Stabilizing enzymes from mesophiles of industrial interest is one of the greatest challenges of protein engineering. The ancestral mutation method, which introduces inferred ancestral residues into a target enzyme, has previously been developed and used to improve the thermostability of thermophilic enzymes. In this report, we studied the ancestral mutation method to improve the chemical and thermal stabilities of Phanerochaete chrysosporium lignin peroxidase (LiP), a mesophilic fungal enzyme. A fungal ancestral LiP sequence was inferred using a phylogenetic tree comprising Basidiomycota and Ascomycota fungal peroxidase sequences. Eleven mutant enzymes containing ancestral residues were designed, heterologously expressed in Escherichia coli and purified. Several of these ancestral mutants showed higher thermal stabilities and increased specific activities and/or kcat/KM than those of wild-type LiP.
The Sullivan model is adopted for the interaction parameters of fluid-fluid and wall-fluid potentials in binary mixtures. The capillary condensation in 'two-coexisting-phases' and 'three-coexisting-phases' open system confined between WO parallel walls ss studied analytically by a dynamical approach. The result shows that not only a first-order bul also a continuous hmsition is possible. The two systems sludied in the present paper have no apparent diffetmce in theit wetting behaviours, and their phase diagram conshucted in terms of the two components of the wall-fluid potential is a segment of an ellipse.
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