Purification and characterisation of an extracellular beta-glucosidase with transglycosylation and exo-glucosidase activities from Fusarium oxysporum.

An extracellular beta-glucosidase from Fusarium oxysporum was purified to homogeneity by gel-filtration and ion-exchange chromatographies. The enzyme, a monomeric protein of 110 kDa, was maximally active at pH 5.0-6.0 and at 60 degrees C. It hydrolysed 1-->4-linked aryl-beta-glucosides and 1-->4-linked, 1-->3-linked and 1-->6-linked beta-glucosides. The apparent Km and kcat values for p-nitrophenyl beta-D-glucopyranoside (4-NpGlcp) and cellobiose were 0.093 (Km), 1.07 mM (kcat) and 1802 (Km), 461.5 min-1 (kcat), respectively. Glucose and gluconolactone inhibited the enzyme competitively with Ki values of 2.05 mM and 3.03 microM, respectively. Alcohols activated the enzyme; butanol showed maximum effect (2.2-fold at 0.5 M) while methanol increased the activity by 1.4-fold at 1 M. The enzyme catalysed the synthesis of methylglucosides, ethylglucoside and propylglucosides, as well as trisaccharides in the presence of different alcohols and disaccharides, respectively. In addition, the enzyme hydrolysed the unsubstituted and methylumbelliferyl cello-oligosaccharides [MeUmb(Glc)n] but the rate of hydrolysis decreased with increasing chain length. Analysis of products released from MeUmb(Glc)n as a function of time revealed that the enzyme attacked these substrates in a stepwise manner and from both ends. Thus, beta-glucosidase from F. oxysporum, with the above interesting properties, could be of commercial interest.

[1]  R. Maheshwari,et al.  Purification and characterization of an extracellular β-glucosidase from the thermophilic fungus Sporotrichum thermophile and its influence on cellulase activity , 1993 .

[2]  M. Rossi,et al.  Exo‐glucosidase activity and substrate specificity of the beta‐glycosidase isolated from the extreme thermophile Sulfolobus solfataricus , 1993, Biotechnology and applied biochemistry.

[3]  H. Schrempf,et al.  Characterization of an intracellular β-glucosidase from Streptomyces reticuli , 1993 .

[4]  J. Eyzaguirre,et al.  Beta‐glucosidase from Penicillium purpurogenum: purification and properties.“ , 1992, Biotechnology and applied biochemistry.

[5]  R. Calza,et al.  Purification and characterization of an extracellular β-glucosidase from the rumen fungus Neocallimastix frontalis EB188 , 1991 .

[6]  J. Woodward,et al.  Cellulase production by Neurospora crassa: purification and characterization of cellulolytic enzymes. , 1990, Enzyme and microbial technology.

[7]  J. Copa-Patiño,et al.  Purification and properties of a β-glucosidase from Penicillium oxalicum autolysates , 1990 .

[8]  Paul Christakopoulos,et al.  Direct fermentation of cellulose to ethanol by Fusarium oxysporum , 1989 .

[9]  A. Arnaud,et al.  Purification and properties of a BT‐glucosidase of Hanseniaspora vineae Van der Walt and Tscheuschner with the view to its utilization in fruit aroma liberation , 1989 .

[10]  D. Dube,et al.  Extracellular cellulolytic enzyme system of Aspergillus japonicus: 2. Purification and characterization of an inducible extracellular β-glucosidase , 1988 .

[11]  S. Shimizu,et al.  β-Glucosidase from Ruminococcus albus , 1988 .

[12]  T. Wood,et al.  METHODS FOR MEASURING CELLULASE ACTIVITIES , 1988 .

[13]  R. Maheshwari,et al.  Sporotrichum thermophile Growth, Cellulose Degradation, and Cellulase Activity , 1987, Applied and environmental microbiology.

[14]  V. Deshpande,et al.  Direct conversion of cellulose/hemicellulose to ethanol by Neurospora crassa , 1986 .

[15]  Kitpreechavanich,et al.  Aspergillus fumigatusの菌体外β‐キシロシダーゼとβ‐グルコシダーゼの精製と性質 , 1986 .

[16]  S. Nagai,et al.  Purification and characterization of extracellular β-xylosidase and β-glucosidase from Aspergillus fumigatus , 1986 .

[17]  A. Arnaud,et al.  Purification and properties of an exocellular β-glucosidase of Candida molischiana (Zikes) Meyer and Yarrow capable of hydrolyzing soluble cellodextrins , 1985 .

[18]  S. Murao,et al.  Enzymic Properties of Three β-Glucosidases from Aspergillus aculeatus No. F-50 , 1985 .

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

[20]  S. Murao,et al.  Enzymic Properties of Three β-Glucosidases from Aspergillus aculeatus , 1985 .

[21]  N. Sharon,et al.  Binding of 4-methylumbelliferyl beta-D-galactosyl-(1 leads to 3)-N-acetyl-beta-D-galactosaminide to peanut agglutinin. Characterisation and application in substitution titrations. , 1983, European journal of biochemistry.

[22]  I. W. Sutherl.  An enzyme system hydrolysing the polysaccharides of Xanthomonas species , 1982 .

[23]  T. Wood,et al.  Purification and Some Properties of the Extracellular β-d-Glucosidase of the Cellulolytic Fungus Trichoderma koningii , 1982 .

[24]  D. Day,et al.  Purification and Properties of β-Glucosidase from Aspergillus terreus , 1982, Applied and environmental microbiology.

[25]  A. Wiseman,et al.  Fungal and other β-d-glucosidases — Their properties and applications , 1982 .

[26]  J. Shewale,et al.  Purification, characterization, and properties of β-glucosidase enzymes from Sclerotium rolfsii , 1981 .

[27]  J. Quail,et al.  The reaction between 2,2-diphenyl-1-picrylhydrazyl and bromine , 1980 .

[28]  M. Mandels,et al.  Cellulases: Biosynthesis and applications , 1980 .

[29]  T. Wood,et al.  The cellulase of Trichoderma koningii. Purification and properties of some endoglucanase components with special reference to their action on cellulose when acting alone and in synergism with the cellobiohydrolase. , 1978, The Biochemical journal.

[30]  D. Sternberg.  Beta-glucosidase of Trichoderma: its biosynthesis and role in saccharification of cellulose , 1976, Applied and environmental microbiology.

[31]  B. Lugtenberg,et al.  Electrophoretic resolution of the ‘major outer membrane protein’ of Escherichia coli K12 into four bands , 1975, FEBS letters.

[32]  T. Wood.  The cellulase of Fusarium solani. Resolution of the enzyme complex. , 1969, The Biochemical journal.

[33]  T. Wood.  Cellulolytic enzyme system of Trichoderma koningii. Separation of components attacking native cotton. , 1968, The Biochemical journal.

[34]  R. Mccomb,et al.  Colorimetric estimation of d-glucose and 2-deoxy-d-glucose with glucose oxidase , 1958 .

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

[36]  E. Friesen,et al.  Rotational Relationships of Alkyl Glucosides , 1942 .