A Novel Ca2+-Dependent Phospholipase D from Streptomyces tendae, Possessing Only Hydrolytic Activity
A Novel Ca2+-Dependent Phospholipase D from Streptomyces tendae, Possessing Only Hydrolytic Activity
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DOI：
10.1007/s12272-009-2017-0
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发表时间：
2009-10-01
影响因子：
6.7
通讯作者：
Yoo, Jin Cheol
中科院分区：
文献类型：
--
作者：
Mander, Poonam;Simkhada, Jaya Ram;Yoo, Jin Cheol
用SepharoseCL-6 B和DEAE-SepharoseCL-6 B柱层析法从Streptomycettendae中分离纯化了一种胞外磷脂酶D（PLDSt）。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳估计PLDSt的分子量约为43 kDa。最大活性是在pH 8和60摄氏度，和酶是稳定的或低于60摄氏度和pH 8和10之间，当测定后1.5和24小时，分别。该酶的活性对Ca 2+有绝对的需求，在2 mM CaCl 2时酶的活性最高。磷脂酰胆碱的Km和Vmax值分别为0.95 mM和810 μ mol min（-1）mg（-1）。更重要的是，PLDSt不能催化甘油、L-丝氨酸、肌醇和乙醇胺的转磷脂酰化，而这些已经被广泛用于评价活性。该结果有力地表明PLDSt不具有转磷脂酰化活性，从而使其成为第一个仅具有水解活性的链霉菌PLD。因此，PLDSt可能是一种新型的PLD酶。
An extracellular phospholipase D (PLDSt) was purified from Streptomyces tendae by two successive chromatographic steps on Sepharose CL-6B and DEAE-Sepharose CL-6B. Molecular weight of the PLDSt was estimated to be approximately 43 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Maximal activity was at pH 8 and 60 degrees C, and the enzyme was stable at or below 60 degrees C and between pH 8 and 10, when assayed after 1.5 and 24 h, respectively. The enzyme activity had an absolute requirement of Ca2+, and the maximum activity was at 2 mM CaCl2. The Km and Vmax values for phosphatidyl choline were 0.95 mM and 810 mu mol min(-1) mg(-1), respectively. More importantly, PLDSt could not catalyze transphosphatidylation of glycerol, L-serine, myo-inositol and ethanolamine, which have been extensively used to evaluate the activity. The result strongly suggests that PLDSt does not have the transphosphatidylation activity, thereby making it the first Streptomyces PLD possessing only hydrolytic activity. PLDSt may therefore be a novel type of PLD enzyme.