Hydrolysis Products Analysis

Thin layer chromatography was employed for the examination of sugar products liberated in the hydrolysis of LBG by recombinant enzyme. In this regard, 100 μL of LBG (0.5%) dissolved in PI buffer (pH 5.5) and 100 μL purified enzyme (0.364 mg/mL) were mixed together and incubated at optimum temperature for 15, 30, 45, and 60 min. Then, the mixture was boiled for 10 min, and centrifuged at 7000 × g for 5 min, and the supernatant was frozen till volume reached 10 μL.

The TLC plate (Silica gel-coated aluminum plate, Merck, Germany) was spotted with 1.0 μL of concentrated hydrolyzed sample and standards of mannose, mannobiose and mannotriose. The concentration of the standards was 1 mg/mL in ddH₂O. The plate was developed using acetonitrile: water (65:35 v/v) as the mobile phase. After drying, the plate was stained with 0.3% N-(1-napthyl)ethylenediamine dihydrochloride dissolved in methanol containing 5% H₂SO₄ (Bounias, 1980) to detect the sugars.