| Determination of Fructokinase Activity from Zobellia galactanivorans
|
|
Author:
Date:
2015-11-05
[Abstract] Mannitol is a polyol that occurs in a wide range of living organisms, where it fulfills different physiological roles. Several pathways have been described for the metabolism of mannitol by bacteria, including the phosphoenolpyruvate-dependent phosphotransferase system (PST) and a M2DH-based catabolic pathway. The latter involves two enzymes, a mannitol-2-dehydrogenase (EC 1.1.1.67) and a fructokinase (EC 2.7.1.4), and has been identified in different bacteria, e.g.,, the marine Bacteroidetes Zobellia galactanivorans (Zg) which had recently gained interest to study the degradation of macroalgal polysaccharides. This protocol describes the biochemical characterization of a recombinant fructokinase (FK) of Zobellia galactanivorans. The ZgFK ...
[摘要] 甘露醇是在多种活生物体中发生的多元醇，其中它实现不同的生理作用。已经描述了用于由细菌代谢甘露醇的几种途径，包括磷酸烯醇丙酮酸依赖性磷酸转移酶系统（PST）和基于M2DH的分解代谢途径。后者涉及两种酶，甘露醇-2-脱氢酶（EC 1.1.1.67）和果糖激酶（EC 2.7.1.4），并且已经在不同的细菌中鉴定，例如海洋拟杆菌Zobellia galactanivorans （ Zg ），其最近获得了研究大分子多糖降解的兴趣。该方案描述了Zobellia 半乳糖厌氧杆菌的重组果糖激酶（FK）的生物化学表征。 < strong> [原理] 果糖激酶（FK）活性通过以下方法确定：果糖激酶（FK）活性通过使用ATP作为辅因子来催化果糖转化成果糖-6-磷酸。酶偶联测定（图1）。通过FK反应（即果糖磷酸化成果糖-6-磷酸（F6P））形成的ADP由将磷酸烯醇丙酮酸（PEP）转化为丙酮酸的丙酮酸激酶（PK）使用。然后，乳酸脱氢酶（LDH）使用NADH作为辅因子将丙酮酸转化为乳酸。通过跟随在340nm处的吸光度的降低来测量FK活性，其对应于NADH转化为NAD + 。
...
|
|
|
| Determination of Mannitol-2-dehydrogenase Activity
|
|
Author:
Date:
2015-11-05
[Abstract] Mannitol is a polyol that occurs in a wide range of living organisms, where it fulfills different physiological roles. Several pathways have been described for the metabolism of mannitol by bacteria, including the phosphoenolpyruvate-dependent phosphotransferase system (PST) and a M2DH-based catabolic pathway. The latter involves two enzymes, a mannitol-2-dehydrogenase (EC 1.1.1.67) and a fructokinase (EC 2.7.1.4), and has been identified in different bacteria, e.g., the marine Bacteroidetes Zobellia galactanivorans (Zg) which had recently gained interest to study the degradation of macroalgal polysaccharides. This protocol describes the biochemical characterization of a recombinant mannitol-2-dehydrogenase (M2DH) of Zobellia galactanivorans. The Zg ...
[摘要] 甘露醇是在多种活生物体中发生的多元醇，其中它实现不同的生理作用。已经描述了用于由细菌代谢甘露醇的几种途径，包括磷酸烯醇丙酮酸依赖性磷酸转移酶系统（PST）和基于M2DH的分解代谢途径。后者涉及两种酶，甘露醇-2-脱氢酶（EC 1.1.1.67）和果糖激酶（EC 2.7.1.4），并且已经在不同的细菌中鉴定，例如， 海洋拟杆菌（Zobellia galactanivorans）（ Zg ），最近对研究大分子多糖降解有兴趣。该方案描述了半乳聚糖微生物的重组甘露醇-2-脱氢酶（M2DH）的生物化学表征。使用NAD +辅助作为辅因子，M2DH酶催化甘露醇向果糖的可逆转化。 在两个方向上测定M2DH活性，即果糖还原和甘露醇氧化。
可逆反应：
D-甘露醇+ NAD + ↔D-果糖+ NADH + H +
|
|
|