The triolein/aqueous interface and lipase activity studied by spectroscopic ellipsometry and coarse grained simulations

Stamm, Arne; Svendsen, Allan; Skjold-Jørgensen, Jakob; Vissing, Thomas; Berts, Ida; Nylander, Tommy

doi:10.1016/j.chemphyslip.2017.10.011

Cited by 12 publications

(16 citation statements)

References 41 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…3C and D). Addition of the S146A mutant resulted in an even lower rate of 12.60 土 12.18 I/s, ~ 6 times lower than the native enzyme as expected for the practically inactive variants 40,45 (see Fig. S7 for all individual histograms of rates).…”

Section: Figure 3 Parallel Imaging Of Lipase Activity On Individual Surface-tethered Liposomes Using Tirfm A) Illustration Of Liposomes Cmentioning

confidence: 58%

“…Several control experiments confirmed that the reported behavior indeed originate from lipase function. We initially tested the effect the S146A lipase variant that is structurally identical to native TLL contains a single mutation to the active serine and have been shown to bind similarly to native TLL on lipid interfaces 23,43,44 but show minute activity 45 (Fig. 2A, red trace).…”

Section: Recordings Of Conversion Of Triglycerides To Fatty Acids By Tllmentioning

confidence: 99%

See 1 more Smart Citation

Label-Free Fluorescence Quantification of Hydrolytic Enzyme Activity on Native Substrates Reveals How Lipase Function Depends on Membrane Curvature

et al. 2020

View full text Add to dashboard Cite

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..

show abstract

Section: Figure 3 Parallel Imaging Of Lipase Activity On Individual Surface-tethered Liposomes Using Tirfm A) Illustration Of Liposomes Cmentioning

confidence: 58%

Section: Recordings Of Conversion Of Triglycerides To Fatty Acids By Tllmentioning

confidence: 99%

Label-Free Fluorescence Quantification of Hydrolytic Enzyme Activity on Native Substrates Reveals How Lipase Function Depends on Membrane Curvature

et al. 2020

View full text Add to dashboard Cite

show abstract

“…These transformations from normal emulsions to fatty acid and monoglyceride-based inverse liquid crystalline structures and further to vesicles and direct (normal) micelles also imply that water molecules start to diffuse into the droplets' interiors and create water pockets, which are eventually transformed into a network of hydrophilic domains in the internal oil-continuous liquid crystalline structures of the formed colloidal objects, see illustration in Figure 3. The lipase-driven transfer of water into the triglyceride (oil) phase at the macroscopic oilewater interface was recently demonstrated experimentally with ellipsometry [72]. The location and activity of the lipase may be triggered by the formation of nanostructures with the extensive water domains inside The triolein digestion products sn-2 monoolein and oleic acid.…”

Section: Dynamic Self-assembly During Emulsion Digestion Studied In Situmentioning

confidence: 99%

Supramolecular structures in lipid digestion and implications for functional food delivery

Salentinig

2019

Current Opinion in Colloid & Interface Science

View full text Add to dashboard Cite

“…Consequently, studying model-thin lipid films upon exposure to an aqueous environment is of fundamental importance as it allows biological processes, such as the action of lipases, to be better understood. Although the lipase-catalyzed hydrolysis of thin triglyceride films has previously been investigated (Snabe and Petersen, 2003;Stamm et al, 2018), the influence of the reaction products such as glycerol, mono-and diglycerides, and free fatty acids (or their salts) on this process is less understood.…”

Section: Introductionmentioning

confidence: 99%

“…From here on, this will be referred to as pH 7 buffer or pH 8.5 buffer. The lipase solutions were prepared at a concentration of 2 ppm TLL, which enabled the kinetics of the digestion to be sufficiently slow to be followed throughout the experiments and is consistent with our previous studies (Stamm et al, 2018). Buffer solutions were stored at 4 °C until required and used within 1 week of preparation.…”

Section: Introductionmentioning

confidence: 99%

The Influence of pH on the Lipase Digestion of Nanosized Triolein, Diolein and Monoolein Films

Humphreys

Campos‐Terán

Arnold

et al. 2022

Front. Soft. Matter

Self Cite

View full text Add to dashboard Cite

Herein we studied the processes at the liquid aqueous interface at pH 7 and 8.5 during Thermomyces lanuginosus lipase-catalyzed hydrolysis of nanosized tri-, di- and mono-olein films deposited on a planar substrate. By employing a combination of ellipsometry, QCM-D and ATR-FTIR, we were able to reveal the physical properties of the thin films at high time resolution throughout the initial hydration and subsequent digestion, as well as the main chemical species present before and after lipolysis. The ATR-FTIR results showed that the degree of digestion and protonated state of the oleic acid produced in the reaction are highly dependent on the pH of the aqueous solvent. Furthermore, the ellipsometry and QCM-D results reveal that the duration of the lag phase observed before lipolysis was detected and the magnitude and type of changes to the physical properties of the thin films throughout digestion was influenced by whether the initial substrate consisted of tri-, di- or mono-olein.

show abstract

The triolein/aqueous interface and lipase activity studied by spectroscopic ellipsometry and coarse grained simulations

Cited by 12 publications

References 41 publications

Label-Free Fluorescence Quantification of Hydrolytic Enzyme Activity on Native Substrates Reveals How Lipase Function Depends on Membrane Curvature

Label-Free Fluorescence Quantification of Hydrolytic Enzyme Activity on Native Substrates Reveals How Lipase Function Depends on Membrane Curvature

Supramolecular structures in lipid digestion and implications for functional food delivery

The Influence of pH on the Lipase Digestion of Nanosized Triolein, Diolein and Monoolein Films

Contact Info

Product

Resources

About