Xue Qing-Gang, Schey Kevin L, Volety Aswani K, Chu Fu-Lin E, La Peyre Jerome F
Cooperative Aquatic Animal Health Research Program, Department of Veterinary Science, Louisiana State University Agricultural Center, 111 Dalrymple Building, Baton Rouge, LA 70803, USA.
Comp Biochem Physiol B Biochem Mol Biol. 2004 Sep;139(1):11-25. doi: 10.1016/j.cbpc.2004.05.011.
Lysozyme was purified from the plasma of eastern oysters (Crassostrea virginica) using a combination of ion exchange and gel filtration chromatographies. The molecular mass of purified lysozyme was estimated at 18.4 kDa by SDS-PAGE, and its isoelectric point was greater than 10. Mass spectrometric analysis of the purified enzyme revealed a high-sequence homology with i-type lysozymes. No similarity was found however between the N-terminal sequence of oyster plasma lysozyme and N-terminal sequences of other i-type lysozymes, suggesting that the N-terminal sequences of the i-type lysozymes may vary to a greater extent between species than reported in earlier studies. The optimal ionic strength, pH, cation concentrations, sea salt concentrations, and temperature for activity of the purified lysozyme were determined, as well as its temperature and pH stability. Purified oyster plasma lysozyme inhibited the growth of Gram-positive bacteria (e.g., Lactococcus garvieae, Enterococcus sp.) and Gram-negative bacteria (e.g., Escherichia coli, Vibrio vulnificus). This is a first report of a lysozyme purified from an oyster species and from the plasma of a bivalve mollusc.
采用离子交换色谱法和凝胶过滤色谱法相结合的方法，从美国东海岸牡蛎（Crassostrea virginica）的血浆中纯化溶菌酶。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）估计纯化后的溶菌酶分子量为18.4 kDa，其等电点大于10。对纯化后的酶进行质谱分析，结果显示其与i型溶菌酶具有高度的序列同源性。然而，牡蛎血浆溶菌酶的N端序列与其他i型溶菌酶的N端序列之间未发现相似性，这表明i型溶菌酶的N端序列在不同物种间的差异程度可能比早期研究报道的更大。测定了纯化后溶菌酶活性的最佳离子强度、pH值、阳离子浓度、海盐浓度和温度，以及其温度和pH稳定性。纯化后的牡蛎血浆溶菌酶能够抑制革兰氏阳性菌（如格氏乳球菌、肠球菌属）和革兰氏阴性菌（如大肠杆菌、创伤弧菌）的生长。这是首次从牡蛎物种以及双壳贝类软体动物的血浆中纯化出溶菌酶的报道。
