Résumé:
Immobilization and chemical modification of the free or immobilized enzyme are among the most proposed solutions to overcome same enzyme limitations and improve the performance. In this work, Ficin extract was immobilized on glyoxyl, aminated and glutaraldehyde-activated agarose. In the other hand, we studied the effects of chemical modification using glutaraldehyde, carbodiimide and ethylenediamine on the properties of immobilized/stabilized ficin on glyoxyl agarose and free ficin. Finally, we use the immobilized ficin for the milk coagulation. Immobilization of ficin on glyoxyl agarose was performed at pH 10 and 25°C for 3 hours maintaining 60% of activity using BANA (Benzoyl-arginine-p-nitroanilide) and casein at 55 ºC. The obtained biocatalyst was active and stable over a large range of pH and temperatures. The obtained biocatalyst was three times more active compared to the free enzyme in presence of 2 M at 55°C and 65°C and could be reused for fives cycles at 55 ºC without losing activity in hydrolysis of casein. Using aminated supports immobilization yield was very low using both substrates and no effect in enzyme stability. Using aminated supports activated with glutaraldehyde, immobilization was optimal at pH 7 with full immobilisation and 40% of maintained activity using BANA and 30% using casein. Stabilization of ficin was observed after immobilization. The maximum loading obtained was 70 mg/g of supports and more than 30 mg/g activity versus casein decrease using casein and not affected using BANA (due to the steric hindrances). The effect of amination using carbodiimide and ethylenediamine was reported at pH 9, where activity increased over 10% using casein and decreased more than 5 folds using BANA. The specificity of the enzyme has been strongly affected (stability was increased at pH 5 and decreased at pH9). On the other hand, free aminated ficin, retained around 80% of activity versus BANA and 90% versus casein. After optimization a new biocatalyst was compared to the obtained using the non-aminated enzyme. The activity decreased versus BANA, but it increased versus casein. Stability of the new biocatalyst was increased more than the non-aminated ficin biocatalyst mainly at pH 7. This permitted to have more hydrolysis of casein at pH 7 and 66 °C compared to the unmodified enzyme. Glutaraldehyde treatment decreased the activity versus BANA while it improved the activity versus casein. This treatment improves also the thermostability of ficin at pH 7. Besides that, ficin immobilized over glyoxyl agarose with different loading was used in milk coagulation, the remaining activity versus casein was 60%, 58%, 41% and 14%, respectively for 3, 10, 30 and 85mg ficin per gram supports due to casein diffusional limitations. Using the biocatalyst in the two-step milk clotting (enzymatic step at 4ºC to prevent aggregation of hydrolysed casein and then at 40ºC), the yield was 9%, 24%, 27% and 20% for the 10mg/g, 30 mg/g and 85 mg/g immobilized ficin and free ficin respectively, under similar ficin concentrations. These results make these ficin biocatalysts very promising for several application.
