Monitoring Cellulase Protein Adsorption and Recovery Using SDS-PAGE

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was employed to study the sorption behaviors of cellulases on microcrystalline celluloses and hardwood pulp. The adsorption and recovery of cellulases from Aspergillus niger and Trichoderma reesei were investigated at 25 °C. Cellulase recovery was conducted by rinsing adsorbed enzymes with sodium acetate buffer, Milli-Q water, and sodium hydroxide solution. The initial, equilibrium, and recovered enzymes were analyzed using SDS-PAGE gels. Gels were scanned and analyzed using ImagePro software. The molecular weights of cellulase proteins were determined using a protein marker having seven known proteins. The cellulase system from Trichoderma reesei had a higher adsorption on all substrates studied than the cellulase system from Aspergillius niger, and higher pH favored desorption from the substrates studied. Experimental results also demonstrated that adsorption and desorption amounts determined by SDS-PAGE were proportional to protein concentrations in their crude mixtures.

[1]  O. Rojas,et al.  In situ monitoring of cellulase activity by microgravimetry with a quartz crystal microbalance. , 2009, The journal of physical chemistry. B.

[2]  Gang Hu.  Adsorption and activity of cellulase enzymes on various cellulose substrates , 2009 .

[3]  Charles E. Wyman,et al.  An improved method to directly estimate cellulase adsorption on biomass solids , 2008 .

[4]  Xavier Turon,et al.  Enzymatic kinetics of cellulose hydrolysis: a QCM-D study. , 2008, Langmuir : the ACS journal of surfaces and colloids.

[5]  John A. Heitmann,et al.  Feedstock pretreatment strategies for producing ethanol from wood, bark, and forest residues , 2008, BioResources.

[6]  A. Sundberg,et al.  Polysaccharides in some industrially important hardwood species , 2005, Wood Science and Technology.

[7]  I. S. Pretorius,et al.  Microbial Cellulose Utilization: Fundamentals and Biotechnology , 2002, Microbiology and Molecular Biology Reviews.

[8]  Johan Börjesson,et al.  Mechanism of surfactant effect in enzymatic hydrolysis of lignocellulose , 2002 .

[9]  Charles E. Wyman,et al.  BIOMASS ETHANOL: Technical Progress, Opportunities, and Commercial Challenges , 1999 .

[10]  Bernard Henrissat,et al.  Cellulases and their interaction with cellulose , 1994 .

[11]  A. Converse,et al.  Recovery of cellulase from lignaceous hydrolysis residue , 1993 .

[12]  A. Converse,et al.  Effect of Substrate Concentration on Multicomponent Adsorption , 1992 .

[13]  K. Schügerl,et al.  Adsorption and reuse of cellulases during saccharification of cellulosic materials , 1991 .

[14]  A. Converse,et al.  Adsorption of cellulase from Trichoderma reesei on cellulose and lignacious residue in wood pretreated by dilute sulfuric acid with explosive decompression , 1990, Biotechnology and bioengineering.

[15]  C. MacKenzie,et al.  Effect of physical parameters on the adsorption characteristics of fractionated Trichoderma reesei cellulase components , 1988 .

[16]  Dong Won Kim,et al.  Adsorption of cellulase fromTrichoderma viride on microcrystalline cellulose , 1988, Applied Microbiology and Biotechnology.

[17]  T. K. Ghose.  Measurement of cellulase activities , 1987 .

[18]  P. K. Smith,et al.  Measurement of protein using bicinchoninic acid. , 1985, Analytical biochemistry.

[19]  H. Grethlein,et al.  The Effect of Pore Size Distribution on the Rate of Enzymatic Hydrolysis of Cellulosic Substrates , 1985, Bio/Technology.

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

[21]  H. Ooshima,et al.  Adsorption of cellulase from Trichoderma viride on cellulose , 1983, Biotechnology and bioengineering.

[22]  Sun Bok Lee,et al.  Adsorption of cellulase on cellulose: Effect of physicochemical properties of cellulose on adsorption and rate of hydrolysis , 1982, Biotechnology and bioengineering.

[23]  P. Carniti,et al.  Cotton cellulose: enzyme adsorption and enzymatic hydrolysis , 1982 .

[24]  C. Wilke,et al.  Adsorption and recovery of cellulases during hydrolysis of newspaper , 1980 .

[25]  L. Fan,et al.  Mechanism of the enzymatic hydrolysis of cellulose: Effects of major structural features of cellulose on enzymatic hydrolysis , 1980 .

[26]  M. Mandels,et al.  Adsorption of Trichoderma cellulase on cellulose. , 1977, Biotechnology and bioengineering.

[27]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[28]  Oliver H. Lowry,et al.  Protein measurement with the Folin phenol reagent. , 1951, The Journal of biological chemistry.

[29]  D. Argyropoulos,et al.  Measurement of cellulase activity with piezoelectric resonators , 2007 .

[30]  R. Matsuno,et al.  Elucidation of adsorption processes of cellulases during hydrolysis of crystalline cellulose , 2004, Applied Microbiology and Biotechnology.

[31]  Douglas E. Eveleigh,et al.  Characteristics of fungal cellulases , 1991 .

[32]  C. MacKenzie,et al.  Detection and characterization of the specific and nonspecific endoglucanases of Trichoderma reesei: Evidence demonstrating endoglucanase activity by cellobiohydrolase II , 1987 .

[33]  D. Rickwood,et al.  Gel electrophoresis of proteins: a practical approach , 1981 .

[34]  L. T. Fan,et al.  Major chemical and physical features of cellulosic materials as substrates for enzymatic hydrolysis , 1980 .