Trade-off between carbohydrates and metal ions regulates the chemotactic directionality of alkaline phosphatase.

Herein we report the chemotactic behaviour of alkaline phosphatase (ALP) in the gradients of carbohydrates (glucose, fructose and sucrose) and metal ions, including cofactors (Zn2+ and Mg2+), under microfluidic conditions. We found that ALP migrates marginally away from the carbohydrate gradient, whereas for divalent metal ions, the direction is opposite and more prominent. This differential phoresis is due to the Hofmeister effect driven change in the ALP surface zeta potential and osmotic pressure imbalance. Gaining control over the chemotactic extent and direction of an enzyme in response to purely non-catalytic conditions will have potential application in designing environment-responsive nanomachines.

Inhibitory effect of nucleotides on acetylcholine esterase activity and its microflow-based actuation in blood plasma.

The inhibitory effect of nucleotides on the catalytic activity of acetylcholine esterase (AChE) was rationalized and a similar inhibition trend was observed when analyzing the macroscopic fluid flow generated by surface immobilized AChE. Additionally, the demonstration of enzymatic micropumping by showing adenine-nucleotide responsive AChE actuated fluid flow from blood plasma paved the way for designing future lab-on-a-chip devices in complex biological environments with potential clinical applications.

Macromolecular Crowding Effect on the Activity of Liposome-Bound Alkaline Phosphatase: A Paradoxical Inhibitory Action.

The cytoplasm of a cell is extremely crowded, with 20-30% being large biomolecules. This crowding enforces a significant amount of the physical and chemical barrier around biomolecules, so understanding any biomolecular event within the cellular system is challenging. Unsurprisingly, enzymes show a diverse kind of catalytic behavior inside a crowded environment and thus have remained an area of active interest in the last few decades. The situation can become even more complex and exciting in the case of understanding the behavior of a membrane-bound enzyme (almost 25-30% of enzymes are membrane-bound) in such a crowded environment that until now has remained unexplored. Herein, we have particularly investigated how a membrane-bound enzyme (using liposome-bound alkaline phosphatase) can behave in a crowded environment comprising polymer molecule-like poly(ethylene glycol) (PEG) of different weights (PEG400, PEG4000, and PEG9000) and Ficoll 400. We have compared the activity using a polymer microbead conjugated enzyme and have found that liposome-bound alkaline phosphatase had much higher activity in crowded environments, showing the importance and superiority of soft-deformable particles (i.e., vesicles) over hard spheres in macro-molecularly crowded media. Interstingly, we have found a paradoxical behavior of inhibitors in terms of both their extent and pathway of inhibitory action. For instance, phosphates, known as competitive inhibitors in buffer, behave as uncompetitive inhibitors in liposome-bound enzymes in crowded media with an ∼5-fold less inhibitory effect, whereas phenyl alanine (an uncompetitive inhibitor in buffer) did not show any inhibitory potential when the enzyme was membrane-bound and in crowded media containing PEG9000 (30 wt %). Overall, this demonstration elucidates aspects of membrane-bound enzymes in crowded media in terms of both catalytic behavior and inhibitory actions and can lead to further studies of the understanding of enzymatic behavior in such complex crowded environments having a dampening effect in regular diffusive transport.

Sucrose-mediated heat-stiffening microemulsion-based gel for enzyme entrapment and catalysis.

Formation of a thermally stiffening microemulsion-based gel showing a nanoconfinement effect of carbohydrates in terms of microviscosity and hydrodynamic diameter of the reverse micelle (specifically with sucrose) is reported. The advantage of this gel as an efficient batch bioreactor for entrapped enzymes (horseradish peroxidase and thermophilic α-glucosidase) was shown, and illustrated its potential biocatalytic application at high temperatures.