Abstract:To obtain strains producing thermostable α-amylase，this study isolated bacteria with thermostable α-amylase producing potency from the samples derived from heat oil immersed soil further identified the strains by morphological，physiological and biochemical analysis as well as 16S rRNA sequencing.A set of techniques were established at pilot-scale for upstream fermentation and downstream enzyme isolation by ammonium sulfate precipitation.The thermal stability of the purified thermostable amylase extract was evaluated.Moreover，the gene encoding the thermostable enzyme was cloned and successfully expressed in Escherichia coli BL21 by heterologous expression，while the amylase was analyzed by bioinformatics methods.The results showed that a strain of amylase-producing Geobacillus thermodenitrificans Y62 was finally obtained after screening.The enzymatic activity was 112.7 U/mL after 48 h of pilot amplification，while the crude enzyme recovery rate was 90.2%.This amylase had excellent thermostability，as it could maintain 37.8% of the activity at a high temperature up to 100 ℃.The enzyme was detected in the form of inclusion bodies in heterologous expression and further solubilized by adjusting the induction conditions.The purified product was identified as α-amylase by mass spectrometry.Bioinformatics analysis showed that the thermostable α-amylase was composed of 511 amino acids with a molecular weight of 59.86 kDa.It contained two transmembrane regions，three catalytic sites and two Ca2+-binding sites，which were similar to the previously reported α-amylase GTA.