Development of chitin cross-linked enzyme aggregates of L-methioninase for upgraded activity, permanence and application as efficient therapeutic formulations

In this study, L-methioninase (METs) was precipitated from Pseudomonas putida MTCC 9782 and was cross-linked with a cross-linking agent glutaraldehyde to obtain a catalytically active insoluble enzyme. Among the various precipitants tested, ammonium sulfate displayed the highest precipitating (80%) efficiency. A double-response statistical concept, software that provides 20 different runs, was employed to assess the role of precipitant, concentration of cross-linking agent, and duration of cross linking. From the different 20 runs performed, the highest enzyme activity was observed in run 6 (88.17 U): the aggregate size was 11.57 μm, the concentration of saturated ammonium sulfate was 80% and glutaraldehyde 2 mM, and the incubation period was 12 h. R2 values of 0.9754 (enzyme activity) and 0.9203 (aggregate size) were obtained, which showed an enhanced association between the experimental and predicted values of enzyme activity. Enzyme molecules covalently cross-linked with chitin beads showed increased activities compared to free enzymes and enzymes cross-linked with glutaraldehyde. FTIR spectra confirmed the secondary structural alterations between CLEA-METs and chitin-cross-linked CLEA-METs. Thermal stability assays showed that chitin cross-linked CLEA-METs and CLEA-METs retained maximum enzyme activities of 95% and 80% at temperatures 55 °C and 60 °C, respectively. Storage stability assays showed that CLEA-METs retained 65% of their initial activity and chitin-immobilized CLEAs retained 88% of their activity. Moreover, scanning electron microscopy, transmission electron microscopy, and high content screening imaging technique revealed that chitin-immobilized CLEA-MET microspheres showed good monodispersity and mesoporous structure with the amorphous clusters of CLEA with few pores. Cytotoxicity analysis demonstrated that chitin-immobilized CLEA-MET significantly inhibited the proliferation of A549 cells up to 96.66% compared to free enzyme (72%) and CLEA-METs (76%).