Immobilization of Enzymes on Nanoporous, Silica Composites

Abstract

In this work, the sol-gel process is used to produce a chemically surface modified gel (CSMG) with a high modification density. Sodium silicate, an inexpensive alternative to tetraethoxysilicate, is used as the silica source. The produced N-CSMG material is shown to have a high amino functional group loading of 3.6 mmol/g SiO2; significantly higher than commercially available matrices. One aspect of this work deals with retention of an open morphology to provide access of interior surface areas. Activation of N-CSMG, in the wet state, with glutaraldehyde produces GA-N-CSMG which is used for the immobilization of enzymes. Nitrogen adsorption (BET), SEM, and thermogravimetric analysis were used to study material properties. Invertase was used as a model enzyme to measure the immobilizing character of the GA-N-CSMG material. Using an optimized immobilization protocol, a very high loading of 700 mg invertase per gram GA-N-CSMG is obtained; significantly higher than other published results. The immobilized activity of 246,000 U/g GA-N-CSMG is also greater than any other in literature. Immobilized invertase showed almost 99% retention of free enzyme activity and no loss in catalytic efficiency. A major part of this work consists of a scale-up in production of low molecular weight protamine (LMWP), a peptide with possible pharmaceutical applications. Since LMWP is produced by the enzymatic hydrolysis of native protamine, the production of LMWP was improved upon through the immobilization of the enzyme thermolysin. The immobilized thermolysin proved to be quite robust and was integrated into a continuous flow-through system for LMWP production. The purification of LMWP was also improved upon by optimization of a heparin-affinity chromatographic process. Optimization allowed for a scale-up of LMWP production from 5 μg/min to 167 μg/min; a 33-fold increase in production with improved product purity and recovery. The last part of this work improves on the N-CSMG material by developing particles of controllable size and regular shape. Directions for further improvement of N-CSMG are discussed, including addition of the pH sensitive biopolymer chitosan. The improved surface coverage of mesoporous silica gels will provide significant benefits in the areas of catalysis, water treatment, sensors, drug delivery, and tissue engineering.