Kinetic parameters and mode of action of the cellobiohydrolases produced by Talaromyces emersonii.

Three forms of cellobiohydrolase (EC 3.2.1.91), CBH IA, CBH IB and CBH II, were isolated to apparent homogeneity from culture filtrates of the aerobic fungus Talaromyces emersonii. The three enzymes are single sub-unit glycoproteins, and unlike most other fungal cellobiohydrolases are characterised by noteworthy thermostability. The kinetic properties and mode of action of each enzyme against polymeric and small soluble oligomeric substrates were investigated in detail. CBH IA, CBH IB and CBH II catalyse the hydrolysis of microcrystalline cellulose, albeit to varying extents. Hydrolysis of a soluble cellulose derivative (CMC) and barley 1,3;1,4-beta-D-glucan was not observed. Cellobiose (G2) is the main reaction product released by CBH IA, CBH IB, and CBH II from microcrystalline cellulose. All three CBHs are competitively inhibited by G2; inhibition constant values (K(i)) of 2.5 and 0.18 mM were obtained for CBH IA and CBH IB, respectively (4-nitrophenyl-beta-cellobioside as substrate), while a K(i) of 0.16 mM was determined for CBH II (2-chloro-4-nitrophenyl-beta-cellotrioside as substrate). Bond cleavage patterns were determined for each CBH on 4-methylumbelliferyl derivatives of beta-cellobioside and beta-cellotrioside (MeUmbG(n)). While the Tal. emersonii CBHs share certain properties with their counterparts from Trichoderma reesei, Humicola insolens and other fungal sources, distinct differences were noted.

[1]  W. Cleland,et al.  Statistical analysis of enzyme kinetic data. , 2006, Methods in enzymology.

[2]  R. Ganju,et al.  Purification and characterization of two cellobiohydrolases from Chaetomium thermophile var. coprophile. , 1989, Biochimica et biophysica acta.

[3]  T. Enari,et al.  Enzymatic hydrolysis of cellulose: is the current theory of the mechanisms of hydrolysis valid? , 1987, Critical reviews in biotechnology.

[4]  T. Wood,et al.  The cellulase of Penicillium pinophilum. Synergism between enzyme components in solubilizing cellulose with special reference to the involvement of two immunologically distinct cellobiohydrolases. , 1986, The Biochemical journal.

[5]  A. McHale,et al.  THE CELLULOLYTIC SYSTEM OF TALAROMYCES-EMERSONII - PURIFICATION AND CHARACTERIZATION OF THE EXTRACELLULAR AND INTRACELLULAR BETA-GLUCOSIDASES , 1981 .

[6]  M. Schülein.  Enzymatic properties of cellulases from Humicola insolens. , 1997, Journal of biotechnology.

[7]  D. Kilburn,et al.  Cellobiohydrolase B, a second exo-cellobiohydrolase from the cellulolytic bacterium Cellulomonas fimi. , 1995, The Biochemical journal.

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

[9]  T. Wood.  Properties of cellulolytic enzyme systems. , 1985, Biochemical Society transactions.

[10]  F. Henrique-Silva,et al.  Cellulase Induction in Trichoderma reesei by Cellulose Requires Its Own Basal Expression* , 1997, The Journal of Biological Chemistry.

[11]  H. van Tilbeurgh,et al.  Separation of endo‐ and exo‐type cellulases using a new affinity chromatography method , 1984 .

[12]  C. Kubicek,et al.  Carbon Source Control of Cellobiohydrolase I and II Formation by Trichoderma reesei , 1991, Applied and environmental microbiology.

[13]  J. Knowles,et al.  Stereochemical course of the action of the cellobioside hydrolases I and II of Trichoderma reesei , 1988 .

[14]  H. van Tilbeurgh,et al.  Detection and differentiation of cellulase components using low molecular mass fluorogenic substrates , 1985 .

[15]  P. Broda,et al.  The identification, molecular cloning and characterisation of a gene from Phanerochaete chrysosporium that shows strong homology to the exo-cellobiohydrolase I gene from Trichoderma reesei. , 1988, Gene.

[16]  G. Kleywegt,et al.  The active site of Trichoderma reesei cellobiohydrolase II: the role of tyrosine 169. , 1996, Protein engineering.

[17]  L. Fägerstam,et al.  The primary structure of a 1,4‐β‐glucan cellobiohydrolase from the fungus Trichoderma reesei QM 9414 , 1984 .

[18]  M. Claeyssens,et al.  Domain structure of cellobiohydrolase II as studied by small angle X-ray scattering: close resemblance to cellobiohydrolase I. , 1988, Biochemical and biophysical research communications.

[19]  B. Henrissat,et al.  Imaging the Enzymatic Digestion of Bacterial Cellulose Ribbons Reveals the Endo Character of the Cellobiohydrolase Cel6A from Humicola insolens and Its Mode of Synergy with Cellobiohydrolase Cel7A , 2000, Applied and Environmental Microbiology.

[20]  M. Coughlan,et al.  Synergistic hydrolysis of cellulose by components of the extracellular cellulase system of Talaromyces emersonii , 1980, FEBS letters.

[21]  Y. Amano,et al.  Fine substrate specificities of four exo-type cellulases produced by Aspergillus niger, Trichoderma reesei, and Irpex lacteus on (1-->3), (1-->4)-beta-D-glucans and xyloglucan. , 1996, Journal of biochemistry.

[22]  M. Siika‐aho,et al.  Hydrolytic properties of two cellulases of Trichoderma reesei expressed in yeast , 1993, Biotechnology and applied biochemistry.

[23]  E. Hehre,et al.  Stereochemical course of hydrolysis and hydration reactions catalysed by cellobiohydrolases I and II from Trichoderma reesei , 1990, FEBS letters.

[24]  M. Tuohy,et al.  Production of thermostable xylan-degrading enzymes by Talaromyces emersonii☆ , 1992 .

[25]  S. Hastrup,et al.  Crystal structure of the catalytic core domain of the family 6 cellobiohydrolase II, Cel6A, from Humicola insolens, at 1.92 A resolution. , 1999, The Biochemical journal.

[26]  Hydrolyses of α- and β-cellobiosyl fluorides by cellobiohydrolases of Trichoderma reesei , 1993 .

[27]  P. Kraulis,et al.  Determination of the three-dimensional solution structure of the C-terminal domain of cellobiohydrolase I from Trichoderma reesei. A study using nuclear magnetic resonance and hybrid distance geometry-dynamical simulated annealing. , 1989, Biochemistry.

[28]  B. Henrissat,et al.  Synergism of Cellulases from Trichoderma reesei in the Degradation of Cellulose , 1985, Bio/Technology.

[29]  D. Fracheboud,et al.  Isolation, purification, and properties of the exocellulase from Sporotrichum (Chrysosporium) thermophile , 1989 .

[30]  H. van Tilbeurgh,et al.  Fungal cellulase systems. Comparison of the specificities of the cellobiohydrolases isolated from Penicillium pinophilum and Trichoderma reesei. , 1989, The Biochemical journal.

[31]  T. Wood,et al.  Purification and properties of a cellobiohydrolase from Penicillium pinophilum , 1986 .

[32]  T. Reinikainen,et al.  Site‐directed mutagenesis of the putative catalytic residues of Trichoderma reesci cellobiohydrolase I and endoglucanase I , 1990, FEBS letters.

[33]  S. Covert,et al.  Structure, organization, and transcription of a cellobiohydrolase gene cluster from Phanerochaete chrysosporium , 1992, Applied and environmental microbiology.

[34]  M. Siika‐aho,et al.  A comparative study of two retaining enzymes of Trichoderma reesei: transglycosylation of oligosaccharides catalysed by the cellobiohydrolase I, Cel7A, and the β‐mannanase, Man5A , 1999 .

[35]  D. Schmidhalter,et al.  Purification and characterization of two exo-cellobiohydrolases from the brown-rot fungus Coniophora puteana (Schum ex Fr) Karst. , 1993, Archives of biochemistry and biophysics.

[36]  K. Poutanen,et al.  Interlaboratory testing of methods for assay of xylanase activity , 1992 .

[37]  B Henrissat,et al.  Specificity mapping of cellulolytic enzymes: Classification into families of structurally related proteins confirmed by biochemical analysis , 1992, Protein science : a publication of the Protein Society.

[38]  B. Henrissat,et al.  A Bifunctionalized Fluorogenic Tetrasaccharide as a Substrate to Study Cellulases* , 1997, The Journal of Biological Chemistry.

[39]  J. Knowles,et al.  Three-dimensional structure of cellobiohydrolase II from Trichoderma reesei. , 1990, Science.

[40]  T. Reinikainen,et al.  Hydrolysis of barley (1→3), (1→4)-β-d-glucan by a cellobiohydrolase II preparation from Trichoderma reesei , 1995 .

[41]  J. Vandekerckhove,et al.  Studies of the cellulolytic system of Trichoderma reesei QM 9414. Analysis of domain function in two cellobiohydrolases by limited proteolysis. , 1988, European journal of biochemistry.

[42]  M. Coughlan,et al.  The saccharifying ability of the cellulase complex of Talaromyces emersonii and comparison with that of other fungal species. , 1979, The International journal of biochemistry.

[43]  N. Gilkes,et al.  Cellulose hydrolysis by bacteria and fungi. , 1995, Advances in microbial physiology.

[44]  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.

[45]  B Henrissat,et al.  Cellulase families revealed by hydrophobic cluster analysis. , 1989, Gene.

[46]  S. Kauppinen,et al.  Homologous domains in Trichoderma reesei cellulolytic enzymes: gene sequence and expression of cellobiohydrolase II. , 1987, Gene.

[47]  S. Covert,et al.  Genomic organization of a cellulase gene family in Phanerochaete chrysosporium , 1992, Current Genetics.

[48]  T. Wood,et al.  Isolation and characterization of the 1,4-beta-D-glucan glucanohydrolases of Talaromyces emersonii. , 1985, The Biochemical journal.

[49]  T. Reinikainen,et al.  Low-level endoglucanase contamination in a Trichoderma reesei cellobiohydrolase II preparation affects its enzymatic activity on β-glucan , 1995 .

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

[51]  H. van Tilbeurgh,et al.  The use of 4‐methylumbelliferyl and other chromophoric glycosides in the study of cellulolytic enzymes , 1982 .

[52]  K. Eriksson,et al.  An assay for selective determination of exo-1,4,-beta-glucanases in a mixture of cellulolytic enzymes. , 1984, Analytical biochemistry.

[53]  G. Pettersson,et al.  The 1,4-beta-D-glucan cellobiohydrolases from Phanerochaete chrysosporium. I. A system of synergistically acting enzymes homologous to Trichoderma reesei. , 1991, Journal of biotechnology.

[54]  M. Claeyssens,et al.  The xylan-degrading enzyme system of Talaromyces emersonii: novel enzymes with activity against aryl beta-D-xylosides and unsubstituted xylans. , 1993, The Biochemical journal.