title: A novel thermoacidophilic and thermostable endo-β-1,4-glucanase from Phialophora sp. G5: its thermostability influenced by a distinct β-sheet and the carbohydrate-binding module.
abstract: An endo-β-1,4-glucanase gene, egG5, was cloned from the fungus Phialophora sp. G5. The 1,290-bp open reading frame encodes a bimodular cellulase composed of an N-terminal family 1 carbohydrate-binding module (CBM) and a C-terminal family 5 glycoside hydrolase catalytic module. Recombinant EgG5 produced in Pichia pastoris exhibited maximal activity at pH 4.0-5.0 and 70 °C, retained 40% of the maximal activity at pH 2.0, and was stable at pH 2.0-10.0. When compared with its closest homolog in Trichoderma sp. C-4 (70.6% identity), EgG5 had better thermostability (51.6% activity at 65 °C for 12 h vs 10% activity at 60 °C for 20 min). Sequence-structure analysis indicated that the distinct β-sheet in EgG5 in place of a linking loop in Trichoderma sp. C-4 endoglucanase might be the reason. To verify its function, two mutants, EgG5-Mut (disrupting the β-sheet with four amino acid substitutions) and EgG5-CBM (removing the CBM), were constructed, expressed in P. pastoris, and characterized. Both mutants had similar pH optima (pH 4.0) and temperature optima (70 °C) but varied in pH stabilities (pH 2.0-10.0 and pH 2.0-7.0, respectively) and thermostabilities. The thermostability of EgG5-Mut (13.4% activity vs 52.5% of EgG5 at 65 °C for 12 h) confirmed the effect of β-sheet on enzyme thermostability. EgG5-CBM was more thermostable (94.9% activity at 65 °C for 12 h and 15.5% activity at 80 °C for 30 min) and had higher specific activity (711.6 vs 60.3 U mg(-1) of EgG5). This study presents an excellent endoglucanase with potential use in the bioconversion of lignocellulosic materials and provides good ideas for the improvement of enzyme thermostability.
authors.affiliation: Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, No. 12 Zhongguancun South Street, Beijing, People's Republic of China.
authors.forename: Junqi
authors.initials: J
authors.lastname: Zhao
authors.forename: Pengjun
authors.initials: P
authors.lastname: Shi
authors.forename: Huoqing
authors.initials: H
authors.lastname: Huang
authors.forename: Zhongyuan
authors.initials: Z
authors.lastname: Li
authors.forename: Tiezheng
authors.initials: T
authors.lastname: Yuan
authors.forename: Peilong
authors.initials: P
authors.lastname: Yang
authors.forename: Huiying
authors.initials: H
authors.lastname: Luo
authors.forename: Yingguo
authors.initials: Y
authors.lastname: Bai
authors.forename: Bin
authors.initials: B
authors.lastname: Yao
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D050260:Carbohydrate Metabolism; D002480:Cellulase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004271:DNA, Fungal; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010676:Phialophora / Q000201:enzymology*; D013379:Substrate Specificity
chemical_list: D004271:DNA, Fungal; D002480:Cellulase