Towards a Green Detergent Discovery by cell-free protein synthesis and biochemical characterization of thermostable glycerol oxidases O2 H2O2 Lars L. Santema, Laura Rotilio, Ruite Xiang, Gwen Tjallinks, Victor Guallar, Andrea Mattevi & Marco W. Fraaije A sticky situation The market size of detergents in 2020 was roughly 7 billion USD[1]. A substantial portion of these detergents ultimately enters waste- water streams, and owing to their limited biodegradability, they contribute to the decline of natural water quality[2]. Especially chemicals used for antibacterial properties are harmful for the environment. To avoid the use of these chemicals, we sought out to find new glycerol oxidases to create hydrogen peroxide as bactericidal agent in detergents. By integrating in silico biopros- pecting, cell-free protein synthesis and activity screening, an effective pipeline was developed to rapidly identify glycerol oxidases. Characterization [1] Liquid Laundry Detergent Market Size Report ID: FBI102962 [2] Mousavi S. A. & Khodadoost F. (2019) Effects of detergent on natural exosystems and wastewater treatment processes: a review. Environmental Science and Pollution research 26, 26439 - 26448 [3] Heuts, D. P. H. M., Hellemond, E. W. van, Janssen, D. B., & Fraaije, M. W. (2007). Discovery, characterization, and kinetic analysis of an alditol oxidase from Streptomyces coelicolor. J Biol Chem 282:20283–20291. https://doi.org/10.1074/jbc.M610849200 [4] Winter, R. T., Heuts, D. P. H. M., Rijpkema, E. M. A., van Bloois, E., Wijma, H. J., & Fraaije, M. W. (2012). Hot or not? Discovery and characterization of a thermostable alditol oxidase from Acidothermus cellulolyticus 11B. Appl Microbiol Biotechnol 95:389–403. https://doi.org/10.1007/s00253-011-3750-0 Steady-state kinetics are everything Blast Search Substrate simulations In vitro expression coupled with an HRP assay Activity ‘proven’ in a few hours! Neg Tf St Ab Ch 6 7 8 9 6 5 7 0 7 5 8 0 8 5 9 0 M e ltin g te m p e ra tu re s pH T e m p e ra tu re ( C ) A ldO A b A ld O C h A ldO S t A ldO Tf V 2 5 8 L _ P 2 5 9 I Organism of origin Yield (mg/L culture) KM (mM) kcat (s-1) kcat/ KM (M-1 s-1) references xylitol glycerol xylitol glycerol xylitol glycerol AldOSc S. coelicolor 350 (but a low thermostability) 0.32 350 13 1.6 4.1 x 104 4.6 Heuts et al. 2007[3] AldOAc A. cellulolyticus 2,5 0.07 270 1.9 1.3 2.7 x 104 4.8 Winter et al. 2012[4] AldOAb A. bacterium 75 0.03 184 4.2 2.6 14 x 104 14 this study AldOCh T. chromogena 30 0.04 143 3.5 2.0 12 x 104 14 this study AldOSt S. thermoviolaceus 71 0.02 523 1.9 4.2 9.5 x 104 8 this study AldOTf T. flexuosa 300 0.03 50 3.1 1.6 10 x 104 32 this study V258L_P259I AldOTf T. flexuosa 300 0.04 41 4.3 4.0 11 x 104 98 this study V257L_P258I AldOAb A. bacterium 72 n.d. 157 n.d. 1.4 n.d. 9 this study 1.5 Å resolution structure of AldOAb Room for improvement Conclusion Our Lab About me Read details soon! Manuscript submitted Protein Energy Landscape Exploration V258L P259I • A new pipeline was developed by integrating computational bioprospecting, in vitro expression and an oxidase assay to quickly discover novel glycerol oxidases. • Three thermostable alditol oxidases active on glycerol where found (from Actinobacteria bacterium, Streptomyces thermoviolaceus, and Thermostaphylospora chromogena). • A high resolution crystal structure of the alditol oxidase from the Actinobacteria isolate was obtained. • A structure-inspired double mutant of the alditol oxidase from Thermopolyspora flexuosa was engineered and found to be the most efficient glycerol oxidase known. More!