Investigating the hydrolysis reaction of Thermomyces languinosa lipase activity at the triolein/aqueous interface : Influence of solution pH and surfactants on lipase triolein digestion

Abstract

The aim of this study is to reveal how the oil-in-water interface of triolein emulsions in aqueous solutions is affected by the lipase activity when using five different surfactants as emulsifiers. For this purpose Dynamic Light Scattering (DLS) and Small Angle X-ray Scattering (SAXS) were applied. Lipases are enzymes that catalyses the hydrolysis of ester bonds of lipids, i.e lipolysis, and act at the interface between water and oil. Surfactants, along with energy from sonication, can be used to create a stable oil in water emulsion that will significantly increase the interface surface area accessible to the lipase. Surfactant and lipase need to be compatible to not reduce enzyme activity due to lipase denaturation and/or by preventing the lipase to adsorb to the water oil interface. Two different types of Thermomyces languinosa lipase (TLL), wildtype and mutant that has very low catalytic activity, were used along with the five different surfactants as well as at two different pH’s of the Tris Buffer that affect the degree protonation of the fatty acid products and hence their solubility.

Three of the surfactants used, the biosurfactant Rhamnolipid, Model detergent B and J, proved to be a good surfactant where TLL maintained some activity, which was reflected in the observed increase in particle size by DLS and structural changes observed by SAXS. Also, the results showed some pH effects, where less effects are shown at higher pH when oleate rather than the protonated fatty acid is formed. The Sodium Dodecyl Sulfate, SDS, didn’t seem to be compatible with TLL at either pH, which is likely due to the exclusion of lipase from the interface. The other environmentally friendly biosurfactants called Sophorolipid proved to be a surfactant that only allowed enzyme at a higher pH. This might have to do with the pH sensitivity of the Sophorolipid.

Popular Abstract

Lipids are a large and diverse group of organic compounds serving as building blocks that gives structural support, function, signalling and energy storing of living cells. Acylglycerides are a subgroup of lipids where triglyceride are the most common one in edible fats and therefore used in this study. Triglycerides or oil are amphiphilic which means that they consist of a large “water fearing” and very small “water loving” part called the hydrophobic and hydrophilic part. Triglycerides in the presence of a solvent such as water tends to self-assemble aggregate to minimize exposure of its hydrophobic part with the aqueous solution. This creates an interface between the lipids and the aqueous phase important in digestion of lipids catalysed by lipase activated when close to the interface. Surfactants, which are compounds with hydrophobic part and larger hydrophilic part than triglycerides adsorb at the water-oil interface and decrease the surface tension and makes it easier to form oil in water emulsions. Five different surfactants are used in this study at pH 7 or pH 8,5 along with two different types of enzymes, one active and one inactive variant of the Thermomyces langinosa lipase (TLL).

The digestion of triglyceride with lipase results into components such as glycerol, fatty acids, di- and monoglycerides that can accumulate at the interface. Interfacial processes are therefore important to understand biological processes and to develop environmentally friendly industrial processes and ways of cleaning. The aim is to reduce the amount of chemicals with sustainably produced and environmentally friendlier, biodegradable options i.e biosurfactants. The two biosurfactants used in this study are named Rhamnolipid and Sophorolipid.

To reveal the effect of the digestion of triglycerides catalysed by the presence of Thermomyces langinosa lipase together with different surfactants, the structure and size of oil in water emulsions have been done with scattering techniques such as Dynamic Light Scattering (DLS) and Small Angle X-ray Scattering (SAXS). DLS is a technique used in order to determine the average size and size distribution of particles or aggregates in a sample. The DLS measurements indicated a general change and increase of size of emulsion soprlets with the active enzyme for the samples prepared with a majority of the surfactants. SAXS is a technique used to investigate the structural changes as well as average size and shape by analysing scattering that occurs when X-rays passing through a sample. The SAXS results proved that the samples with active enzyme showed structural changes that arise from an increase of products from lipid digestion. Preliminary modelling of the data suggests that these changes involve formation of a shell surrounding the lipid droplets with the majority of surfactants. The results from both techniques showed that TLL proved to be a suitable lipase with all the surfactants at some or both pH-value except with surfactant Sodium Dodecyl Sulfate (SDS). Regarding the exchange to a biosurfactant, only Rhamnolipid proved to be suitable to allow for lipid digestion at both pH-values.

• author: Nildén, Alice
• supervisor: Tommy Nylander
• organization: Department of Chemistry
• course: KEMK10 20212
• year: 2022
• type: M2 - Bachelor Degree
• subject: Chemistry
• keywords: DLS, Lipase, Lipids, SAXS, Surfactants, Physical Chemistry
• language: English
• id: 9081840
• date added to LUP: 2022-06-23 10:52:35
• date last changed: 2022-06-23 10:52:35