novel phospholipase B from Streptomyces sp NA684-purification, characterization, gene cloning, extracellular production and prediction of the catalytic residues
novel phospholipase B from Streptomyces sp NA684-purification, characterization, gene cloning, extracellular production and prediction of the catalytic residues
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DOI：
10.1111/febs.12366
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发表时间：
2013-08-01
期刊：
影响因子：
5.4
通讯作者：
Sugimori, Daisuke
中科院分区：
文献类型：
--
作者：
Matsumoto, Yusaku;Mineta, Shingo;Sugimori, Daisuke
链霉菌一种新的金属离子非依赖性磷脂酶B(PLB684)菌株NA684从培养上清液中纯化了264倍，回收率为2.85%(6330U mg蛋白~(-1))。PLB684可降解溶血磷脂酰胆碱和二酰基磷脂酰胆碱，溶血磷脂酰胆碱主要在磷脂酰胆碱水解的早期阶段产生。二肉豆蔻基磷脂酸的K-m、V-max和k(CAT)分别为14.5 mM、15.8mmolmin(-1)和1.02×10~(-4)mgProtein(-1)和1.02x10(4)S(-1)。用气相色谱研究了SN-甘油-3-磷酸胆碱的水解性。在反应平衡中，释放的脂肪酸摩尔比(n-1：n-2)为45：55。该基因开放阅读框全长1239bp，编码一个30个氨基酸的信号肽和一个382个氨基酸的成熟酶。PLB684与链霉菌AGR0001(UniProt登录号：J1RQY0)的一个未知蛋白有60%的同源性。利用pUC702表达载体，以变铅青链霉菌为宿主，获得了PLB684。突变分析表明，Ser12对于PLB684的催化功能是必不可少的，其活性部位可能包括Ser330和His332残基。
A novel metal ion-independent phospholipase B (PLB684) from Streptomyces sp. strain NA684 was purified 264-fold from the culture supernatant with 2.85% recovery (6330Umg protein(-1)). PLB684 hydrolyzed lysophosphatidylcholine and diacylphosphatidylcholine, and lysophosphatidylcholine was primarily produced during the early stages of phosphatidylcholine hydrolysis. K-m, V-max and k(cat) for hydrolysis of dimyristoyl phosphatidic acid were 14.5mm, 15.8mmolmin(-1)mgprotein(-1) and 1.02x10(4)s(-1), respectively. sn-glycero-3-phosphocholine hydrolysis was investigated using GC. In the reaction equilibrium, the molar ratio of released fatty acids (sn-1:sn-2) was 45:55. ORF of the gene is 1239bp in length and codes for a 30-amino acid signal peptide and a 382-amino acid mature enzyme. PLB684 shows 60% identity to a uncharacterized protein of Streptomycesauratus AGR0001 (UniProt accession number: J1RQY0). PLB684 was achieved using a pUC702 expression vector and Streptomyceslividans as the host. Mutagenesis analysis showed that Ser12 is essential for the catalytic function of PLB684 and that the active site may include residues Ser330 and His332.