Calcium sulphate-based soybean salting-out urease purification for enzyme-induced carbonate precipitation in road base stabilisation

Plant-based biocementation via enzyme-induced carbonate precipitation (EICP) presents a promising alternative to conventional soil stabilisation. Crude urease extracted from soybeans contains substantial organic matter, which can reduce its effectiveness for soil stabilisation. This study investigates the extraction and purification of soybean urease for stabilising crushed rock base (CRB) material in road construction. A calcium sulphate (CaSO₄) salting-out method was proposed and compared with conventional purification techniques, including filtration and centrifugation, in terms of organic matter removal, turbidity reduction and calcium carbonate (CaCO₃) precipitation behaviour. The purified enzyme was subsequently applied to CRB under varying soybean concentrations, cementation solution (CS) concentrations, and treatment cycles. Mechanical performance of CRB-treated samples was assessed using unconfined compressive strength (UCS) testing, while microstructural characteristics were examined using scanning electron microscopy (SEM) and X-ray diffraction (XRD). Results show that CaSO₄ salting-out effectively removes excess organic matter while maintaining enzymatic activity for EICP, enabling enzyme recovery without energy-intensive centrifugation at practical soybean concentrations. Although the CaSO₄ salting-out enzyme produced lower total CaCO₃ mass than less-refined extracts, it achieved more uniform precipitation and improved strength efficiency per unit of CaCO₃. The controlled addition of skim milk further enhanced the compressive strength without affecting urea conversion. SEM revealed irregular and spherical CaCO₃ particles from all urease extracts, while skim milk addition promoted spherical deposition around and in between the CRB particles. XRD confirmed calcite as the main phase governing strength development.