A thermotolerant beta-glucosidase isolated from an endophytic fungi, Periconia sp., with a possible use for biomass conversion to sugars.

A fungal strain, BCC2871 (Periconia sp.), was found to produce a thermotolerant beta-glucosidase, BGL I, with high potential for application in biomass conversion. The full-length gene encoding the target enzyme was identified and cloned into Pichia pastoris KM71. Similar to the native enzyme produced by BCC2871, the recombinant beta-glucosidase showed optimal temperature at 70 degrees C and optimal pH of 5 and 6. The enzyme continued to exhibit high activity even after long incubation at high temperature, retaining almost 60% of maximal activity after 1.5h at 70 degrees C. It was also stable under basic conditions, retaining almost 100% of maximal activity after incubation for 2h at pH8. The enzyme has high activity towards cellobiose and other synthetic substrates containing glycosyl groups as well as cellulosic activity toward carboxymethylcellulose. Thermostability of the enzyme was improved remarkably in the presence of cellobiose, glucose, or sucrose. This beta-glucosidase was able to hydrolyze rice straw into simple sugars. The addition of this beta-glucosidase to the rice straw hydrolysis reaction containing a commercial cellulase, Celluclast 1.5L (Novozyme, Denmark) resulted in increase of reducing sugars being released compared to the hydrolysis without the beta-glucosidase. This enzyme is a candidate for applications that convert lignocellulosic biomass to biofuels and chemicals.

[1]  M. Bhat,et al.  Cellulose degrading enzymes and their potential industrial applications. , 1997, Biotechnology advances.

[2]  R. Glew,et al.  The dual effects of alcohols on the kinetic properties of guinea pig liver cytosolic beta-glucosidase. , 1989, The Journal of biological chemistry.

[3]  P. Vithayathil,et al.  Degradation of larchwood xylan by enzymes of a thermophilic fungus, Thermoascus aurantiacus. , 1989, Archives of biochemistry and biophysics.

[4]  B Henrissat,et al.  A classification of glycosyl hydrolases based on amino acid sequence similarities. , 1991, The Biochemical journal.

[5]  Z. Prágai,et al.  Cloning and expression of a β-1,4-endoglucanase gene from Cellulomonas sp. CelB7 in Escherichia coli; purification and characterization of the recombinant enzyme , 1996 .

[6]  M. Ueda,et al.  Synergistic Saccharification, and Direct Fermentation to Ethanol, of Amorphous Cellulose by Use of an Engineered Yeast Strain Codisplaying Three Types of Cellulolytic Enzyme , 2004, Applied and Environmental Microbiology.

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

[8]  E Owen,et al.  Biochemical characterization and mechanism of action of a thermostable beta-glucosidase purified from Thermoascus aurantiacus. , 2001, The Biochemical journal.

[9]  K. Hayashi,et al.  Agrobacterium tumefaciens β-glucosidase is also an effective β-xylosidase, and has a high transglycosylation activity in the presence of alcohols , 1998 .

[10]  Pernilla Turner,et al.  A novel variant of Thermotoga neapolitana beta-glucosidase B is an efficient catalyst for the synthesis of alkyl glucosides by transglycosylation. , 2007, Journal of biotechnology.

[11]  B. Saha,et al.  Production, purification, and characterization of a highly glucose-tolerant novel beta-glucosidase from Candida peltata , 1996, Applied and environmental microbiology.

[12]  M. Ueda,et al.  Direct and Efficient Production of Ethanol from Cellulosic Material with a Yeast Strain Displaying Cellulolytic Enzymes , 2002, Applied and Environmental Microbiology.

[13]  R. Glew,et al.  Hydrolysis of a naturally occurring beta-glucoside by a broad-specificity beta-glucosidase from liver. , 1986, The Biochemical journal.

[14]  P. Barré,et al.  Purification, Characterization, and Substrate Specificity of a Novel Highly Glucose-Tolerant β-Glucosidase fromAspergillus oryzae , 1998, Applied and Environmental Microbiology.

[15]  C. Skory,et al.  Properties of an intracellular β-glucosidase purified from the cellobiose-fermenting yeast Candida wickerhamii , 1996, Applied Microbiology and Biotechnology.

[16]  J. Svasti,et al.  Expression and purification of dalcochinase, a beta-glucosidase from Dalbergia cochinchinensis Pierre, in yeast and bacterial hosts. , 2006, Protein expression and purification.

[17]  G. L. Miller Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar , 1959 .

[18]  S. Hari Krishna,et al.  Simultaneous saccharification and fermentation of lignocellulosic wastes to ethanol using a thermotolerant yeast. , 2001, Bioresource technology.

[19]  Markku Saloheimo,et al.  Expression in Trichoderma reesei and characterisation of a thermostable family 3 beta-glucosidase from the moderately thermophilic fungus Talaromyces emersonii. , 2004, Protein expression and purification.

[20]  L. Eurwilaichitr,et al.  Purification, Biochemical Characterization, and Gene Cloning of a New Extracellular Thermotolerant and Glucose Tolerant Maltooligosaccharide-Forming α-Amylase from an Endophytic Ascomycete Fusicoccum sp. BCC4124 , 2007, Bioscience, biotechnology, and biochemistry.

[21]  P. Christakopoulos,et al.  Purification and characterisation of an extracellular beta-glucosidase with transglycosylation and exo-glucosidase activities from Fusarium oxysporum. , 1994, European journal of biochemistry.

[22]  Asano Krisana,et al.  Endo-1,4-beta-xylanase B from Aspergillus cf. niger BCC14405 isolated in Thailand: purification, characterization and gene isolation. , 2005, Journal of biochemistry and molecular biology.

[23]  N. Juge,et al.  Functional expression of human liver cytosolic β‐glucosidase in Pichia pastoris , 2002 .

[24]  H. O. D. op den Camp,et al.  A highly expressed family 1 beta-glucosidase with transglycosylation capacity from the anaerobic fungus Piromyces sp. E2. , 2002, Biochimica et biophysica acta.

[25]  T. L. Graham,et al.  Partial purification and characterization of a soybean beta-glucosidase with high specific activity towards isoflavone conjugates. , 2001, Phytochemistry.

[26]  J. Visser,et al.  β-Glucosidase multiplicity from Aspergillus tubingensis CBS 643.92: purification and characterization of four β-glucosidases and their differentiation with respect to substrate specificity, glucose inhibition and acid tolerance , 2001, Applied Microbiology and Biotechnology.

[27]  S. Brunak,et al.  Improved prediction of signal peptides: SignalP 3.0. , 2004, Journal of molecular biology.

[28]  F. Govers,et al.  A beta-glucosidase/xylosidase from the phytopathogenic oomycete, Phytophthora infestans. , 2002, Phytochemistry.

[29]  G. Legler,et al.  Reaction of β‐d‐Glucosidase A3 from Aspergillus wentii with d‐Glucal , 1979 .

[30]  L. F. Martins,et al.  Comparison of Penicillium echinulatum and Trichoderma reesei cellulases in relation to their activity against various cellulosic substrates. , 2008, Bioresource technology.

[31]  V. Bisaria,et al.  Purification and characterization of recombinant Escherichia coli-expressed Pichia etchellsii β-glucosidase II with high hydrolytic activity on sophorose , 2005, Applied Microbiology and Biotechnology.

[32]  J. A. Jorge,et al.  Beta-glucosidase activity from the thermophilic fungus Scytalidium thermophilum is stimulated by glucose and xylose. , 2004, FEMS microbiology letters.

[33]  H. Yeoh,et al.  Kinetic properties of β-glucosidase from Aspergillus ornatus , 1986, Applied Microbiology and Biotechnology.

[34]  A. I. Antonov,et al.  Design of highly efficient cellulase mixtures for enzymatic hydrolysis of cellulose , 2007, Biotechnology and bioengineering.

[35]  J. Brenchley,et al.  Characterization of an Unusual Cold-Active β-Glucosidase Belonging to Family 3 of the Glycoside Hydrolases from the Psychrophilic Isolate Paenibacillus sp. Strain C7 , 2005, Applied and Environmental Microbiology.

[36]  Saroj K. Mishra,et al.  Purification and characterization of two beta-glucosidases from a thermo-tolerant yeast Pichia etchellsii. , 2003, Biochimica et biophysica acta.

[37]  T. Tani,et al.  Production and Characterization of Recombinant Phanerochaete chrysosporium β-Glucosidase in the Methylotrophic Yeast Pichia pastoris , 2003 .

[38]  Jang-Su Park,et al.  Substrate specificity and transglycosylation catalyzed by a thermostable β-glucosidase from marine hyperthermophile Thermotoga neapolitana , 2005, Applied Microbiology and Biotechnology.

[39]  J. Copa-Patiño,et al.  A Phanerochaete chrysosporium β-d-glucosidase/β-d-xylosidase with specificity for (1 → 3)-β-d-glucan linkages , 1994 .

[40]  A. Esen,et al.  Analysis of rice glycosyl hydrolase family 1 and expression of Os4bglu12 β-glucosidase , 2006, BMC Plant Biology.

[41]  A. Harkki,et al.  The bgl1 gene of Trichoderma reesei QM 9414 encodes an extracellular, cellulose‐inducible β‐glucosidase involved in cellulase induction by sophorose , 1995, Molecular microbiology.

[42]  S. Withers,et al.  Cloning, Expression, Characterization, and Nucleophile Identification of Family 3, Aspergillus nigerβ-Glucosidase* , 2000, The Journal of Biological Chemistry.

[43]  P Béguin,et al.  Molecular biology of cellulose degradation. , 1990, Annual review of microbiology.