Heat-resisting glycosidase gene and soluble expression and application method thereof
A glycosidase and heat-resistant technology, applied in the fields of genetic engineering and biomass utilization, can solve the problems of inability to provide high-efficiency and low-cost biological enzymes
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Embodiment 1
[0045] Embodiment 1: Construction of recombinant plasmid pET-28a-bgl
[0046] 1.1 Cultivation of Thermotoga thermarum DSM 5069
[0047] T. thermarum DSM 5069 was purchased from DSMZ Culture Collection Center (www.dsmz.de) with the number 5069. The medium formula is: 5g / L soluble starch, 1g / L yeast powder, 1.5g / L KH 2 PO 4 , 4.2g / L Na 2 HPO 4 x 12H 2 O, 3.4g / L NaCl, 1g / L MgSO 4 x 7H 2 O, 0.76g / L EDTA, 1mL / L trace elements, 0.5g / L Na 2 S·9H 2 O, 0.5g / L Cysteine HCl, 1mg / L resazurin, adjust the pH to 7.0, boil with nitrogen, remove oxygen, put the culture medium into an anaerobic bottle for sterilization under anaerobic conditions. Trace element (1000×) formula: FeCl 3 2.0g / L;H 3 BO 3 0.05g / L; ZnCl 2 0.05g / L; CuCl 2 2H 2 O 0.03g / L; MnCl 2 4H 2 O 0.05g / L; (NH 4 ) 2 MoO 4 0.05g / L; AlKSO 4 2H 2 O0.05g / L. ) was inoculated with a syringe according to 0.5% inoculum volume, cultured statically at 82° C. for 24 hours, and the cells were collected.
[0048] 1.2 G...
Embodiment 2
[0063] Example 2: Soluble expression and purification of recombinant extremely thermostable glycosidase
[0064] 2.1 Soluble expression of recombinant extreme thermostable glycosidase
[0065] The recombinant plasmid pET-28a-bgl was transformed into Escherichia coli JM109 (DE3) host bacteria (Novagen), on LB plates containing kanamycin (50 μg / mL) (LB medium: tryptone 10g / L, yeast extract 5g / L, NaCl 5g / L, agar 15g / L) after culturing overnight at 37°C, pick the transformants into 200mL LB medium (50μg / mL kanamycin) at 37°C, shake at 200rpm until OD 600 When the temperature is 0.6, add the final concentration of 0-0.5mM isopropyl β-D-thiogalactopyranoside (IPTG) inducer, induce culture at 37°C, 30°C or 25°C for 6h, and use a high-speed refrigerated centrifuge Centrifuge the culture solution at 13,000rpm for 15min at 4°C to collect the bacteria, remove the supernatant and add sterile water, break the cells by ultrasonic, and collect the supernatant and precipitate by centrifugat...
Embodiment 3
[0083] Embodiment 3: Qualitative analysis of recombinant extremely thermostable glycosidase
[0084] 3.1 Enzyme activity assay
[0085] Add 100 μL 100 mmol / L citric acid-disodium hydrogen phosphate buffer (pH 6.0) and appropriate amount of water to 20 μL 10 mmol / L ginsenoside Rb1 in the reaction system of 200 μL, first incubate at 90 °C for 5 min, then add 10 μL enzyme solution (diluted to Appropriate multiple) for 10 minutes. Evaporate to dryness immediately after the reaction, add methanol, and measure the amount of Rd by TLC or HPLC. The conditions of TLC and HPLC were performed according to the literature (The Journal of Micobiology, 2005, 43:456-462). Enzyme activity unit (U) is defined as: under the assay conditions, the amount of enzyme used to produce 1 μmol ginsenoside Rd per minute is 1 enzyme activity unit.
[0086] 3.2 Determination of the optimum reaction temperature
[0087] In the range of 50-100°C, the enzyme activity was measured every 5°C. The buffer is ...
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