In this work, magnetosomes produced by microorganisms were chosen as a suitable magnetic carrier for covalent immobilization of thermostable
trypsin conjugates with an expected applicability for efficient and rapid digestion of
proteins at elevated temperatures. First, a biogenic
magnetite was isolated from Magnetospirillum gryphiswaldense and its free surface was coated with the natural
polysaccharide chitosan containing free amino and hydroxy groups. Prior to covalent immobilization, bovine
trypsin was modified by conjugating with α-, β- and γ-
cyclodextrin. Modified
trypsin was bound to the magnetic carriers via amino groups using 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide and
N-hydroxysulfosuccinimide as coupling
reagents. The magnetic
biomaterial was characterized by magnetometric analysis and electron microscopy. With regard to their biochemical properties, the immobilized
trypsin conjugates showed an increased resistance to elevated temperatures, eliminated
autolysis, had an unchanged pH optimum and a significant storage stability and reusability. Considering these parameters, the presented enzymatic system exhibits properties that are superior to those of
trypsin forms obtained by other frequently used approaches. The proteolytic performance was demonstrated during in-
solution digestion of model
proteins (
horseradish peroxidase,
bovine serum albumin and hen egg white
lysozyme) followed by mass spectrometry. It is shown that both magnetic immobilization and chemical modification enhance the characteristics of
trypsin making it a promising tool for protein digestion.