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. 2001 Jun 1;356(Pt 2):453–460. doi: 10.1042/0264-6021:3560453

Thermostability of endo-1,4-beta-xylanase II from Trichoderma reesei studied by electrospray ionization Fourier-transform ion cyclotron resonance MS, hydrogen/deuterium-exchange reactions and dynamic light scattering.

J Jänis 1, J Rouvinen 1, M Leisola 1, O Turunen 1, P Vainiotalo 1
PMCID: PMC1221856  PMID: 11368772

Abstract

Endo-1,4-beta-xylanase II (XYNII) from Trichoderma reesei is a 21 kDa enzyme that catalyses the hydrolysis of xylan, the major plant hemicellulose. It has various applications in the paper, food and feed industries. Previous thermostability studies have revealed a significant decrease in enzymic activity of the protein at elevated temperatures in citrate buffer [Tenkanen, Puls and Poutanen (1992) Enzyme Microb. Technol. 14, 566-574]. Here, thermostability of XYNII was investigated using both conventional and nanoelectrospray ionization Fourier-transform ion cyclotron resonance MS and hydrogen/deuterium (H/D)-exchange reactions. In addition, dynamic light scattering (DLS) was used as a comparative method to observe possible changes in both tertiary and quaternary structures of the protein. We observed a significant irreversible conformational change and dimerization when the protein was exposed to heat. H/D exchange revealed two distinct monomeric protein populations in a narrow transition temperature region. The conformational change in both the water and buffered solutions occurred in the same temperature region where enzymic-activity loss had previously been observed. Approx. 10-30% of the protein was specifically dimerized when exposed to the heat treatment. However, adding methanol to the solution markedly lowered the transition temperature of conformational change as well as increased the dimerization up to 90%. DLS studies in water confirmed the change in conformation observed by electrospray ionization MS. We propose that the conformational change is responsible for the loss of enzymic activity at temperatures over 50 degrees C and that the functioning of the active site in the enzyme is unfeasible in a new, more labile solution conformation.

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