TY  - JOUR
TI  - Extracellular Production and Purification of the β-glucanase in Pichia pastoris Expression System
AB  - In this study, Rhizomucor miehei -1,3-1,4-glucanase gene was expressed under the regulation of AOX1 promoter in the Pichia pastoris expression system, and the clone providing the highest production level was determined. The codon-optimized gene was ligated into expression vector pPICZA and transferred into competent P. pastoris KM71H cells. Thirty transformants were selected from plates containing different concentrations of zeocin and cultured in test tubes for protein production. Glucanase enzyme activities in supernatant samples were measured and ten clones showing the highest glucanase activity were determined. The proteins in supernatant samples were also analyzed by SDS-PAGE and the glucanase enzyme was observed in 2 bands, approximately 34 and 38 kDa. Protein production was performed for 72 hours in 200 mL induction medium (BMMY) with the clone providing the highest glucanase enzyme production and the recombinant glucanase enzyme was purified by Ni-NTA affinity chromatography. After purification, it was determined that the analyzed clone reached 79.6 mg/L glucanase production level. SDS-PAGE analysis of samples from each step of the purification procedure showed that the two protein bands also observed in the supernatant samples represent glucanase enzyme.
AU  - KARAOĞLAN, Mert
AU  - Erden Karaoğlan, Fidan
DO  - 10.18185/erzifbed.972627
PY  - 2021
JO  - Erzincan Üniversitesi Fen Bilimleri Enstitüsü Dergisi
VL  - 14
IS  - 2
SN  - 1307-9085
SP  - 620
EP  - 630
DB  - TRDizin
UR  - http://search/yayin/detay/479711
ER  -