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. R 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%).