Krondorfer Iris, Lipp Katharina, Brugger Dagmar, Staudigl Petra, Sygmund Christoph, Haltrich Dietmar, Peterbauer Clemens K
Food Biotechnology Laboratory, Department of Food Science and Technology, University of Natural Resources and Life Sciences, Vienna, Austria.
Food Biotechnology Laboratory, Department of Food Science and Technology, University of Natural Resources and Life Sciences, Vienna, Austria; University of Applied Sciences Wiener Neustadt - Campus Tulln, Tulln, Austria.
PLoS One. 2014 Mar 10;9(3):e91145. doi: 10.1371/journal.pone.0091145. eCollection 2014.
Pyranose dehydrogenase (PDH), a member of the GMC family of flavoproteins, shows a very broad sugar substrate specificity but is limited to a narrow range of electron acceptors and reacts extremely slowly with dioxygen as acceptor. The use of substituted quinones or (organo)metals as electron acceptors is undesirable for many production processes, especially of food ingredients. To improve the oxygen reactivity, site-saturation mutagenesis libraries of twelve amino acids around the active site of Agaricus meleagris PDH were expressed in Saccharomyces cerevisiae. We established high-throughput screening assays for oxygen reactivity and standard dehydrogenase activity using an indirect Amplex Red/horseradish peroxidase and a DCIP/D-glucose based approach. The low number of active clones confirmed the catalytic role of H512 and H556. Only one position was found to display increased oxygen reactivity. Histidine 103, carrying the covalently linked FAD cofactor in the wild-type, was substituted by tyrosine, phenylalanine, tryptophan and methionine. Variant H103Y was produced in Pichia pastoris and characterized and revealed a five-fold increase of the oxygen reactivity.
吡喃糖脱氢酶（PDH）是黄素蛋白GMC家族的成员之一，它对糖类底物具有非常广泛的特异性，但仅限于狭窄范围的电子受体，并且与作为受体的氧气反应极其缓慢。对于许多生产过程，尤其是食品成分的生产过程，使用取代醌或（有机）金属作为电子受体是不可取的。为了提高对氧气的反应活性，在酿酒酵母中表达了姬松茸PDH活性位点周围12个氨基酸的位点饱和诱变文库。我们使用间接的Amplex Red/辣根过氧化物酶和基于DCIP/D-葡萄糖的方法，建立了针对氧气反应活性和标准脱氢酶活性的高通量筛选测定法。活性克隆数量较少证实了H512和H556的催化作用。仅发现一个位置显示出对氧气的反应活性增加。在野生型中携带共价连接的FAD辅因子的组氨酸103被酪氨酸、苯丙氨酸、色氨酸和甲硫氨酸取代。变体H103Y在毕赤酵母中产生并进行了表征，结果显示其对氧气的反应活性提高了五倍。
