Discovery of pectin-degrading enzymes and directed evolution of a novel pectate lyase for processing cotton fabric
Discovery of pectin-degrading enzymes and directed evolution of a novel pectate lyase for processing cotton fabric
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DOI：
10.1074/jbc.m411838200
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发表时间：
2005-03-11
影响因子：
4.8
通讯作者：
Kerovuo, J
中科院分区：
文献类型：
--
作者：
Solbak, AI;Richardson, TH;Kerovuo, J
在纺织工业中，作为棉织物预处理的一部分，越来越需要更经济和对环境负责的方法来改进煮练工艺。使用果胶降解酶的酶促方法在这一努力中是潜在的有价值的候选者，因为它们可以减少目前使用的有毒碱性化学品的量。通过对复杂环境DNA文库的高通量筛选，发现了40多种新型微生物果胶酸裂解酶，并对其酶学性质进行了表征。发现几种候选酶具有最适pH和对棉纤维中果胶物质的特异活性，与它们在精练过程中的使用相容。然而，没有一个显示出所需的温度特性。因此，选择候选酶进行进化。利用基因位点饱和诱变技术，获得了36个单位点耐热突变体。然后通过使用基因重组（TM）技术产生源自12个表现最佳的单位点突变体的组合文库。产生了具有进一步改善的耐热性的十九种变体。测试这些变体在生物精练应用中的改进的耐热性和性能。表现最好的变体（CO14）含有8个突变，并且具有比野生型酶高16 ℃的解链温度，同时在50 ℃下保持相同的比活性。该酶的最适温度为70 ℃，比野生型高20 ℃。用进化的酶获得的煮练结果显著优于用化学煮练获得的结果，使得有可能取代传统的和对环境有害的化学煮练工艺。
There is a growing need in the textile industry for more economical and environmentally responsible approaches to improve the scouring process as part of the pretreatment of cotton fabric. Enzymatic methods using pectin-degrading enzymes are potentially valuable candidates in this effort because they could reduce the amount of toxic alkaline chemicals currently used. Using high throughput screening of complex environmental DNA libraries more than 40 novel microbial pectate lyases were discovered, and their enzymatic properties were characterized. Several candidate enzymes were found that possessed pH optima and specific activities on pectic material in cotton fibers compatible with their use in the scouring process. However, none exhibited the desired temperature characteristics. Therefore, a candidate enzyme was selected for evolution. Using Gene Site Saturation Mutagenesis(TM) technology, 36 single site mutants exhibiting improved thermotolerance were produced. A combinatorial library derived from the 12 best performing single site mutants was then generated by using Gene Reassembly(TM) technology. Nineteen variants with further improved thermotolerance were produced. These variants were tested for both improved thermotolerance and performance in the bioscouring application. The best performing variant (CO14) contained eight mutations and had a melting temperature 16 degreesC higher than the wild type enzyme while retaining the same specific activity at 50 degreesC. Optimal temperature of the evolved enzyme was 70 degreesC, which is 20 degreesC higher than the wild type. Scouring results obtained with the evolved enzyme were significantly better than the results obtained with chemical scouring, making it possible to replace the conventional and environmentally harmful chemical scouring process.