Kinetic Investigation and modeling of cellulase enzyme using Non-crystalline cellulose and Cello-oligosaccharides

Abstract

Enzymatic hydrolysis of Cellulose depends on many factors; physical properties of substrate (composition, crystallinity, degree of polymerization etc.), enzyme synergy (origin, composition etc.), mass transfer (substrate adsorption, bulk and pore diffusion etc.) and intrinsic kinetics. Most of these effects occur concurrently, therefore cannot be distinguished from each other. Accurate assessment of intrinsic kinetics requires pure form of cellulosic substrates unhindered by mass transfer resistances, or physical factors of substrate. Non-crystalline cellulose (NCC) and Cello-oligosaccharides (COS) are the products of our laboratory which were used as substrates to study the enzymes. The most notable difference seen in this study is that the activity measured by initial rates against NCC is two orders of magnitude higher than that against crystalline cellulose. Since removal of physical barrier primarily increases the hydrolysis by Endo-glucanase, a significant amount of cello-oligosaccharides and cellobiose was seen to accumulate in hydrolysis of NCC. Cellobiose gradually disappeared whereas cello-oligosaccharides remained constant throughout the enzymatic hydrolysis. The actions of Endo-Glucanases and Exo-Glucanases during the synergism were much more easily distinguished when NCC was used as the substrate. From the experiments conducted on COS, it became apparent that Exo-glucanases cannot act on the soluble substrates. On the other hand, ß-glucosidase acts on the cellobiose as well as the cello-oligosaccharides. To find the inhibitory effects, hydrolysis intermediates and products (NCC, cello-oligosaccharides, cellobiose and glucose) are externally supplied at the initial stages of hydrolysis. The time course data on cellulose, COS, cellobiose, and glucose were taken and incorporated into a comprehensive kinetic model that accounts for inhibitory effects of reaction intermediates and products (cello-oligosaccharides, cellobiose and glucose) to determine the kinetic parameters. The model has shown a good agreement with experimental data.