Institute of Technical Microbiology, Hamburg University of Technology, Hamburg, Germany.
PLoS One. 2014 Jan 14;9(1):e85844. doi: 10.1371/journal.pone.0085844. eCollection 2014.
Glucose hydrolyzing enzymes are essential to determine blood glucose level. A high-throughput screening approach was established to identify NAD(P)-dependent glucose dehydrogenases for the application in test stripes and the respective blood glucose meters. In the current report a glucose hydrolyzing enzyme, derived from a metagenomic library by expressing recombinant DNA fragments isolated from hay infusion, was characterized. The recombinant clone showing activity on glucose as substrate exhibited an open reading frame of 987 bp encoding for a peptide of 328 amino acids. The isolated enzyme showed typical sequence motifs of short-chain-dehydrogenases using NAD(P) as a co-factor and had a sequence similarity between 33 and 35% to characterized glucose dehydrogenases from different Bacillus species. The identified glucose dehydrogenase gene was expressed in E. coli, purified and subsequently characterized. The enzyme, belonging to the superfamily of short-chain dehydrogenases, shows a broad substrate range with a high affinity to glucose, xylose and glucose-6-phosphate. Due to its ability to be strongly associated with its cofactor NAD(P), the enzyme is able to directly transfer electrons from glucose oxidation to external electron acceptors by regenerating the cofactor while being still associated to the protein.
葡萄糖水解酶对于确定血糖水平至关重要。建立了一种高通量筛选方法，以鉴定 NAD(P)依赖性葡萄糖脱氢酶，用于测试条和相应的血糖仪。在本报告中，通过表达从干草浸液中分离的重组 DNA 片段，从宏基因组文库中鉴定出一种葡萄糖水解酶。表现出葡萄糖作为底物活性的重组克隆显示出一个 987 bp 的开放阅读框，编码 328 个氨基酸的肽。分离出的酶使用 NAD(P)作为辅助因子，具有典型的短链脱氢酶序列基序，与来自不同芽孢杆菌属的特征葡萄糖脱氢酶的序列相似性为 33%至 35%。鉴定出的葡萄糖脱氢酶基因在大肠杆菌中表达、纯化并随后进行了表征。该酶属于短链脱氢酶超家族，具有广泛的底物范围，对葡萄糖、木糖和葡萄糖-6-磷酸具有高亲和力。由于其能够与辅因子 NAD(P)强烈结合，该酶能够通过在与蛋白质结合的同时将辅因子再生，直接将葡萄糖氧化产生的电子从葡萄糖转移到外部电子受体。
