Juneja L R, Kazuoka T, Yamane T, Shimizu S
Department of Food Science and Technology, School of Agriculture, Nagoya University, Japan.
Biochim Biophys Acta. 1988 Jun 15;960(3):334-41. doi: 10.1016/0005-2760(88)90041-0.
Transphosphatidylation from phosphatidylcholine (PC) to phosphatidylethanolamine (PE) was conducted by seven phospholipase D preparations from different sources, one of which was of cabbage origin and other six of Streptomyces origin. The reactions were carried out at 30 degrees C using mixture of ethyl acetate containing PC and buffer containing ethanolamine and phospholipase D. To obtain the activity ratio of transphosphatidylation and hydrolysis at various ratios of ethanolamine and water concentrations, the apparent rate constant ratios of transphosphatidylation and hydrolysis, (k'3/k3)app, were calculated, keeping the concentration of PC constant (17.8 mM). Among the seven enzymes examined, five showed good transphosphatidylation, in which 100% of PC could be converted to PE at ethanolamine concentrations ranging from 0.3 to 1.0 M, while two showed poor transphosphatidylation activity. At higher ethanolamine concentrations, the reaction rate was decreased due to substrate inhibition. Hydrolytic reactions were conducted at 30 degrees C with respective enzymes by using mixture of ethyl acetate containing PC or PE and buffer containing phospholipase D. The ratio of (kcat/Km)PE/(kcat/Km)PC was calculated to determine the substrate specificity of various phospholipase D enzymes. The values of (k'3/k3)app varied with the origin of the enzymes, whereas the values of (kcat/Km)PE/(kcat/Km)PC were not so different. The results obtained show that (k'3/k3)app is a good parameter to select an enzyme and that the timing of stopping the reaction is also important to avoid the hydrolysis of the product.
采用来自不同来源的7种磷脂酶D制剂进行了从磷脂酰胆碱（PC）到磷脂酰乙醇胺（PE）的转磷脂酰基化反应，其中一种来自卷心菜，另外6种来自链霉菌。反应在30℃下进行，使用含有PC的乙酸乙酯混合物以及含有乙醇胺和磷脂酶D的缓冲液。为了获得不同乙醇胺与水浓度比例下转磷脂酰基化和水解的活性比，在PC浓度保持恒定（17.8 mM）的情况下，计算了转磷脂酰基化和水解的表观速率常数比（k'3/k3）app。在所检测的7种酶中，有5种表现出良好的转磷脂酰基化能力，在乙醇胺浓度为0.3至1.0 M的范围内，100%的PC可转化为PE，而另外2种表现出较差的转磷脂酰基化活性。在较高的乙醇胺浓度下，由于底物抑制，反应速率降低。水解反应在30℃下分别使用含有PC或PE的乙酸乙酯混合物以及含有磷脂酶D的缓冲液与相应的酶进行。计算（kcat/Km）PE/（kcat/Km）PC的比值以确定各种磷脂酶D酶的底物特异性。（k'3/k3）app的值随酶的来源而变化，而（kcat/Km）PE/（kcat/Km）PC的值差异不大。所得结果表明，（k'3/k3）app是选择酶的一个良好参数，并且停止反应的时机对于避免产物水解也很重要。
