title: Thermostabilization of extremophilic Dictyoglomus thermophilum GH11 xylanase by an N-terminal disulfide bridge and the effect of ionic liquid [emim]OAc on the enzymatic performance.
abstract: In the present study, an extremophilic GH11 xylanase was stabilized by an engineered N-terminal disulphide bridge. The effect of the stabilization was then tested against high temperatures and in the presence of a biomass-dissolving ionic liquid, 1-ethyl-3-methylimidazolium acetate ([emim]OAc). The N-terminal disulfide bridge increased the half-life of a GH11 xylanase (XYNB) from the hyperthermophilic bacterium Dictyoglomus thermophilum by 10-fold at 100°C. The apparent temperature optimum increased only by ∼5°C, which is less than the corresponding increase in mesophilic (∼15°C) and moderately thermophilic (∼10°C) xylanases. The performance of the enzyme was increased significantly at 100-110°C. The increasing concentration of [emim]OAc almost linearly increased the inactivation level of the enzyme activity and 25% [emim]OAc inactivated the enzyme almost fully. On the contrary, the apparent temperature optimum did not decrease to a similar extent, and the degree of denaturation of the enzyme was also much lower according to the residual activity assays. Also, 5% [emim]OAc largely counteracted the benefit obtained by the stabilizing disulfide bridge in the temperature-dependent activity assays, but not in the stability assays. Km was increased in the presence of [emim]OAc, indicating that [emim]OAc interfered the substrate-enzyme interactions. These results indicate that the effect of [emim]OAc is targeted more to the functioning of the enzyme than the basic stability of the hyperthermophilic GH11 xylanase.
authors: Aalto University, School of Chemical Technology, Department of Biotechnology and Chemical Technology, P.O. Box 16100, 00076 Aalto, Finland.
authors: He
authors: H
authors: Li
authors: Anna
authors: A
authors: Kankaanpää
authors: Hairong
authors: H
authors: Xiong
authors: Michael
authors: M
authors: Hummel
authors: Herbert
authors: H
authors: Sixta
authors: Heikki
authors: H
authors: Ojamo
authors: Ossi
authors: O
authors: Turunen
mesh_terms: D019943:Amino Acid Substitution; D001419:Bacteria / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D004220:Disulfides / Q000737:chemistry; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability / Q000187:drug effects; D006207:Half-Life; D007093:Imidazoles / Q000494:pharmacology; D052578:Ionic Liquids / Q000494:pharmacology; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013696:Temperature; D001617:beta-Glucosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D004220:Disulfides; D007093:Imidazoles; D052578:Ionic Liquids; D011994:Recombinant Proteins; D001617:beta-Glucosidase; D043364:Endo-1,4-beta Xylanases; C090001:XynB xylanase; C518739:1-ethyl-3-methylimidazolium
keywords: Dictyoglomus thermophilum; GH11 xylanase; Ionic liquid; N-terminal disulphide bridge; Thermostability; [emim]OAc