Alkalinealpha- amylase, coding gene and uses thereof
An amylase, alkaline technology, applied in genetic engineering, plant genetic improvement, enzymes, etc., to achieve high-efficiency hydrolysis
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Embodiment 1
[0026] Extraction of Total DNA from Alkaliphiles N10 (CGMCC NO.0463)
[0027] Adopt the alkaliphilic bacteria N10 isolated from Chahan Naojian Lake, Inner Mongolia, China, get 20 grams of fresh wet thallus, suspend in 10 milliliters of 50mM Tris buffer (pH8.0), add a small amount of lysozyme and 8 milliliters of 0.25m MEDTA ( pH8.0), mix well and place at 37°C for 20min, then add 2 ml of 10% SDS, place at 55°C for 5min, extract with equal volume of phenol and chloroform respectively, take the last supernatant solution, add 2 times Volume ethanol, recover DNA, wash with 70% and absolute ethanol respectively, dissolve the precipitate in 0.5 ml TE buffer solution (pH8.0, 10 mM Tris, 1 mM EDTA), add 10 mg / ml RNase 3 μl, incubate at 37 ° C for 1 hour, use etc. The volume of phenol and chloroform were extracted once, and the supernatant was added with 2 times the volume of ethanol to recover the DNA, washed with 70% and absolute ethanol respectively, dried in vacuum, and dissolved i...
Embodiment 2
[0034] Purification and Characterization of Recombinant Amylases
[0035] The cells of the recombinant bacterium E.coli JM109AMY2 were suspended in 10 mM glycine buffer (pH 10), the cells were disrupted by ultrasonic waves, and the centrifuged supernatant was the crude enzyme solution of the recombinant α-amylase. The supernatant enzyme solution was purified by DEAE-Sephadex A-25 ion exchange column chromatography, hydroxyapatite adsorption column chromatography and PAGE preparative electrophoresis, and the obtained enzyme preparation showed a band on SDS-PAGE. The basic properties of this recombinant alpha-amylase were determined using standard methods known in protein chemistry. The molecular weight of the recombinase measured by SDS-PAGE is 60000 Daltons, which is similar to the theoretically calculated molecular weight (66000 Daltons); the isoelectric point pI of the recombinase measured by PAGEIEF is 4.9; The optimum pH is 10 and the optimum temperature is 50°C. It is c...
Embodiment 3
[0037] Hydrolysis of starch by recombinant amylase for oligosaccharide conversion
[0038] 0.45L of 2% soluble starch solution (prepared with pH 10, 0.1M glycine-NaOH buffer solution) was placed in a 1.0L reactor, 0.05L of enzyme solution was added, and the temperature was raised to 40°C for 12 hours. The reaction mixture was desalted through Sephadex G-10 column, concentrated, and chromatographically on Xinhua filter paper, the results showed that the resulting product was glucose, maltose and other oligosaccharides ( image 3 ).
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