Identification of a β-glucosidase from the Mucor circinelloides genome by peptide pattern recognition.

Mucor circinelloides produces plant cell wall degrading enzymes that allow it to grow on complex polysaccharides. Although the genome of M. circinelloides has been sequenced, only few plant cell wall degrading enzymes are annotated in this species. We applied peptide pattern recognition, which is a non-alignment based method for sequence analysis to map conserved sequences in glycoside hydrolase families. The conserved sequences were used to identify similar genes in the M. circinelloides genome. We found 12 different novel genes encoding members of the GH3, GH5, GH9, GH16, GH38, GH47 and GH125 families in M. circinelloides. One of the two GH3-encoding genes was predicted to encode a β-glucosidase (EC 3.2.1.21). We expressed this gene in Pichia pastoris KM71H and found that the purified recombinant protein had relative high β-glucosidase activity (1.73U/mg) at pH5 and 50°C. The Km and Vmax with p-nitrophenyl-β-d-glucopyranoside as substrate was 0.20mM and 2.41U/mg, respectively. The enzyme was not inhibited by glucose and retained 84% activity at glucose concentrations up to 140mM. Although zygomycetes are not considered to be important degraders of lignocellulosic biomass in nature, the present finding of an active β-glucosidase in M. circinelloides demonstrates that enzymes from this group of fungi have a potential for cellulose degradation.

[1]  R. E. Huber,et al.  Physical and Kinetic Properties of the Family 3 β-Glucosidase from Aspergillus niger Which Is Important for Cellulose Breakdown , 2004, The Protein Journal.

[2]  X. Zhou,et al.  Parallel metatranscriptome analyses of host and symbiont gene expression in the gut of the termite Reticulitermes flavipes , 2009, Biotechnology for biofuels.

[3]  Lene Lange,et al.  Function-Based Classification of Carbohydrate-Active Enzymes by Recognition of Short, Conserved Peptide Motifs , 2013, Applied and Environmental Microbiology.

[4]  G. Bergstrom,et al.  Arsenal of plant cell wall degrading enzymes reflects host preference among plant pathogenic fungi , 2011, Biotechnology for biofuels.

[5]  Q. Shen,et al.  Characterization of a thermostable β-glucosidase from Aspergillus fumigatus Z5, and its functional expression in Pichia pastoris X33 , 2012, Microbial Cell Factories.

[6]  M. Nei,et al.  MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. , 2011, Molecular biology and evolution.

[7]  P. Christakopoulos,et al.  Cloning, expression and characterization of an ethanol tolerant GH3 β-glucosidase from Myceliophthora thermophila , 2013, PeerJ.

[8]  J. Haldane,et al.  Graphical Methods in Enzyme Chemistry , 1957, Nature.

[9]  J. Thompson,et al.  The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. , 1997, Nucleic acids research.

[10]  P. Busk,et al.  Cellulolytic potential of thermophilic species from four fungal orders , 2013, AMB Express.

[11]  Mikko Arvas,et al.  Re-annotation of the CAZy genes of Trichoderma reesei and transcription in the presence of lignocellulosic substrates , 2012, Microbial Cell Factories.

[12]  E. Fawzi.  Highly Thermostable Xylanase Purified from Rhizomucor Miehei NRRL 3169 , 2011, Acta biologica Hungarica.

[13]  M. Jeya,et al.  Characterization of β-glucosidase from a strain of Penicillium purpurogenum KJS506 , 2010, Applied Microbiology and Biotechnology.

[14]  Leping Li,et al.  Accurate anchoring alignment of divergent sequences , 2006, Bioinform..

[15]  B. Henrissat,et al.  Post-genomic analyses of fungal lignocellulosic biomass degradation reveal the unexpected potential of the plant pathogen Ustilago maydis , 2012, BMC Genomics.

[16]  Mikko Arvas,et al.  Array comparative genomic hybridization analysis of Trichoderma reesei strains with enhanced cellulase production properties , 2010, BMC Genomics.

[17]  F. Squina,et al.  Aspergillus niger β-Glucosidase Has a Cellulase-like Tadpole Molecular Shape , 2013, The Journal of Biological Chemistry.

[18]  Bernard Henrissat,et al.  Carbohydrate-active enzymes from the zygomycete fungus Rhizopus oryzae: a highly specialized approach to carbohydrate degradation depicted at genome level , 2011, BMC Genomics.

[19]  Rina Rani Ray,et al.  Current Commercial Perspective of Rhizopus oryzae: A Review , 2011 .

[20]  Wendy Schackwitz,et al.  Tracking the roots of cellulase hyperproduction by the fungus Trichoderma reesei using massively parallel DNA sequencing , 2009, Proceedings of the National Academy of Sciences.

[21]  Feng Xu,et al.  Biomass Converting Enzymes as Industrial Biocatalysts for Fuels and Chemicals: Recent Developments , 2012 .

[22]  J. O. Baker,et al.  Isolation and characterization of two forms of β-d-glucosidase fromAspergillus niger , 1993, Applied biochemistry and biotechnology.

[23]  E. Myers,et al.  Basic local alignment search tool. , 1990, Journal of molecular biology.

[24]  L. Lynd,et al.  Toward an aggregated understanding of enzymatic hydrolysis of cellulose: Noncomplexed cellulase systems , 2004, Biotechnology and bioengineering.

[25]  A. Ali,et al.  The cloning, expression and characterization of a cellobiase gene encoding a secretory enzyme from Cellulomonas biazotea. , 1998, Gene.

[26]  C. Vágvölgyi,et al.  Characterization of a β-glucosidase with transgalactosylation capacity from the zygomycete Rhizomucor miehei. , 2012, Bioresource technology.

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

[28]  Q. Yan,et al.  High level expression of extracellular secretion of a β-glucosidase gene (PtBglu3) from Paecilomyces thermophila in Pichia pastoris. , 2012, Protein expression and purification.

[29]  T. C. McIlvaine,et al.  A BUFFER SOLUTION FOR COLORIMETRIC COMPARISON , 1921 .

[30]  J. Ni,et al.  Identification of activity related amino acid mutations of a GH9 termite cellulase. , 2010, Bioresource technology.

[31]  J. Koga,et al.  Alternative Splicing Produces Two Endoglucanases with One or Two Carbohydrate-Binding Modules in Mucor circinelloides , 2005, Journal of bacteriology.

[32]  R. D. de Vries,et al.  Transcriptome analysis of Aspergillus niger grown on sugarcane bagasse , 2011, Biotechnology for biofuels.

[33]  L. Xia,et al.  Enzymatic hydrolysis of maize straw polysaccharides for the production of reducing sugars , 2008 .

[34]  Chaoguang Tian,et al.  Deciphering Transcriptional Regulatory Mechanisms Associated with Hemicellulose Degradation in Neurospora crassa , 2012, Eukaryotic Cell.

[35]  Frédéric Monot,et al.  Comparative kinetic analysis of two fungal β-glucosidases , 2010, Biotechnology for biofuels.

[36]  Brandi L. Cantarel,et al.  The Carbohydrate-Active EnZymes database (CAZy): an expert resource for Glycogenomics , 2008, Nucleic Acids Res..

[37]  B. Saha,et al.  Production, purification and properties of endoglucanase from a newly isolated strain of Mucor circinelloides , 2004 .

[38]  L. Nagy,et al.  A new β-glucosidase gene from the zygomycete fungus Rhizomucor miehei , 2009, Antonie van Leeuwenhoek.

[39]  O. Sinitsyna,et al.  Characterization of a GH family 3 β-glycoside hydrolase from Chrysosporium lucknowense and its application to the hydrolysis of β-glucan and xylan. , 2012, Bioresource technology.

[40]  Song Fan,et al.  Thermoanaerobacterium thermosaccharolyticum β-glucosidase: a glucose-tolerant enzyme with high specific activity for cellobiose , 2012, Biotechnology for Biofuels.

[41]  David K. Johnson,et al.  Biomass Recalcitrance: Engineering Plants and Enzymes for Biofuels Production , 2007, Science.

[42]  W. Qin,et al.  Overexpression of an exotic thermotolerant β-glucosidase in trichoderma reesei and its significant increase in cellulolytic activity and saccharification of barley straw , 2012, Microbial Cell Factories.