Immobilization of enzyme on detonation nanodiamond for highly efficient proteolysis.
Immobilization of enzyme on detonation nanodiamond (dND, 3-10nm) and its application for efficient proteolysis have been demonstrated. By evaluation of the Michaelis constant (K(m)) and maximum velocity (V(max)) of immobilized enzyme, its activity was not impaired significantly by immobilization. And enzyme immobilized on dNDs exhibited much better thermal and chemical stabilities than its free counterpart and maintained high activity even after 10 times reuse. The efficient proteolysis by trypsin immobilized on dNDs (dND-trypsin) is demonstrated with the digestion of myoglobin (or other model protein) in a short time (5min). Large numbers of identified peptides obtained by dNDs-trypsin enable a higher degree of sequence coverage and more positive identification of proteins than those obtained by in-solution digestion and the commercial immobilized trypsin beads, respectively. Moreover, immobilization of peptide-N-glycosidase F (PNGase F) on dNDs was realized and resulted in faster sequential glycosidase digestion of glycopeptides in less than 10min.
Wei
Liming
L
Department of Chemistry & Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, China. weiliming@fudan.edu.cn
Zhang
Wei
W
Lu
Haojie
H
Yang
Pengyuan
P
D000595:Amino Acid Sequence; D000818:Animals; D002264:Carboxylic Acids / Q000737:chemistry; D002417:Cattle; D018130:Diamond / Q000737:chemistry*; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000378:metabolism*; D006020:Glycopeptides / Q000378:metabolism; D007094:Imides / Q000737:chemistry; D007700:Kinetics; D008969:Molecular Sequence Data; D009211:Myoglobin / Q000378:metabolism; D049329:Nanostructures / Q000737:chemistry*; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D010449:Peptide Mapping; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000737:chemistry / Q000378:metabolism; D012710:Serum Albumin, Bovine / Q000378:metabolism; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism; D014867:Water / Q000737:chemistry
D002264:Carboxylic Acids; D004800:Enzymes, Immobilized; D006020:Glycopeptides; D007094:Imides; D009211:Myoglobin; D010446:Peptide Fragments; D014867:Water; D012710:Serum Albumin, Bovine; D018130:Diamond; D014357:Trypsin; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase