Expression and Characterization of Rice Sucrose Synthase RSuS2 in Pichia pastoris

• Main Authors: Tz-Li Chen, 陳姿利
• Other Authors: 王愛玉
• Format: Others
• Language: zh-TW
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/64488981166206675297

碩士 === 國立臺灣大學 === 微生物與生化學研究所 === 93 === Sucrose synthase (SuS) catalyzes the reversible conversion of sucrose and UDP into fructose and UDPG. There are three Sus isogenes in rice (Oryza sativa) (RSus1, RSus2 and RSus3). Among these three isogenes, RSus1 and RSus2 are ubiquitously expressed in various tissues, while RSus3 is expressed predominantly in rice seed. In this research, recombinant RSuS2 expressed and purified from Pichia pastoris was used to study on the enzyme function of RSuS2. The RSus2 cDNA was introduced into yeast Pichia pastoris for expression. The transformed cells accumulated high level of RSuS2 protein and SuS activity after 60 hours of methanol induction. The recombinant RSuS2 was purified and characterized on its general properties. The Michaelis constant (Km) of the recombinant RSuS2 for sucrose, UDP, fructose, and UDPG were 104.8 mM, 0.099 mM, 6.65 mM, and 0.146 mM, respectively. The synthetic activity could be stimulated by Mg2+ and Ca2+. Moreover, the cleavage activity of RSuS2 was inhibited by fructose 6-phosphate, but was activated by fructose 2,6 bisphosphate. On the other hand, membrane-bound RSuS was observed in the crude extract of recombinant RSuS2. The cleavage activity of purified RSuS2 was activated by Triton X-100, while the synthetic activity was activated by phospholipids.