A simple method for obtaining reusable reactors containing immobilized trehalase: Characterization of a crude trehalase preparation immobilized on chitin particles

We propose a simple method for obtaining a stable, specific and reusable immobilized trehalase reactor for specific quantification of trehalose. Periplasmic trehalase was extracted from transformed Escherichia coli cells (strain Mph2 carrying the plasmid pTRE11 which harbored the trehalase gene TreA+) by osmotic shock. Periplasmic protein molecules, obtained by osmotic shock, were immobilized on chitin particles (Tyler 35) without any further step of purification. The maximal percentage of trehalase activity retained on chitin was 86% when 2.48 U of trehalase were immobilized on 0.1 g chitin. At pH 5.5 (pH optimum) the optimal temperature was 50 °C. For immobilized trehalase, the apparent Michaelis constant (Km app.) was 0.5 mM trehalose at pH 5.5 and 30 °C with a Vm app. of 0.036 μmol of glucose min−1. On the other hand, for soluble trehalase Km was 1.32 mM at pH 5.5 and 30 °C, with a Vm of 0.011 μmol of glucose min−1. The reactors stored in 50 mM sodium maleate buffer, pH 6.0, at 10 °C, for 55 days and reused 10 times, had no significant loss of activity. Furthermore, the stability of the immobilized conjugate was also tested in a columnar reactor showing no loss of activity during 40 h of continuous operation, at pH 6.0 and 30 °C.