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Institute
A clearing assay for lipolytic enzymes has been realized in 96-well microtiter plates. A thin layer containing emulsified tributyrin as turbidity-generating substrate was placed on a thicker supporting aqueous layer. Both layers were stabilized by a gel-forming agent. Enzyme addition leads to clearing of the emulsion detected with a standard microtiter plate reader as a decrease of extinction. Dependencies of the signal kinetics on the substrate and enzyme concentrations were studied. For 0.5–1 % tributyrin content the reaction rate is not substrate-limited. An initial slope of the signal kinetics is proportional to the lipase activity. A detailed characterization of the assay was performed. Lipolysis of tributyrin was confirmed by glycerol detection. Various gel-forming agents were compared and diffusion conditions in these gels were analyzed. Agar and agarose were found to be the most suitable gel-forming agents, which do not affect enzyme diffusion whereas polyacrylamide gels block lipase diffusion and therefore are not suitable for the assay. The optimized assay prepared from 1 % tributyrin emulsion in 2 % agar gel was tested with six microbial lipases and porcine pancreatic lipase. The detection limit is 20–60 ng/well which is equivalent to 30 μU/well for T. lanuginosus lipase.
For the first time, commercial macroporous melamine formaldehyde foam Basotect® (BT) was used as a basic carrier material for both adsorptive and covalent enzyme immobilization. In order to access inherent amino groups, the Basotect® surface was pretreated with hydrochloric acid. The resulting material revealed 6 nmol of superficial amino groups per milligram Basotect®. Different optimized strategies for tethering the laccase from Trametes versicolor and the lipase from Thermomyces lanuginosus onto the pre-treated Basotect® surface were studied. Particularly, for covalent immobilization, two different strategies were pursued: lipase was tethered via a cross-linking method using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and laccase was bound after functionalizing Basotect® with hydrophilic copolymer poly(ethylene-alt-maleic anhydride) (PEMA). Prior to laccase immobilization, the PEMA coating of Basotect® was verified by ATR-FTIR analysis. Subsequent quantification of available high-reactive PEMA anhydride moieties revealed an amount of 1028 ± 73 nmol per mg Basotect®. The surface-bound enzyme amounts were quantified as 4.1–5.8 μg per mg Basotect®. A theoretical surface-covered enzyme mass for the ideal case that an enzyme monolayer was immobilized onto the Basotect® surface was calculated and compared to the amount of adsorptive and covalently bound enzymes before and after treatment with SDS. Furthermore, the enzyme activities were determined for the different immobilization approaches, and the stability during storage over time and against sodium dodecyl sulfate treatment was monitored. Additionally, PEMA-BT-bound laccase was tested for the elimination of anthropogenic micropollutant bisphenol A from contaminated water in a cost-effective and environmentally-friendly way and resulted in a degradation rate higher than 80%.
Hierarchically structured, porous carbon materials (PCM) were synthesized by sucrose infiltration into template material and subsequent carbonization. Three porous carbon materials were prepared using porous concrete (PCM-01) or silica gel (PCM-02 and 03) as template. Carbon particles from 125 to 200 μm were generated. Surface carboxylic group density was determined with 1.1 mmol/g dry material for each variant. Firstly, tailor-made PCM was evaluated as suitable support for lipase immobilization. Recombinant produced lipase of Thermomyces lanuginosus (TLL) was used as model enzyme. Two independent crude immobilization strategies were applied. Residual activities of up to 8.6 U/g and 31 U/g dry material for adsorptive and covalent immobilization (linkage via EDC) were achieved, respectively. Additionally, TLL was immobilized on commercially available polymethacrylate support showing similar residual lipase activities. Secondly, covalent immobilization was optimized to generate reproducible, highly stable and active immobilizates. Optimized covalent immobilizates showed residual activities of up to 10 U∙g⁻¹ dry carbon material using p-nitrophenyl-palmitate assay and protein loads of up to 45 mg g⁻¹dry carbon material. Covalent bound TLL-PCM showed storage stability for 12 months, remaining 100% of the initial activity. Operational stability resulted in stable and 100% active immobilizates over five consecutive cycles of use. Experiments showed that tailor-made porous carbon material is a promising support for lipase immobilization, which is adaptable in shape and dimension.
Zur Immobilisierung von Enzymen kann man Träger kaufen oder diese selbst herstellen. In früheren Arbeiten wurde ein Verfahren entwickelt, um hochporöse Kohlenstoffmaterialien in beliebigen Dimensionen herzustellen. Als Modell-Enzym zur Immobilisierung wurde die Thermomyces lanuginosus Lipase (TLL) gewählt, die adsorptiv und kovalent an das Material sowie an zwei käufliche Trägermaterialien
auf Polymethacryl-Basis (Funktionalitäten: Ethylendiamin- bzw. Diolgruppen)gebunden werden sollten. Es wurden Kohlenstoffgranulat (d = 125 –200 mm) und zylindrische Monolithe (h = 4 cm; d = 1 cm) synthetisiert. Die adsorptive Immobilisierung ergab am Kohlenstoffgranulat Aktivitäten von bis zu 2,5Ug⁻¹ gegenüber p-Nitrophenyl Palmitat (pNPP). Bei den zwei käuflichen Materialien
wurden Aktivitäten von bis zu 1,8Ug⁻¹ erreicht. Mit der kovalenten Immobilisierung konnten Aktivitäten von bis zu 10Ug⁻¹ am Kohlenstoffmaterial sowie dem Polymethacryl-Material (–OH funktionalisiert) gegenüber pNPP erreicht werden. Das Kohlenstoffmaterial ist ein geeigneter Träger für die TLL, der im Vergleich mit gängigen Materialien Aktivitäten in der gleichen Größenordnung zulässt. Das Porenvolumen des Materials sowie die
Makroporosität könnten erhöht werden, um Diffusions- und Durchströmungsbarrieren im Monolithen abzubauen.