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Heterologous Expression and Characterization of a GH3 β-Glucosidase from Thermophilic Fungi Myceliophthora thermophila in Pichia pastoris
- Zhao, Junqi, Guo, Chao, Tian, Chaoguang, Ma, Yanhe
- Applied biochemistry and biotechnology 2015 v.177 no.2 pp. 511-527
- thermal stability, molecular models, Komagataella pastoris, Myceliophthora thermophila, cellobiose, nucleotides, amino acids, thermophilic fungi, genes, pH, enzyme substrates, heterologous gene expression, glycosides, beta-glucosidase, gentiobiose
- 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.