Thermophilic alkali alpha amylase and its coding gene
An amylase, alkaline technology, applied in genetic engineering, plant genetic improvement, enzymes, etc., can solve problems such as production problems, and achieve the effect of high thermal stability
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Embodiment 1
[0036] 1. Extraction of total DNA from Bacillus thermodenitrophilus NG80-2 (CGMCC No.1228)
[0037] Studies have shown that the α-amylase produced by Geobacillus thermodenitrificans NG80-2 is particularly suitable for hydrolyzing starch polysaccharide molecules under high temperature and alkaline conditions. Therefore, in this embodiment, the thermophilic denitrophilic Bacillus NG80-2 obtained from the oil well formation water separation of Guan 69-8 block, Dagang Oilfield, Tianjin, China (this bacterial strain has been preserved in the General Microorganism Center of China Committee for the Preservation of Microbial Cultures) , its preservation number is CGMCC No.1228), take 3ml of its overnight cultivated fresh culture bacterial liquid, centrifuge and collect the bacterial cells, the bacterial cells are suspended in 250 microliters of 50mM Tris buffer (pH8.0), add 10 microliters of 0.4 MEDTA (pH8.0), mix well and keep warm at 37°C for 20min, then add 30μl 20mg / ml lysozyme, m...
Embodiment 2
[0047] Embodiment two Purification and Characterization of Recombinant α-Amylase
[0048] Insert the above-mentioned recombinant strain E.Coli BL21AMY monoclonal into 20ml of LB medium containing 50μg / ml Kan, culture at 37°C, 180rpm / min for 12 hours, and then insert the culture at 1% (V / V) inoculum 200ml LB medium containing 50μg / ml Kan (total 10 shake flasks), 37°C, 220rpm / min culture A600 is 0.6, add IPTG to a final concentration of 0.2mM, 45°C, 150rpm / min induction for 4 hours. The cells were collected by centrifugation, suspended in 20 mM sodium phosphate (pH 8.0) buffer, and the cells were disrupted by ultrasonic waves. The centrifuged supernatant was the crude extract of the recombinant α-amylase. The supernatant was purified by chelating Sepharose nickel affinity column chromatography, 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....
Embodiment 3
[0049] Embodiment Three Determination of starch hydrolysis activity of recombinant thermophilic alkaline α-amylase
[0050] A 20ml screw cap test tube contains 4ml of the following reaction system: 1ml of 0.1% starch solution, 20mM Na 2 HPO 4 (pH8.0), 1 mg of the purified enzyme preparation of the recombinant thermophilic alkaline α-amylase prepared in the above-mentioned Example 2. React at 60°C for 10 minutes. Add 1ml 0.1M H 2 SO 4 Stop the reaction. Take 2ml of the total reaction solution and add 0.5ml 0.5% KI-I 2 Solution, mix well, and measure the UV spectrophotometric value at 620nm by absorptiometry.
[0051] (1) Optimum enzyme activity temperature
[0052] Under standard reaction conditions, measure the activity of above-mentioned recombinant thermophilic alkaline α-amylase in the scope of 25-80 ℃ (such as figure 2 shown), indicating that the optimum temperature for maintaining the enzyme activity was 60°C.
[0053] (2) Optimum pH value
[0054] Under stan...
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