In this work, the PQQGDH gene was integrated into the chromosome of Escherichia coli EcNR2 and then modulated by multiplex-site in situ engineering to achieve improved substrate specificity and thermal stability.
They figure prominently in directed evolution experiments, where libraries of enzyme variants are screened for improved substrate specificity, thermal stability, or other beneficial properties.
Figure 3b shows the results of single cell lysate kinase activity assay using Akt as a substrate (PKC and PKA inhibitors added to improve substrate specificity without affecting Akt activity).
This type of flexibility is a good starting point for directed evolution and protein-engineering approaches to improve substrate specificity toward biotechnologically relevant sugars, e.g. animal-like β-1,4-galactosylated and α-1,3-fucosylated chito-oligosaccharides (Boer et al. 2004).
We designed a new alkaline phosphatase (ALP -sensitive fluorogenic probe in which ALP -sensitiveive spacer group, p-hydroxybenzyl alcohol, is linked to a profluorogenic comprobe to inprove substrate specificity.
These include altering the reaction mechanism of the enzyme to catalyse new reactions, switching substrate specificity, expanding substrate specificity, and improving substrate specificity, such as enantioselectivity in kinetic resolutions.
The L255W mutation improves substrate specificity, but, at the same time, sacrifices structural stability.
The S237A substitution in CTX-M-14 shifted the substrate specificity to improve catalytic efficiency for benzylpenicillin hydrolysis and decrease cefotaxime hydrolysis (p-value of 0.001 when compared to wild-type), consistent with an alanine at position 237 in the TEM-1/SHV-type enzymes that hydrolyze cefotaxime slowly (Table 2 and Supporting Information Figure S1).
This again emphasizes the importance of substrate specificity to enzyme activation.
