Ferrari Alessandro R, Lee Misun, Fraaije Marco W
Molecular Enzymology Group, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, The Netherlands.
Biotechnol Bioeng. 2015 Jun;112(6):1074-80. doi: 10.1002/bit.25532. Epub 2015 Mar 2.
Chitooligosaccharide oxidase from Fusarium graminearum (ChitO) oxidizes N-acetyl-D-glucosamine (GlcNAc) and its oligomers with high efficiency at the C1-hydroxyl moiety while it shows poor or no activity with other carbohydrates. By sequence and structural comparison with other known carbohydrate oxidases (glucooligosaccharide oxidase from Acremonium strictum and lactose oxidase from Microdochium nivale) eleven mutants were designed to redirect the catalytic scope of ChitO for improved oxidation of lactose, cellobiose and maltose. The catalytic properties of the most interesting mutants were further improved by combining single mutations. This has resulted in the creation of a set of ChitO variants that display totally different substrate tolerances. One ChitO variant shows a dramatic improvement in catalytic efficiency towards oxidation of glucose, cellobiose, lactose, and maltose. We also describe a ChitO variant with the highest catalytic efficiency in GlcNAc oxidation so far reported in the literature.
禾谷镰刀菌的壳寡糖氧化酶（ChitO）能高效氧化N-乙酰-D-葡萄糖胺（GlcNAc）及其寡聚物的C1-羟基部分，而对其他碳水化合物的活性较差或无活性。通过与其他已知碳水化合物氧化酶（曲霉属的葡寡糖氧化酶和雪腐微座囊菌的乳糖氧化酶）进行序列和结构比较，设计了11个突变体，以改变ChitO的催化范围，从而改善对乳糖、纤维二糖和麦芽糖的氧化作用。通过组合单突变进一步改善了最具潜力突变体的催化特性。这导致创建了一组具有完全不同底物耐受性的ChitO变体。一种ChitO变体对葡萄糖、纤维二糖、乳糖和麦芽糖氧化的催化效率有显著提高。我们还描述了一种ChitO变体，其在GlcNAc氧化方面具有迄今为止文献报道的最高催化效率。
