Study of individual domains' functionality in fused lipolytic biocatalysts based on Geobacillus lipases and esterases

The prospects of industrial uses of microbial enzymes have increased greatly during the 21st century. Fused lipolytic enzymes (where one or both fused domains possess lipolytic activity) is a rapidly growing group of industrial biocatalysts. However, themost effective fusion strategy, catalytic behavior of each domain and influence of added linkers on physicochemical and kinetic characteristics of such biocatalysts has not been yet explored. In this study the functionality of individual domains in fused lipolytic enzymes, while using GDEst-lip, GDLip-lip and GDEst-est enzymes as a model system, is analyzed for the first time. Analysis of mutant GDEst-lip, GDLiplip and GDEst-est variants, where one domain is inactive, showed that both domains retained their activity, although the reduction in specific activity of individual domains has been detected. Moreover, experimental data proposed that the N-terminal domain mostly influenced the thermostability, while the C-terminal domain was responsible for thermal activity. GDEst-lip variants fused by using rigid (EAAELAAE) and flexible (GGSELSGG) linkers indicated that a unique restriction site or a rigid linker is the most preferable fusion strategy to develop new chimeric biocatalysts with domains of Geobacillus lipolytic enzymes. (C) 2020 Elsevier B.V. All rights reserved.

Automated summary

The study analyzed the functionality of individual domains in fused lipolytic enzymes for the first time, showing that even if one domain is inactive, the other domain retains its activity, albeit with reduced specific activity. Experimental data suggested that the N-terminal domain mainly influenced thermostability, while the C-terminal domain was responsible for thermal activity.