Glucose cii test wako

βGlc(1→4)AF was dissolved and made to 1 mM with the following buffers: 50 mM sodium phosphate buffer (pH 8.0, 7.0, and 6.0) and 50 mM sodium acetate buffer (pH 5.0 and 4.0). The solutions (1.0 mL) were incubated at temperatures of 50, 40, 30, and 20 °C. For each measurement time, aliquots (100 μL) of the reaction mixtures were diluted with 20 mM sodium acetate buffer (900 μL, pH 4.0). Glucose produced by the decomposition was quantified using a glucose oxidase-peroxidase method with a Glucose CII-test Wako (Wako Pure Chemicals), with the following modification. The phosphate buffer solution containing 500 mg/L phenol (pH 7.1) in the kit was substituted with 50 mM 2-morpholinoethanesulfonic acid (MES) buffer (pH 6.0) containing 1 mM N-ethyl-N-(3-sulfopropyl)-3-methylaniline sodium salt (TOPS, Dojindo Laboratories, Mashiki, Japan). The working reagent was mixed with an equal volume of the sample and incubated at 30 °C for 10 min, followed by measurement of the absorbance at 550 nm. The concentration of βGlc(1→4)AF was calculated by subtracting the concentration of Glc measured from the initial concentration of βGlc(1→4)AF. The rate constant (k) was determined through liner regression of the following equation (1).
The half-life (t_1/2) was calculated as ln 2/k.
The activation energy (E_a) at each pH was determined through linear regression of the Arrhenius equation (2).

Culture supernatants of WT or mutant Pc Cel6A (10 µL) were incubated with 100 µg of PASC or cellulose III _I prepared as described previously ¹²⁾¹³⁾ in 100 mM sodium acetate buffer (pH 5.0) with a total volume of 200 µL using 96-well plates at 50 or 60 °C with shaking at 1,000 rpm. After two hours of incubation, the solutions were filtered using 96-well plates with a 0.22 µm filter (MultiScreen ^® Filter Plates, Merck Millipore, MA, USA). Then, 5 µL of 40 U/mL Aspergillus niger β-glucosidase (Megazyme Ltd.) was added to 100 µL of filtrate, and the plates were incubated at 60 °C for 48 h with shaking at 1,000 rpm. After hydrolysis, the solutions were heated at 98 °C for 3 min and filtered again. The concentration of glucose in filtrate was quantified using Glucose CII-Test Wako (FUJIFILM Wako Pure Chemical Corporation) by measuring the absorbance at 492 nm with a Thermo Scientific Multiscan ^® FC (Thermo Fisher Scientific).

To obtain sample solutions,
human insulin, AY-VE-insulin, and YT1-S-insulin
(2.3 mg insulin) were dispersed in 100 mL of PBS (pH 7.4) and dissolved
by adding 1 N or 10 N HCl solution. After adjusting the pH to 7.4
with 1 N or 10 N NaOH solution, the samples were subcutaneously administrated
(38 μg/kg insulin) to 16 h-fasted healthy ddy mice (6 weeks
old, male) (Japan SLC, Inc). At appropriate intervals (0, 1, 2, 4,
6, 8, and 12 h), blood was collected from the jugular veins of the
mice anesthetized with isoflurane. The blood glucose concentration
was determined using the Glucose-CII-Test Wako (Wako Pure Chemical
Industries).