Effect of beta-galactosidase on the spin-coated lipid layer of a lipid sponge phase as a carrier for enzyme by ATR-FTIR
(2019) KEMR17 20182Department of Chemistry
- Abstract
- Transport in and through biological membranes that consist of lipid bilayers has a crucial role in many applications in the food or pharmaceutical industries. How to deliver an enzyme through the membranes without damaging the enzymatic activity or the "shelf-life" of the enzyme has been of large interest since 1970 [29-30].
In this work, we have shown that a thin lipid sponge phase (L3) layer can be formed on adiamond crystal of an attenuated total reflectance (ATR) accessory for Fourier transform infrared (FTIR).The lipid system used was composed of diglycerolmonooleate (DGMO), glycerol monooleate - Capmul GMO-50 (GMO-50) and the nanoparticle stabilizer polysorbate 80 (P80) in ethanol (EtOH).
In this coated lipid layer, the enzyme... (More) - Transport in and through biological membranes that consist of lipid bilayers has a crucial role in many applications in the food or pharmaceutical industries. How to deliver an enzyme through the membranes without damaging the enzymatic activity or the "shelf-life" of the enzyme has been of large interest since 1970 [29-30].
In this work, we have shown that a thin lipid sponge phase (L3) layer can be formed on adiamond crystal of an attenuated total reflectance (ATR) accessory for Fourier transform infrared (FTIR).The lipid system used was composed of diglycerolmonooleate (DGMO), glycerol monooleate - Capmul GMO-50 (GMO-50) and the nanoparticle stabilizer polysorbate 80 (P80) in ethanol (EtOH).
In this coated lipid layer, the enzyme beta-galactosidase (beta-gal) could be immobilized and characterized by the catalysis of lactose. Beta-galactosidase is an important enzyme in the food industry that is used to hydrolyze lactose to glucose and galactose for making lactose-free milk or other dairy products that are important for lactose intolerant peoples.
We investigated by ATR-FTIR the catalytic response of the enzyme in lipid layers coated at different spinning times on the crystals and different concentrations of enzyme.
The FTIR signals that are related to the catalytic process of beta-gal were identified at561 cm-1 and 582 cm-1 in the Infrared spectrum. The obtained signal belonged to the produced glucose. Furthermore, to understand the effect of spin-coating and enzyme activity as well as the best condition in term of maximizing the glucose formation- and lipid signals decrease rates for the system and technique, we designed a factorial experiment combining different spinning times (drop-casting, 20and 300 s) and enzyme dilution factors (10, 50 and 100 times). The optimal condition for the highest catalytic rate and lowest disturbance of the lipid signal was further used to investigate the re-usability of the encapsulated enzyme. No conclusions about the re-usability of the encapsulated enzyme could not be drawn from the experiment due to the unreliable collected data. (Less) - Popular Abstract (Swedish)
- Transporten i och genom de biologiska membranerna är väldigt viktig och har många applikationer i mat- och läkemedel industrier. Dessa membranerna består av dubbel-lager av lipid molekyler. Genom att blanda två olika sorter av lipiderna diglycerolmonooleate (DGMO) och glycerol monooleate (GMO-50) med nano-partikelstabilisatorpolysorbate 80 (P80) i överskott av vatten skapar det en lipidblandning med porer i som kallas för ”sponge-fas”. En tidigare studie av Valldeperas visar att de porerna kan fungera som ”bäraren” för enzymet beta-galaktosidas med syftet för att minska nedbrytning av enzymet genom membranerna. Hur som helst, det saknar fortfarande en förståelse för hur enzymet fungerar i lipidblandningen och om enzymet ändrar struktur på... (More)
- Transporten i och genom de biologiska membranerna är väldigt viktig och har många applikationer i mat- och läkemedel industrier. Dessa membranerna består av dubbel-lager av lipid molekyler. Genom att blanda två olika sorter av lipiderna diglycerolmonooleate (DGMO) och glycerol monooleate (GMO-50) med nano-partikelstabilisatorpolysorbate 80 (P80) i överskott av vatten skapar det en lipidblandning med porer i som kallas för ”sponge-fas”. En tidigare studie av Valldeperas visar att de porerna kan fungera som ”bäraren” för enzymet beta-galaktosidas med syftet för att minska nedbrytning av enzymet genom membranerna. Hur som helst, det saknar fortfarande en förståelse för hur enzymet fungerar i lipidblandningen och om enzymet ändrar struktur på lipidblandningen.
Beta-galaktosidas är ett vanligt enzym som används för att bryta ner laktos till glukos och galaktos. Enzymet är viktig för produktionen av laktos-fritt mjölk och andra mejeriprodukter. Denna funktion ska användas för att studera strukturen av lipidblandningen i närvaran av β-gal. Enzymet ska blandas in i lipidformuleringen innan en tunn lager av mixen skapas med hjälp av ”spin-coating” tekniken. Olika kombinationer av ”spinning” tid och enzymets koncentrationmåste testas för att få fram den optimala förhållande vad gällande maxglukosmängd bildad och reaktionshastigheten.
I detta projekt har mätningar av aktivitet av enzymet, beta-galaktosidas,som kapslas in i lipids porerna gjorts med spektroskopiska mätmetoden, FTIR-ATR (Fourier Transform Infared Spectroscopy-Attenuated Total Reflection) som mäter vibrationerna av molekyler när en röntgenstrålning passerar genom dom. Mätningarna visar att aktiviteten av enzymet kan tämmas av FTIR-ATR och enzymet är fortfarande aktivt även när det är kapslat av lipidblandningen. Signifikanta signaler av produkten glukos kan också detekteras efter två timmars reaktionenstid. Tyvärr,glukos bildad hastigheten kan inte förutsägas med bara två faktorerna ”spinning tid” och ”enzymets koncentration”, ytterligare faktorer behövs för att kunna fatta en konkret slutsats om hastigheten. (Less)
Please use this url to cite or link to this publication:
http://lup.lub.lu.se/student-papers/record/8976661
- author
- le, Daphne LU
- supervisor
- organization
- course
- KEMR17 20182
- year
- 2019
- type
- H2 - Master's Degree (Two Years)
- subject
- keywords
- Lipid sponge phase (L3), DGMO/GMO-50/P80, beta-galactosidase, enzymatic activity, spin-coating, ATR-FTIR, physical chemistry, fysikalisk kemi
- language
- English
- id
- 8976661
- date added to LUP
- 2019-07-04 13:29:57
- date last changed
- 2019-07-04 13:29:57
@misc{8976661,
abstract = {{Transport in and through biological membranes that consist of lipid bilayers has a crucial role in many applications in the food or pharmaceutical industries. How to deliver an enzyme through the membranes without damaging the enzymatic activity or the "shelf-life" of the enzyme has been of large interest since 1970 [29-30].
In this work, we have shown that a thin lipid sponge phase (L3) layer can be formed on adiamond crystal of an attenuated total reflectance (ATR) accessory for Fourier transform infrared (FTIR).The lipid system used was composed of diglycerolmonooleate (DGMO), glycerol monooleate - Capmul GMO-50 (GMO-50) and the nanoparticle stabilizer polysorbate 80 (P80) in ethanol (EtOH).
In this coated lipid layer, the enzyme beta-galactosidase (beta-gal) could be immobilized and characterized by the catalysis of lactose. Beta-galactosidase is an important enzyme in the food industry that is used to hydrolyze lactose to glucose and galactose for making lactose-free milk or other dairy products that are important for lactose intolerant peoples.
We investigated by ATR-FTIR the catalytic response of the enzyme in lipid layers coated at different spinning times on the crystals and different concentrations of enzyme.
The FTIR signals that are related to the catalytic process of beta-gal were identified at561 cm-1 and 582 cm-1 in the Infrared spectrum. The obtained signal belonged to the produced glucose. Furthermore, to understand the effect of spin-coating and enzyme activity as well as the best condition in term of maximizing the glucose formation- and lipid signals decrease rates for the system and technique, we designed a factorial experiment combining different spinning times (drop-casting, 20and 300 s) and enzyme dilution factors (10, 50 and 100 times). The optimal condition for the highest catalytic rate and lowest disturbance of the lipid signal was further used to investigate the re-usability of the encapsulated enzyme. No conclusions about the re-usability of the encapsulated enzyme could not be drawn from the experiment due to the unreliable collected data.}},
author = {{le, Daphne}},
language = {{eng}},
note = {{Student Paper}},
title = {{Effect of beta-galactosidase on the spin-coated lipid layer of a lipid sponge phase as a carrier for enzyme by ATR-FTIR}},
year = {{2019}},
}