Akademik Veri Yönetim Sistemi
JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC, cilt.64, sa.3-4, ss.129-134, 2010 (SCI-Expanded)
Pichia pastoris is a methanol utilizing yeast which does not naturally produce starch degrading enzymes. In this study, the gene encoding the alpha-amylase enzyme in Bacillus subtilis PY22 was amplified by PCR, sequenced and cloned into P. pastoris KM71H host strain using the vector pPICZ alpha A 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 et-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 degrees 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. (C) 2009 Elsevier B.V. All rights reserved.