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Enzymatic characterization of a chemically modified human sulfamidase and comparison with the recombinant enzyme
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH).
2018 (English)Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
Abstract [en]

Mucopolysaccharidosis (MPS) represents a large group of recessively inherited lysosomal storage diseases, which results from a deficiency of enzymes responsible for the degradation of glycosaminoglycans. One subtype of MPS; type IIIA, is characterized by a deficiency in the gene encoding for the enzyme sulfamidase. This causes accumulation of heparan sulfate resulting in degeneration of the central nervous system commencing during early childhood. Development delays are the first signs followed by severe behavioral problems and mental deterioration. Patients often die at an early stage in life and today there is no effective treatment. The company Swedish Orphan Biovitrum develops modified sulfamidase variants with the aim of creating a successful enzyme-replacement treatment of this disease. Since a chemically modified sulfamidase is a promising drug candidate for patients with MPS IIIA, an enzymatic characterization on activity level is of interest, as well as a comparison with the recombinant sulfamidase. The characterization in this study was based on finding optimum temperature and pH for enzyme activity under current assay conditions, which led to a thermostability study using nano differential scanning fluorimetry. Also, the use of alternative substrates in the enzymatic activity assay and the inhibiting effects of phosphate was investigated. The resulting data indicated an activity optimum at a temperature of 70 °C for the chemically modified sulfamidase and 7 4 °C for the native enzyme. These results could be confirmed with a thermostability study, which gave TM values of 93.5 °C and 96.8 °C respectively. No significant difference in pH optima could be observed; both enzymes had a calculated optimum at pH 6. The results show that both enzymes are very thermostable and inhibiting effects of phosphate was demonstrated. 

Place, publisher, year, edition, pages
2018.
National Category
Natural Sciences Engineering and Technology
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URN: urn:nbn:se:kth:diva-235807OAI: oai:DiVA.org:kth-235807DiVA, id: diva2:1253525
Available from: 2018-10-05 Created: 2018-10-05 Last updated: 2018-10-05Bibliographically approved

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