Abstract: Cellobiose dehydrogenase activity (range 0.25 to 1 iu mL-1) is monitored by oxidation of cellobiose to cellobionolactone. The cellobiose substrate, the 2,6-dichlorophenolindophenol(I) reagent solution in Tris buffer (pH 7.5) and the gluconolactone(II) solution in acetate buffer are mixed before addition of the enzyme. After incubation at 50°C for 20 s the absorbance is measured at 605 nm. The decrease in absorbance is a measure of enzyme activity, because I is reduced to a colorless compound. The presence of II prevents reaction of any β-glucosidase. With a sample throughput of 120 h-1, the correlation coefficient between the flow and manual procedures is 0.96 (n = 40).

Abstract: The sample was diluted with 0.1 M phosphate buffer of pH 7 containing 0.1 M ammonium sulfate and 1.5 g/l of Triton X-405. A portion (40 µL) of the solution was injected into a carrier stream (1.2 mL/min) of 0.2 M phosphate buffer of pH 7 containing 0.1 M (NH4)2SO4 and 1.5 g/l of Triton X-405. Reagent streams (both 0.8 mL/min) of 7.1 mM H2O2 and 1.6 mM 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) were mixed in a 30 cm coil before merging with the carrier stream. The mixture was passed through a 30 cm coil before being incubated at 40°C in a 200 cm coil. The solution was then passed through a 60 cm coil and the absorbance was measured at 418 nm. A diagram of the manifold used is given. Detection and determination limits were 0.005 and 0.05 iu/ml of peroxidase, respectively. Inter- and intra-day RSD (n = 63 and 14, respectively) were 1.1 and 4.8%, respectively. The throughput was ~e;100 samples/h. An automated flow injection method has been developed for the determination of microbial peroxidase activity. The substrate used was hydrogen peroxide and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate (ABTS) was used as the electron donor. In the presence of hydrogen peroxide, peroxidase catalyses the dehydrogenation of ABTS, resulting in the formation of a resonance-stabilized radical cation of ABTS. The green-blue color formed, recorded at 418 nm, is taken as a measure of the peroxidase activity. The general technical conditions and the general enzymatic kinetics have been optimized. Conditions for activation and stabilization of the enzyme were found, e.g., ammonium sulfate acts as a peroxidase activator. The resulting method has a good precision, sensitivity and speed.

Abstract: The method is based on the hydrolysis of 4-nitrophenyl-α-D-glucopyranoside(I) by the cited enzyme, glucan 1,4-α-glucosidase(II), and spectrophotometric determination of the 4-nitrophenol released. The sample (30 µL) is injected into a water carrier stream (1.4 mL min-1), which is mixed with a reagent stream (0.53 mL min-1) containing 2 g L-1 of I in 0.1 M acetate buffer (pH 4.3). The solution is incubated for 20 s at 50°C, passed through a reaction coil at 60°C, and mixed with 0.1 M Na2CO3 (0.53 mL min-1) before the absorbance is measured at 400 nm. Similar procedures have been developed for the determination of α-L-arabinofuranosidase and α-galactosidase activity with 4-nitrophenyl-α-L-arabinofuranoside and 4-nitrophenyl-α-D-galactopyranoside as chromophores, respectively.