Abstract
Soil microorganisms play critical roles in the degradation of micro-and nano-pollutants, and the corresponding proteins and enzymes play roles in pollutant recognition, transportation, and degradation. Our ability to study these pathways from soil samples is often complicated by the complex processes involved in extracting proteins from soil matrices. This study aimed to develop a new protein soil extraction protocol that yielded active, intracellular enzymes from the perchlorate degradation pathway, particularly perchlorate reductase. An indirect method, which focused on first separating the cells from the soil matrix, followed by cell lysis and enzyme extraction, was evaluated. The optimized indirect method achieved a final extraction efficiency of the active enzyme and total protein of 15.7 % and 3.3 %, respectively. The final step of separating enzymes from residual soil components resulted in the highest activity and protein losses of 67.7 % ± 14.8 % and 91.8 % ± 1.8 %, respectively. Five buffers, each at different concentrations (0.01 M, 0.05 M, and 0.1 M), were tested to enhance enzyme extraction efficiency. The best extractant requires careful consideration between the highest activity and the quality of the recovered enzymes. Coextraction of humic substances could be minimized by using 0.1 M as compared to 0.01 M and 0.05 M of sodium pyrophosphate; however, this resulted in less recovered activity compared to lower extractant concentrations.