title: Preparation and evaluation of dual-enzyme microreactor with co-immobilized trypsin and chymotrypsin.
abstract: The preparation of capillary microfluidic reactor with co-immobilized trypsin and chymotrypsin with the use of a low-cost commercially available enzymatic reagent (containing these proteases) as well as the evaluation of its usefulness in proteomic research were presented. The monolithic copolymer synthesized from glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EDMA) was used as a support. Firstly, the polymerization conditions were optimized and the monolithic bed was synthesized in the fused silica capillary modified with 3-(trimethoxysilyl)propyl methacrylate (γ-MAPS). The polymer containing epoxy groups was then modified with 1,6-diaminohexane, followed by the attachment of glutaraldehyde and immobilization of enzymes. The efficiency of the prepared monolithic Immobilized Enzyme Microreactor (μ-IMER) with regard to trypsin activity was evaluated using the low-molecular mass compound (Nα-benzoyl-l-arginine ethyl ester, BAEE). The activities of both enzymes were investigated using a macromolecular protein (human transferrin, Tf) as a substrate. In the case of BAEE, the reaction product was separated from the substrate using the capillary liquid chromatography and the efficiency of the reaction was determined by the peak area of the substrate. The hydrolysis products of transferrin were analyzed with MALDI-TOF which allows for the verification of the prepared enzymatic system applicability in the field of proteomic research.
authors.affiliation: Chair of Environmental Chemistry and Bioanalytics, Faculty of Chemistry, Nicolaus Copernicus University in Toruń, Gagarina 7, 87-100 Toruń, Poland.
authors.forename: Kinga
authors.initials: K
authors.lastname: Meller
authors.affiliation: Chair of Environmental Chemistry and Bioanalytics, Faculty of Chemistry, Nicolaus Copernicus University in Toruń, Gagarina 7, 87-100 Toruń, Poland; Interdisciplinary Centre of Modern Technologies, Nicolaus Copernicus University in Toruń, Wileńska 4, 87-100 Toruń, Poland.
authors.forename: Paweł
authors.initials: P
authors.lastname: Pomastowski
authors.affiliation: Chair of Environmental Chemistry and Bioanalytics, Faculty of Chemistry, Nicolaus Copernicus University in Toruń, Gagarina 7, 87-100 Toruń, Poland; Interdisciplinary Centre of Modern Technologies, Nicolaus Copernicus University in Toruń, Wileńska 4, 87-100 Toruń, Poland.
authors.forename: Damian
authors.initials: D
authors.lastname: Grzywiński
authors.affiliation: Chair of Environmental Chemistry and Bioanalytics, Faculty of Chemistry, Nicolaus Copernicus University in Toruń, Gagarina 7, 87-100 Toruń, Poland; Interdisciplinary Centre of Modern Technologies, Nicolaus Copernicus University in Toruń, Wileńska 4, 87-100 Toruń, Poland. Electronic address: michu@chem.umk.pl.
authors.forename: Michał
authors.initials: M
authors.lastname: Szumski
authors.affiliation: Chair of Environmental Chemistry and Bioanalytics, Faculty of Chemistry, Nicolaus Copernicus University in Toruń, Gagarina 7, 87-100 Toruń, Poland; Interdisciplinary Centre of Modern Technologies, Nicolaus Copernicus University in Toruń, Wileńska 4, 87-100 Toruń, Poland.
authors.forename: Bogusław
authors.initials: B
authors.lastname: Buszewski
mesh_terms: D001120:Arginine / Q000031:analogs & derivatives / Q000737:chemistry; D019149:Bioreactors; D002853:Chromatography, Liquid; D002918:Chymotrypsin / Q000737:chemistry*; D004800:Enzymes, Immobilized / Q000378:metabolism*; D004852:Epoxy Compounds / Q000737:chemistry; D006801:Humans; D008689:Methacrylates / Q000737:chemistry; D017646:Organosilicon Compounds / Q000737:chemistry; D058105:Polymerization; D011506:Proteins / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D012821:Silanes / Q000737:chemistry; D012822:Silicon Dioxide / Q000737:chemistry; D014357:Trypsin / Q000737:chemistry*
chemical_list: D004800:Enzymes, Immobilized; D004852:Epoxy Compounds; D008689:Methacrylates; D017646:Organosilicon Compounds; D011506:Proteins; D012821:Silanes; C017492:methacryloxypropyltrimethoxysilane; D012822:Silicon Dioxide; C004919:ethylene dimethacrylate; D001120:Arginine; C009727:benzoylarginine ethyl ester; D002918:Chymotrypsin; D014357:Trypsin; C505504:3-(trimethoxysilyl)propyl methacrylate; C007870:glycidyl methacrylate
keywords: Capillary liquid chromatography; Chymotrypsin; Immobilized enzyme microreactor; Proteomics; Transferrin; Trypsin