Characterization of a recombinant aryl β-glucosidase from Neosartorya fischeri NRRL181

Dayanand Kalyani, Kyoung-Mi Lee, Manish Kumar Tiwari, Priyadharshini Ramachandran, Hoon Kim, In-Won Kim, Marimuthu Jeya, Jung-Kul Lee

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

An isolated gene from Neosartorya fischeri NRRL181 encoding a β-glucosidase (BGL) was cloned, and its nucleotide sequence was determined. DNA sequence analysis revealed an open reading frame of 1,467 bp, capable of encoding a polypeptide of 488 amino acid residues. The gene was over-expressed in Escherichia coli, and the protein was purified using nickel-nitrilotriacetic acid chromatography. The purified recombinant BGL showed a high level of catalytic activity, with V max of 886 μmol min−1 mg-protein−1 and a K m of 68 mM for p-nitrophenyl-β-d-glucopyranoside (pNPG). The optimal temperature for enzyme activity was about 40°C, and the optimal pH was about 6.0. A homology model of N. fischeri BGL1 was constructed based on the X-ray crystal structure of Phanerochaete chrysosporium BGLA. Molecular dynamics simulation studies of the enzyme with the pNPG and cellobiose shed light on the unique substrate specificity of N. fischeri BGL1 only towards pNPG.
Original languageEnglish
JournalApplied Microbiology and Biotechnology
Volume94
Issue number2
Pages (from-to)413-423
ISSN0175-7598
DOIs
Publication statusPublished - 2012
Externally publishedYes

Keywords

  • BIOTECHNOLOGY
  • BASIDIOMYCETE PHANEROCHAETE-CHRYSOSPORIUM
  • THERMOPHILIC FUNGUS
  • THERMOASCUS-AURANTIACUS
  • GLYCOSYL HYDROLASES
  • TRICHODERMA-REESEI
  • CRYSTAL-STRUCTURE
  • PURIFICATION
  • EXPRESSION
  • CLONING
  • STRAIN
  • Characterization
  • beta-Glucosidase
  • Glycoside hydrolase family 1
  • Homology modeling
  • Substrate specificity
  • Facultatively Anaerobic Gram-Negative Rods Eubacteria Bacteria Microorganisms (Bacteria, Eubacteria, Microorganisms) - Enterobacteriaceae [06702] Escherichia coli species expression system
  • Fungi Plantae (Fungi, Microorganisms, Nonvascular Plants, Plants) - Ascomycetes [15100] Neosartorya fischeri species strain-NRRL181
  • Fungi Plantae (Fungi, Microorganisms, Nonvascular Plants, Plants) - Basidiomycetes [15300] Phanerochaete chrysosporium species
  • Neosartorya fischeri BGL gene [Ascomycetes] Neosartorya fischeri beta-glucosidase gene expression
  • cellobiose 528-50-7
  • p-nitrophenyl-beta-D-glucopyranoside 2492-87-7
  • recombinant aryl beta-glucosidase
  • 03502, Genetics - General
  • 03504, Genetics - Plant
  • 04500, Mathematical biology and statistical methods
  • 10062, Biochemistry studies - Nucleic acids, purines and pyrimidines
  • 10068, Biochemistry studies - Carbohydrates
  • 10515, Biophysics - Biocybernetics
  • 10802, Enzymes - General and comparative studies: coenzymes
  • 31000, Physiology and biochemistry of bacteria
  • 31500, Genetics of bacteria and viruses
  • 39008, Food microbiology - General and miscellaneous
  • 51518, Plant physiology - Enzymes
  • Bioprocess Engineering
  • Enzymology
  • Models and Simulations
  • Molecular Genetics
  • enzyme activity
  • substrate specificity
  • Biochemistry and Molecular Biophysics
  • Computational Biology
  • AAF74209 GenBank, EMBL, DDJB nucleotide sequence
  • AAL34084 GenBank, EMBL, DDJB nucleotide sequence
  • BAE87008 GenBank, EMBL, DDJB nucleotide sequence
  • cloning laboratory techniques, genetic techniques
  • DNA sequence analysis laboratory techniques, genetic techniques
  • homology modeling mathematical and computer techniques
  • molecular dynamics simulation mathematical and computer techniques
  • nickel-nitrilotriacetic acid chromatography laboratory techniques, chromatographic techniques
  • X-ray crystallography laboratory techniques, crystallographic techniques
  • Amino Acid Sequence
  • Chromatography, Affinity
  • Cloning, Molecular
  • DNA, Fungal
  • Enzyme Stability
  • Escherichia coli
  • Gene Expression
  • Hydrogen-Ion Concentration
  • Kinetics
  • Models, Molecular
  • Molecular Sequence Data
  • Neosartorya
  • Open Reading Frames
  • Protein Conformation
  • Recombinant Proteins
  • Sequence Analysis, DNA
  • Sequence Homology, Amino Acid
  • Temperature
  • EC 3.2.1.21 beta-Glucosidase
  • Amino acids
  • Cloning
  • Encoding (symbols)
  • Enzyme activity
  • Gene encoding
  • Molecular dynamics
  • Optimization
  • Purification
  • Recombinant proteins
  • beta glucosidase
  • amino acid
  • catalysis
  • chromatography
  • coliform bacterium
  • fungus
  • gene
  • homology
  • optimization
  • pathogen
  • peptide
  • pH
  • protein
  • recombination
  • substrate
  • article
  • controlled study
  • crystal structure
  • DNA sequence
  • enzyme kinetics
  • fungal gene
  • gene identification
  • gene overexpression
  • hydrolysis
  • molecular docking
  • Neosartorya fischeri
  • nonhuman
  • nucleotide sequence
  • Phanerochaete
  • site directed mutagenesis
  • temperature
  • Phanerochaete chrysosporium
  • Amino acid residues
  • Cellobiose
  • DNA sequence analysis
  • Glucopyranoside
  • Glucosidase
  • Homology models
  • Molecular dynamics simulations
  • Nickel-nitrilotriacetic acid chromatography
  • Nucleotide sequences
  • Open reading frame
  • Optimal temperature
  • X ray crystal structures
  • β-Glucosidase
  • ENZYMES
  • GENES
  • GENETICS
  • GLYCOSIDASES
  • KINETICS
  • PHYSICAL PROPERTIES
  • PHYSICOCHEMICAL PROPERTIES
  • RECOMBINANT ENZYMES
  • β-GLUCOSIDASES
  • Biotechnology
  • Enzyme systems

Cite this

Kalyani, D., Lee, K-M., Tiwari, M. K., Ramachandran, P., Kim, H., Kim, I-W., Jeya, M., & Lee, J-K. (2012). Characterization of a recombinant aryl β-glucosidase from Neosartorya fischeri NRRL181. Applied Microbiology and Biotechnology, 94(2), 413-423. https://doi.org/10.1007/s00253-011-3631-6