α-Synuclein (α-syn) accumulates as insoluble amyloid but also forms soluble α-syn oligomers (αSOs), thought to be even more cytotoxic than fibrils. To detect and block the unwanted activities of these αSOs, we have raised 30 monoclonal antibodies (mAbs) against different forms of αSOs, ranging from unmodified αSOs to species stabilized by lipid peroxidation products and polyphenols, αSOs formed by C-terminally truncated α-syn, and multivalent display of α-syn on capsid virus-like particles (cVLPs). While the mAbs generally show a preference for αSOs, they also bind fibrils, but to variable extents. Overall, we observe great diversity in the mAbs’ relative affinities for monomers and αSOs, varied requirements for the C-terminal extension of α-syn, and only a modest effect on α-syn fibrillation. Several mAbs show several orders of magnitude preference for αSOs over monomers in in-solution studies, while the commercial antibody MJF14 only bound 10-fold more strongly to αSOs than monomeric α-syn. Gratifyingly, seven mAbs almost completely block αSO permeabilization of membrane vesicles. Five selected mAbs identified α-syn-related pathologies like Lewy bodies (LBs) and Lewy Neurites, as well as Glial Cytoplasmic Inclusions in postmortem brains from people diagnosed for PD, dementia with LBs or multiple system atrophy, although to different extents. Three mAbs were particularly useful for pathological evaluation of postmortem brain human tissue, including early stages of PD. Although there was no straightforward connection between the mAbs’ biophysical and immunohistochemical properties, it is encouraging that this comprehensive collection of mAbs able to recognize different aggregated α-syn species in vitro also holds diagnostic potential.

Cold-active enzymes hold promise for energy-efficient processes. Amylases are widely used in household and industrial applications, but only a few are cold-active. Here we describe three novel secreted amylases, Rho13, Ika2 and I3C6, all from bacteria growing in the cold and alkaline ikaite columns in Greenland. They all hydrolyzed starch to smaller malto-oligomers, but only Rho13 and Ika2 hydrolyzed cyclodextrins, and only Ika2 displayed transglycosylation activity. Ika2 forms a stable dimer, while both Rho13 and I3C6 are mainly monomeric. They all have optimal active temperatures around 30–35 °C and significant enzymatic activity below 20 °C, but Rho13 and I3C6 had an alkaline optimal pH, while Ika2 was markedly acidophilic. They showed complex dependence on Ca²⁺ concentration, with the activity of Rho13 and I3C6 following a bell-shaped curve and Ika2 being unaffected; however, removal of Ca²⁺ reduced the stability of all three enzymes. Loss of structure occurred well above the temperature of optimal activity, showing the characteristic psychrophilic divorce between activity and stability. MD simulations showed that Ika2 did not have a well-defined Ca²⁺ binding site, while Rho13 and I3C6 both maintained one stably bound Ca²⁺ ion. We identified psychrophilic features as higher levels of backbone fluctuations compared to mesophilic counterparts, based on a lower number of internal hydrogen bonds and salt bridges. This increased fluctuation was also found in regions outside the active site and may provide easier substrate access and accommodation, as well as faster barrier transitions. Our work sheds further light on the many ways in which psychrophilic enzymes adapt to increased catalysis at lower temperatures.

Better enzymes are needed to develop sustainable methods to recycle plastics with C‐X heterobonds such as polyurethane (PUR) and nylon, for which no industrial‐scale solutions exist. Current methods rely largely on sequence mining based on a small number of known enzymes. Here, we expand the pool of PURases and nylonases by bioprospecting legacy plastic waste with fluorophore plastic mimics combined with fluorescence‐assisted cell sorting (FACS). We identify 29 plastic‐degrading bacteria, from which 12 enzymes are identified by mass spectrometry and homology searches. Compared to existing enzymes, these enzymes show higher thermostability and hydrolytic activities against different high‐molecular weight PUR polymers and nylon textiles compared to previously described wildtype enzymes. To our knowledge, this is the first reported example of enzymes capable of hydrolyzing longer chains of untreated PUR and nylon as well as crosslinked PUR. This study significantly increases the number of known PURases and nylonases and provides starting points for optimization campaigns through protein engineering and for in silico discovery.

Better enzymes are needed to develop sustainable methods to recycle plastics with C‐X heterobonds such as polyurethane (PUR) and nylon, for which no industrial‐scale solutions exist. Current methods rely largely on sequence mining based on a small number of known enzymes. Here, we expand the pool of PURases and nylonases by bioprospecting legacy plastic waste with fluorophore plastic mimics combined with fluorescence‐assisted cell sorting (FACS). We identify 29 plastic‐degrading bacteria, from which 12 enzymes are identified by mass spectrometry and homology searches. Compared to existing enzymes, these enzymes show higher thermostability and hydrolytic activities against different high‐molecular weight PUR polymers and nylon textiles compared to previously described wildtype enzymes. To our knowledge, this is the first reported example of enzymes capable of hydrolyzing longer chains of untreated PUR and nylon as well as crosslinked PUR. This study significantly increases the number of known PURases and nylonases and provides starting points for optimization campaigns through protein engineering and for in silico discovery.

Cold-active enzymes hold promise in energy-efficient processes. Amylases are widely used in household and industrial applications, but only a few are cold-active. Here we describe three novel secreted amylases Rho13, Ika2 and I3C6, all from bacteria growing in the cold and alkaline ikaite columns in Greenland. They all hydrolysed starch to smaller malto-oligomers, but only Rho13 and Ika2 hydrolysed cyclodextrins and only Ika2 displayed transglycosylation activity. Ika2 forms a stable dimer, while both Rho13 and I3C6 are mainly monomeric. They all have optimal temperatures around 30-35℃ and significant activity below 20℃, but Rho13 and I3C6 had an alkaline pH optimum while Ika2 was markedly acidophilic. They show complex dependence on Ca2+ concentration, with activity of Rho13 and I3C6 following a bell-shaped dependence and Ika2 being unaffected; however, removal of Ca2+ reduces stability of all 3 enzymes. Loss of structure occurs well above the optimal activity, showing the characteristic psychrophilic divorce be-tween activity and stability. MD simulations showed that Ika2 did not have a well-defined Ca2+ binding site, while Rho13 and I3C6 both maintain one stably bound Ca2+ ion. We identified psychrophilic features as higher levels of backbone fluctuations compared to mesophilic counterparts, founded in a lower number of internal hydrogen bonds and salt bridges. This increased fluctuation is also found in regions outside the active site and may provide easier substrate access and ac-commodation and faster barrier transitions. Our work sheds further light on the many ways in which psychrophilic enzymes adapt to increased catalysis at lower temperatures.