C. L. A.

Universidade Federal do Estado do Rio de Janeiro (UNIRIO), Rio de Janeiro, Rio de Janeiro, Brazil

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Publications (1)2.97 Total impact

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    ABSTRACT: 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 2.48U 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 0.5mM trehalose at pH 5.5 and 30 ◦C with a Vm app. of 0.036 �mol of glucose min−1. On the other hand, for soluble trehalase Km was 1.32mM at pH 5.5 and 30 ◦C, with a Vm of 0.011 �mol of glucose min−1. The reactors stored in 50mM 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.
    Enzyme and Microbial Technology 06/2006; 36:Page 486 - 492. · 2.97 Impact Factor

Publication Stats

1 Citation
2.97 Total Impact Points

Institutions

  • 2006
    • Universidade Federal do Estado do Rio de Janeiro (UNIRIO)
      Rio de Janeiro, Rio de Janeiro, Brazil