Chitin is an abundant polysaccharide used by many organisms for structural rigidity and water repulsion. As such, the insoluble crystalline structure of chitin poses significant challenges for enzymatic degradation. Acidic mammalian chitinase, a processive glycosyl hydrolase, is the primary enzyme involved in the degradation of environmental chitin in mammalian lungs. Mutations to acidic mammalian chitinase have been associated with asthma, and genetic deletion in mice increases morbidity and mortality with age. We initially set out to reverse this phenotype by engineering hyperactive acidic mammalian chitinase variants. Using a screening approach with commercial fluorogenic substrates, we identified mutations with consistent increases in activity. To determine whether the activity increases observed were consistent with more biologically relevant chitin substrates, we developed new assays to quantify chitinase activity with insoluble chitin, and identified a one-pot fluorogenic assay that is sufficiently sensitive to quantify changes to activity due to the addition or removal of a carbohydrate-binding domain. We show that the activity increases from our directed evolution screen were lost when insoluble substrates were used. In contrast, naturally occurring gain-of-function mutations gave similar results with oligomeric and insoluble substrates. We also show that activity differences between acidic mammalian chitinase and chitotriosidase are reduced with insoluble substrate, suggesting that previously reported activity differences with oligomeric substrates may have been driven by differential substrate specificity. These results highlight the need for assays against physiological substrates when engineering metabolic enzymes, and provide a new one-pot assay that may prove to be broadly applicable to engineering glycosyl hydrolases.

中文翻译：

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哺乳动物几丁质酶对可溶性和不溶性底物的几丁质水解活性的差异。

几丁质是一种丰富的多糖，被许多生物用于结构刚性和防水性。因此，几丁质的不溶性晶体结构对酶促降解提出了重大挑战。酸性哺乳动物几丁质酶是一种进行性糖基水解酶，是参与降解哺乳动物肺中环境几丁质的主要酶。酸性哺乳动物几丁质酶的突变与哮喘有关，小鼠的基因缺失会随着年龄的增长而增加发病率和死亡率。我们最初着手通过改造高活性酸性哺乳动物几丁质酶变体来逆转这种表型。使用商业荧光底物的筛选方法，我们确定了活性持续增加的突变。为了确定观察到的活性增加是否与更多生物相关的几丁质底物一致，我们开发了新的测定方法来量化不溶性几丁质的几丁质酶活性，并确定了一种单锅荧光测定法，该测定法足够灵敏，可以量化由于添加或去除碳水化合物结合域。我们表明，当使用不溶性底物时，我们定向进化筛选中的活性增加会丢失。相比之下，自然发生的功能获得性突变对寡聚和不溶性底物产生了类似的结果。我们还表明酸性哺乳动物几丁质酶和壳三糖苷酶之间的活性差异因不溶性底物而减少，表明先前报道的与寡聚底物的活性差异可能是由不同的底物特异性驱动的。这些结果强调了在设计代谢酶时需要对生理底物进行检测，并提供了一种新的单锅检测方法，可能被证明广泛适用于工程糖基水解酶。