Label free fluorescence quantification of hydrolytic enzyme activity on native substrates reveal how lipase function depends on membrane curvature

Abstract

Lipases are important hydrolytic enzymes used in a spectrum of technological applications, such as the pharmaceutical and detergent industry. Due to their versatile nature and ability to accept a broad range of substrates they have been extensively used for biotechnological and industrial applications. Current assays to measure lipase activity primarily rely on low sensitivity measurement of pH variations or visible changes on material properties, like hydration, and often require high amount of proteins. Fluorescent readouts on the other hand offer high contrast and even single molecule sensitivity, albeit they are reliant on fluorogenic substrates that structurally resemble the native ones. Here we present a method that combines the highly sensitive readout of fluorescent techniques while reporting enzymatic lipase function on native substrates. The method relies on embedding the environmentally sensitive fluorescent dye pHrodo and native substrates into the bilayer of liposomes. The charged products of the enzymatic hydrolysis alter the local membrane environment and thus the fluorescence intensity of pHrodo. The fluorescence can be accurately quantified and directly assigned to product formation and thus enzymatic activity. We illustrated the capacity of the assay to report function of diverse lipases and phospholipases both in a microplate setup and at the single particle level on individual nanoscale liposomes using Total Internal Reflection Fluorescence (TIRF). The parallelized sensitive readout of microscopy combined with the inherent polydispersity in sizes of liposomes allowed us to screen the effect of membrane curvature on lipase function and identify how mutations in the lid region control the membrane curvature dependent activity. We anticipate this methodology to be applicable for sensitive activity readouts for a spectrum of enzymes where the product of enzymatic reaction is charged.

Direct Observation of Sophorolipid Micelle Docking in Model Membranes and Cells by Single Particle Studies Reveals Optimal Fusion Conditions

“…Liposomes were prepared by freeze-and-thaw method as we did recently [ 26 , 31 , 32 ]. Briefly, after adding the lipid to vial, it was kept under nitrogen flow for 10 min to remove all solvent, and finally, kept in high vacuum for at least one hour.…”

“…All experiments were done using the same experimental setup. Briefly, liposomes was tethered to the glass support as described earlier [ 26 , 31 , 32 ] and visualized by exposing them with a 640 nm laser beam for 50 ms. Similar measurements were recorded with Blue laser 488 to make sure that the background of the experiment was free of crosstalk.…”

“…Similar sets of experiments were performed over all three different pH buffer liposomes. All the data were collected and analyzed by a homemade python script adapted from an earlier publications by our group [ 31 , 32 , 33 ]. The interactions of SL micelles were recorded as docking and kiss-and-run type events on the liposomes.…”