Physics And Chemistry Basis Of Biotechnology - De Cuyper - tiera.ru

D. Georlette et al
V 196F, engineering a salt bridge (N 1 50D) and an aromatic interaction (V 196F), caused
decreased activity and significantly increased melting temperature (44°C to 46.4°C),
establishing a correlation between increased stability and lowered activity (D' Amico et
al., personal communication). Some other site-directed mutagenesis experiments have
been carried on subtilisin [45] and a moderately stable thermolysin like protease [76].
Both studies revealed that the stability of the mutated enzymes was drastically
improved due to a small number of mutations, while increasing or retaining the original
catalytic properties of the enzymes, which does not correspond with the stabilityspecific
activity-flexibility assumption. Moreover, a recent in vitro evolution
experiment has proved that stability and catalytic activity are not systematically
inversely related, since random mutagenesis on the Bacillus subtilis p-nitrobenzyl
esterase led to an increase in stability (>14°C increase in Tm) without compromising
catalytic activity [77]. In fact, reduced stability may not necessarily arise from a general
reduction in strength of intramolecular forces, but from weakened interactions in one or
a few important regions of the structure [78].
Figure 1. Thermal unfolding of the psychrophilic Pseudomonas sp. TACII 18 (left and
yeast (right) phosphoglycerate kinase (PGK). a to d: baseline-subtracted thermograms
recorded by DSC. a and b: free enzymes (the deconvolution in :wo transitions is dotted). c
and d: PGK in the presence of 5 mM 3-phosphoglyceric acid and 5 mM Mg-ADP (solid
lines). In c and d, the thermogram for free enzyme is given as a dashed line for
comparison. HLd, heat-labile domain; HSd, heat-stable domain.
184