Immobilization of nitrilase by different immobilization methods and investigation of its uses in the synthesis of carboxylic acids

In the thesis study, the immobilization of various recombinant nitrilase (NIT) enzymes with unknown substrate specificity by different methods, the determination of the most appropriate immobilization method, and their use in the synthesis of corresponding carboxylic acids from nitrile compounds for which immobilized nitrilases are specific were investigated. It was determined that the most suitable nitrilase for the synthesis of acrylic acid, benzoic acid and mandelic acid were NIT-104, NIT-105, and NIT-106 and NIT-P1-118, respectively. Among the applied immobilization methods, the entrapments of NIT-104 and NIT-105 enzymes in PVA gel and the entrapments of NIT-P1-118 in PVA gel and alginate (ALG) gel were the most suitable methods. However, a suitable immobilization method could not be determined for NIT-106. The optimum pH value of all immobilized enzyme samples was found to be 7.5. The optimum temperature values were determined as 30 °C for PVA-NIT-104, PVA-NIT-P1-118 and ALG-NIT-P1-118, and 35 °C for PVA-NIT-105. The Km values for PVA-NIT-104, PVA-NIT-105, PVA-NIT-P1-118 and ALG-NIT-P1-118 were determined as 11.7, 4.9, 26.20 and 26.18 mM, respectively, their corresponding Vmax values were 1.6, 2.0, 6.35 and 1.96 U/mg protein. It was determined that the thermal and storage stability of all immobilized enzyme samples were considerably higher than their free counterparts. ALG-NIT-P1-118 which retained approximately 95% of its initial activity after 20 reuses and PVA-NIT-104 which retained 84% of its initial activity after 15 reuses, were determined as the immobilized nitrilases with the highest reuse stability in a batch reactor. It has been evaluated that PVA-NIT-104 will be quite suitable for acrylic acid synthesis in industrial applications, since it can convert all acrylonitrile at an initial concentration of 0.1 M into acrylic acid within 30 minutes under the determined optimum conditions.