Identification and Characterization of a Novel Salt-Tolerant Esterase from the Deep-Sea Sediment of the South China Sea.
Identification and Characterization of a Novel Salt-Tolerant Esterase from the Deep-Sea Sediment of the South China Sea.
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南海深海沉积物中新型耐盐酯酶的鉴定和表征
DOI：
10.3389/fmicb.2017.00441
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发表时间：
2017
影响因子：
5.2
通讯作者：
Li PY
中科院分区：
文献类型：
--
作者：
Zhang Y;Hao J;Zhang YQ;Chen XL;Xie BB;Shi M;Zhou BC;Zhang YZ;Li PY
海洋酯酶在海洋有机碳的降解和循环中起着重要的作用。从海洋中提取的耐盐酯酶在要求高盐的工业过程中可能有很好的潜力。虽然已经鉴定了大量的海洋酯酶，但有关耐盐酯酶的报道还很少。在这里，一个包含7,200个克隆的化石金字塔图书馆是从南中国海的深海沉积物样本中构建的。通过功能筛选，从该文库中鉴定出一个编码酯酶的基因H8，并在大肠杆菌中表达。系统发育分析表明，H8是细菌脂解酶V家族的新成员。H8能有效地降解短链单酯(C4-C10)，其中对对硝基苯基己酸酯的催化活性最高。H8的最适温度为35℃，最适pH为10.0。H8具有很高的耐盐性，在4.5M的氯化钠中保持稳定，表明H8对海洋盐碱环境有很好的适应能力，具有一定的工业潜力。与已报道的高酸碱性残基比和低等电点的嗜盐/耐盐酶不同，H8含有大量的碱性残基，导致其高碱性/酸性残基比和高预测等电点(9.09)。此外，在数据库中发现了10多个具有相似碱性/酸性残基比例和预测等电点的同源序列，表明H8及其同源物代表了一组新的耐盐酯酶。我们还通过定点突变研究了碱性残基在H8耐盐性中的作用。Arg195、Arg203或Arg236突变为酸性谷氨酸显著降低了H8在高盐下的活性和/或稳定性，表明这些碱性残基在H8的耐盐性中发挥了作用。这些结果对海洋细菌酯酶和耐盐酶的研究具有重要意义。
Marine esterases play an important role in marine organic carbon degradation and cycling. Halotolerant esterases from the sea may have good potentials in industrial processes requiring high salts. Although a large number of marine esterases have been characterized, reports on halotolerant esterases are only a few. Here, a fosmid library containing 7,200 clones was constructed from a deep-sea sediment sample from the South China Sea. A gene H8 encoding an esterase was identified from this library by functional screening and expressed in Escherichia coli. Phylogenetic analysis showed that H8 is a new member of family V of bacterial lipolytic enzymes. H8 could effectively hydrolyze short-chain monoesters (C4–C10), with the highest activity toward p-nitrophenyl hexanoate. The optimal temperature and pH for H8 activity were 35°C and pH 10.0, respectively. H8 had high salt tolerance, remaining stable in 4.5 M NaCl, which suggests that H8 is well adapted to the marine saline environment and that H8 may have industrial potentials. Unlike reported halophilic/halotolerant enzymes with high acidic/basic residue ratios and low pI values, H8 contains a large number of basic residues, leading to its high basic/acidic residue ratio and high predicted pI (9.09). Moreover, more than 10 homologous sequences with similar basic/acidic residue ratios and predicted pI values were found in database, suggesting that H8 and its homologs represent a new group of halotolerant esterases. We also investigated the role of basic residues in H8 halotolerance by site-directed mutation. Mutation of Arg195, Arg203 or Arg236 to acidic Glu significantly decreased the activity and/or stability of H8 under high salts, suggesting that these basic residues play a role in the salt tolerance of H8. These results shed light on marine bacterial esterases and halotolerant enzymes.