Institute for Biology and Biotechnology of Plants, University of Münster, Münster, Germany.
Biotechnol Bioeng. 2018 Apr;115(4):863-873. doi: 10.1002/bit.26533. Epub 2018 Jan 19.
Partially acetylated chitosan oligosaccharides (paCOS) have various potential applications in agriculture, biomedicine, and pharmaceutics due to their suitable bioactivities. One method to produce paCOS is partial chemical hydrolysis of chitosan polymers, but that leads to poorly defined mixtures of oligosaccharides. However, the effective production of defined paCOS is crucial for fundamental research and for developing applications. A more promising approach is enzymatic depolymerization of chitosan using chitinases or chitosanases, as the substrate specificity of the enzyme determines the composition of the oligomeric products. Protein-engineering of these enzymes to alter their substrate specificity can overcome the limitations associated with naturally occurring enzymes and expand the spectrum of specific paCOS that can be produced. Here, engineering the substrate specificity of Bacillus sp. MN chitosanase is described for the first time. Two muteins with active site substitutions can accept N-acetyl-D-glucosamine units at their subsite (-2), which is impossible for the wildtype enzyme.
部分乙酰化壳寡糖（paCOS）由于其适宜的生物活性，在农业、生物医药和制药领域具有各种潜在的应用。生产 paCOS 的一种方法是壳聚糖聚合物的部分化学水解，但这会导致寡糖的混合物定义不明确。然而，有效生产明确的 paCOS 对于基础研究和开发应用至关重要。使用几丁质酶或壳聚糖酶进行酶解是一种更有前途的方法，因为酶的底物特异性决定了寡聚物产物的组成。通过对这些酶进行蛋白质工程改造以改变其底物特异性，可以克服天然存在的酶相关的局限性，并扩展可以生产的特定 paCOS 的范围。本文首次描述了细菌 MN 壳聚糖酶的底物特异性工程。两个具有活性位点取代的突变体可以在其亚位点（-2）接受 N-乙酰-D-葡萄糖胺单元，而野生型酶则不可能。
