Purification, and Biochemical and Biophysical Characterization of Cellobiohydrolase I from Trichoderma harzianum IOC 3844 = Purification, and Biochemical and Biophysical Characterization of Cellobiohydrolase I from Trichoderma harzianum IOC 3844

다국어 초록 (Multilingual Abstract)

Because of its elevated cellulolytic activity, the filamentous fungus Trichoderma harzianum has a considerable potential in biomass hydrolysis applications. Trichoderma harzianum cellobiohydrolase I (ThCBHI), an exoglucanase, is an important enzyme in the process of cellulose degradation. Here, we report an easy single-step ion-exchange chromatographic method for purification of ThCBHI and its initial biophysical and biochemical characterization. The ThCBHI produced by induction with microcrystalline cellulose under submerged fermentation was purified on DEAE-Sephadex A-50 media and its identity was confirmed by mass spectrometry. The ThCBHI biochemical characterization showed that the protein has a molecular mass of 66 kDa and pI of 5.23. As confirmed by smallangle X-ray scattering (SAXS), both full-length ThCBHI and its catalytic core domain (CCD) obtained by digestion with papain are monomeric in solution. Secondary structure analysis of ThCBHI by circular dichroism revealed α- helices and β-strands contents in the 28% and 38% range, respectively. The intrinsic fluorescence emission maximum of 337 nm was accounted for as different degrees of exposure of ThCBHI tryptophan residues to water. Moreover, ThCBHI displayed maximum activity at pH 5.0 and temperature of 50℃ with specific activities against Avicel and p-nitrophenyl-β-D-cellobioside of 1.25 U/mg and 1.53 U/mg, respectively.

참고문헌 (Reference)

1 Eftink, M. R, "Use of fluorescence spectroscopy as thermodynamics tool" 323 : 459-473, 2000

2 Zhang, Y. H. P, "Toward an aggregated understanding of enzymatic hydrolysis of cellulose: Noncomplexed cellulase systems" 88 : 797-824, 2004

3 Fischer, H., "The molecular weight of protein in solution can be determined for a single SAXS measurement on a relative scale" 43 : 101-109, 2010

4 Wolfenden, R, "The depth of chemical time and the power of enzymes as catalysts" 34 : 938-945, 2001

5 Cantarel, B. L, "The Carbohydrate-Active EnZymes database (CAZy): An expert resource for glycogenomics" 37 : 233-238, 2009

6 Bailey, S, "The CCP4 suite : Programs for protein crystallography" 50 : 760-763, 1994

7 Divne, C, "The 3-dimensional crystal-structure of the catalytic core of cellobiohydrolase-I from Trichoderma reesei" 265 : 524-528, 1994

8 Nieves, R. A., "Survey and analysis of commercial cellulase preparations suitable for biomass conversion to ethanol" 14 : 301-304, 1998

9 Mansfield, S. D, "Substrate and enzyme characteristics that limit cellulose hydrolysis" 15 : 804-816, 1999

10 Davies, G, "Structures and mechanisms of glycosyl hydrolases" 3 : 853-859, 1995

11 Carpita, N. C, "Structural models of primary-cell walls in flowering plants: Consistency of molecular structure with the physical properties of the wall during growth" 3 : 1-30, 1993

12 Raghothama, S., "Solution structure of the CBM10 cellulose binding module from Pseudomonas xylanase A" 39 : 978-984, 2000

13 Biochemistry IUo, "Report of the Commission on Enzymes" Pergamon Press Oxford 1961

14 Bradford, M. M, "Rapid and sensitive method for quantification of microgram quantities of protein utilizing the principle of protein-dye binding" 72 : 248-254, 1976

15 Gusakov, A. V, "Purification, cloning and characterisation of two forms of thermostable and highly active cellobiohydrolase I (Cel7A) produced by the industrial strain of Chrysosporium lucknowense" 36 : 57-69, 2005

16 Bakare, M. K, "Purification and characterization of cellulase from the wild-type and two improved mutants of Pseudomonas fluorescens" 4 : 898-904, 2005

17 Aboul- Enein, F. A, "Purification and characterization of a novel thermoactive cellulase from thermophilic actinomycetes isolated from soil sample of Egypt" 2 : 81-86, 2010

18 신게움, "Purification and Characterization of a Thermostable Cellobiohydrolase from Fomitopsis pinicola" 한국미생물·생명공학회 20 (20): 1681-1688, 2010

19 Mandels, M, "Production of cellulases" 95 : 391-398, 1969

20 Jager, G., "Practical screening of purified cellobiohydrolases and endoglucanases with alpha-cellulose and specification of hydrodynamics" 3 : 18-, 2010

21 Pollastri, G, "Porter: A new, accurate server for protein secondary structure prediction" 21 : 1719-1720, 2005

22 Zhang, Y. H. P, "Outlook for cellulase improvement: Screening and selection strategies" 24 : 452-481, 2006

23 Margeot, A, "New improvements for lignocellulosic ethanol" 20 : 372-380, 2009

24 Henrissat, B, "New families in the classification of glycosyl hydrolases based on amino-acid-sequence similarities" 293 : 781-788, 1993

25 Wood, T. M, "Methods for measuring cellulase activities" 160 : 87-112, 1988

26 Claeyssens, S., "Metabolic changes in rats fed a low protein-diet during post-weaning growth" 39 : 676-681, 1990

27 Aden, A, "Lignocellulosic Biomass to Ethanol Process Design and Economics Utilizing Co-current Dilute Acid Prehydrolysis and Enzymatic Hydrolysis for Corn Stover" National Renewable Energy Laboratory Golden, Colorado. 2002

28 Jeoh, T, "Implications of cellobiohydrolase glycosylation for use in biomass conversion" 1 : 10-, 2008

29 Zhou, J., "Identification and purification of the main components of cellulases from a mutant strain of Trichoderma viride T 100-14" 99 : 6826-6833, 2008

30 Teeri, T. T., "Hydrolysis of crystaline cellulose by native and engineered Trichoderma reesei cellulases" 207 : 21-AGFD, 1994

31 Castro, A, "High-yield endoglucanase production by Trichoderma harzianum IOC- 3844 cultivated in pretreated sugarcane mill byproduct" 2010

32 Divne, C, "High-resolution crystal structures reveal how a cellulose chain is bound in the 50 angstrom long tunnel of cellobiohydrolase I from Trichoderma reesei" 275 : 309-325, 1998

33 Cosgrove, D. J, "Growth of the plant cell wall" 6 : 850-861, 2005

34 Munoz, I. G, "Family 7 cellobiohydrolases from Phanerochaete chrysosporium: Crystal structure of the catalytic module of Cel7D (CBH58) at 1.32 angstrom resolution and homology models of the isozymes" 314 : 1097-1111, 2001

35 Stals, I, "Factors influencing glycosylation of Trichoderma reesei cellulases. I: Postsecretorial changes of the O- and N-glycosylation pattern of Cel7A" 14 : 713-724, 2004

36 Hammersley, A. P, "FIT2D: An Introduction and Overview" ESRF Internal Report. 1997

37 Prasad, S, "Ethanol as an alternative fuel from agricultural, industrial and urban residues" 50 : 1-39, 2007

38 Sreerama, N, "Estimation of protein secondary structure from circular dichroism spectra: Comparison of CONTIN, SELCON, and CDSSTR methods with an expanded reference set" 287 : 252-260, 2000

39 Biochemistry IUo, "Enzyme Nomenclature: Recommendations 1964 of the International Union of Biochemistry" Elsevier. Amesterdam 1965

40 von Ossowski, I, "Engineering the exo-loop of Trichoderma reesei cellobiohydrolase, Ce17A. A comparison with Phanerochaete chrysosporium Cel7D" 333 : 817-829, 2003

41 Kraulis, P. J., "Determination of the 3-dimensional solutions structure of the C-terminal domain of cellobiohydrolase-I from Trichoderma reesei: A study using nuclear magnetic ressonance and hybrid distance geometry dynamical simulated annealing" 28 : 7241-7257, 1989

42 Valaskova, V, "Degradation of cellulose and hemicelluloses by the brown rot fungus Piptoporus betulinus: Production of extracellular enzymes and characterization of the major cellulases" 152 : 3613-3622, 2006

43 Tarentino, A. L, "Deglycosylation of asparagine-linked glycans: Purification, properties, and specificity of oligosaccharide-cleaving enzymes from Flavobacterium meningosepticum" 230 : 44-57, 1994

44 Teeri, T. T, "Crystalline cellulose degradation: New insight into the function of cellobiohydrolases" 15 : 160-167, 1997

45 Wang, L. S, "Comparison of domains function between cellobiohydrolase I and endoglucanase I from Trichoderma pseudokoningii S-38 by limited proteolysis" 24 : 27-38, 2003

46 Laemmli, U. K, "Cleavage of structrural proteins during the assembly of the head of bacteriophage T4" 227 : 680-691, 1970

47 Boer, H, "Characterization of Trichoderma reesei cellobiohydrolase CeI7A secreted from Pichia pastoris using two different promoters" 69 : 486-494, 2000

48 Gama, F. M., "Cellulose morphology and enzymatic reactivity: A modified solute exclusion technique" 43 : 381-387, 1994

49 Henrissat, B, "Cellulases and their interaction with cellulose" 1 : 169-196, 1994

50 Himmel, M. E, "Biomass recalcitrance: Engineering plants and enzymes for biofuels production" 316 : 804-807, 2007

51 Lahjouji, K., "Biochemical and molecular characterization of a cellobiohydrolase from Trametes versicolor" 75 : 337-346, 2007

52 Deshpande, M. V, "An assay for selective determination of exo-1,4,-beta-glucanase in a mixture of cellulolytic enzymes" 138 : 481-487, 1984

53 Irwin, D. C, "Activity studies of 8 purified cellulases: Specificity, synergism, and binding domain effects" 42 : 1002-1013, 1993

54 Stahlberg, J., "Activity studies and crystal structures of catalytically deficient mutants of cellobiohydrolase I from Trichoderma reesei" 264 : 337-349, 1996

55 Arantes, V, "Access to cellulose limits the efficiency of enzymatic hydrolysis: The role of amorphogenesis" 3 : 1-11, 2010

56 Wu, S. T., "Ab initio modeling of small proteins by iterative TASSER simulations" 5 : 17-, 2007

57 Stahlberg, J., "A new model for enzymatic hydrolysis of cellulose based on the 2- domains structure of cellobiohydrolase-I" 9 : 286-290, 1991

58 Henrissat, B, "A classification of glycosyl hydrolases based on amino-acid-sequence similarities" 280 : 309-316, 1991