Lipases at Solid Surfaces ­ An Adsorption and Activity Study

Abstract

The adsorption of lipases to solid surfaces was studied by means of in situ ellipsometry. In addition, the activities of the adsorbed lipases were measured in situ, by the hydrolysis of p-nitro phenyl acetate. Adsorption was made to methylated silica surfaces with varying degree of wettability as well as to clean silica surfaces.



Lipases from Humicola lanuginosa (HLL) ­ the wild-type (WT) and a mutant with increased hydrophobicity in the active site region, and lipase B from Candica antarctica (CALB) were investigated. HLL is known to be activated in the presence of an interface. By the displacement of a 'lid', covering the active site in the closed form, the hydrophobic active site region is revealed. CALB does not have this lid but the active site is, however, situated in the middle of a large hydrophobic area.



Studies were performed with respect to concentration, pH, ionic strength and temperature. For HLL-WT and -mutant the effects of surfactants, in mixture with the lipase or sequentially added, on the adsorption and activities of the adsorbed lipases were also investigated. The surfactants used were SDS, C12E5 and Na-dodecanoate.



The adsorbed amounts for all lipases in the study decreased with the hydrophobicity of the surface, the decrease was most pronounced, however, for HLL-WT. The adsorbed amounts were significantly higher for HLL-mutant and CALB than for HLL-WT. Measurements of the activities of the adsorbed lipases show that the specific activity, especially for HLL-WT, increased with increasing wettability of the surface.



It is indicated that the lipase adsorbs with its active site region directed towards the surface when adsorbed to a surface with high hydrophobicity. As the surface hydrophobicity decreases, conformational/orientational changes of the adsorbed lipase presumably occur. These changes probably lead to an increased exposure of the active site to the solution, i. e. access for the water soluble substrate, resulting in increased activity. From the results it is shown that HLL-WT is significantly interfacially activated.



Further, the adsorbed amounts of lipases were low when adsorbed from mixtures with surfactants (conc > cmc). The desorption effects of the surfactants at low concentrations (< cmc) were significant when sequentially added, the non ionic surfactant being more efficient than the anionic ones. No significant effects on the lipase activities were found of SDS or C12E5, at the actual concentrations. The presence of Na-dodecanotae, however, increased the specific activities of the lipases.

Abstract (Swedish)

Popular Abstract in Swedish

Lipasadsorption till fasta ytor studerades med hjälp av in situ ellipsometri. Samtidigt mättes aktiviteten av det adsorberade lipaset in situ genom hydrolys av p-nitrofenylacetat. Adsorption mättes till metylerade kiseloxidytor med varierande grad av metylering samt till rent kiseloxid. Olika lipaser studerades; lipas från Humicola lanuginosa (HLL)­vildtypen (WT) och en mutant med ökad hydrofobicitet i 'active site' området, och lipas B från Candida antarctica (CALB).



Det är känt att HLL aktiveras i närvaro av en yta genom att ett 'lock', som döljer 'active site' i det stängda läget, öppnas och därvid exponerar det hydrofoba 'active site'. CALB saknar förmodligen 'lock mekanismen' men 'active site' är här omgivet av en stor hydrofob region.



Studier gjordes med avseende på koncentration, pH, jonstyrka och temperatur. Inverkan av ytaktiva ämnen på adsorption och aktivitet av adsorberat lipas studerades för HLL-WT och -mutant, i blandning med lipas eller sekventiellt tillsatt. SDS, C12E5 och Na-dodekanoat användes i studien.



Adsorberad mängd minskade med ytans hydrofobicitet för alla tre lipaserna. Minskningen var störst för HLL-WT. Den adsorberade mängden var större för HLL-mutant och CALB än för HLL-WT. Aktivetetsmätningar av adsorberat lipas visar att den specifika aktiviteten, speciellt för HLL-WT, ökar med ökad vätbarhet av ytan. Resultaten indikerar att lipasadsorption till en hydrofob yta sker med 'active site' riktat mot ytan. När ythydrofobiciteten minskar, ändrar sannolikt lipaset orientering/konformation på ytan med en ökad exponering av 'active site' mot lösningen som följd. Härigenom ökar också tillgängligheten för det vattenlösliga substratet och därmed aktiviteten.



Betydelsen av en yta för aktiviteten visades tydligt för HLL-WT. Lipas adsorberade i låga mängder från blandningar med ytaktiva ämnen (konc > cmc).



Desorptionseffekterna av de ytaktiva ämnena (konc < cmc) var tydliga då de tillsattes sekventiellt, störst effekt hade den nonjoniska tensiden.Varken SDS eller C12E5 hade någon inverkan på det adsorberade lipasets aktivitet vid de aktuella koncentrationerna. Närvaron av Na-dodekanoat däremot, ökade lipasaktiviteten.

• author: Wannerberger, Kristin
• supervisor: [unknown] [unknown]
• opponent: Docent Gölander, Carl-Gustaf, Pharmacia Pharmaceuticals, Uppsala, Sweden
• publishing date: 1996
• type: Thesis
• publication status: published
• subject: Food Engineering
• keywords: surfactants, surface wettability, solid surfaces, activity, adsorption, Ellipsometry, lipase, Food and drink technology, Livsmedelsteknik
• pages: 64 pages
• defense location: Chemical Center, Lecture Hall A
• defense date: 1996-03-08 10:15:00
• external identifiers: other:LUTKDH/TKL0-1024/1-64
• language: English
• LU publication?: no
• id: abd70899-2973-435d-89cc-5f0baad6730f (old id 28233)
• date added to LUP: 2016-04-04 09:58:11
• date last changed: 2018-11-21 20:55:56