Exploitation of protocols for assaying cellulose synthase activity by using light-scattering and spectroscopy

2014 Fiscal Year Final Research Report

• Project/Area Number: 25650033
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Multi-year Fund
• Research Field: Functional biochemistry
• Research Institution: Kyoto University
• Principal Investigator: IMAI Tomoya 京都大学, 生存圏研究所, 准教授 (90509142)
• Project Period (FY): 2013-04-01 – 2015-03-31
• Keywords: 酵素反応速度論 / 非放射性同位元素 / セルロース合成酵素 / 光散乱 / 濁度測定 / セルロース大腸菌合成系 / アンスロン硫酸法 / Direct Red-23

Outline of Final Research Achievements

Some methods to estimate the enzymatic activity of cellulose synthase, without using radioisotopes, were tested with (i) in vitro system using crude native enzyme and (ii) recombinant cellulose synthase reconstituted in E. coli. For the system (i), the turbidity, which is due to light-scattering by aggregation of cellulose product, was used for seeing the progress of reaction and a kinetic analysis was successfully made. For the system (ii), a classical protocol of anthrone-sulfuric acid method was improved for the adequate quantification of cellulose, which quantitatively demonstrated the production of cellulose by recombinant cellulose synthase in the reconstitution system with E. coli.

Free Research Field

膜タンパク質