In this work, an electrostatic-induced interfacial assembly of porcine pancreatic lipase (PPL) with the nanosheets of layered double hydroxide (LDHNSs) is designed to rationally control the orientation of bound PPL. The PPL orientation in the bidimensional confinement spacing alters relying on the PPL loading, with the majority of active sites facing the LDH layer at low PPL loading and facing the adjacent protein molecule at high PPL loading. The biocatalytic activity of the bound PPL significantly depends on its orientation. Remarkable enhancement of the bio-activity has been observed when the PPL/LDHNSs mass ratio is less than 9, and a maximum activity is met with at PPL/LDHNSs = 0.5. In addition, the thermal stability of PPL-LDHNSs bioactivity has been obviously improved in comparison with soluble PPL. (C) 2010 American Institute of Chemical Engineers AIChE J, 56: 2677-2686, 2010