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RESEARCH STUDY ON THE DISTRIBUTION OF D-AMINO ACIDS AND D-AMINO ACID RACEMASE IN MICROALGAE

微藻中D-氨基酸和D-氨基酸消旋酶的分布研究

基本信息

批准号：
12660189
负责人：
NAGAHISA Eizo
金额：
$ 1.98万
依托单位：
Kitasato University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2002
项目状态：
已结题

项目摘要

Distribution of free and combined D-amino acids in marine diatoms and freshwater microalgae was measured. And variation in D-amino acids content under various culture conditions of diatom, Asterionella sp. was examined. Furthermore, the origin of D-amino acids in microalgae was researched.1. The presence of free D-aspartate and D-alanine was verified in diatoms, Asterionella sp., Thalassiola sp., Nitzschia sp., and Pseudo-nitzschia sp. D-Aspartate was also found in fresh water microalgae, Botrydiopsos alpina, Chlorella pyeniodos, C.vulgaris and Scenedesumus obliquus, but D-alanine was not detected in these species.2. The content of D-alanine varied by growth phase : D-Alanine content of Asterionella sp. increased in stationary phase and decreased in extinction phase.3. Combined form of D-aspartate, D-glutamate and D-alanine were detected in the residue of diatoms after extracting with 80% ethanol, but D-alanine was not detected in freshwater microalgae.4. Alanine racemase activity was detected in the marine diatom, Thalassiosira sp. which was cultured germ-free. Optimal pH of this enzyme was extremely high, about 9.5 and the Km value (substrate L-alanine) was 19.7 mM. The enzyme did not require pyridoxal 5'-phosphate (PLP) as a cofactor in the reaction. These results suggest that alanine racemase from Thalassiosira sp. bindes PLP more tightly as a cofactor compared with bacterial enzyme.

测定了游离态和结合态D-氨基酸在海洋硅藻和淡水微藻中的分布。并研究了硅藻Asterionella sp.在不同培养条件下D-氨基酸含量的变化。并对微藻中D-氨基酸的来源进行了研究.游离D-天冬氨酸和D-丙氨酸的存在在硅藻、星状藻属、Thalassiola sp.，菱形藻，在淡水微藻、高山葡萄藻、小球藻、普通小球藻和栅藻中也检测到D-天冬氨酸，但未检测到D-丙氨酸. D-丙氨酸的含量随生长期的不同而变化：在稳定期，小球藻的D-丙氨酸含量增加，在绝灭期，小球藻的D-丙氨酸含量降低. 80%乙醇提取后的硅藻残渣中检测到D-天冬氨酸、D-谷氨酸和D-丙氨酸的结合形式，而淡水微藻中未检测到D-丙氨酸.在无菌培养的海洋硅藻海链藻中检测到丙氨酸消旋酶活性。该酶的最适pH极高，约为9.5，Km值（底物L-丙氨酸）为19.7 mM。该酶在反应中不需要吡哆醛5 '-磷酸（PLP）作为辅因子。这些结果表明，与细菌酶相比，来自海链藻的丙氨酸消旋酶作为辅因子更紧密地结合PLP。