Michlmayr Herbert, Varga Elisabeth, Malachova Alexandra, Nguyen Nhung Thi, Lorenz Cindy, Haltrich Dietmar, Berthiller Franz, Adam Gerhard
Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna (BOKU), Tulln, Austria
Christian Doppler Laboratory for Mycotoxin Metabolism and Center of Analytical Chemistry, Department of Agrobiotechnology (IFA-Tulln), University of Natural Resources and Life Sciences, Vienna (BOKU), Tulln, Austria.
Appl Environ Microbiol. 2015 Aug;81(15):4885-93. doi: 10.1128/AEM.01061-15. Epub 2015 May 15.
Glycosylation plays a central role in plant defense against xenobiotics, including mycotoxins. Glucoconjugates of Fusarium toxins, such as deoxynivalenol-3-O-β-d-glucoside (DON-3G), often cooccur with their parental toxins in cereal-based food and feed. To date, only limited information exists on the occurrence of glucosylated mycotoxins and their toxicological relevance. Due to a lack of analytical standards and the requirement of high-end analytical instrumentation for their direct determination, hydrolytic cleavage of β-glucosides followed by analysis of the released parental toxins has been proposed as an indirect determination approach. This study compares the abilities of several fungal and recombinant bacterial β-glucosidases to hydrolyze the model analyte DON-3G. Furthermore, substrate specificities of two fungal and two bacterial (Lactobacillus brevis and Bifidobacterium adolescentis) glycoside hydrolase family 3 β-glucosidases were evaluated on a broader range of substrates. The purified recombinant enzyme from B. adolescentis (BaBgl) displayed high flexibility in substrate specificity and exerted the highest hydrolytic activity toward 3-O-β-d-glucosides of the trichothecenes deoxynivalenol (DON), nivalenol, and HT-2 toxin. A Km of 5.4 mM and a Vmax of 16 μmol min(-1) mg(-1) were determined with DON-3G. Due to low product inhibition (DON and glucose) and sufficient activity in several extracts of cereal matrices, this enzyme has the potential to be used for indirect analyses of trichothecene-β-glucosides in cereal samples.
糖基化在植物抵御包括霉菌毒素在内的外源化合物的过程中发挥着核心作用。镰刀菌毒素的葡萄糖共轭物，如脱氧雪腐镰刀菌烯醇-3-O-β-d-葡萄糖苷（DON-3G），经常与其母体毒素同时存在于谷物类食品和饲料中。迄今为止，关于糖基化霉菌毒素的存在情况及其毒理学相关性的信息有限。由于缺乏分析标准以及直接测定需要高端分析仪器，因此有人提出先对β-葡萄糖苷进行水解裂解，然后分析释放出的母体毒素，以此作为一种间接测定方法。本研究比较了几种真菌和重组细菌β-葡萄糖苷酶水解模型分析物DON-3G的能力。此外，还在更广泛的底物范围内评估了两种真菌和两种细菌（短乳杆菌和青春双歧杆菌）糖苷水解酶家族3β-葡萄糖苷酶的底物特异性。来自青春双歧杆菌的纯化重组酶（BaBgl）在底物特异性方面表现出高度灵活性，对单端孢霉烯族毒素脱氧雪腐镰刀菌烯醇（DON）、雪腐镰刀菌烯醇和HT-2毒素的3-O-β-d-葡萄糖苷具有最高的水解活性。以DON-3G测定的Km为5.4 mM，Vmax为16 μmol min(-1) mg(-1)。由于产物抑制作用低（DON和葡萄糖）且在几种谷物基质提取物中具有足够的活性，这种酶有潜力用于谷物样品中单端孢霉烯族毒素-β-葡萄糖苷的间接分析。
