Behaviour and stability of E. coli BL21 whole cells immobilized on modified polyethylene for the synthesis of purine nucleosides

LETICIA BENTANCOR, JORGE A.TRELLES, MARIANO GRASSELLI, ELIZABETH S. LEWKOWICZ, AND ADOLFO M. IRIBARREN,

Darmstadt

Congreso; V Simposio Internacional de Biocatalisis y Biotransformaciones (BIOTRANS 2001); 2001

Universidad de Darmstadt

Nucleoside analogues have been extensively used in cancer and antiviral therapies and, more recently, as building blocks for antisense strategies. Traditionally, nucleosides are prepared by chemical methods which often involve difficult, inefficient and time-consuming multistage processes. Enzymatic and microbial preparations of both natural and unnatural nucleosides are alternative methodologies, which offer regio and stereoselectivity as well as simple experimental conditions. Recently, we have demonstrated the high efficiency of E. coli BL21,[1] in the synthesis of adenosine. This process is based on the action of phosphorylases that catalyze the interconversion of a pyrimidine nucleoside into the corresponding purine one. In order to develop an appropriate procedure to synthesize modified nucleosides we have carried out the immobilization of E. coli BL21 whole cells on current supports as calcium alginate, agar, agarose and polyacrilamide,[2]. Amine containing modified polyethylene macroporous sheets (thickness: 1.5 mm, porosity: 40%, average pore diameter: 120 m) was used as a novel matrix. The immobilization procedure is simple: the support and the cells suspended in phosphate buffer were shaked for 24 h at 30 ºC. Assessment of activity, stability and re-use of the immobilized cells was carried out using the synthesis of adenosine as control reaction. The results showed that this biocatalyst maintained highest activity for more than 45 days at both 4 ºC and 37 ºC, while at 60 ºC the activity dropped off within 2 days. Though the addition of glutaraldehyde as a cell trapping agent, increases stability, its use is not recommended since activity is seriously affected and support recycling (steam sterilization and subsequent treatment with 0.5M NaOH) is not viable.