Gel Assay to Introduce Polymer Biodegradation in the Undergraduate Laboratory Austin L. Bolay, William Hiester, Nicole Y. Davis, and Mitch H. Weiland* Cite This: J. Chem. Educ. 2020, 97, 2302-2307 Read Online ACCESS Metrics & More Article Recommendations * sı Supporting Information ABSTRACT: Polymers are ubiquitous and essential to modern society, which is why the American Chemical Society has mandated inclusion of polymer chemistry in the undergraduate curriculum. To meet this requirement, we have chosen to weave a polymeric theme through multiple laboratory courses beginning with organic chemistry, where students use aspartic acid to synthesize poly(aspartic acid), an ecofriendly alternative to non- biodegradable poly(carboxylates). Subsequently, these student-synthesized polymers serve as substrates for the enzyme poly(aspartic acid) hydrolase-1 in our biochemistry course. This experiment introduces the concept of biodegradation through a gel assay that allows students to visualize enzyme-mediated polymer degradation. Students learn the difference between mono- and polydisperse polymers, how biodegradation affects the size of a polymer through analysis of mobility shifts in an agarose gel, and how to use densitometry software to calculate enzyme activity. Finally, keeping the same polymeric theme provides a source of continuity in our curriculum while expanding students’ understanding of polymer chemistry from the viewpoint of different chemistry disciplines. KEYWORDS: Upper-Division Undergraduate, Biochemistry, Polymer Chemistry, Hands-On Learning/Manipulatives, Enzymes, Laboratory Instruction ■ INTRODUCTION Biochemistry courses inherently cover an array of natural polymers; however, rarely are synthetic polymers discussed along with their biodegradation. Guided by the recent changes in the ACS Guidelines and Evaluation Procedures for Bachelor’s Degree Programs, 1 we aimed to incorporate a polymeric theme into our curriculum that would bridge natural and synthetic polymers using poly(aspartic acid) (PAA). PAA is a green alternative to nonbiodegradable polymers like poly(carboxylates) with applications ranging from industrial antiscaling agents to functioning as a superabsorbent material in hygiene products. 2 Additionally, PAA has found utility as a scaffold in biomedical applications with reduced human toxicity due to being composed of a naturally occurring amino acid. 3 Synthesis of PAA is accomplished by heating monomeric aspartic acid to yield a polysuccinamide (PSI). The PSI ring is then opened through the addition of sodium hydroxide to yield thermal PAA (tPAA), which has been well-studied and contains ∼70% β-Asp units, Figure 1. 4 Additionally, tPAA has racemized aspartates and irregular end groups. 5 This simplistic procedure was described as an organic chemistry laboratory exercise to introduce students to a green approach to polymer synthesis 6 and further expanded to include additional characterization of the polymer by IR and NMR. 7 Because the latter experiment is currently used on our campus, we aimed to continue this polymeric theme through develop- ment of a laboratory experiment that introduces students to biodegradation with emphasis on the green chemistry principle of “use of renewable feedstocks.” 8 tPAA is an ecofriendly polymer that can be recycled back to monomeric aspartate by the addition of two PAA hydrolases (PAAH), PAAH-1 and PAAH-2, first isolated from the freshwater bacteria Sphingomonas sp. KT-1. 9 Characterization of the isolated enzymes showed that PAAH-1 cleaves tPAA through the β-linkage to liberate oligo(aspartic acid) (OAA), and PAAH-2 facilitates the breakdown of OAA to mono- mers. 10,11 While others have described undergraduate bio- polymer degradation 12 and biodegradation experiments using soil/compost, 13-16 an undergraduate laboratory experiment using an isolated enzyme has not been discussed. Here, we describe a two-week laboratory exercise that begins with students revisiting concepts from a polymer synthesis laboratory they performed in organic chemistry 7 and using these previously produced synthetic products as substrates for PAAH-1 biodegradation. In week 1, students pour agarose gels and visualize tPAA degradation by PAAH-1. Enzymatic Received: December 27, 2019 Revised: June 18, 2020 Published: July 21, 2020 Laboratory Experiment pubs.acs.org/jchemeduc © 2020 American Chemical Society and Division of Chemical Education, Inc. 2302 https://dx.doi.org/10.1021/acs.jchemed.9b01204 J. Chem. Educ. 2020, 97, 2302-2307 Downloaded via GARDNER WEBB UNIV on November 17, 2020 at 19:17:56 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.