Identification of a new subfamily of salt-tolerant esterases from a metagenomic library of tidal flat sediment
Identification of a new subfamily of salt-tolerant esterases from a metagenomic library of tidal flat sediment
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DOI：
10.1007/s00253-011-3433-x
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发表时间：
2012-01-01
影响因子：
5
通讯作者：
Lee, Jung-Hyun
中科院分区：
文献类型：
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作者：
Jeon, Jeong Ho;Lee, Hyun Sook;Lee, Jung-Hyun
为了寻找新的脂溶酶，从韩国江华岛潮滩沉积物中建立了一个宏基因组文库。利用甘油三酯琼脂平板进行功能筛选，从fosmid文库的80,050个克隆中筛选出3个克隆。序列分析显示，这些克隆含有不同的开放阅读框，与数据库中推测的脂溶酶氨基酸同源性为50-57%。基于系统发育分析，他们被鉴定为编码新成员，在细菌脂溶酶家族IV中形成一个独特的新亚家族。一致的序列GT(S)SA(G)G，包含酶的活性位点丝氨酸，与GDSAG基序不同，在其他亚家族中保守。这些基因在大肠杆菌中表达，重组蛋白被纯化为活性可溶性形式。这些酶对对戊酸对硝基苯（C5）的活性最高，在中温和微碱性条件下表现出最佳的活性，特别是在3 M NaCl（或KCl）存在下，酶的最大活性保持在50%以上。在这项研究中，我们证明了利用海洋潮滩沉积物作为DNA来源的宏基因组方法扩大了脂溶酶编码基因的多样性。
To search for novel lipolytic enzymes, a metagenomic library was constructed from the tidal flat sediment of Ganghwa Island in South Korea. By functional screening using tributyrin agar plates, 3 clones were selected from among the 80,050 clones of the fosmid library. The sequence analysis revealed that those clones contained different open reading frames, which showed 50-57% amino acid identity with putative lipolytic enzymes in the database. Based on the phylogenetic analysis, they were identified to encode novel members, which form a distinct and new subfamily in the family IV of bacterial lipolytic enzymes. The consensus sequence, GT(S)SA(G)G, encompassing the active site serine of the enzymes was different from the GDSAG motif, conserved in the other subfamily. The genes were expressed in Escherichia coli and recombinant proteins were purified as active soluble forms. The enzymes showed the highest activity toward p-nitrophenyl valerate (C5) and exhibited optimum activities at mesophilic temperature ranges and slightly alkaline pH. In particular, the enzymes displayed salt tolerance with over 50% of the maximum activity remained in the presence of 3 M NaCl (or KCl). In this study, we demonstrated that the metagenomic approach using marine tidal flat sediment as a DNA source expanded the diversity of lipolytic enzyme-encoding genes.