Thin-Layer Chromatography for Maltase Assay

To verify substrate hydrolysis and detect the products formed, MAL1 WT and mutant Asp199Ala reaction mixtures were separated on TLC plates (Silica Gel 60 F₂₅₄) with concentrating zones (Merck, Germany). The reactions were conducted in maltase buffer at 37°C, and at certain time points aliquots were withdrawn and heated for 5 min at 96°C to inactivate the enzyme. Concentrations of 1‐kestose, nystose, melezitose and maltotriose in the reaction mixture were 50 mm, 6‐kestose was used at 10 mm, and malt extract and IMOs were used at 2% w/v. Palatinose and maltose were used at 100 mm in the assay of catalytic ability of the Asp199Ala mutant. Maltase was added at 1 U/ml reaction mixture, or at 247 µg/ml in the case of the Asp199Ala mutant. The amount of protein equivalent to 1 U was determined through activity assays on 1 mmα‐pNPG; 0.5 µl of each of the stopped reaction mixtures were spotted onto TLC plates and sugars were separated with two runs in chloroform:acetic acid:water (6:7:1 v/v/v) (Stingele et al., 1999). Sugars were visualized by immersion of the plates in aniline–diphenylamine reagent and subsequent heating of the dried plates at 120°C (Jork et al., 1990).