mg 2+ It promotes the activity of 7-β-xyl enzyme, of which Na + Its effect is most significant. And Ca... 2+ Fe 3+ Zn 2+ It has an inhibitory effect on 7-β-xyl enzyme activity.

[0098] Experimental Example 4: Optimization of Fermentation Conditions

[0099] I. Experimental Procedure:

[0100] 1. Initial fermentation conditions: A single colony of strain WA11-1-1 was inoculated into a liquid medium with a xylan concentration of 0.2% and cultured at 28℃ and 180 rpm for 3 days for enrichment culture. Then, 15 mL of seed culture was transferred to a 500 mL shake flask containing 300 mL of 0.2% xylan liquid medium (initial fermentation medium) and fermented at 28℃ and 180 rpm for 4 days. After fermentation, the culture was centrifuged at 4℃ and 12000 rpm for 15 min. The supernatant filtered through gauze was used to detect β-xylosidase activity.

[0101] 2.7 Method for determining β-xyl enzyme activity: Same as step 3 in Experimental Example 3.

[0102] 3. Single-factor experiments to optimize fermentation conditions: Based on the principle of single-factor experimental design, the effects of xylan concentration, fermentation time, fermentation temperature, and liquid volume on the production of 7-β-xyl by strain WA11-1-1 were investigated, while keeping the composition of the fermentation medium and other culture conditions constant. Each experiment was conducted in triplicate, and the average value was taken as the result.

[0103] 3.1 Effect of xylan concentration on 7-β-xylase activity

[0104] Single colonies were picked and inoculated into xylan liquid medium with a xylan concentration of 0.2% and cultured at 28℃ and 180 rpm for 3 days for bacterial enrichment culture. Then, 15 mL of seed culture was transferred to 300 mL of xylan liquid medium with different concentrations (0.2%, 0.4%, 0.6%, 0.8%, 1.0% and 1.2%) and fermented at 28℃ and 180 rpm for 4 days. After centrifugation at 4℃ and 12000 rpm for 15 min, the supernatant was used to detect 7-β-xyl enzyme activity.

[0105] 3.2 Effect of fermentation time on 7-β-xyl enzyme activity

[0106] Single colonies were picked and inoculated into 0.2% xylan liquid medium and cultured at 28℃ and 180 rpm for 3 days for bacterial enrichment. Then, 15 mL of the seed culture was transferred to 300 mL of 0.2% xylan liquid medium and fermented at 28℃ and 180 rpm for 2, 3, 4, 5, 6 and 7 days respectively. After centrifugation at 4℃ and 12000 rpm for 15 min, the supernatant was collected to detect 7-β-xyl enzyme activity.

[0107] 3.3 Effect of fermentation temperature on 7-β-xyl enzyme activity

[0108] Single colonies were picked and inoculated into 0.2% xylan liquid medium and cultured at 28℃ and 180 rpm for 3 days for bacterial enrichment. Then, 15 mL of the seed culture was transferred to 300 mL of 0.2% xylan liquid medium and cultured at 22℃, 25℃, 28℃, 31℃, 34℃ and 37℃ for another 4 days at 180 rpm. After centrifugation at 4℃ and 12000 rpm for 15 min, the supernatant was collected to detect 7-β-xyl enzyme activity.

[0109] 4.3.3.4 Effect of liquid volume on 7-β-xyl enzyme activity

[0110] Single colonies were picked and inoculated into 0.2% xylan liquid medium and cultured at 28℃ and 180 rpm for 3 days for bacterial enrichment. Then, 15 mL of the seed culture was transferred to 100 mL, 150 mL, 200 mL, 250 mL, 300 mL and 350 mL of 0.2% xylan liquid medium, respectively, and fermented at 28℃ and 180 rpm for 4 days. After centrifugation at 4℃ and 12000 rpm for 15 min, the supernatant was used to detect 7-β-xyl enzyme activity.

[0111] 4.3.4 Orthogonal experiment to optimize fermentation conditions

[0112] Based on the analysis of the results of the single-factor experiments, orthogonal design assistant was used to design orthogonal experiments L9(4)(3) with different xylan concentrations (0.2%, 0.4%, 0.6%, 0.8%, 1.0% and 1.2%), fermentation times (2d, 3d, 4d, 5d, 6d and 7d), fermentation temperatures (22℃, 25℃, 28℃, 31℃, 34℃ and 37℃) and liquid volumes (100mL, 150mL, 200mL, 250mL, 300mL and 350mL) as variables to determine the optimal fermentation conditions for the production of 7-β-xyl by strain WA11-1-1.

[0113] 4.3.5 Determination of 10-DAT conversion rate of strain WA11-1-1 before and after optimization of fermentation conditions for 7-β-xyl production

[0114] Fermentation was carried out under both pre- and post-optimization conditions, with 7-DAT added to a final concentration of 0.04 mg / ml. The conversion rate of 10-DAT was measured after 4 days. The experiment was repeated three times.

[0115] II. Experimental Results:

[0116] 1. Effects of various parameters on 7-β-xylase activity: Experimental results are as follows Figure 10 As shown, Figure 10In this text, A represents xylan concentration; B represents fermentation time; C represents fermentation temperature; and D represents liquid volume. Figure 10 It can be seen that within the xylan concentration range of 0.2% to 0.6%, 7-β-xyl enzyme activity increases with increasing xylan concentration, reaching its maximum at 0.6% xylan concentration; within the xylan concentration range of 0.6% to 1.2%, 7-β-xyl enzyme activity decreases with increasing xylan concentration; therefore, the xylan concentration levels for the orthogonal experiment are 0.4%, 0.6%, and 0.8% (…). Figure 10 A).

[0117] Figure 10 B shows that within the fermentation time range of 2d to 3d, the activity of 7-β-xyl enzyme increases with increasing fermentation time, reaching its maximum at 3d fermentation time; within the fermentation time range of 3d to 7d, the activity of 7-β-xyl enzyme decreases with increasing fermentation time; therefore, the fermentation time levels for the orthogonal experiment are 2d, 3d, and 4d.

[0118] Figure 10 C shows that within the fermentation temperature range of 22℃ to 28℃, the activity of 7-β-xyl enzyme increases with increasing fermentation temperature, reaching its maximum at 28℃; within the fermentation temperature range of 28℃ to 37℃, the activity of 7-β-xyl enzyme decreases with increasing fermentation temperature; therefore, the fermentation temperature levels for the orthogonal experiment are 25℃, 28℃, and 31℃.

[0119] Figure 10 D showed that within the liquid volume range of 100 mL to 350 mL, the activity of 7-β-xyl enzyme increased with increasing liquid volume. Therefore, the liquid volume levels for the orthogonal experiment were 250 mL, 300 mL, and 350 mL.

[0120] 2. Enzyme activity assay: Based on the results of single-factor experiments, orthogonal experiments were conducted to optimize four parameters: xylan concentration, fermentation time, fermentation temperature, and liquid volume. Figure 11 As shown in Table 5, experiment number 9 exhibited the highest enzyme activity, reaching 0.623 ± 0.061 U / mL, representing a significant improvement compared to the enzyme activity before optimization. Analysis of the range (R) revealed that the influence of the four factors on the production of 7-β-xyl by strain WA11-1-1, from strongest to weakest, was: xylan concentration, fermentation time, fermentation temperature, and liquid volume. Analysis of the mean (K) determined the optimal combination of the four factors to be: xylan concentration 0.8%, fermentation time 4 days, fermentation temperature 28℃, and liquid volume 250 mL. These are the optimal fermentation conditions for the production of 7-β-xyl by strain WA11-1-1. Detailed analysis results are shown in Table 6. Figure 12 .

[0121] Table 5 Orthogonal Experiment L9(4)(3) Table

[0122]

[0123] Table 6. Variance Analysis of Orthogonal Experiments

[0124]

[0125] 3. HPLC chromatograms of 10-DAT before and after optimization of fermentation conditions for 7-β-xyl production by strain WA11-1-1 are shown in [reference needed]. Figure 13 .Depend on Figure 13 It can be seen that the 10-DAT conversion rate of strain WA11-1-1 before and after optimization of fermentation conditions for producing 7-β-xyl was determined as follows: Under the optimal fermentation combination conditions for producing 7-β-xyl by strain WA11-1-1 (xylan concentration 0.8%, fermentation time 4d, fermentation temperature 28℃, liquid volume 250mL), the 10-DAT conversion rate reached 40.72±1.08%, which was 1.85 times higher than before.

[0126] Finally, it should be noted that the above embodiments are merely illustrative of the principles, performance, and effects of the present invention, and are not intended to limit the present invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A strain of Streptomyces louchei ( Streptomyces rochei WA11-1-1, characterized in that, The Streptomyces Wheterii WA11-1-1 was isolated from the American cockroach and is deposited at the Guangdong Provincial Microbial Culture Collection Center with accession number GDMCC NO.61629; the Streptomyces Wheterii WA11-1-1 is used to prepare 10-deacetylpaclitaxel.

2. The Streptomyces loucherei WA11-1-1 as described in claim 1, characterized in that, The 10-deacetylated paclitaxel was prepared by converting 7-xylose-10-deacetylated paclitaxel during fermentation by Streptomyces WA11-1-1.

3. The Streptomyces loucherei WA11-1-1 as described in claim 2, characterized in that, The fermentation conditions were: xylan concentration 0.8%, fermentation time 4 days, fermentation temperature 28℃, and liquid volume 250mL.

4. The Streptomyces loucherei WA11-1-1 as described in claim 1, characterized in that, The 10-deacetylated paclitaxel is used to prepare paclitaxel.

5. The Streptomyces WA11-1-1 as described in claim 4, characterized in that, The paclitaxel is used to prepare a drug composition for treating tumors.

6. The Streptomyces loucherei WA11-1-1 as described in claim 5, characterized in that, The tumors include ovarian cancer, breast cancer, and non-small cell lung cancer.

7. The Streptomyces loucherei WA11-1-1 as described in claim 6, characterized in that, The pharmaceutical composition also includes physiological saline and other pharmaceutically acceptable carriers.

8. The use of Streptomyces WA11-1-1 as described in claim 1 in the production of 7-β-xyl enzyme.

9. The application as described in claim 8, characterized in that, The reaction temperature of the 7-β-xyl enzyme is 35℃, and the reaction pH is 6; the storage temperature is 20℃~30℃, and the pH is 7.

Images