The flexibility in tuning the structure and charge properties of PNIPAm microgels during their synthesis makes them a suitable choice for various biological applications. Two-step free radical polymerization, a common method employed for synthesis of core-shell microgel has been well adopted to obtain cationic poly(N-isopropylacrylamide-aminoethyl methacrylate) (PNIPAm-AEMA) shell and PNIPAm core. Scanning electron microscopy (SEM), dynamic light scattering (DLS), zeta potential, and ninhydrin assay suggests nearly monodispersed particles of cationic nature. Amino groups on the microgel provides suitable attachment point for covalent immobilization of acetyl coenzyme A synthetase (Acs) via 1-ethyl-3-(3-N,N- dimethylaminopropyl) carbodiimide (EDC) chemistry. On immobilization, 61.55% of initial activity of Acs has been retained, while Michaelis-Menten kinetics of the immobilized Acs indicates identical K(m) (Michaelis constant) but decrease in the V(max) (maximum substrate conversion rate) compared to free enzyme. Immobilized Acs shows an improvement in activity at wide temperature and pH range and also demonstrates good thermal, storage, and operational stability. The Acs-microgel bioconjugate has been successfully reused for four consecutive operation cycles with more than 50% initial activity.