In the accompanying paper, we described evolving a lipase to the point where variants were soluble, stable and capable of degrading C8 TAG and C8 esters. These variants were tested for their ability to survive in an environment that might be encountered in a washing machine. Unfortunately, they were inactivated both by treatment with a protease used in laundry detergents and by very low concentrations of sodium dodecyl sulfate (SDS). In addition, all the variants had very low levels of activity with triglycerides with long aliphatic chains and with naturally occurring oils, like olive oil. Directed evolution was used to select variants with enhanced properties. In the first 10 rounds of evolution, the primary screen was selected for variants capable of hydrolyzing olive oil whereas the secondary screen was selected for enhanced tolerance towards a protease and SDS. In the final six rounds of evolution, the primary and secondary screens identified variants that retained activity after treatment with SDS. Sixteen cycles of evolution gave variants with greatly enhanced lipolytic activity on substrates that had both long (C16 and C18) as well as short (C3 and C8) chains. We found variants that were stable for more than 3 hours in protease concentrations that rapidly degrade the wild-type enzyme. Enhanced tolerance towards SDS was found in variants that could break down naturally occurring lipid and resist protease attack. The amino acid changes that gave enhanced properties were concentrated in the cap domain responsible for substrate binding.

中文翻译：

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不断发展脂肪酶以水解天然甘油三酸酯，同时增强了对蛋白酶和表面活性剂的耐受性。

在随附的论文中，我们描述了将脂肪酶发展到变体可溶，稳定并能够降解C8 TAG和C8酯的程度。测试了这些变体在洗衣机可能遇到的环境中生存的能力。不幸的是，它们既可以用洗衣粉中使用的蛋白酶处理，也可以用极低浓度的十二烷基硫酸钠（SDS）灭活。另外，所有变体对具有长脂族链的甘油三酸酯和天然存在的油（如橄榄油）的活性都非常低。使用定向进化来选择具有增强特性的变体。在进化的前十轮中，选择初级筛选用于能够水解橄榄油的变体，而选择次级筛选以增强对蛋白酶和SDS的耐受性。在进化的最后六轮中，主要和次要筛选确定了使用SDS处理后保留活性的变体。进化的十六个循环使具有长链（C16和C18）以及短链（C3和C8）的底物的脂解活性大大增强。我们发现在蛋白酶浓度下可稳定降解超过3小时的变体，可迅速降解野生型酶。在可以破坏天然脂质并抵抗蛋白酶攻击的变体中发现对SDS的耐受性增强。赋予增强特性的氨基酸变化集中在负责底物结合的帽结构域中。