Impact of the tryptophan residues of Humicola lanuginosa lipase on its thermal stability

Thermal stability of wild type Humicola lanuginosa lipase (wt HLL) and its two mutants, W89L and the single Trp mutant W89m (W117F, W221H, and W260H), were compared. Differential scanning calorimetry revealed unfolding of HLL at T-d = 74.4 degreesC whereas for W89L and W89m this endotherm was decrea sed to 68.6 and 62 degreesC, respectively, demonstrating significant contri bution of the above Trp residues to the structural stability of HLL. Fluore scence emission spectra revealed the average microenvironment of Trps of wt HLL and W89L to become more hydrophilic at elevated temperatures whereas t he opposite was true for W89m. These changes in steady-state emission were sharp, with midpoints (T-m) at approx. 70.5, 61.0, and 65.5 degreesC for wt HLL, W89L, and W89m, respectively. Both steady-state and time resolved flu orescence spectroscopy further indicated that upon increasing temperature, the local movements of tryptophan(s) in these lipases were first attenuated . However. faster mobilities became evident when the unfolding temperatures (T-m) were exceeded, and the lipases became less compact as indicated by t he increased hydrodynamic radii. Even at high temperatures (up to 85 degree sC) a significant extent of tertiary and secondary structure was revealed b y circular dichroism. Activity measurements are in agreement with increased amplitudes of conformational fluctuations of HLL. with temperature. Our re sults also indicate that the thermal unfolding of these lipases is not a tw o-state process but involves intermediate states. Interestingly, a heating and cooling cycle enhanced the activity of the lipases, suggesting the prot ein to be trapped in an intermediate, higher energy state. The present data show that the mutations. especially W89L in the lid, contribute significan tly to the stability, structure and activity of HLL. (C) 2001 Elsevier Scie nce B.V. All rights reserved.