Interrelationships between cellulase activity and cellulose particle morphology

Abstract

It is well documented that the enzymatic hydrolysis of cellulose follows a reaction pattern where an initial phase of relatively high activity is followed by a gradual slow-down over the entire course of the reaction. This phenomenon is not readily explained by conventional factors like substrate depletion, product inhibition or enzyme instability. It has been suggested that the underlying reason for the loss of enzyme activity is connected to the heterogeneous structure of cellulose, but so far attempts to establish quantitative measures of such a correlation remain speculative. Here, we have carried out an extensive microscopy study of Avicel particles during extended hydrolysis with Hypocrea jecorina cellobiohydrolase 1 (CBH1) and endoglucanase 1 and 3 (EG1 and EG3) alone and in mixtures. We have used differential interference contrast microscopy and transmission electron microscopy to observe and quantify structural features at lm and nm resolution, respectively. We implemented a semi-automatic image analysis protocol, which allowed us to analyze almost 3000 individual micrographs comprising a total of more than 300,000 particles. From this analysis we estimated the temporal development of the accessible surface area throughout the reaction. We found that the number of particles and their size as well as the surface roughness contributed to surface area, and that within the investigated degree of conversion (\30 %) this measure correlated linearly with the rate of reaction. Based on this observation we argue that cellulose structure, specifically surface area and roughness, plays a major role in the ubiquitous rate loss observed for cellulases.

The Role of Evolving Interfacial Substrate Properties on Heterogeneous Cellulose Hydrolysis Kinetics

“…Furthermore, neither depletion of surface area nor shortening of chains explains long term hydrolysis slowdown 8 . Previous studies have linked structural effects of enzyme degradation to accessibility 41,42,48,[52][53][54][55] , but to our knowledge, the results shown here are the first specifically quantifying the accessibility of cellulose by means of productive binding capacity measurements throughout the duration of hydrolysis.…”

Direct kinetic comparison of the two cellobiohydrolases Cel6A and Cel7A from Hypocrea jecorina

“…All experiments were performed in 50 mM NaAcetate pH 5.0 at 25°C and Avicel (PH101, Sigma Aldrich 11365), that had initially been washed and precipitated five times in buffer, was used as substrate. Avicel consists of microcrystalline cellulose, and the product used here has a typical particle size of 10-50 µm [23]. The quenched flow instruments works better with smaller particles, and the substrate used here was dispersed for 10 min with an ultra Turrax T25 Basic (IKA, Staufen, Germany) coaxial homogenizer with a nominal final particle size of 5 µm.…”

“…microcrystalline-and amorphous cellulose [55,56]. Avicel particles have a complex structure with a high degree of roughness [23], which probably present a diversity of attack sites for the enzymes.…”

A two-phase substrate model for enzymatic hydrolysis of lignocellulose: application to batch and continuous reactors

“…Bansal et al [3] performed a systematic study and found that approximately 90% of the cause of rate retardation was due to substrate depletion, accessibility, and “hydrolysability” (propensity of cellulose to be hydrolyzed by enzymes), while the intrinsic reactivity remained effectively unchanged. Olsen et al [21] found a correlation between hydrolysis rate and surface area (and roughness) of cellulose particles, which decrease with the extent of conversion.…”