Obayashi Yumiko, Wei Bong Chui, Suzuki Satoru
Center for Marine Environmental Studies, Ehime University, Matsuyama, Japan.
Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia.
Front Microbiol. 2017 Oct 10;8:1952. doi: 10.3389/fmicb.2017.01952. eCollection 2017.
Microbial extracellular hydrolytic enzymes that degrade organic matter in aquatic ecosystems play key roles in the biogeochemical carbon cycle. To provide linkages between hydrolytic enzyme activities and genomic or metabolomic studies in aquatic environments, reliable measurements are required for many samples at one time. Extracellular proteases are one of the most important classes of enzymes in aquatic microbial ecosystems, and protease activities in seawater are commonly measured using fluorogenic model substrates. Here, we examined several concerns for measurements of extracellular protease activities (aminopeptidases, and trypsin-type, and chymotrypsin-type activities) in seawater. Using a fluorometric microplate reader with low protein binding, 96-well microplates produced reliable enzymatic activity readings, while use of regular polystyrene microplates produced readings that showed significant underestimation, especially for trypsin-type proteases. From the results of kinetic experiments, this underestimation was thought to be attributable to the adsorption of both enzymes and substrates onto the microplate. We also examined solvent type and concentration in the working solution of oligopeptide-analog fluorogenic substrates using dimethyl sulfoxide (DMSO) and 2-methoxyethanol (MTXE). The results showed that both 2% (final concentration of solvent in the mixture of seawater sample and substrate working solution) DMSO and 2% MTXE provide similarly reliable data for most of the tested substrates, except for some substrates which did not dissolve completely in these assay conditions. Sample containers are also important to maintain the level of enzyme activity in natural seawater samples. In a small polypropylene containers (e.g., standard 50-mL centrifugal tube), protease activities in seawater sample rapidly decreased, and it caused underestimation of natural activities, especially for trypsin-type and chymotrypsin-type proteases. In conclusion, the materials and method for measurements should be carefully selected in order to accurately determine the activities of microbial extracellular hydrolytic enzymes in aquatic ecosystems; especially, low protein binding materials should be chosen to use at overall processes of the measurement.
在水生生态系统中降解有机物的微生物细胞外水解酶在生物地球化学碳循环中发挥着关键作用。为了在水生环境中将水解酶活性与基因组或代谢组学研究联系起来，需要一次性对许多样本进行可靠的测量。细胞外蛋白酶是水生微生物生态系统中最重要的酶类之一，海水中的蛋白酶活性通常使用荧光模型底物进行测量。在这里，我们研究了海水中细胞外蛋白酶活性（氨肽酶、胰蛋白酶型和糜蛋白酶型活性）测量中的几个问题。使用具有低蛋白结合特性的荧光酶标仪，96孔微孔板产生了可靠的酶活性读数，而使用常规聚苯乙烯微孔板产生的读数明显偏低，尤其是对于胰蛋白酶型蛋白酶。从动力学实验结果来看，这种低估被认为是由于酶和底物都吸附到微孔板上所致。我们还使用二甲基亚砜（DMSO）和2-甲氧基乙醇（MTXE）研究了寡肽类似物荧光底物工作溶液中的溶剂类型和浓度。结果表明，对于大多数测试底物，2%（海水样品与底物工作溶液混合物中溶剂的终浓度）的DMSO和2%的MTXE都能提供同样可靠的数据，但有一些底物在这些测定条件下不能完全溶解。样品容器对于维持天然海水样品中的酶活性水平也很重要。在小的聚丙烯容器（如标准50 mL离心管）中，海水样品中的蛋白酶活性迅速下降，导致对天然活性的低估，尤其是对于胰蛋白酶型和糜蛋白酶型蛋白酶。总之，为了准确测定水生生态系统中微生物细胞外水解酶的活性，应仔细选择测量的材料和方法；特别是，在测量的整个过程中应选择低蛋白结合材料。
