Haloalkane hydrolysis with an immobilized haloalkane dehalogenase

Haloalkane dehalogenase from Rhodococcus rhodochrous was covalently immobil ized onto a polyethyleneimine impregnated gamma -alumina support, The dehal ogenating enzyme was found to retain greater than 40% of its original activ ity after immobilization, displaying an optimal loading (max. activity/supp orted protein) of 70 to 75 mg/g with an apparent maximum (max. protein/supp ort) of 156 mg/g. The substrate, 1,2,3-triichloropropane, was found to favo rably partition (adsorb) onto the inorganic alumina carrier (10 to 20 mg/g) , thereby increasing the local reactant concentration with respect to the c atalyst's environment, whereas the product, 2,3-dichloropropan-1-ol, demons trated no affinity. Additionally, the inorganic alumina support exhibited n o adverse effects because of solvent/ component incompatibilities or deteri oration due to pH variance (pH 7.0 to 10.5). As a result of the large surfa ce area to volume ratio of the support matrix and the accessibility of the bound protein, the immobilized biocatalyst was not subject to internal mass transfer limitations. External diffusional restrictions could be eliminate d with simple agitation (mixing speed: 50 rpm; flux: 4.22 cm/min). The pH-d ependence of the immobilized dehalogenase was essentially the same as that for the native enzyme. Finally, both the thermostability and resistance tow ard inactivation by organic solvent were improved by more than an order of magnitude after immobilization, (C) 2001 John Wiley & Sons, Inc.