Original Framework for Enzyme Engineering of Nigerian Actinomycete-Derived Industrial Enzymes with Enhanced Stability and Activity

Actinomycetes from Nigerian ecological niches are an underexplored source of industrial enzymes with potential superior catalytic and stability properties compared to commercially established enzyme sources, and their systematic discovery and rational engineering represents an original contribution to biotechnology relevant to Nigeria's industrial development. This dissertation develops an original framework for the discovery and enzyme engineering of Actinomycete-derived industrial enzymes from Nigerian soil ecosystems. Soil samples from twelve ecologically diverse Nigerian sites representing forest, savanna, mangrove, and arid zone habitats were used for targeted Actinomycete isolation by humic acid agar selective culture. Isolates producing lipase, amylase, cellulase, and protease activities were screened by high-throughput activity-based plate assays. Promising enzyme producers were characterized at the species level by 16S rRNA gene sequencing and multilocus phylogenetic analysis. The three most active amylase-producing isolates from Jos Plateau savanna soils underwent genome sequencing, with amylase gene identification by annotation and heterologous expression in Escherichia coli. Crystal structures of two novel amylases were solved at 1.8 Angstrom resolution, revealing a distinctive calcium-coordinating loop architecture not present in characterized amylases. Structure-guided mutagenesis guided by molecular dynamics simulation and free energy perturbation calculations targeted this loop region to engineer enhanced thermostability variants. Directed evolution by error-prone PCR and DNA shuffling of the most promising variant lineage produced a double-mutant enzyme with forty-three-fold increased half-life at 70 degrees Celsius and twenty percent improved specific activity at neutral pH. The dissertation constructs an original enzyme engineering framework combining structural characterisation, computational guidance, and directed evolution applicable across the Nigerian Actinomycete enzyme discovery pipeline. Keywords: enzyme engineering, Actinomycetes, Nigerian soil, amylase, directed evolution.