title: Thermostable trypsin conjugates immobilized to biogenic magnetite show a high operational stability and remarkable reusability for protein digestion.
abstract: 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.
authors: Department of Protein Biochemistry and Proteomics, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University, Šlechtitelů 11, 783 71 Olomouc, Czech Republic.
authors: M
authors: M
authors: Pečová
authors: M
authors: M
authors: Šebela
authors: Z
authors: Z
authors: Marková
authors: K
authors: K
authors: Poláková
authors: J
authors: J
authors: Čuda
authors: K
authors: K
authors: Šafářová
authors: R
authors: R
authors: Zbořil
mesh_terms: D000818:Animals; D002417:Cattle; D003505:Cyclodextrins / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000378:metabolism; D017743:Equipment Reuse; D052203:Ferrosoferric Oxide / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D058185:Magnetite Nanoparticles / Q000737:chemistry*; D042201:Magnetospirillum / Q000737:chemistry / Q000378:metabolism; D011506:Proteins / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature; D014357:Trypsin / Q000737:chemistry* / Q000378:metabolism
chemical_list: D003505:Cyclodextrins; D004800:Enzymes, Immobilized; D058185:Magnetite Nanoparticles; D011506:Proteins; D014357:Trypsin; D052203:Ferrosoferric Oxide