Expression of Thermobifida fusca thermostable raw starch digesting alpha-amylase in yeast and its application in raw starch hydrolysis

• Main Authors: Cheng-Yu Chen, 陳政佑
• Other Authors: Chao-Hsun Yang
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/93077159306651563238

碩士 === 靜宜大學 === 化粧品科學系碩士班 === 98 === A thermostable raw starch digesting α-amylase gene (tfa) from thermophilic actinomycete Thermobifida fusca NTU22 was expressed in Pichia pastoris X-33 with pGAPZαA and in Yarrowia lipolytica Po1g with pYLSC1. The extracellular amylase activity of Pichia pastoris transformant (pGAPZα-tfa) was 0.51 U/mL after cultivation in 100 mL YPD broth in 500-mL Hinton flasks and shaken (150 rpm) at 28℃ for 72 h. The amylase of P. pastoris transformant (pGAPZα-tfa) was purified 20.21 fold through ultrafiltration concentration and Ni Sepharose™ High Performance Column Chromatography. The overall yield of the purified amylase was 30.47%. The extracellular amylase activity of Y. lipolytica transformant (pYLSC1-tfa) was 0.73 U/mL after cultivation in 100 mL YPD broth in 500-mL Hinton flasks and shaken (200 rpm) at 28℃ for 60 h. The amylase of Y. lipolytica (pYLSC1-tfa) was purified 10.76 fold through ultrafiltration concentration, DEAE-Sepharose CL-6B chromatography and Sepharose CL-6B chromatography. The overall yield of the purified amylase was 21.87 %. Both purified amylases for P. pastoris (pGAPZα-tfa) and Y. lipolytica (pYLSC1-tfa) showed a single band at about 65 kDa by SDS-polyacrylamide gel electrophoresis. The purified amylase was application in raw sago starch hydrolysis. After 72-h treatment, the starch hydrolysis rate was 29%. The DPw (weight-average degree of polymerization) of raw sago starch obviously decreased from 830,945 to 237,092. The surface of starch granules was rough, and some granules displayed deep cavities.