Purification and characterization of the second Streptomyces phospholipase A2 refolded from an inclusion body
Purification and characterization of the second Streptomyces phospholipase A2 refolded from an inclusion body
复制标题
DOI：
10.1016/j.pep.2006.05.009
复制
发表时间：
2006-11-01
影响因子：
1.6
通讯作者：
Sugiyama, Masanori
中科院分区：
文献类型：
--
作者：
Jovel, Santa Romero;Kumagai, Takanori;Sugiyama, Masanori
从Streptomyces violaceoruber A-2688中发现的分泌型磷脂酶A(2) （PLA(2)）是我们在原核生物中首次发现的PLA(2)。coeficolor Streptomyces coeficolor A3(2)的基因组序列数据表明，该细菌携带两个编码假想PLA(2)S的基因，分别与S. violaceoruber PLA(2)具有100%和78%的同源性。在本研究中，我们将前者和后者分别命名为第一和第二PLA(2)s。第二个PLA(2)在大肠杆菌细胞中表达时，形成包涵体。本研究展示了一种纯化方法，使其均匀而不失去酶活性：用脱氧胆酸钠洗涤包涵体，在pH为12的2 M尿素存在下溶解，然后用稀释法重新折叠。圆二色光谱证实了酶的再折叠作用。纯化后的PLA(2)与S. violaceoruber PLA(2)具有相同的比活性，产量约为6.8 mg/L。第二种PLA(2)表现出与S. violaceoruber PLA(2)相似的酶学性质，除了前者酶不利用磷脂酸作为底物。通过计算机同源性建模建立的S. coelieolor PLA(2)模型的表面静电电位表明，酶的正电荷表面不影响底物特异性。(c) 2006爱思唯尔公司版权所有。
A secreted phospholipase A(2) (PLA(2)) from Streptomyces violaceoruber A-2688, previously identified by us, is the first PLA(2) identified in prokaryotes. Genome sequence data of Streptomyces coeficolor A3(2) indicates that the bacterium carries two genes encoding hypothetical PLA(2)S, which exhibit 100 and 78% identity, respectively, to the S. violaceoruber PLA(2). In this study, we named the former and latter proteins as the first and second PLA(2)s, respectively. When the second PLA(2) was expressed in Escherichia coli cells, it formed an inclusion body. The present study demonstrates a method to purify it to homogeneity without the disappearance of the enzymatic activity: the inclusion body was washed with sodium deoxycholate and dissolved in the presence of 2 M urea at pH 12, then refolded by the dilution method. The refolding of enzyme was confirmed by the circular dichroism spectrum. The second PLA(2) purified to homogeneity had the same specific activity as that of the S. violaceoruber PLA(2) and the yield was approximately 6.8 mg/L culture. The second PLA(2) exhibits similar enzymatic properties to the S. violaceoruber PLA(2), except that the former enzyme does not utilize phophatidic acid as a substrate. The surface electrostatic potential of the S. coelieolor PLA(2) model, which is created by the computer-homology modeling, suggests that the positively charged surface of the enzyme does not affect the substrate specificity. (c) 2006 Elsevier Inc. All rights reserved.