Understanding the interactions between biological macromolecules and material surfaces is important for various biotechnological applications. In this context, QCM-D analysis can offer insight and shed light on relevant interfacial processes. In this blog post, we present an example where QCM-D was used to optimize the enzymatic hydrolysis of cellulose, including the study motivation, measurement concepts, data analysis, and future possibilities.
The enzymatic conversion of lignocellulose – the most abundant carbohydrate on the planet – into sugar monomers is an important need for biorefinery and clean energy applications. The binding of cellulase enzymes to cellulose is an important step in the enzymatic process, however, it can be difficult for cellulose to bind effectively, which in turn hinders enzymatic efficiency. There is interest in developing methods to improve efficiency and one promising strategy involves adding nonenzymatic expansin proteins, which can loosen the cellulose structure to aid cellulase binding. The effect of expansin on improving cellulase efficiency has been debated in the scientific literature and QCM-D technology has provided unique capabilities to study the real-time adsorption of cellulase to cellulose-coated surfaces in the presence of expansins along with resulting enzyme activity details
The real-time enzymatic hydrolysis of thin cellulose films prepared on cationic polymer-modified gold surfaces was studied by the QCM-D technique. The measurement protocol involved first incubating the cellulose-coated surfaces in aqueous buffer to allow the cellulose films to swell until reaching equilibrium condition as indicated by stable QCM-D signals. Then, expansin and one of two cellulase types were injected into the measurement chamber containing the swollen cellulose film, and different expansin concentrations were tested. It was possible to track cellulase and expansin adsorption onto the cellulose film, followed by material desorption due to hydrolytic processes. Various quantitative parameters were evaluated, including initial adsorption rate, maximum adsorption capacity, and hydrolysis rate. The Voigt model was applied to QCM-D data collected at multiple overtones to estimate changes in film thickness and viscosity, and kinetic modeling was also performed
The QCM-D data demonstrated that protein adsorption occurred first, followed by net mass desorption from the sensor surface. When one cellulase type and expansin were added together, it was discovered that an optimal 1:1 ratio of the two proteins had the highest adsorption rate constant and hydrolytic rate whereas a higher expansin ratio demonstrated competitive binding that impeded enzymatic efficiency (Fig. 1). A similar trend in the adsorption rate constant and hydrolytic rate values was observed with the second cellulase type, reinforcing that the presence of expansin can improve the rate of cellulase activity by up to five-fold compared to without expansin but also that excess expansin can lead to sub-optimal rate enhancement.
Figure 1. Effect of expansin protein on lignocellulose hydrolysis by cellulase enzyme. Expansin proteins bind to cellulose in order to loosen structure and aid enzyme binding as well. Measured levels of cellulase enzyme-mediated mass change rates, adsorption capacities, and hydrolysis rates in the presence of different expansin protein concentrations.
The QCM-D technique provided a label free approach to quantify multiple steps of complex enzymatic processes in order to optimize processing conditions. In this case study, the main focus was on directly verifying the enhancing effect of expansins on cellulase enzymes and also unraveling which mechanistic factors drive the synergism. Importantly, it was possible to discover that expansins work optimally at relatively low concentrations but higher concentrations can impede cellulase binding due to competitive adsorption. These mechanistic details could be discerned readily based on the experimental design and additional parameters like solution pH, temperature, and flow conditions are easy to study with the QCM-D approach
There are a wide range of enzymatic processes involving different classes of biomacromolecules that can be studied using the QCM-D technique. Additional examples include proteolytic degradation of protein films and phospholipase hydrolysis of lipid films as well as replicases for genome replication. Such studies can be useful to elucidate fundamental molecular mechanisms while increasing attention has been placed on using the QCM-D approach to optimize processing conditions to maximize enzymatic performance. In some cases, it is also possible to test potential inhibitors of certain enzymes in order to determine potency and mechanisms of action, which can be relevant to medical and biotechnology applications.
Download the overview to learn more about how QCM-D can be used to measure molecular interaction at surfaces and interfaces in biotechnology and biophysics.
Malin graduated in engineering physics in 2006, where her research focused on the QCM-D technology. Since then, she has been scrutinizing the how’s and why’s of the world in general, and the world of QCM-D in particular.
