The application of proline-specific endoproteinases
(i.e., Brewers Clarex[R], DSM, France) that target on the degradation of haze-active proteins (i.e., hordeins) reduces the formation of storage haze in final beer product.
For the proteolysis, we used Glu-C endoproteinase
from Staphylococcus aureus V8, which cleaves proteins C-terminally from Glu residues.
Abbreviations: CNBr, cyanogen bromide; Lys-C, endoproteinase
Lys-C; MS, mass spectrometry; OCC, oral cavity cancer; RPLC-MS, reverse phase liquid chromatography-mass spectrometry; SCX, strong cation exchange chromatography.
Peptides with endoproteinase
Arg-C digestion in at least one of the termini were considered, with 2 missed cleavages allowed.
Native lectin protein (0.5 mg) was reduced by dithiothreitol, S-carbox-amidom ethylated and digested with endoproteinase
C (substrate:enzyme = 50:1, w/w) in 40 [micro]l of 10 mM Tris- HCl (pH 7.5) at 37[degrees]C for 2 h.
Peptide mapping and evaluation of glycopeptide microheterogeneity derived from endoproteinase
digestion of erythropoietin by affinity high-performance capillary electrophoresis.
Next, the hemolysate is incubated with the endoproteinase
Glu-C for 18 hours at 37[degrees]C to cleave Hb into peptides, whereby the specific glycated and non-glycated N-terminal peptides of the [beta]-chain of Hb are measured by either capillary electrophoresis or electrospray ionization mass spectrometry.
Amin I, Jinap S, Jamilah B (1998) Proteolytic activity (aspartic endoproteinase
and carboxypeptidase) of cocoa bean during fermentation.
The [sup.32]P[O.sub.4]-labeled sample was then subjected to proteolytic digestion with endoproteinase
Asp-N, which cleaves at aspartic acid residues, or aminopeptidase I, which nonspecifically removes amino acid residues inwards from N-terminus, for I hour at room temperature and followed by immunoprecipitation.
Foliar chips were incubated for 48 h in 0.5 ml of phosphate buffer (0.05 M, pH 7.5) that contained 1.95 units of carboxypeptidase B (Sigma), 2 units of endoproteinase
glu-C (Boehringer-Mannheim), or 1.5 units of subtilisin (Boehringer-Mannheim).
For digestion, 2.5 [micro]L of serum was diluted with 50 [micro]L of digestion buffer; the digestion buffer contained 25 mmol/L Tris-HCl pH 7.4, 10 mmol/L dithiothreitol, 9.5 [micro]mol/L isotopic peptide internal calibrator [purified synthetic peptide, amino acid sequence RQIKKQTALVE with isotopic L-leucine (13C6, 15 N) incorporated at position 9, synthetized by Primm Biotech], and 0.1 g/L glutamyl endoproteinase
activity was quantified by using mass spectrometry (3).