PURIFICATION AND CHARACTERIZATION OF HYPERTHERMOSTABLE RECOMBINANT Pyrococcus furiosus ?-AMYLASE EXPRESSED IN Pichia pastoris KM71

• Main Author: Delfahedah, Yulia
• Format: Theses
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/38274
• Institution: Institut Teknologi Bandung

?-Amylase (EC 3.2.1.1) hydrolyzes ?-1,4-glycosidic bond in starch producing linear and branch oligosaccharides. In starch processing industry, thermostable ?- amylase was generally used to convert starch into oligosaccharides. Hyperthermostable Pyrococcus furiosus ?-amylase (PFA) has an activity at high temperature. The aims of this study were to purify and characterize the hyperthermostable recombinant PFA expressed in Pichia pastoris KM71. This research was conducted into several stages, namely transformation of P. pastoris KM71 with pPICZ?A-PFA, optimization of incubation time and concentration of methanol for production of PFA, purification of PFA by Co-NTA affinit y chromatography under denaturing condition, and characterization of purified PFA. The recombinant plasmid pPICZ?A-PFA was integrated into chromosome of P. pastoris KM71 via single cross-over homologous recombination. The integration of PFA gene in the P. pastoris KM71 chromosome was confirmed by PCR colony as indicated by the presence of 1.7 kb DNA fragment on agarose gel analysis. Furthermore, the P. pastoris KM71 transformants were subjected to grow at high concentration of zeocin (2000 mg/mL) for selection of high copy number of integration. In this construct, the PFA gene was regulated by AOX1 promoter and its coding sequence was fused with an oligonucleotide for pre-pro-? factor signal peptide. Therefore, the expression of PFA required methanol as an inducer and the PFA will be secreted into culture medium. The expression of PFA in the P. pastoris KM71 transformants which can grow on high concentration of zeocin was induced by addition of methanol at 30 °C. It was found that the optimum methanol concentration was 3 % with incubation period of 120 hours. SDS-PAGE analysis and zymogram analysis showed that the molecular weight of PFA was ~56 kDa. The crude enzyme extract was concentrated by ultrafiltration and then purified by Co-NTA affinity chromatography under denatured condition. The PFA can degrade soluble starch with activity of 1.8x105 U/mg as determined b y dinitrosalycilic acid (DNS) method. The PFA has been purified with a purification level of 63-fold and purified PFA has optimum pH of 5.0 and temperature of 90 oC.