Mr. Jóhann Grétar Kröyer Gizurarson, Science Institute, University of Iceland, presents his Masters Thesis in Biochemistry
The improvement of protein stability is important in relation to the use of enzymes as practical biocatalysts and in the use of proteins as pharmaceuticals. All proteins are unstable, especially when in solution. Among the processes that can affect protein stability are proteolytic degradation in vitro or in vivo, thermal denaturation, and antigenicity after injection. Attempts to improve protein stability include protein engineering, immobilization, chemical modification and the use of co-solutes.
In this study bovine trypsin was modified by coupling the enzyme to either D-glucosamine or a partially acetylated chitoologosaccharide via binary carbodiimide/succinimide ester conjugation. D-glucosamine was found to conjugate, on average, to 12 residues on trypsin. The oligosaccharide coupling produced cross-linked polydisperse complexes with hydrodynamical radii from, on average, from 218 to 330 nm for 1:5 and 1:10 cross-linked trypsin, repectively. The physical properties of the enzyme species were studied by electrophoresis, gel filtration, circular dichroism, nanoprticle tracking analysis and MALDI-TOF mass spectrometry. The stability of these enzyme species was studied by activity assays, urea denaturation, diffferential scanning calorimetry and autolysis.
The modified trypsin showed increased resistance against thermal inactivation and autolysis, better storage stability but stability against urea inactivation was unchanged. The proteolytic activity against azo-casein improved for the crosslinked trypsins but was slightly reduced for D-glucosamine conjugated trypsin. The species were found to become basophilic upon conjugation and crosslinking. D-glucosamine conjugated trypsin was found to be slightly structurally altered which and as a consequence displayed 1.2 times higher catalytic efficiency (kcat/Km) than native trypsin against the substrate L-BAPNA.
Thesis Advisor: Dr. Hörður Filippusson, Professor in Biochemistry
Examinor: Dr. Baldur Símonarson, Docent Emeritus in Biochemistry