Directed Evolution and Structural Analysis of Alkaline Pectate Lyase from the Alkaliphilic Bacterium Bacillus sp. Strain N16-5 To Improve Its Thermostability for Efficient Ramie Degumming
Directed Evolution and Structural Analysis of Alkaline Pectate Lyase from the Alkaliphilic Bacterium Bacillus sp. Strain N16-5 To Improve Its Thermostability for Efficient Ramie Degumming
复制标题
DOI：
10.1128/aem.01017-15
复制
发表时间：
2015-06
影响因子：
4.4
通讯作者：
Cheng Zhou;J. Ye;Yanfen Xue;Yanhe Ma
中科院分区：
文献类型：
--
作者：
Cheng Zhou;J. Ye;Yanfen Xue;Yanhe Ma
摘要耐热碱性果胶酸裂解酶作为苎麻化学脱胶工艺的替代品，在纺织工业中具有潜在的应用价值。特别地，来自芽孢杆菌属菌株N16-5（BspPelA）的碱性果胶酸裂解酶具有酶促苎麻脱胶的潜力，因为其在极碱性条件下的高比活性而不需要额外的Ca 2+。然而，BspPelA显示出较差的热稳定性，并且在50°C下孵育仅30分钟后无活性。在此，定向进化用于改善BspPelA的热稳定性，以实现高效和稳定的脱胶。经过两轮易错PCR和> 12，000个突变体的筛选，获得了10个具有改善的热稳定性的突变体。序列分析和定点突变表明，单一的E124 I，T178 A，和S271 G取代负责提高热稳定性。结构和分子动力学模拟分析表明，疏水簇和新氢键网络的形成是这三种取代基提高热稳定性的关键因素。最耐热的组合突变体EAET在t50上表现出140倍的增加，在50°C下的t50值（酶失去其初始活性的50%的时间），伴随着与野生型BspPelA相比活性降低84.3%，而最有利的组合突变体EA在50°C下表现出t50值增加24倍，活动增加了23.3%。苎麻脱胶与EA突变体比野生型BspPelA更有效。总的来说，我们的研究结果表明，EA突变体，表现出显着的改善，在热稳定性和活性，具有潜在的应用在苎麻脱胶在纺织工业。
ABSTRACT Thermostable alkaline pectate lyases have potential applications in the textile industry as an alternative to chemical-based ramie degumming processes. In particular, the alkaline pectate lyase from Bacillus sp. strain N16-5 (BspPelA) has potential for enzymatic ramie degumming because of its high specific activity under extremely alkaline conditions without the requirement for additional Ca2+. However, BspPelA displays poor thermostability and is inactive after incubation at 50°C for only 30 min. Here, directed evolution was used to improve the thermostability of BspPelA for efficient and stable degumming. After two rounds of error-prone PCR and screening of >12,000 mutants, 10 mutants with improved thermostability were obtained. Sequence analysis and site-directed mutagenesis revealed that single E124I, T178A, and S271G substitutions were responsible for improving thermostability. Structural and molecular dynamic simulation analysis indicated that the formation of a hydrophobic cluster and new H-bond networks was the key factor contributing to the improvement in thermostability with these three substitutions. The most thermostable combined mutant, EAET, exhibited a 140-fold increase in the t 50 (time at which the enzyme loses 50% of its initial activity) value at 50°C, accompanied by an 84.3% decrease in activity compared with that of wild-type BspPelA, while the most advantageous combined mutant, EA, exhibited a 24-fold increase in the t 50 value at 50°C, with a 23.3% increase in activity. Ramie degumming with the EA mutant was more efficient than that with wild-type BspPelA. Collectively, our results suggest that the EA mutant, exhibiting remarkable improvements in thermostability and activity, has the potential for applications in ramie degumming in the textile industry.