Enzymes Protocols

Microplate Enzyme Assay Using Fluorescence-Based MUB & MUC-Linked Substrates Download
Fluorescence-based protocol used by the Wallenstein lab at NREL for measuring β-D-cellubiosidase, α-glucosidase, β-glucosidase, leucine aminopeptidase (LAP), N-acetyl-β-glucoasminidase (NAG), phosphatase, and β-xylosidase in soil. This method uses a microplate format and thus requires a microplate reader.
Submitted by: Shawna McMahon and Meg Steinweg
Originally Submitted: June 19, 2009
Last Updated: January 09, 2012 (Note new incubation time notes for 4°C and raw-data calculation examples)
Fluorescence-Based Protocol Using MUB & MUC-Linked Substrates Download
This protocol includes an extensive list of MUC (methylcoumarin) and MUB (methylumbelliferone) which can be used to measure hydrolytic enzymes including aminopeptidases, esterases such as phosphatase & sulfatase, and glycosidases such as β-cellobiosidase and β-xylosidase. In total, substrates are listed for 25 different enzymes, with the suggestion that there are several dozen other coumarin-linked substrates available. This method also uses a microplate format and includes instructions for soil, litter and water samples.
Submitted by: Bob Sinsabaugh
Originally Submitted: July 15, 2009
Last Updated: July 15, 2009
Absorbance-Based PhenOx-Perox Protocol Download
The purpose of this assay is to measure the activity of enzymes that can oxidize phenols. Such enzymes are classified by what they use as an oxidant. Oxygenases use molecular oxygen; peroxidases use hydrogen peroxide. Phenol oxidases and peroxidases are involved in the breakdown of lignin and other aromatic compounds and in the production and degradation of humic substances. This protocol is extensive, including instructions analysis of soil, litter and water in both tube-based and microplate-based methods.
Submitted by: Bob Sinsabaugh
Date Submitted: July 15, 2009
Last Updated: July 15, 2009
Absorbance-Based Protocol Using p-Nitrophenyl-Linked Substrates Download
Absorbance-based protocol used in the Sinsabaugh lab at the University of New Mexico for measuring phosphatase, β-glucosidase, cellobiohydrolase (CBH), chitinase, α-glucosidase, β-xylosidase, β-N-acetylglucosaminidase (NAG), leucine aminopeptidase, glycine aminopeptidase and "trypsin" peptidase. This method uses substrates that are linked with p-Nitrophenyl (pNP), some of which can be quite expensive. This is not a microplate-based method and simply requires a spectrophotometer for analysis.
Submitted by: Bob Sinsabaugh
Date Posted: July 15, 2009
Last Updated: July 15, 2009