Expression and characterization of Bacillus subtilis PY22 α-amylase in Pichia pastoris

Pichia pastoris is a methanol utilizing yeast which does not naturally produce starch degrading enzymes. In this study, the gene encoding the α-amylase enzyme in Bacillus subtilis PY22 was amplified by PCR, sequenced and cloned into P. pastoris KM71H host strain using the vector pPICZαA allowing methanol induced expression and secretion of the protein. Recombinant expression resulted in high levels of extracellular amylase production, as high as 22 mg/L in the shake flask culture supernatant. Clones containing various copy numbers of the gene were screened for α-amylase production and two-copy clone was determined to be the best producer at shake flask conditions. The clone capable of the highest production was selected for further study involving the small-scale production and partial purification of the recombinant enzyme. The partially purified enzyme showed the highest activity at 60 °C and pH 7, retaining 78% activity when kept at this temperature and pH for 1 h. The presence of Ca2+ ions in the reaction medium resulted in a 41% increase in the amylase activity.