Appeal No. 1996-1960
Application No. 07/975,167
49; col. 3, lines 36-38). Such substrates include p-nitrophenyl derivatives of mono and
oligosaccharides, wherein in the case of oligosaccharides, the p-nitrophenol radical is attached to the
terminal glucose unit (col. 2, lines 16-19). Preferred substrates include (a) p-nitrophenyl-"-
maltopentaoside and p-nitrophenyl-"-maltohexaoside for determining "-amylase activity, (b)
p-nitrophenyl-"-glucoside and p-nitrophenyl-"-maltoside for determining "-glucosidase activity and (c)
p-nitrophenyl-ß-glucoside and p-nitrophenyl-ß-maltoside for determining ß-glucosidase activity (col. 2,
lines 38-47).
Farnham describes methods for preparing nitroaromatic glycosides useful as substrates in
determining "-amylase activity (col. 1, lines 14-18), comprising a series of acetylation, nitration and
deacetylation steps (col. 1, line 65 - col. 2, line 54). The nitroaromatic residue may be substituted with
halogen (col. 2, lines 14-34).
Blair describes "-amylase assays using oligosaccharide substrates of 4-10 glucose units with a
chromogen, e.g., p-nitrophenol, on the reducing end, wherein the substrate is cleaved into smaller
fragments by "-amylase (an endo-enzyme) and the smaller fragments are cleaved with an exo-enzyme,
e.g., "-glucosidase, ß-glucosidase or glucoamylase, to liberate p-nitrophenol (col. 1, lines 9-24). Blair
discloses an "-amylase substrate comprising an oligosaccharide having at least 3 glucose units, its
reducing end glucose bonded, via a bond cleavable by "- or ß-glucosidase, to a chromogen and its
non-reducing end or terminal glucose unit bonded to a blocking group which inhibits cleavage by exo-
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