Highly stable adsorptive and covalent immobilization of Thermomyces lanuginosus lipase on tailor-made porous carbon material

Abstract

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−1 dry 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.