High stability and low competitive inhibition of thermophilic Thermopolyspora flexuosa GH10 xylanase in biomass-dissolving ionic liquids

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Researchers

Research units

  • VTT Technical Research Centre of Finland

Abstract

Thermophilic Thermopolyspora flexuosa GH10 xylanase (TfXYN10A) was studied in the presence of biomass-dissolving hydrophilic ionic liquids (ILs) [EMIM]OAc, [EMIM]DMP and [DBNH]OAc. The temperature optimum of TfXYN10A with insoluble xylan in the pulp was at 65–70 °C, with solubilised 1 % xylan at 70–75 °C and with 3 % xylan at 75–80 °C. Therefore, the amount of soluble substrate affects the enzyme activity at high temperatures. The experiments with ILs were done with 1 % substrate. TfXYN10A can partially hydrolyse soluble xylan even in the presence of 40 % (v/v) ILs. Although ILs decrease the apparent temperature optimum, a surprising finding was that at the inactivating temperatures (80–90 °C), especially [EMIM]OAc increases the stability of TfXYN10A indicating that the binding of IL molecules strengthens the protein structure. Earlier kinetic studies showed an increased Km with ILs, indicating that ILs function as competitive inhibitors. TfXYN10A showed low increase of Km, which was 2-, 3- and 4-fold with 15 % [EMIM]OAc, [DBNH]OAc and [EMIM]DMP, respectively. One reason for the low competitive inhibition could be the high affinity to the substrate (low Km). Xylanases with low Km (~1 mg/mL) appear to show higher tolerance to ILs than xylanases with higher Km (~2 mg/mL). Capillary electrophoresis showed that TfXYN10A hydrolyses xylan to the end-products in 15–35 % ILs practically as completely as without IL, also indicating good binding of the short substrate molecules by TfXYN10A despite of major apparent IL binding sites above the catalytic residues. Substrate binding interactions in the active site appear to explain the high tolerance of TfXYN10A to ILs.

Details

Original languageEnglish
Pages (from-to)1487-1498
Number of pages12
JournalApplied Microbiology and Biotechnology
Volume101
Issue number4
Publication statusPublished - 1 Feb 2017
MoE publication typeA1 Journal article-refereed

    Research areas

  • Active site modelling, Competitive inhibition, GH10 xylanase, Hydrophilic ionic liquids, Stability, Substrate interaction

ID: 11074181