Introduction

1. Kinetic Investigation of Cellulase Enzyme Using Non-Crystalline Cellulose and Cello-Oligosaccharaides Suma Peri and Y.Y.Lee Department of Chemical Engineering, Auburn University,AL,USA. ABSTRACT Hydrolysis of cellulose by cellulase enzyme is a solid-liquid heterogeneous reaction. As such the reaction is strongly affected by the physical resistances caused most notably by the crystalline structure. Recently we have invented a method to produce non-crystalline cellulose (NCC) from a commercial α-cellulose or cotton by a relatively simple process. This material is drastically different from natural cellulose in that the crystalline structure is disrupted. We have used this material for the kinetic study of cellulose hydrolysis. Among the major findings is that a significant amount of oligomers was found to accumulate throughout the reaction. It appears that oligomers are inhibitory to cellulase enzyme, especially the endo-glucanase. We have also produced cello-oligosaccharides (beta 1-4 glucan) from alpha-cellulose and used as the substrate for cellulase. When treated separately, the oligomers can be easily hydrolyzed to glucose by sulfuric acid, but was not hydrolyzed significantly cellulase enzyme. Introduction Acid and Enzymatic Hydrolysis of Cello-oligosaccharides • The crystallinity of cellulose is a major factor hindering the enzymatic reaction by cellulase. Under the influence of the crystallinity, it is difficult to obtain the intrinsic kinetic information. • We have produced non-crystalline cellulose (NCC) from alpha cellulose by treatment with concentrated sulfuric acid. • The main objective of this work is to investigate the kinetic behavior of enzymatic hydrolysis against NCC and seek additional information that may not be available from crystalline cellulose. Glucose Cellobiose Materials and Methods COS • NonCrystallineCellulose(NCC), and Cello-Oligosaccharides(COS) were prepared in our laboratory. • Cellulase enzyme : Spezyme CP, Genencor, .Lot No. 301-00348-25 • The conditions for acid hydrolysis of cello-oligosaccharideswere 121°C, 20 min., 4%H2SO4. • The enzymatic hydrolysis conditions: 1%(w/v) glucan concentration, pH 4.8, 50ºC and 150 rpm Acid Hydrolysis DP5 DP4 DP3 Cellobiose DP6 Glucose Glucose Glucose Product distribution from the enzymatic hydrolysis of Avicel cellulose and NCC Cellobiose DP4 DP3 Enzymatic Hydrolysis, 15FPU/g-glucan 1 FPU/g- glucan (96 hours) 1 FPU/g- glucan (6 hour) Glucose Cellobiose • Acid hydrolysis of COS resulted in 93% glucose yield in 20 min • Enzymatic hydrolysis gave 17.7% of glucose yield Cellobiose Glucose Avicel Avicel Glucose Enzymatic Hydrolysis of COS and Avicel Cellobiose Glucose Cellobiose NCC NCC Cello-oligosaccharides are more difficult to hydrolyze than Avicel. oligomers oligomers Profiles of glucose, cellobiose, andoligomers in hydrolysis of NCC Summary • Hydrolysis of NCC exhibits extremely high initial rate. The reaction then essentially ceases after 10 hours. • In the enzymatic hydrolysis of NCC, a significant amount of cellobiose and COS are formed as reaction intermediates. • Cellobiose gradually disappeared whereas COS remained constant throughout the enzymatic hydrolysis. • COS are easily hydrolyzed by sulfuric acid, but are not significantly hydrolyzed by cellulase. • Hydrolysis of COS by Spezyme CP was slower than that of Avicel. • COS appear to be inhibitory to endo-glucanase. Oligomers were not degraded throughout the reaction.