Surface characterization and interfacial activity of chitinase chi18-5 against chitosan in langmuir monolayers

One of the challenges for producing active chitinase formulations relies on the gap between the laboratory tests and the biological scenarios where the enzyme will perform its function. In this work, we have employed different Langmuir monolayer arrays to evaluate the interfacial behavior of a recently purified recombinant chitinase, Chi18-5. We have demonstrated that two conformations exist for the chitinase at pH values close to its pI, showing very distinct structural properties at the air/aqueous interface. Enzyme activity was assessed by implementing different kinetic approaches and using a chitosan-1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) mixed film as organized substrate model membrane. Combining these strategies, we demonstrated that better catalytic efficiencies can be obtained for Chi18-5 at pH 5. Moreover, the chitinase activity at the air/aqueous interface can be tuned by introducing in situ pH modifications over the surrounding milieu. We also studied the changes in the topography at the mesoscale level using Brewster Angle Microscopy (BAM). We found that Chi18-5 segregated onto the chitosan domains of the membrane, showing differences in homogeneity depending on the pH imposed. Alternatively, pure Chi18-5 was tested for immobilization onto a hydrophilic activated solid support using the Langmuir-Blodgett technique. Atomic Force Microscopy (AFM) analyses showed successfully stabilization and preservation of molecular features attributed to the pH at which the enzyme deposition was performed.