Seidl Verena, Druzhinina Irina S, Kubicek Christian P
Research Area Gene Technology and Applied Biochemistry, Institute of Chemical Engineering, TU Vienna, Getreidemarkt 9/166-5, A-1060 Vienna, Austria.
Microbiology (Reading). 2006 Jul;152(Pt 7):2003-2012. doi: 10.1099/mic.0.28897-0.
To identify carbon sources that trigger beta-N-acetylglucosaminidase (NAGase) formation in Hypocrea atroviridis (anamorph Trichoderma atroviride), a screening system was designed that consists of a combination of Biolog Phenotype MicroArray plates, which contain 95 different carbon sources, and specific enzyme activity measurements using a chromogenic substrate. The results revealed growth-dependent kinetics of NAGase formation and it was shown that NAGase activities were enhanced on carbon sources sharing certain structural properties, especially on alpha-glucans (e.g. glycogen, dextrin and maltotriose) and oligosaccharides containing galactose. Enzyme activities were assessed in the wild-type and a H. atroviridis Deltanag1 strain to investigate the influence of the two NAGases, Nag1 and Nag2, on total NAGase activity. Reduction of NAGase levels in the Deltanag1 strain in comparison to the wild-type was strongly carbon-source and growth-phase dependent, indicating the distinct physiological roles of the two proteins. The transcript abundance of nag1 and nag2 was increased on carbon sources with elevated NAGase activity, indicating transcriptional regulation of these genes. The screening method for the identification of carbon sources that induce enzymes or a gene of interest, as presented in this paper, can be adapted for other purposes if appropriate enzyme or reporter assays are available.
为了鉴定能触发绿僵菌（无性型为绿色木霉）中β-N-乙酰氨基葡萄糖苷酶（NAGase）形成的碳源，设计了一种筛选系统，该系统由Biolog表型微阵列板（包含95种不同碳源）和使用显色底物的特定酶活性测量方法组成。结果揭示了NAGase形成的生长依赖性动力学，并且表明在具有某些结构特性的碳源上NAGase活性增强，特别是在α-葡聚糖（如糖原、糊精和麦芽三糖）和含半乳糖的寡糖上。在野生型和绿僵菌Deltanag1菌株中评估酶活性，以研究两种NAGase（Nag1和Nag2）对总NAGase活性的影响。与野生型相比，Deltanag1菌株中NAGase水平的降低强烈依赖于碳源和生长阶段，表明这两种蛋白质具有不同的生理作用。在NAGase活性升高的碳源上，nag1和nag2的转录丰度增加，表明这些基因的转录调控。如果有合适的酶或报告分析方法，本文中介绍的用于鉴定诱导酶或感兴趣基因的碳源的筛选方法可适用于其他目的。
