Haernvall Karolina, Fladischer Patrik, Schoeffmann Heidemarie, Zitzenbacher Sabine, Pavkov-Keller Tea, Gruber Karl, Schick Michael, Yamamoto Motonori, Kuenkel Andreas, Ribitsch Doris, Guebitz Georg M, Wiltschi Birgit
Acib-Austrian Centre of Industrial Biotechnology, Graz, Austria.
Institute of Molecular Biotechnology, Graz University of Technology, Graz, Austria.
Front Bioeng Biotechnol. 2022 Jan 26;10:769830. doi: 10.3389/fbioe.2022.769830. eCollection 2022.
Environmentally friendly functionalization and recycling processes for synthetic polymers have recently gained momentum, and enzymes play a central role in these procedures. However, natural enzymes must be engineered to accept synthetic polymers as substrates. To enhance the activity on synthetic polyesters, the canonical amino acid methionine in lipase (TTL) was exchanged by the residue-specific incorporation method for the more hydrophobic non-canonical norleucine (Nle). Strutural modelling of TTL revealed that residues Met-114 and Met-142 are in close vicinity of the active site and their replacement by the norleucine could modulate the catalytic activity of the enzyme. Indeed, hydrolysis of the polyethylene terephthalate model substrate by the Nle variant resulted in significantly higher amounts of release products than the Met variant. A similar trend was observed for an ionic phthalic polyester containing a short alkyl diol (C5). Interestingly, a 50% increased activity was found for TTL [Nle] towards ionic phthalic polyesters containing different ether diols compared to the parent enzyme TTL [Met]. These findings clearly demonstrate the high potential of non-canonical amino acids for enzyme engineering.
合成聚合物的环境友好型功能化和回收工艺近来发展势头迅猛，酶在这些工艺中起着核心作用。然而，必须对天然酶进行改造，使其能够将合成聚合物作为底物。为提高对合成聚酯的活性，通过残基特异性掺入法，将脂肪酶（TTL）中的典型氨基酸甲硫氨酸替换为疏水性更强的非典型正亮氨酸（Nle）。TTL的结构建模显示，残基Met-114和Met-142靠近活性位点，用正亮氨酸替换它们可能会调节酶的催化活性。事实上，与Met变体相比，Nle变体对聚对苯二甲酸乙二酯模型底物的水解产生的释放产物量显著更高。对于含有短链烷基二醇（C5）的离子型邻苯二甲酸聚酯，也观察到了类似趋势。有趣的是，与亲本酶TTL [Met]相比，发现TTL [Nle]对含有不同醚二醇的离子型邻苯二甲酸聚酯的活性提高了50%。这些发现清楚地证明了非典型氨基酸在酶工程中的巨大潜力。
