Thermal Proteome Profiling and Meltome Analysis of a Thermophilic Bacterial Strain, Geobacillus thermoleovorans ARTRW1: Toward Industrial Applications Merve Oztug, 1–3 Evren Kilinc, 4 Muslum Akgoz, 3 and Nevin Gul Karaguler 1,2 Abstract Thermophilic microorganisms that thrive in extreme environments are of great importance because they express heat-resistant enzymes with the potential to serve as biocatalysts in industrial applications. Thermal proteome profiling (TPP) is a multiplexed quantitative mass spectrometry method for analyses of structural information and melting behavior of thousands of proteins, simultaneously determining the thermal denaturation profiles of each protein. We report, in this study, TPP applied to a thermophilic bacterial proteome, a recently isolated strain of Geobacillus thermoleovorans named as ARTRW1. The proteome was investigated in terms of ther- mostable enzymes that are relevant to industrial applications. In this study, we present the thermostability profiles of its 868 proteins. The majority of G. thermoleovorans proteome was observed to melt between 62.5°C and 72°C, with melting point (T m ) mean value of 68.1°C – 6.6°C. Unfolding characteristics of several enzymes, including amylase, protease, and lipase, were demonstrated which are highly informative in terms of their applicability to specific industrial processes. A significant correlation was observed between protein melting temperature and the structural features such as molecular weight and abundance, whereas correlations were modest or weak in relation to the a-helix structure percentages. Taken together, we demonstrated a system-wide melting profile analysis of a thermal proteome and listed proteins with elevated T m values that are highly promising for applications in medicine, food engineering, and cosmetics in particular. The extracted T m values were found similar to those obtained by biophysical methods applied to purified proteins. TPP analysis has significant industrial and biomedical potentials to accelerate thermophilic enzyme research and innovation. Keywords: thermal proteome profiling, proteomics, meltome, mass spectrometry, Geobacillus thermoleovorans ARTRW1, bioengineering Introduction E nzymes have played a crucial role in many bio- technological processes since ancient times and are widely used in various industrial fields due to their economic and sustainability advantages over chemical catalysts (Choi et al., 2015; Wenda et al., 2011). These advantages mainly appear as lower production costs, higher product efficiency, improved product quality, less energy use, chemical use, and a significant reduction in toxic byproducts and waste disposal (Arbige et al., 2019). The current enzyme market size is es- timated to be in the range of $ 7 billion in the fields of medicine, diagnosis, research, therapeutic, and industrial use. The industrial enzyme market size, by itself, is estimated to be about $ 4.5 billion and annual growth is projected to be 4% (Arbige et al., 2019). Despite the high potential of enzymes, their industrial applications have been mostly restricted due to undesirable properties in terms of thermostability, catalytic efficiency, and specificity (Choi et al., 2015). Thermostability, which is the unique property of an en- zyme, is its ability to resist structural changes caused by the applied heat. Since the reaction conditions used in the industry 1 Department of Molecular Biology and Genetics, Faculty of Science and Letters, Istanbul Technical University, Istanbul, Turkey. 2 Dr. Orhan O ¨ calgiray Molecular Biology-Biotechnology and Genetics Research Center, Istanbul Technical University, Istanbul, Turkey. 3 National Metrology Institute, TUBITAK UME, Gebze, Turkey. 4 Department of Biophysics, School of Medicine, Acıbadem Mehmet Ali Aydınlar University, _ Istanbul, Turkey. OMICS A Journal of Integrative Biology Volume 24, Number 12, 2020 ª Mary Ann Liebert, Inc. DOI: 10.1089/omi.2020.0115 756 Downloaded by 52.91.53.142 from www.liebertpub.com at 01/10/21. For personal use only.