Comparison of glutaraldehyde cross linking versus direct Schiff base reaction for conjugation of L-asparaginase to nano-chitosan and improvement of enzyme physicochemical properties

The bacterial L-asparaginase (ASNase) has been used in the treatment of asparagine-associated tumors; however, the instability of the enzyme increases the number of injections as well as the side effects. In the present study, ASNase was conjugated to nanochitosan (ASNase -CSNPS) by direct (shiff-base) and indirect (glutaraldehyde linker) methods. In order to get the optimal conjugation, ASNase/CSNPS ratio was first investigated. The physicochemical properties (optimum pH, temperature, residual activity), enzyme kinetics (Michaelis constants; Km and maximal velocity; Vmax) and stability (against freezing, proteolysis, and chemical denaturation) were determined. The results showed that the highest residual enzyme activity (>85%) was obtained using a combination of ASNase and CSNPS at 1:5 mass ratio in both conjugation methods. ASNase -CSNPS prepared by glutaraldehyde linker had higher Km and Vmax values (69.7 μM, 20.6 mol/mL/minμ), wider range of optimum pH and higher temperature stability compared to ASNase-CSNPS produced by Schiff-base (Km: 105.8 μM, Vmax 14.5 mol/ml/minμ) method. ASNase-CSNPS produced by indirect method had more stability against freezing-thawing, and proteolysis when compared with ASNase-CSNPS prepared using direct method. The results showed that application of glutaraldehyde coupling was superior to Schiff base cross linking for conjugating of ASNase to CSNPS and for production of ASNase with better physicochemical properties for future cancer therapy.