Structural characterization, functional analysis and computational annotation of a metagenome-derived Glucoamylase enzyme: Effect of temperature, pH, metal Ions, and surfactants on enzyme activity

Glucoamylase (GluAmy) holds significant industrial relevance, particularly in starch processing industries, owing to its ability to efficiently hydrolyze complex carbohydrates into glucose under diverse environmental conditions. In this study, a thermostable and alkalophilic GluAmy gene, 510 bp in length, was amplified from a hot spring metagenome. The gene was initially cloned into the pJET 1.2 vector and transformed into Escherichia coli DH5α, followed by heterologous expression using the pET28a vector in E. coli BL21 (DE3) cells. Purification via Ni-His affinity chromatography yielded GluAmy (19.2 kDa), which was biochemically characterized for activity and stability across a wide pH range (3.0–12.0) and temperatures (10°C–110 °C). The enzyme's activity was influenced by metal ions (Mn2+, Mg2+, Ca2+, Zn2+, Fe2+, Na+, Co2+, Cu2+, Ni2+) at 5–10 mM concentrations, as well as surfactants (Tween-20, Tween-80, Triton X-100, SDS) at 5–10 %. GluAmy demonstrated optimal activity at 80 °C and pH 9.0. Co2+ and Ca2+ enhanced activity by 115.2 % and 105.6 %, respectively, whereas Tween-20 reduced activity to 56.3 %. The purified enzyme exhibited the highest specific activity against starch (12.94 U/mg), followed by dextrin (11.67 U/mg) at 1 % substrate concentration. Computational analysis revealed a protein structure predominantly composed of random coils (53.25 %), contributing to its thermal stability. These findings underscore the potential of GluAmy as a robust biocatalyst for industrial applications, particularly in processes requiring high temperature and alkaline conditions.