Construction method and application of a kind of alkali-resistant alpha-amylase and its genetic engineering bacteria
A technology of genetically engineered bacteria and a construction method, which is applied in the construction field of alkali-resistant α-amylase and its genetically engineered bacteria, can solve the problems of low enzyme expression and difficulty in obtaining production strains that meet the needs of industrial production, and achieve good resistance The effect of alkaline properties
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Embodiment 1
[0060] Alkaline resistant α-amylase gene clone:
[0061] Specifically include:
[0062] A. Chromosomal DNA extraction of Bacillus sp. LS-04
[0063] Using the SDS extraction method, the specific steps are as follows:
[0064] Alkaline bacillus in liquid culture medium (every L culture medium contains: yeast extract 5g, peptone 10g, sodium chloride 5g, KH 2 PO 4 1g, using Na 2 CO 3 Adjust the pH to 9.5) after culturing for 12 hours, collect 3 mL of bacteria by centrifugation and use 0.25 mL of 20 mg / mL lysozyme solution to reselect the cells; place in a water bath at 37 °C for 30 min, then add 55 μL of 10% SDS (sodium dodecyl sulfate) The solution was incubated in a water bath at 65°C for 60 min; extracted twice with equal volume of phenol:chloroform and then once with equal volume of chloroform, the supernatant was taken and mixed with 2 times the volume of absolute ethanol, and then precipitated at room temperature for 20 min. The nucleic acid precipitate was collected...
Embodiment 2
[0074] Construction of Alkali-resistant α-amylase Genetic Engineering Bacteria:
[0075] Specifically include:
[0076] A. Cloning of alkaline α-amylase gene without signal peptide coding sequence
[0077] Get the recombinant plasmid pMD-Alamy04 that obtains in embodiment 1 as template, with primer AlamyF2 (5'-AC GAATTC GCAACTCCACAGAACGGTACGATGATGCA-3') and AlamyR2 (5'-TA GCGGCCGC TTATTGTTGTGTATAGATGGAA-3') was used as amplification primer to obtain amylase gene without signal peptide coding sequence. The PCR reaction system is the same as described in Example 1.
[0078] B, construction of alkaline-resistant α-amylase gene expression vector
[0079] Gel recovered the PCR product in the above step and digested it with restriction endonucleases EcoRI and NotI at 37°C for 1h, then heated at 65°C for 20min; The processed expression plasmid pPIC9k was connected to obtain the recombinant plasmid pPIC-Alamy04. The connection method and the screening method of positive clones...
Embodiment 3
[0083] Example 3 Recombinant Yeast Fermentation Production of Alkali-resistant α-Amylase and Research on Its Properties
[0084]Pick the recombinant yeast and streak culture on the starch detection plate for 40 hours, inoculate a single colony into 20 mL of liquid YEPD medium, culture at 30°C and 200 rpm for 20 hours, collect the cells by centrifugation, and transfer all the cells to 30 mL of BMGY medium , continue to cultivate at 30°C and 200rpm for 12 hours, then centrifuge to collect the bacteria and transfer them all to 40mL BMMY medium, culture at 30°C and 200rpm, add 0.2ml of methanol to the shaker flask every 12h, and ferment for 96h , the alkaline-resistant α-amylase activity in the fermentation broth reached 3120U / mL, and its activity unit was about 44 times that of the original strain, indicating that the recombinant alkaline-resistant α-amylase had achieved heterologous high-efficiency expression. The optimum temperature for the recombinant alkaline-resistant α-amyl...
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