Spadiut Oliver, Pisanelli Ines, Maischberger Thomas, Peterbauer Clemens, Gorton Lo, Chaiyen Pimchai, Haltrich Dietmar
Department of Food Sciences and Technology, BOKU - University of Natural Resources and Applied Life Sciences, Vienna, Austria.
J Biotechnol. 2009 Feb 5;139(3):250-7. doi: 10.1016/j.jbiotec.2008.11.004. Epub 2008 Nov 27.
Pyranose 2-oxidase (P2Ox) has several proposed biotechnological applications such as a bio-component in biofuel cells or for carbohydrate transformations. To improve some of the catalytic properties of P2Ox from Trametes multicolor, we selected a semi-rational approach of enzyme engineering, saturation mutagenesis of active-site residues and subsequent screening of mutant libraries for improved activity. One of the active-site mutants with improved catalytic characteristics identified was V546C, which showed catalytic constants increased by up to 5.7-fold for both the sugar substrates (D-glucose and D-galactose) and alternative electron acceptors (1,4-benzoquinone, BQ and ferricenium ion, Fc(+)], albeit at the expense of increased Michaelis constants. By combining V546C with other amino acid replacements, we obtained P2Ox variants that are of interest for biofuel cell applications due to their increased k(cat) for both BQ and Fc(+), e.g., V546C/E542K showed 4.4- and 17-fold increased k(cat) for BQ compared to the wild-type enzyme when D-glucose and D-galactose, respectively, were the saturating substrates, while V546C/T169G showed approx. 40- and 50-fold higher k(cat) for BQ and Fc(+), respectively, with D-galactose in excess. This latter variant also shows significantly modulated sugar substrate selectivity, due to an increase in k(cat)/K(M) for D-galactose and a decrease in k(cat)/K(M) for D-glucose when oxygen is the electron acceptor, as well as improved catalytic efficiencies for d-galactose, regardless of the electron acceptor used. While the wild-type enzyme strongly prefers D-glucose over D-galactose as its substrate, V546C/T169G converts both sugars equally well as was shown by the kinetic constants determined as well as by biotransformation experiments.
吡喃糖2-氧化酶（P2Ox）有几种潜在的生物技术应用，比如作为生物燃料电池中的生物组件或用于碳水化合物转化。为改善来自变色栓菌的P2Ox的某些催化特性，我们选择了一种酶工程的半理性方法，即对活性位点残基进行饱和诱变，随后筛选突变文库以提高活性。鉴定出的具有改善催化特性的活性位点突变体之一是V546C，它对糖底物（D-葡萄糖和D-半乳糖）和替代电子受体（1,4-苯醌，BQ和铁离子，Fc(+)）的催化常数增加了高达5.7倍，尽管米氏常数有所增加。通过将V546C与其他氨基酸替换相结合，我们获得了对生物燃料电池应用有意义的P2Ox变体，因为它们对BQ和Fc(+)的催化常数（k(cat)）增加，例如，当分别以D-葡萄糖和D-半乳糖为饱和底物时，V546C/E542K对BQ的催化常数（k(cat)）与野生型酶相比分别增加了4.4倍和17倍，而V546C/T169G在D-半乳糖过量时，对BQ和Fc(+)的催化常数（k(cat)）分别高出约40倍和50倍。后一种变体还显示出糖底物选择性的显著调节，这是因为当氧气作为电子受体时，D-半乳糖的催化常数与米氏常数之比（k(cat)/K(M)）增加，而D-葡萄糖的该比值降低，并且无论使用何种电子受体，对D-半乳糖的催化效率都有所提高。虽然野生型酶强烈偏好D-葡萄糖而非D-半乳糖作为底物，但V546C/T169G对两种糖的转化效果相同，这通过动力学常数测定以及生物转化实验得以证明。
