在使用高通量微孔板测定水，土壤，沉积物和微生物测定胞外酶活性的
Summary
基于微板程序描述了用于胞外酶活性的比色或荧光分析。这些程序允许在大量环境样品等活动的管理的时间范围内的快速检测。
Abstract
大部分的养分循环和碳处理的自然环境是通过微生物释放胞外酶的活性。因此，这些胞外酶的活性的测定可以给见解生态系统水平的过程，如有机物分解或氮和磷的矿化速率。环境样品中的胞外酶活性的测定通常涉及暴露样品到人工比色或荧光底物和跟踪底物水解的速率。在这里，我们描述了基于微板的方法，这些方法允许对大量样品的一个短的时间内进行分析。样品被允许与人工基质内的96孔微孔板或深孔微孔板块进行反应，并且酶的活性，随后通过吸收所产生的终产物的荧光或使用典型的微孔板里德确定r或荧光。这种高通量的程序不仅有利于空间上不同的网站或生态系统之间的比较，而且还通过减少每个样品所需的试剂整体体积大大减少这种试验的费用。
Introduction
微生物如细菌和真菌通过生产胞外酶的取得从复杂的有机化合物的营养物质和碳。这些酶通常水解的聚合物为可以被取入细胞小亚基。因此，在生态层面上，这些微生物胞外酶是负责大部分的养分矿化和发生在自然的环境中有机物的分解。酶，例如纤维二糖水解（CBH）和β-葡糖苷酶是纤维素降解和工作重要一致来催化纤维素水解成葡萄糖1,2，它提供了对微生物的吸收和同化一个可利用的碳底物。酶磷酸酶释放的有机磷易溶于无机磷酸基团，基本上矿化磷酸盐和使其可用于大多数生物3使用。其他酶，如N-乙酰氨基（NAG酶），是的importan吨中几丁质的降解，可以使碳和氮供微生物采集4。
一项所述的程序，用于在自然环境中的微生物胞外酶活性的测定是使用人工对硝基苯基（ 对 NP）相连的基板，该最初被开发，以检测土壤磷酸酶活5的方法。此方法依赖于有色终产物， 对硝基苯酚，当人工基板通过适当的酶水解时释放的检测。 对硝基苯酚的随后可以通过测量其吸光度约为400-410 nm的比色定量。这种方法已经被应用到检测其它酶如NAG酶6，并已应用于各种研究着眼于在土壤和沉积物7-9微生物胞外酶活性。
这是originall一种替代方法Ý用于评估细胞外糖苷酶的活性在水生环境10,11是利用4 -甲基伞形酮（MUB）相连的底物。释放（4 – 甲基伞形酮）的最终产物是高荧光的，并且可以使用与周围四百六十分之三百六纳米的激发/发射设置一个荧光计来检测。各种MUB联的人工基质的情况下，允许至少为许多酶（ 如 β-葡糖苷酶，纤维二糖水解，NAG酶，磷酸酶）的活性的荧光测定作为可使用p NP-衬底比色步骤进行测定。其它微生物的胞外酶，如蛋白降解的亮氨酸氨肽酶，可以使用7 – 氨基-4 – 甲基香豆素（COU）键合底物荧光计进行测定。既MUB-和COU联基板已被用来确定各种陆地和水中的样品12,13中酶的活性。
虽然以前的研究单位描述离子束增强沉积荧光或比色微孔板方法来确定胞外酶的活性14，有必要建立一个清晰的介绍了如何进行这样的试验。在这里，我们展示了进行高通量微孔板技术的使用比色p NP联基板的做法土壤和沉积物，并在使用荧光MUB联基板技术自然水域胞外酶的活性分析程序。我们专注于β-葡糖苷酶，NAG酶和磷酸酶的活性的测量，因为这些酶可以分别连接到碳，氮，磷和骑自行车。然而，此处描述的步骤可以应用到使用不同的人工基质的其他胞外酶的测定。
Protocol
Representative Results
Discussion
确定各种土壤和沉积物中微生物胞外酶的活性可以有效地洞察养分的矿化与有机物处理17的速率。然而，土壤可以在其水分含量而有所不同，所以标准化活动，以土壤干重是很重要的。这就要求（通常两天）超越了简单的测量酶活性的附加干燥步骤。因此，相对于酶活性的水样，接近瞬时的结果提供了在测定中，在土壤和沉积物酶活性的可靠的检测需要几天。对于一些土壤，它甚至可能是…
Disclosures
The authors have nothing to disclose.
Acknowledgements
资助这项工作的各个方面是由各种来源，包括农业部美国农业部的具体合作协议58-6408-1-595和美国国家科学基金会（奖1049911）提供。
Materials
| REAGENTS AND MATERIALS | |||
| Glacial acetic acid | Various suppliers | ||
| Sodium acetate | Various suppliers | ||
| Sodium hydroxide | Various suppliers | ||
| p-Nitrophenol | Fisher | BP612-1 | Alternates available |
| p-Nitrophenyl (pNP)-phosphate | Sigma | N3234 | pNP-substrate |
| pNP-β-glucopyranoside | Sigma | N7006 | pNP-substrate |
| pNP-β-N-acetylglucosaminide | Sigma | N9376 | pNP-substrate |
| Clear 96-well microplates | Fisher | 12-563-301 | Alternates available |
| 96-well deep well blocks | Costar | 3958 | Alternates available |
| Aluminum weigh pans | Various suppliers | ||
| Sterile 15 ml centrifuge tubes | Various suppliers | ||
| Sterile 50 ml centrifuge tubes | Various suppliers | ||
| 4-Methylumbelliferone | Sigma | M1381 | |
| 4-Methylumbelliferyl (MUB)-phosphate | Sigma | M8883 | MUB-substrate |
| 4-MUB-glucopyranoside | Sigma | M3633 | MUB-substrate |
| 4-MUB-N-acetylglucosaminide | Sigma | M2133 | MUB-substrate |
| Sodium bicarbonate | Various suppliers | ||
| Black 96-well microplate | Costar | 3792 | |
| Pipette reservoir | Various suppliers | ||
| EQUIPMENT | |||
| Centrifuge | Eppendorf | 5810R | |
| Centrifuge rotor | Eppendorf | A-4-81 | For microplates/deep-well blocks |
| Microplate reader | BioTek | Synergy HT | Alternates available |
| Microplate fluorometer | BioTek | FLx 800 | Alternates available |
| 8-channel pipettor | Various suppliers |
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