RESUMO
BACKGROUND: Immobilization is an appropriate tool to ease the handling and recycling of enzymes in biocatalytic processes and to increase their stability. Most of the established immobilization methods require case-to-case optimization, which is laborious and time-consuming. Often, (chromatographic) enzyme purification is required and stable immobilization usually includes additional cross-linking or adsorption steps. We have previously shown in a few case studies that the molecular biological fusion of an aggregation-inducing tag to a target protein induces the intracellular formation of protein aggregates, so called inclusion bodies (IBs), which to a certain degree retain their (catalytic) function. This enables the combination of protein production and immobilization in one step. Hence, those biologically-produced immobilizates were named catalytically-active inclusion bodies (CatIBs) or, in case of proteins without catalytic activity, functional IBs (FIBs). While this strategy has been proven successful, the efficiency, the potential for optimization and important CatIB/FIB properties like yield, activity and morphology have not been investigated systematically. RESULTS: We here evaluated a CatIB/FIB toolbox of different enzymes and proteins. Different optimization strategies, like linker deletion, C- versus N-terminal fusion and the fusion of alternative aggregation-inducing tags were evaluated. The obtained CatIBs/FIBs varied with respect to formation efficiency, yield, composition and residual activity, which could be correlated to differences in their morphology; as revealed by (electron) microscopy. Last but not least, we demonstrate that the CatIB/FIB formation efficiency appears to be correlated to the solvent-accessible hydrophobic surface area of the target protein, providing a structure-based rationale for our strategy and opening up the possibility to predict its efficiency for any given target protein. CONCLUSION: We here provide evidence for the general applicability, predictability and flexibility of the CatIB/FIB immobilization strategy, highlighting the application potential of CatIB-based enzyme immobilizates for synthetic chemistry, biocatalysis and industry.
Assuntos
Enzimas Imobilizadas/metabolismo , Corpos de Inclusão/metabolismo , Biocatálise , Escherichia coli/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Microbiologia Industrial , Agregados Proteicos , Engenharia de Proteínas/métodos , Relação Estrutura-AtividadeRESUMO
After having received adjuvant FOLFOX treatment consisting of oxaliplatin, folinic acid and fluoruracil following hemicolectomy in colon cancer 7 years ago, the findings of non-cirrhotic portal hypertension presented by acute upper gastrointestinal bleeding in a 49-year old woman were interpreted as oxaliplatin-associated. Imaging techniques, hepatic venous pressure measurement and liver biopsy supported pre-sinusoidal damage due to NRH (nodular regenerative hyperplasia) as the underlying cause, even though histological findings were moderate. Following primary endoscopic treatment, a stable condition has thus far been achievable with standard drug therapy.