The L255W mutation improves substrate specificity, but, at the same time, sacrifices structural stability.
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.
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.
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).
Protein engineering is needed to improve the substrate specificity, enantioselectivity, specific activity, thermostability, and heterologous expression in other hosts (especially food grade microorganisms) leading to eventual large scale production and applications.
Immobilization of protocatechuate 3,4-dioxygenase from Stenotrophomonas maltophilia KB2 has been shown to be an effective method for improving the substrate specificity of the enzyme.
Site-directed mutagenesis was carried out on the active site of water-soluble PQQ glucose dehydrogenase (PQQGDH-B) to improve its substrate specificity.
