Alpha-L-rhamnosidase and preparing method and applications thereof
A rhamnosidase and nucleotide technology, which is applied in the field of genetic engineering technology and biomedicine, can solve the problems of reducing the yield of the target product, low reaction temperature, high thermal stability, etc., and achieve high expression and optimal reaction temperature High, excellent thermal stability
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Embodiment 1
[0033] Example 1: Acquisition of α-rhamnosidase gene of the present invention and construction of recombinant plasmid pET-TPERha
[0034] 1.1 Thermotoga petrophila Culture of DSM 13995
[0035] Thermotoga petrophila DSM 13995 was purchased from the DSMZ Culture Collection Center (www.dsmz.de) with the number 13995, and its medium formula was: 10 g / L starch, 5 g / L tryptone, 3 g / L yeast extract, 5 g / L L meat extract, 10 g / L 2-morpholineethanesulfonic acid, 10 mg / L ferric sulfate heptahydrate, 1 mg / L resazurin, and adjust the pH to 7.2. Inoculate with a syringe according to the inoculum volume of 0.5 wt.% medium, culture statically at 85°C for 24 h, and collect the cells.
[0036] 1.2 Extraction of genomic DNA
[0037] (1) After statically culturing Thermotoga petrophila DSM 13995 for about 24 hours, take 30 mL of the bacterial liquid and centrifuge at 4,000 g for 10 min to collect the cells.
[0038] (2) Resuspend the cells in 9.5 mL TE buffer, add 0.5 mL 10 wt.% sodi...
Embodiment 2
[0053] Example 2: Preparation of α-rhamnosidase of the present invention
[0054] The recombinant plasmid pET-TPERha was transformed into Escherichia coli JM109(DE3) host bacteria (purchased from Novagen), and placed on an LB plate containing kanamycin (50 μg / mL) (LB medium: tryptone 10 g / L, yeast Extract 5 g / L, NaCl 5 g / L, agar 15 g / L) after culturing overnight at 37°C, pick the transformants into 200 mL of LB medium (50 μg / mL kanamycin) at 37°C, Shake culture at 200 rpm until OD600 is 0.6, add a final concentration of 0.5 mM isopropyl β-D-thiogalactopyranoside (IPTG) inducer, culture at 30°C for 6 h, and use a high-speed refrigerated centrifuge Centrifuge at 13,000 rpm for 15 min at 4°C to collect the cells.
[0055] Since the recombinant plasmid pET-TPERha contains a His-tag tag, it was purified by His·Bind Purification Kit (purchased from Novagen) to obtain a purified recombinant enzyme. Specific operation process:
[0056] A. Processing of samples
[0057] (1) Resus...
Embodiment 3
[0072] Example 3: Qualitative determination of α-rhamnosidase described in the present invention
[0073] 1. Determination method of enzyme activity
[0074] Reaction system 100 μL, 5 μL 20 mmol / L p-nitrophenyl-α-L-rhamnoside ( p NPR) was added 85 μL 100 mmol / L citric acid-disodium hydrogen phosphate buffer (pH 6.0), incubated at 90°C for 3 min, then added 10 μL enzyme solution (diluted to an appropriate multiple) for 10 min, and the After coloring, 600 μL of 1 mol / L sodium carbonate solution was added to terminate the reaction. Absorbance was measured at 405 nm. Enzyme activity unit (U) is defined as: under assay conditions, produce 1 μmol per minute p - The amount of enzyme required for nitrophenol is 1 enzyme activity unit.
[0075] 2. Determination of the optimum reaction pH
[0076] Under the conditions of different pH (3.5-7.5, 100 mmol / L citric acid-disodium hydrogen phosphate buffer), the enzyme activity was measured at 90°C, and the results were as follows fi...
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