Insertion of Endocellulase Catalytic Domains into Thermostable Consensus Ankyrin Scaffolds: Effects on Stability and Cellulolytic Activity
Insertion of Endocellulase Catalytic Domains into Thermostable Consensus Ankyrin Scaffolds: Effects on Stability and Cellulolytic Activity
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DOI：
10.1128/aem.02121-13
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发表时间：
2013-11-01
影响因子：
4.4
通讯作者：
Barrick, Doug
中科院分区：
文献类型：
--
作者：
Cunha, Eva S.;Hatem, Christine L.;Barrick, Doug
降解纤维素生产生物燃料有望解决重要的环境和经济问题。然而，将纤维素转化为单糖的水解纤维素酶的低活性（因此需要高的酶与底物比）仍然是主要的障碍。作为稳定纤维素酶和增强其活性的潜在策略，我们将极端微生物的纤维素酶嵌入超稳定的α-螺旋共有锚蛋白结构域支架中。我们发现催化结构域CelA（CA，GH 8; Clostridium thermocellum）和Cel 12 A（C12 A，GH 12; Thermotoga maritima）在锚蛋白支架的背景下是稳定的，并且对可溶性和不溶性底物都是活性的。每个融合体中的锚蛋白重复序列是折叠的，尽管似乎对于C12 A催化结构域（CD;其中N和C末端在晶体结构中是远的），两个侧翼锚蛋白结构域是独立的，而对于CA（其中末端是近的），侧翼锚蛋白结构域彼此稳定。虽然CA的活性在锚蛋白支架的情况下没有变化，但C12 A的活性在高温下增加2- 6倍（对于再生的无定形纤维素和羧甲基纤维素底物）。对于C12 A，活性随着侧翼锚蛋白重复序列的数量而增加。这些结果表明，锚蛋白阵列是一个有前途的支架，用于构建设计师cellulosomes，保持或增强酶活性和保持热稳定性。这种模块化结构将使得在单个多肽内以精确的间距排列多个纤维素酶结构域成为可能，从而使我们能够搜索可以优化对重复纤维素晶格的反应性的间距。
Degradation of cellulose for biofuels production holds promise in solving important environmental and economic problems. However, the low activities (and thus high enzyme-to-substrate ratios needed) of hydrolytic cellulase enzymes, which convert cellulose into simple sugars, remain a major barrier. As a potential strategy to stabilize cellulases and enhance their activities, we have embedded cellulases of extremophiles into hyperstable alpha-helical consensus ankyrin domain scaffolds. We found the catalytic domains CelA (CA, GH8; Clostridium thermocellum) and Cel12A (C12A, GH12; Thermotoga maritima) to be stable in the context of the ankyrin scaffold and to be active against both soluble and insoluble substrates. The ankyrin repeats in each fusion are folded, although it appears that for the C12A catalytic domain (CD; where the N and C termini are distant in the crystal structure), the two flanking ankyrin domains are independent, whereas for CA (where termini are close), the flanking ankyrin domains stabilize each other. Although the activity of CA is unchanged in the context of the ankyrin scaffold, the activity of C12A is increased between 2- and 6-fold (for regenerated amorphous cellulose and carboxymethyl cellulose substrates) at high temperatures. For C12A, activity increases with the number of flanking ankyrin repeats. These results showed ankyrin arrays to be a promising scaffold for constructing designer cellulosomes, preserving or enhancing enzymatic activity and retaining thermostability. This modular architecture will make it possible to arrange multiple cellulase domains at a precise spacing within a single polypeptide, allowing us to search for spacings that may optimize reactivity toward the repetitive cellulose lattice.