For AchE purification, size-exclusion chromatography was utilized given that the commercially accessible AchE purchased from Sigma was not completely pure. Following purification, the AchE fractions were concentrated using 50kDa Amicon centrifugal filter. The final concentration ranged from 0.5μM to 2 μM, and stock solutions were prepared accordingly. To evaluate the activity of the AchE enzyme, a freshly prepared Ellman’s assay solution containing 3mL 100mM NaHPO4 (pH = 7.8), along with 40μL of Acetylthiocholine chloride (Millipore – Sigma) and 100μL of 5,5'-dithio-bis-(2-nitrobenzoic acid) DTNB (Ellman’s Reagent, Thermo Scientific) was used. The entire experimental
procedure was carried out using NanodropTM 2000c (Thermo Scientific) spectrophotometer in a UV cuvette of Z=8.5mm (ultra-micro, BRAND). This cuvette was loaded with 200 μL of Ellman’s Assay solution and 400 μL of 1x PBS (filtered, Sigma Aldrich), into which 40 μL of AchE enzyme solution was swiftly introduced within a 1 to 2 second timeframe. The enzyme activity was observed at the
absorbance of Ellman’s Assay (412nm) and at the AchE absorbance (280nm). The measured enzymatic activity was around 1000U – 4000U. The enzyme samples were divided into aliquots based on three distinct conditions, encompassing both with and without lyoprotectants as outlined in the table provided and lyophilized for 3 – 4 hours. Depend on the time like 24H and 48H, the enzyme activity was observed at 50°C.
From Table 1, it has been analyzed that trehalose has enzyme active above 1000 U, whereas sucrose slight retains the enzyme activity (>500U). No protectant showed lowest activity (<500U).