Xylanase II from Trichoderma reesei QM 9414: conformational and catalytic stability to Chaotropes, Trifluoroethanol, and pH changes.
Xylanase II, a key enzyme in the hydrolysis of xylan, was purified from cultures of Trichoderma reesei QM 9414 (anamorph of Hypocrea jecorina) grown on wheat straw as a carbon source. Xylanase treated with increasing guanidinium hydrochloride concentrations was denatured in a cooperative way regarding secondary and tertiary structures with midpoint transitions 5.6 ± 0.1 and 3.7 ± 0.1 M, respectively, whereas the enzymatic activity showed an intermediate state at 2-4 M denaturant. Treatment with urea showed that xylanase secondary structure was stabilized up to 4 M urea to be destabilized thereafter in a cooperative way with a transition midpoint Dm = 5.7 ± 0.2 M, but the ellipticity at 220 nm was greater than control in the presence of urea up to 6 M. Tertiary structure in the presence of urea showed also intermediate states with partial cooperative transitions with a midpoint: Dm = 2.7 ± 0.04 and 6.7 ± 0.3 M, respectively, whereas the enzymatic activity was enhanced about 40% at 2 M and inhibited above 4 M urea. Assays with the fluorescent probe 4,4'-bis-1-phenylamine-8-naphftalene sulfonate (bis-ANS) proved that the intermediate states had the characteristics of molten globule structures. The change of free energy for xylanase in absence of denaturants obtained from the spectral centre of mass (SCM) data at 298 K is = − 17 kJmol⁻¹ . In the presence of increasing trifluoroethanol (TFE), the enzyme gained α-helix content and lose tertiary structure and catalytic activity. Changes in pH (2-9) had practically no effect on the secondary structure of the enzyme, whereas the SCM values indicated that tertiary structure is maintained above pH 4. Bis-ANS binds to xylanase at pH 2 and 2.5 and in the presence of 30-40% TFE (v/v) characterizing molten globule states in those environmental conditions.
López
G
G
Departamento de Bioquímica y Biología Molecular I., Facultad de Biología, Universidad Complutense, 28040, Madrid, Spain.
Bañares-Hidalgo
A
A
Estrada
P
P
D000817:Anilino Naphthalenesulfonates / Q000737:chemistry; D002942:Circular Dichroism; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry; D005656:Fungal Proteins / Q000737:chemistry; D019791:Guanidine / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D011489:Protein Denaturation; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D014242:Trichoderma / Q000201:enzymology*; D014270:Trifluoroethanol / Q000737:chemistry*; D014508:Urea / Q000737:chemistry*
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D005656:Fungal Proteins; C035763:5,5'-bis(8-(phenylamino)-1-naphthalenesulfonate); D014270:Trifluoroethanol; D014508:Urea; D043364:Endo-1,4-beta Xylanases; D019791:Guanidine