Correlating enzyme density, conformation and activity on nanoparticle surfaces in highly functional bio-nanocomposites

Abstract

The biological activity of the immobilized enzyme is crucial for the performance of different nanoparticle mediated enzymatic assays, where enzymatic conversion can be used for label-free analyte detection. In this article we have addressed two significant aspects of enzyme-nanoparticle interactions. First, we have developed copper sulfide (CuS) nanoparticles with an average diameter of 25 nm as a potential enzyme-interface using trypsin protease as a model enzyme. CuS nanoparticles showed high trypsin immobilization capacity of about 14.0 mg m(-2) with the significant retention of native enzymatic activity (75-98%) at room temperature, even beyond the calculated tightly packed monolayer coverage (which is around 4.1 mg m(-2)). Second, we report a quantitative correlation between the structure-functional relationship and the density of immobilized trypsin on a nanoparticle surface. The in situ conformation of immobilized trypsin could be efficiently analyzed by fluorescence, circular dichroism and FT-IR spectroscopic measurements because of the small size of the nanoparticles. Trypsin molecules appear to retain their close-native tertiary and secondary structural features (with a small loss of 1-2% of helical content) in the entire surface density range (2.0-14.0 mg m(-2)) on the CuS nanoparticles. However, interestingly, at a low surface coverage (2.0 mg m(-2)), immobilized trypsin retains almost 98% of its native enzymatic activity, leading to a highly functional bio-nanocomposite. However, at higher surface coverages, the enzyme activity decreases to 77%, indicating the influence of steric crowding. Furthermore, the high functionality of the immobilized trypsin at low surface density on CuS nanoparticle was also confirmed by determining the kinetic parameters of enzymatic activity.

Production of β‐Lactoglobulin hydrolysates by monolith based immobilized trypsin reactors

“…Based on Saha et al. the molecular dimension of native trypsin from porcine pancreas is 4.8 nm x 3.7 nm x 3.2 nm. Assuming that the spatial dimensions of bovine‐derived trypsin are similar and that enzyme molecules are surrounded by a hydration layer of approximately 0.2 nm, one trypsin molecule would occupy between 10 and 20 nm 2 surface.…”

“…Considering BET surface areas and the molar mass of trypsin from bovine pancreas (23.8 kDa), each trypsin molecule in N1 and N2 occupies approximately 17 and 11 nm 2 of surface, respectively. Based on Saha et al [22] the molecular dimension of native trypsin from porcine pancreas is 4.8 nm x 3.7 nm x 3.2 nm. Assuming that the spatial dimensions of bovine-derived trypsin are similar and that enzyme molecules are surrounded by a hydration layer of approximately 0.2 nm, one trypsin molecule would occupy between 10 and 20 nm 2 surface.…”

Physisorption of enzymatically active chymotrypsin on titania colloidal particles

“…Due to these low coverage densities, the formation of multilayers is negligible and lateral interactions between adsorbed enzymes are rather weak. Saha et al [58] showed that the activity of trypsin on CuS nanoparticle depends on surface density. When trypsin molecules form a complete monolayer the 98% of the native enzyme activity was almost retained.…”

A Robust and General Approach to Quantitatively Conjugate Enzymes to Plasmonic Nanoparticles

“…For example, it has been found that the density of enzymes on the nanoparticle surface directly impacts the enzyme activity, which needs to be taken into account when using enzymatic conversions for label-free analyte detection. 62 Enzyme quantity and distribution were also established to be crucial in the performance of enzyme-powered nanomotors, where a certain threshold of enzyme numbers and asymmetric distribution was needed to generate nanoparticle motion. 18,19…”