Development and Optimization of Purification Processes and Protein Stabilization Methods for Enzymes Applied for Biocatalytical N-Acetylneuraminic Acid Synthesis

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Dokumentart: Diplomarbeit, Magisterarbeit, Master Thesis
Institut: Department Biotechnologie
Sprache: Englisch
Erstellungsjahr: 2020
Publikationsdatum:
SWD-Schlagwörter: Entwicklung , Optimierung , Reinigung , Reinigungsverfahren , Proteine , Stabilisierung , Methode , Enzym , Biokatalysator , Biokatalyse , Acetylneura
DDC-Sachgruppe: Biowissenschaften, Biologie

Kurzfassung auf Englisch:

The production of N-acetylneuraminic acid with a sequential biocatalysis using two enzymes has a high potential for high yield synthesis. However, the efficiency of enzymatic catalysis depends on the purity, yield and activity of the enzymes. This thesis focuses on the optimization of the purification of his-tagged N-acetylneuraminic acid lyase (Neu5AC lyase) and N-acetyl-D-glucosamine 2-epimerase (GlcNAc 2-epimerase) using immobilized metal affinity chromatography. Additionally, a method for the stabilization of both enzymes during lyophilization was developed. Binding experiments with pure enzymes reveals higher binding capacities on column materials than stated by the manufacturer on Ni2+ (up to 380% higher) and Co2+ (33 to 400% higher) columns. Co2+ columns showed 30 to 50% higher binding capacities compared to Ni2+ resins. Reduced protein binding arises from metal ion binding properties. Metal ion transfer due to high binding affinity coordination sites on the protein is a main cause. A comparison of different column materials and buffers for GlcNAc 2-epimerase purification showed reduced yields when using Tris buffers. Samples disrupted and purified with Tris generated 36 and 13% lower yields than sodium phosphate buffered samples. Protein elution is promoted by competitive binding of amines in Tris buffers which leads to a reduced enzyme yield. The stability of purified GlcNAc 2-epimerase in solution could be maintained over a period of 49 days by applying sterile storage and handling conditions. Additionally, the time of the filtration process could be reduced by 50% by changing the filter system from cellulose acetate membrane filters to polyethersulfone filter capsules. Studies on maintaining enzyme stability during lyophilization showed enzyme-specific results. Mannose proved to be an effective lyoprotectant for GlcNAc 2-epimerase at a concentration of 20 mM in 20 mM Tris buffer. Due to the structural similarity of mannose and the substrate of GlcNAc 2-epimerase, the native form is stabilized by binding the active site. All Neu5Ac lyase samples showed low recovery after lyophilization in Tris buffer. However, the absence of buffer salts prevents pH shifts arising from freeze concentration in eutectic mixtures. Thus, high recoveries could be achieved by freeze-drying Neu5Ac lyase in distilled water.

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