In this work,we developed a new approach to porous polymer monoliths that permits substantial enhancement in coverage with gold nanoparticles.An enzymatic bioreactor was designed using GNP monoliths.Both porcine lipase and trypsin were reversibly immobilized on a monolithic polymer support functionalized with gold nanoparticles.This flow through enzymatic reactor with porcine lipase was then used to catalyze the hydrolysis of glyceryl tributyrate(tributyrin).The reaction kinetics is characterized by Michaelis-Menten constant,Km = 10.9 mmol/L,and maximum reaction rate,Vmax = 5.0 mmol/L.min.The maximum reaction rate for the immobilized enzyme is 1,000 times faster compared to lipase in solution.The fast reaction rate enabled to achieve 86.7% conversion of tributyrin in mere 2.5 min and an almost complete conversion in 10 min.The reactor lost only less than 10% of its activity even after continuous pumping through it a solution of substrate equaling 1,760 reactor volumes.Finally,potential application of this enzymatic reactor was demonstrated with the transesterification of triacylglycerides from kitchen oil to fatty acid methyl esters thus demonstrating the ability of the reactor to produce biodiesel.The bioreactor immobilized with trypsin was applied for protein digestion of cytochrome C,bovine serum albumin(BSA),and proteins extracted from E.coli cells,which demonstrated efficient protein digestion achieved within 1.06 min.The digested products were measured by HPLC and MALDI-TOF-MS,which elucidates high peptide coverage.Compared with digestion using free trypsin which takes 24 h,our newly designed bioreactor exhibits much higher catalytic efficiency.Furthermore,use of gold nanoparticles enabled rejuvenation of the activity of the deactivated reactor simply by stripping the inactive enzyme from the nanoparticles using 2-mercaptoethanol and subsequent immobilization of fresh enzyme to achieve the reusability of the enzymatic bioreactors.