| The stabilization, immobilization and biocatalysis of hydroperoxide lyase (HPL) of the enzymatic extract, from Penicillium camemberti, in neat organic solvent media (NOSM) were investigated. The effects of lyoprotectants, KCl and dextran 1 kDa, on HPL activity and its stability were studied. The addition of KCl to the HPL enzymatic protein 70:1 (w/w), prior to its lyophilization, enhanced its activity by 6.53-fold, whereas the use of dextran resulted in its inactivation. The thermostability of HPL activity of the enzymatic extract at 80°C, in the presence of KCl, was higher by 2.78-fold than that without it. Selected chemical additives, glycine and 2-mercaptoethanol, as well as surfactant coated-enzyme (SCE) were also employed to increase the catalytic activity and stability of HPL enzymatic extract. The presence of 7% glycine or 2.5% of 2-mercaptoethanol in hexane reaction medium increased the HPL activity by 1.76- and 1.22-fold, respectively, as compared to that without additive. The modification of the enzymatic extract by its coating with Span 65, at a surfactant to protein ratio of 1:1 (w/w), resulted in 2.57-fold increase in HPL activity. The presence of 7.0% glycine or 2.5% of 2-mercaptoethnol resulted also by a higher HPL thermostability, with 3.8- and 3.5-fold, respectively, as compared to that without additive; however, the decrease in HPL thermostability was obtained with Span 65 coated-enzyme. The HPL enzymatic extract was encapsulated within alginate and hydrophobically modified alginate hydrogel, using reverse micellar system (RMS) and ternary micellar system (TMS). The dehydration, with 2-propanol, of the encapsulated enzymatic extract increased the HPL specific activity by 1.83-fold as compared to that without dehydration. Using hexane as the reaction medium, the encapsulated HPL extract in 2.0% (w/v) alginate (Alg) resulted by a 52.5% encapsulation yield and 1.37-fold higher enzyme activity than that of the free one. With ratio of 2.5 alginate to 1 of either RMS or TMS, the partition coefficient of 10-HPOD was 2.40- and 1.67-fold, respectively, higher than that of alginate alone. The HPL activity of the enzymatic extract, encapsulated in Alg:TMS (2.5:1), was enhanced by 1.42-fold, as compared to that with alginate alone; however, the inactivation of HPL activity was obtained with Alg:RMS (2.5:1). The immobilization of HPL enzymatic extract, using selected supports, and its biocatalysis in NOSM were also investigated. DowexRTM50WX4-200 was found to be the most appropriated support, with an increase in HPL activity by 2.66-fold as compared to that of the free one; however, an inactivation of HPL activity was obtained with the use of EupergitRTMC250L-IDA, EupergitRTMC250L-EDA and Silica as supports. The chemical modification of HPL enzymatic extract with succinic anhydride, at a molar ratio of 10:1 of succinic anhydride to lysine, increased the immobilization yield by 2.21-fold, as compared to that of the untreated one. The chemical modification of HPL enzymatic extract by 14.37 to 54.98% enhanced the enzyme activity of the immobilized one by 4.28- and 3.31-fold, respectively. On the other hand, the highest HPL enzyme activity in hexane reaction medium of the free and the Dowex immobilized enzyme extracts was obtained with an initial water activity (aw) of 0.10 and 0.33, respectively. In addition, the optimum reaction temperature for the HPL activity was determined to be 55 and 45°C for the free and immobilized enzymatic extract, respectively. |
