Investigation of β-glucosidase from recombinant Pichia pastoris for the enhanced enzyme production and concentration

• Main Authors: Chien-Yuan Chen, 陳建元
• Other Authors: C.Will Chen
• Format: Others
• Language: zh-TW
• Published: 2018
• Online Access: http://ndltd.ncl.edu.tw/handle/kgp3h9

碩士 === 大同大學 === 生物工程學系(所) === 106 === Cellulase is the key enzyme for saccharization of lignocellulosic materials and the production of future biobased energy. In this study, we used brown sugar 30 g/L in medium, and sodium glutamate as nitrogen source in feed medium to enhance cell growth and to promote protein secretion and β-glucosidase production from a recombinant Pichia pastoris mut 92. After about 220 hrs, fermentation with brown sugar, the best result of the dry cell weight concentration, total protein and pNPG activity was 67.7 g/L, 0.49 mg/mL, and 4.89 pNPG U/mL, respectively, which were 1.17, 1.51 and 1.45 folds, respectively, compared to the condition without adding brown sugar. After centrifugation, a supernatant of broth was purified by 2-step crossflow filtration with 0.2 μm and 50 kDa pore diameter, and finally the enzyme’s pNPG activity was 6 times concentrated, 3 times purified and with 63.2 % recovery. 55 ℃ and pH 5.0 were the optimal condition for enzyme’s activity which were found from the optimal temperature and pH test. Under the optimal condition, 9:1 (volume ratio of cellulase : concentrated β-glucosidase) results in 69.8 FPU/mL , which was 1.22 times higher than the filter paper (FP) activity of pure cellulase in the same condition, thus, shows the potential of concentrated β-glucosidase for the future industrial application.