The Effect of Enzyme Concentration on the Rate of the Hydrolysis of Cellulose W. Sattler zyxwvuts MLKJIHG and H. Esterbauer* zyxwvu MLKJIH Institute zyxwvutsrq EDCBA of Biochemistry, University of Graz, Schubertstrasse 1, A-80 10 Graz, A ustria 0. Glatter Institute of Physical Chemistry, University of Graz, Heinrichstrasse 28 A-80 10 Graz, Austria W. Steiner Institute of Biotechnology, Microbiology and Waste Treatment, Graz University of Technology, Schlogelgasse 9, A-80 10 Graz, Austria Accepted for publication August 10, 1988 zyxwvu EDCBA The relationship among extent of hydrolysis, reaction time, and enzyme dosage was investigated. For this, Sigmacell 50 and pretreated poplar wood (20 g/L) was hydrolyzed with varying dosages of cellulases from three different sources zyxwvuts CBA (5 to 100 FPU/g) for time periods ranging from 2 to 94 h. It was found that the formation of glucose can be described by summation of two paral- lel first order reactions. The extent of hydrolysis at fixed time increases with increasing enzyme dosage in a hy- perbolic function. From the empirical data it is possible to calculate the fractions of easily and difficult hydrolyz- able cellulose and the digestability which could maxi- mally be obtained at infinite enzyme loadings. In the system Sigmacell 50 and Celluclast the easily and diffi- cult hydrolyzable components are 43.0 and 57.0%, re- spectively, and the maximum digestability at 94 h is 82.6%. Poplar wood, steam treated at 200°, 220", and 240°C, showed with Celluclast at 24 h a maximum di- gestability (weight percentage of wood degraded to glu- cose) of 43.9, 64.9, and 68.0%. The relationships derived from experimental data allow one to compare objec- tively the effectiveness of different cellulase enzymes and different pretreatments. INTRODUCTlON zyxwvuts JIHGFED The enzymatic degradation of cellulose is catalyzed by three major enzymes of the cellulase complex: endo- glucanases, exoglucanases, and j3-glucosidases working in a synergistic mechanism. '.* The complex composition of the cellulase system3 and the different physical structure of cellulosic materials complicate the systematic comparison of experimental hydrolysis data. Various kinetic models have been developed to describe the enzymatic hydrolysis of cellulosic and lignocellulosic materials. Some of them are based on the assumption that the reaction rate is proportional to the amount of adsorbed enzyme on the cellulose surface: some of the models are *To whom all correspondence should be addressed based on the structural features of the substrate, like po size distribution, index of crystaIlinity, and specific surfac area,5,6 others are based on the properties of the cellulase enzyme and the mass transfer in the reaction ~ y s t e A theoretical derivation of a hydrolysis model is given b Lee and Fan' and Okazaki and Moo-Young.' Some au- thors'0-12 have developed semiempirical models based on the assumption that the reaction between cellulase and ce lulose can be described by a summation of pseudo-fir order reactions. A distributed parameter model as proposed by Lee and Fan* should take into account the various factors affecting the hydrolysis rate such as structural properties of the su strate, mode of action of the cellulase, adsorption-desorption phenomena, or mass transfer in the system and would al- low a theoretical description of the kinetic behavior of the cellulose-cellulase system. The goal of our investigations was to derive from empiri cal data one mathematical equation which has a minimum of parameters but a wide range of validity and applicabili in respect to degree of hydrolysis, reaction time, type of substrate, and enzyme-substrate ratio. MATERIALS AND METHODS Enzymes Celluclast CCN (lot No. 3000-85/4) was obtained as gift from Novo Industries (Denmark). It is a brownish liquid with a density of 1.1989 g/mL and a protein content of 128.6 mg/g liquid. The measured enzyme activities wer 45 FPU and 6.5 units P-glucosidaselg liquid. Novozym TN 188 (P-glucosidase) was also a gift from Novo. It is a liquid with a density of 1.1884 g/mL and a 6-glucosidase activity of 520 units/g liquid. Biotechnology and Bioengineering, Vol. 33, Pp. 1221-1234 (1989) zyxwvu 0 1989 John Wiley 81 Sons, Inc. CCC 0006-3592/89/0100 122 1 -014$04.00