ORIGINAL RESEARCH published: 06 March 2018 doi: 10.3389/fmicb.2018.00368 Edited by: George S. Bullerjahn, Bowling Green State University, United States Reviewed by: Kaarina Sivonen, University of Helsinki, Finland Izabela Marques Dourado Bastos, University of Brasília, Brazil *Correspondence: Andrew D. Steen asteen1@utk.edu Specialty section: This article was submitted to Aquatic Microbiology, a section of the journal Frontiers in Microbiology Received: 29 September 2017 Accepted: 16 February 2018 Published: 06 March 2018 Citation: Mullen L, Malcolm X Shabazz High School Aquatic Biogeochemistry Team, Boerrigter K, Ferriero N, Rosalsky J, Barrett AB, Murray PJ and Steen AD (2018) Potential Activities of Freshwater Exo- and Endo-Acting Extracellular Peptidases in East Tennessee and the Pocono Mountains. Front. Microbiol. 9:368. doi: 10.3389/fmicb.2018.00368 Potential Activities of Freshwater Exo- and Endo-Acting Extracellular Peptidases in East Tennessee and the Pocono Mountains Lauren Mullen 1 , Malcolm X Shabazz High School Aquatic Biogeochemistry Team 2 , Kim Boerrigter 2,3 , Nicholas Ferriero 2 , Jeff Rosalsky 4 , Abigail van Buren Barrett 1 , Patrick J. Murray 2 and Andrew D. Steen 1 * 1 Department of Earth and Planetary Sciences, University of Tennessee, Knoxville, Knoxville, TN, United States, 2 Malcolm X Shabazz High School, Newark, NJ, United States, 3 Harvard College, Cambridge, MA, United States, 4 Pocono Environmental Education Center, Dingmans Ferry, PA, United States Proteins constitute a particularly bioavailable subset of organic carbon and nitrogen in aquatic environments but must be hydrolyzed by extracellular enzymes prior to being metabolized by microorganisms. Activities of extracellular peptidases (protein- degrading enzymes) have frequently been assayed in freshwater systems, but such studies have been limited to substrates for a single enzyme [leucyl aminopeptidase (Leu-AP)] out of more than 300 biochemically recognized peptidases. Here, we report kinetic measurements of extracellular hydrolysis of five substrates in 28 freshwater bodies in the Delaware Water Gap National Recreation Area in the Pocono Mountains (PA, United States) and near Knoxville (TN, United States), between 2013 and 2016. The assays putatively test for four aminopeptidases (arginyl aminopeptidase, glyclyl aminopeptidase, Leu-AP, and pyroglutamyl aminopeptidase), which cleave N-terminal amino acids from proteins, and trypsin, an endopeptidase, which cleaves proteins mid-chain. Aminopeptidase and the trypsin-like activity were observed in all water bodies, indicating that a diverse set of peptidases is typical in freshwater. However, ratios of peptidase activities were variable among sites: aminopeptidases dominated at some sites and trypsin-like activity at others. At a given site, the ratios remained fairly consistent over time, indicating that they are driven by ecological factors. Studies in which only Leu-AP activity is measured may underestimate the total peptidolytic capacity of an environment, due to the variable contribution of endopeptidases. Keywords: extracellular enzymes, aminopeptidase, endopeptidase, freshwater, trypsin, protein INTRODUCTION Kinetics of extracellular enzymes can give insight into the rates and pathways of organic matter processing in the environment (Schimel and Weintraub, 2003; Arnosti et al., 2014; Sinsabaugh et al., 2014). Diverse classes of extracellular enzymes have been observed in freshwaters, including peptidases, polysaccharide hydrolases, phosphatases, lipases, peroxidases, and laccases (Findlay and Sinsabaugh, 1999). Peptidases can be particularly valuable to microbial communities, because proteins provide organic N as well as C, and because protein-like organic matter is on Frontiers in Microbiology | www.frontiersin.org 1 March 2018 | Volume 9 | Article 368