Gene Cloning And Purification Of Thermo-Stable Acid α-Amylase From Bacillales Bacterium SF-8

As an important industrial enzymeα-amylases have diverse applications in a wide variety of industries such as starch fine processing, fermentation, papermaking and textile. With industrial production and improve the demand ofα-amylases, thus research onα-amylases is particularly significant in both industrial production and scientific investigation.We found strain SF-8 was Gram positive and sporulated in the later phase by series of experiments.It began to produceα-amylases when fermentated after 16 hours, the yield ofα-amylases increased rapidly after 24 hours until to the stable phase .In this research genomic library construction and PCR amplification were used to get the thermostable acid amylase gene.Gene fragments were ligated to plasmid pUC19,then introduced into E.coli DH5αby electroporation. Though got about 5000 transformants finally, we still can’t get the gene sequence.During the progress of purification, the crude enzyme produced by the strain SF-8 was extracted by ammonium sulfate precipitation, purified by Macro-Prep DEAE chromatography, and Sephacryl S-100 gel filtration. The recovery rate and purification value of this enzyme was 36.42% and 7.27 respectively. Molecular weight of the enzyme was estimated to be 58-60 kDa by native staining.Studies on some basic properties of this enzyme shows that the optimal temperature and pH of enzyme degredation were respectively 60℃and 5.0. 66% and 72% enzyme activity was retained when stored at 60℃, pH4.5, it’s fit for industrial application. The presence of Ca²⁺ didn’t promote its activity, the enzyme activity was activated by Mg2+ and Mn²⁺, while inhibited by Fe²⁺ and Cu²⁺ .