Immobilization of a recombinant thermostable esterase (Pf2001) from Pyrococcus furiosus on microporous polypropylene:: Isotherms, hyperactivation and purification

In this study, a recombinant thermostable esterase (Pf2001 Delta 60) from the hyperthermophilic archaea Pyrococcus furiosus was immobilized on microporous polypropylene (Accurel MP 1000). The adsorption was rapid, with 90% total protein and esterase activity being retained in the first 15 min. No desorption of the enzyme was detected in a time course of 180 min after immobilization, which indicates an intense enzyme-support interaction. The adsorption isotherms from the raw protein extract were determined by total protein quantification and esterase activity in the adsorption medium. Two distinct behaviors were observed: For total protein, the experimental data adjusted well to the Langmuir model (q(m) = 35.3 mg g(-1) and K-a = 16.1 mL mg(-1)), indicating the formation of a monolayer; while the experimental data for esterase activity correlated better with the multilayer Langmuir model (q(m) = 4.4 U/g, K-a = 2223.7 mL U-1 and K-aa = 25.7 mL U-1), indicating the possible formation of a bilayer. Effect of enzyme immobilization on activity (hyperactivation) was evaluated by retention activity parameter (R%). This parameter was dependent of the protein/support ratio at the beginning of the immobilization process. Two hundred and thirty-seven percent hyperactivation was observed when the protein/support ratio was 18 mg/g protein. Furthermore, the immobilization process by means of selective adsorption permitted the purification of the esterase from P. furiosus in a single step. (c) 2007 Elsevier B.V. All rights reserved.