Abstract
Enzymes are involved in various types of biological processes. In many cases, they are part of multi-component machineries where enzymes are localized in close proximity to each-other. In such situations, it is still not clear whether inter-enzyme spacing actually plays a role or if the colocalization of complementary activities is sufficient to explain the efficiency of the system. Here, we focus on the effect of spatial proximity when identical enzymes are immobilized onto a surface. By using an innovative grafting procedure based on the use of two engineered protein fragments, Jo and In, we produce model systems in which enzymes are immobilized at surface densities that can be controlled precisely. The enzyme used is a xylanase that participates to the hydrolysis of plant cell wall polymers. By using a small chromogenic substrate, we first show that the intrinsic activity of the enzymes is fully preserved upon immobilization and does not depend on surface density. However, when using beechwood xylan, a naturally occurring polysaccharide, as substrate, we find that the enzymatic efficiency decreases by 10–60% with the density of grafting. This unexpected result is probably explained through steric hindrance effects at the nanoscale that hinder proper interaction between the enzymes and the polymer. A second effect of enzyme immobilization at high densities is the clear tendency for the system to release preferentially shorter oligosaccharides from beechwood xylan as compared to enzymes in solution.
Highlights
Enzymes are proteins with specific catalytic activities, accepting only one type of substrate for catalysis, and that play a crucial role in cell metabolism
The coverage density is given in moles per surface area, where surface area refers to the actual available surface of the beads as measured through the BET method
We investigated the effect of grafting the xylanase NpXyn11A at various densities on porous paramagnetic beads both on the specific activity of the system and the size/quality of the products as compared to free enzymes in solution
Summary
Enzymes are proteins with specific catalytic activities, accepting only one type of substrate for catalysis, and that play a crucial role in cell metabolism. A fascinating example of a multienzyme complex (category (ii)) is the cellulosome described for the first time in the thermophilic bacterium Clostridium thermocellum in the early 80 s and that is involved in the degradation of the plant cell wall polysaccharides, primarily cellulose[21] This enzymatic machinery consists of a non-catalytic scaffoldin protein decorated with cohesin modules, each of them being able to bind tightly to a dockerin module fused to a hydrolytic enzyme. Attempts have been made to immobilize GHs on solid supports in a cellulosome-like design[25,33,39,40,41,42,43,44,45] In this configuration, the protein spatial proximity is not tuneable and the enzymatic activity is characterized only through the amount of product generated while the important issue regarding the effect of enzyme proximity on the chemical nature of the products (size, structure) has clearly been overlooked until now. Our results reveal that the enzyme spatial proximity has a substantial impact on both the kinetics of the reaction and the nature of the products released
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