Evaluation of α-Amylase Assays with 4-Nitrophenyl-α-oligosaccharides as Substrates

Abstract

Measurements of -amylase with 4-nitrophenyl glucosides offer the following advantages over methods that rely on the formation of NADH: a short lag phase, no apparent interference by metabolites and enzymes of the sample and extremely stable Substrates with low blank values. The intrinsic sensitivity of nitrophenyl formation was equal to that of hydrolysis of maltotetraose, but was less than that of glucose-producing methods using oligosaccharides. In contrast to starch, the chromogenic Substrates are more rapidly hydrolysed by salivary than by pancreatic amylase.Disadvantages of these Substrates are:higher turnover by animal than by human amylases, and a marked susceptibility of the chromophore to small changes of pH and protein concentration. Some analytical qualities such äs specificity, accuracy, precision, stability of the Substrate and linear ränge are described in detail and compared with those of other methods. Bewertung von Methoden zur Bestimmung von a-Amylase mit 4-Nitrophenyl-a-oligosacchariden als SubstratenZusammenfassung: -Amylase-Bestimmungen mit 4-NitrophenyIglucosiden sind Methoden, die auf einer Reduktion von NAD beruhen, durch eine kürzere lag phase, fehlende Störung durch Metaboliten und Enzyme der Probe und durch außerordentlich haltbare Substrate mit niedriger Leerreaktion überlegen. Die Empfindlichkeit der Nitrophenolbildung entspricht der Spaltung von Maltotetraose, sie bleibt jedoch hinter dem Entstehen von Glucose aus Oligosacchariden zurück. Im Gegensatz zu Stärke werden die chromogenen Substrate schneller durch Speichel-als durch Pankreasamylase abgebaut.Nachteile ihrer Verwendung sind ein höherer Umsatz durch Amylasen tierischer Herkunft im Vergleich zu menschlichen Enzymen und der starke Einfluß geringer Veränderungen der Proteinkonzentration und des pHWertes im Ansatz. Im einzelnen werden die Spezifität, Richtigkeit und Präzision dieser Verfahren, die Haltbarkeit ihrer Substrate, der lineare Meßbereich und ein Vergleich mit anderen Methoden beschrieben.

Hybrid Living Capsules Autonomously Produced by Engineered Bacteria

“…They have applications in the food, fermentation, textile, paper, detergent and pharmaceutical industries ( 47 ). α-Amylase activity was assessed by incubating the capsules in the presence of a model substrate, 4-nitrophenyl α-D-maltohexaoside, which produces a yellow product upon cleavage by α-amylases ( 48 ). We observed significantly more enzyme activity in capsules containing curli fibers with α-amylase compared to capsules containing wild-type CsgA curli fibers (Fig.…”

Ring-Opening of Cyclodextrins: An Efficient Route to Pure Maltohexa-, Hepta-, and Octaoses

“…The presence of a benzylidene functional group at the non-reducing end of the substrate prevents hydrolysis by α-glucosidase. This observation was made by K. Lorentz in 1983 with the use of unprotected 4-nitrophenyloligosaccharides [48]. Using this substrate in combination with auxiliary enzymes, more than 95% of hydrolyzed substrates in the form of 4-nitrophenol glycosides were obtained.…”

Characterization of the starch surface binding site on Bacillus paralicheniformis α-amylase

“…Kinetic parameters for hydrolysis of the amylase substrate pNP-G6 [56] were first determined for each variant protein and wild type BliAmy and are listed in Table 4, as a confirmation of the integrity of the active site [17]. All variants show almost the same k cat /K m values indicating that they had all folded into the catalytically active form, i.e.…”