title: Heterologous Expression and Characterization of a GH3 β-Glucosidase from Thermophilic Fungi Myceliophthora thermophila in Pichia pastoris.
abstract: A novel β-glucosidase of glycoside hydrolase (GH) family 3 from Myceliophthora thermophila (mtbgl3b) was successfully expressed in Pichia pastoris. The full-length gene consists of 2613 bp nucleotides encoding a protein of 870 amino acids. MtBgl3b showed maximum activity at pH 5.0 and remained more than 70 % relative activity at 3.5-6.0. The enzyme displayed the highest activity at 60 °C and kept about 90 % relative activity for 50-65 °C; besides, the enzyme showed psychrophilic trait and remains 51 % relative activity at 40 °C. MtBgl3b exhibited good stability over a wide pH range of 3.0-10.0 and was thermostable at 60 and 65 °C. The enzyme displayed highest activity towards p-nitrophenyl-β-D-glucopyranoside (pNPG), followed by p-nitrophenyl-D-cellobioside (pNPC), cellotetraose, cellotriose, cellobiose, and gentiobiose. When using 10 % cellobiose (w/v) as the substrate, the enzyme showed transglycosylation activity to produce the cellotriose. The kinetic parametric of K m and V max were 2.78 mM and 927.9 μM mg(-1) min(-1), respectively. Finally, the reaction mode of the enzyme and the substrates were analyzed by molecular docking approach.
authors.affiliation: Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Xiqi Dao 32, Tianjin Airport Economic Area, Tianjin, 300308, China.
authors.forename: Junqi
authors.initials: J
authors.lastname: Zhao
authors.forename: Chao
authors.initials: C
authors.lastname: Guo
authors.forename: Chaoguang
authors.initials: C
authors.lastname: Tian
authors.forename: Yanhe
authors.initials: Y
authors.lastname: Ma
mesh_terms: D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D005800:Genes, Fungal; D006031:Glycosylation / Q000187:drug effects; D006868:Hydrolysis; D007477:Ions; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D010843:Pichia / Q000187:drug effects / Q000235:genetics*; D011994:Recombinant Proteins / Q000302:isolation & purification; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D020037:Sordariales / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D040681:Structural Homology, Protein; D013379:Substrate Specificity / Q000187:drug effects; D001617:beta-Glucosidase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D007477:Ions; D008670:Metals; D011994:Recombinant Proteins; D001617:beta-Glucosidase