A model explaining declining rate in hydrolysis of lignocellulose substrates with cellobiohydrolase I (Cel7A) and endoglucanase I (Cel7B) of Trichoderma reesei

It is commonly observed that the rate of enzymatic hydrolysis of solid cellulose substrates declines markedly with time. In this work the mechanism behind the rate reduction was investigated using two dominant cellulases of Trichoderma reesei: exoglucanase Cel7A (formerly known as CBHI) and endoglucanase Cel7B (formerly EGI). Hydrolysis of steam-pretreated spruce (SPS) was performed with Cel7A and Cel7B alone, and in reconstituted mixtures. Throughout the 48-h hydrolysis, soluble products, hydrolysis rates, and enzyme adsorption to the substrate were measured. The hydrolysis rate for both enzymes decreases rapidly with hydrolysis time. Both enzymes adsorbed rapidly to the substrate during hydrolysis. Cel7A and Cel7B cooperate synergistically, and synergism was approximately constant during the SPS hydrolysis. Thermal instability of the enzymes and product inhibition was not the main cause of reduced hydrolysis rates. Adding fresh substrate to substrate previously hydrolyzed for 24 h with Cel7A slightly increased the hydrolysis of SPS; however, the rate increased even more by adding fresh Cel7A. This suggests that enzymes become inactivated while adsorbed to the substrate and that unproductive binding is the main cause of hydrolysis rate reduction. The strongest increase in hydrolysis rate was achieved by adding Cel7B. An improved model is proposed that extends the standard endo-exo synergy model and explains the rapid decrease in hydrolysis rate. It appears that the processive action of Cel7A becomes hindered by obstacles in the lignocellulose substrate. Obstacles created by disordered cellulose chains can be removed by the endo activity of Cel7B, which explains some of the observed synergism between Cel7A and Cel7B. The improved model is supported by adsorption studies during hydrolysis.

[1]  F. Tjerneld,et al.  Hydrolysis of steam-pretreated lignocellulose , 1999, Applied biochemistry and biotechnology.

[2]  M. Mandels,et al.  Competitive adsorption of cellulase components and its significance in a synergistic mechanism , 1984, Biotechnology and bioengineering.

[3]  M. Vršanská,et al.  The cellobiohydrolase I from Trichoderma reesei QM 9414: action on cello-oligosaccharides , 1992 .

[4]  H. Ooshima,et al.  Kinetic study on enzymatic hydrolysis of cellulose by cellulose from Trichoderma viride , 1983, Biotechnology and bioengineering.

[5]  Tuula T. Teeri,et al.  Crystalline cellulose degradation : new insight into the function of cellobiohydrolases , 1997 .

[6]  J. Buchert,et al.  Modification of hardwood dissolving pulp with purifiedTrichoderma reesei cellulases , 1996 .

[7]  J. Saddler,et al.  The effect of Trichoderma cellulases on the fine structure of a bleached softwood kraft pulp , 1999 .

[8]  B. Ganem,et al.  Identification of two functionally different classes of exocellulases. , 1996, Biochemistry.

[9]  M. Penttilä,et al.  cDNA cloning of a Trichoderma reesei cellulase and demonstration of endoglucanase activity by expression in yeast. , 1997, European journal of biochemistry.

[10]  B. Henrissat,et al.  Structures and mechanisms of glycosyl hydrolases. , 1995, Structure.

[11]  D. Wilson,et al.  Substrate heterogeneity causes the nonlinear kinetics of insoluble cellulose hydrolysis. , 1999, Biotechnology and bioengineering.

[12]  Mark Holtzapple,et al.  Inhibition of Trichoderma reesei cellulase by sugars and solvents , 1990, Biotechnology and bioengineering.

[13]  G. Pettersson,et al.  Acid hydrolysis of bacterial cellulose reveals different modes of synergistic action between cellobiohydrolase I and endoglucanase I. , 1999, European Journal of Biochemistry.

[14]  A. Converse,et al.  Substrate reactivity as a function of the extent of reaction in the enzymatic hydrolysis of lignocellulose. , 1997, Biotechnology and bioengineering.

[15]  E. Hägglund.  Chemistry of wood , 1951 .

[16]  T. A. Jones,et al.  High-resolution crystal structures reveal how a cellulose chain is bound in the 50 A long tunnel of cellobiohydrolase I from Trichoderma reesei. , 1998, Journal of molecular biology.

[17]  T. Reinikainen,et al.  The three-dimensional crystal structure of the catalytic core of cellobiohydrolase I from Trichoderma reesei. , 1994, Science.

[18]  B. Nidetzky,et al.  Specific quantification of trichoderma reesei cellulases in reconstituted mixtures and its application to cellulase–cellulose binding studies , 1994, Biotechnology and bioengineering.

[19]  B. Henrissat,et al.  A scheme for designating enzymes that hydrolyse the polysaccharides in the cell walls of plants , 1998, FEBS letters.

[20]  Recycling of process streams in ethanol production from softwoods based on enzymatic hydrolysis , 1998, Applied biochemistry and biotechnology.

[21]  J. Sugiyama,et al.  Unidirectional processive action of cellobiohydrolase Cel7A on Valonia cellulose microcrystals , 1998, FEBS letters.

[22]  John N. Saddler,et al.  The effect of initial pore volume and lignin content on the enzymatic hydrolysis of softwoods , 1998 .

[23]  J. Ståhlberg,et al.  Trichoderma reesei has no true exo-cellulase: all intact and truncated cellulases produce new reducing end groups on cellulose. , 1993, Biochimica et biophysica acta.

[24]  G J Kleywegt,et al.  The crystal structure of the catalytic core domain of endoglucanase I from Trichoderma reesei at 3.6 A resolution, and a comparison with related enzymes. , 1997, Journal of molecular biology.

[25]  M. Galbe,et al.  The influence of SO2 and H2SO4 impregnation of willow prior to steam pretreatment , 1995 .

[26]  T. Teeri,et al.  The Cellulases Endoglucanase I and Cellobiohydrolase II of Trichoderma reesei Act Synergistically To Solubilize Native Cotton Cellulose but Not To Decrease Its Molecular Size , 1996, Applied and environmental microbiology.

[27]  J N Saddler,et al.  Factors affecting cellulose hydrolysis and the potential of enzyme recycle to enhance the efficiency of an integrated wood to ethanol process , 2000, Biotechnology and bioengineering.

[28]  A. Schechter,et al.  Tritium labeling of proteins to high specific radioactivity by reduction methylation. , 1980, The Journal of biological chemistry.

[29]  Mats Galbe,et al.  Optimisation of steam pretreatment of SO2-impregnated mixed softwoods for ethanol production , 1998 .

[30]  G. Pettersson,et al.  The initial kinetics of hydrolysis by cellobiohydrolases I and II is consistent with a cellulose surface-erosion model. , 1998, European journal of biochemistry.

[31]  C. Divne,et al.  Trichoderma reesei cellobiohydrolases: why so efficient on crystalline cellulose? , 1998, Biochemical Society transactions.

[32]  F. Tjerneld,et al.  Ion-exchange chromatographic purification and quantitative analysis of Trichoderma reesei cellulases cellobiohydrolase I, II and endoglucanase II by fast protein liquid chromatography , 1998 .

[33]  F. Tjerneld,et al.  Hydrolysis of microcrystalline cellulose by cellobiohydrolase I and endoglucanase II from Trichoderma reesei: adsorption, sugar production pattern, and synergism of the enzymes. , 1998, Biotechnology and bioengineering.

[34]  W. Steiner,et al.  Cellulose hydrolysis by the cellulases from Trichoderma reesei: a new model for synergistic interaction. , 1994, The Biochemical journal.