IMMOBILIZATION OF ENZYMES ON NANOPOROUS SILICA COMPOSITES

Diploma

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-NCSMG 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 heparinaffinity 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.


Introduction

Organic-inorganic composites have generated a significant amount of interest because of their inherent properties.  The rigid structure of the inorganic component combined with the functionality of organic groups has yielded advanced materials with improved properties for optics, electronics, protective coatings, sensors, catalysis, and many other fields (Sanchez, Soler-Illia et al. 2001).  Silica-based inorganic composites have received much attention since the development of the M41S periodic mesoporous matrix (Kresge, Leonowicz et al. 1992) in the early 1990s.  Since then a number of protocols have been developed, using a wide array of conditions, to produce a variety of mesoporous silicas (Sayari and Hamoudi 2001).  Two general methods are available for the chemical modification of mesoporous silicas:  (1) post-synthesis grafting (Beck, Vartuli et al. 1992) and (2) co-condensation (Burkett, Sims et al. 1996).  Post-synthesis grafting involves the reaction of organosilanes with silanol groups present on the matrix surface.  Generally this method results in an inhomogeneous surface coverage of the function group with concentration at the outer surface and pore entrances (Lim and Stein 1999). 

In the co-condensation process, organosilanes are mixed with the silica sol prior to gelation and condensation between the sol and organosilanes results in a silica matrix with a chemically active surface.  A homogenous distribution of functional groups can be achieved with the co-condensation process (Lim and Stein 1999) with higher loading densities (Mori and Pinnavaia 2001; Kruk, Asefa et al. 2002).  However, practically all co-condensation processes to date make use of tetraethoxysilane (TEOS) or tetramethoxysilane (TMOS) as the source of silica.  Only recently has a report been published detailing the synthesis of mesoporous organosilicas from sodium silicate in a co-condensation process (Yu, Gong et al. 2004).  Yu et. al. vigorously mixed acidified solutions of sodium silicate and organosilanes and allowed hydrolysis to occur for one hour.  The solution temperature was then raised to 50 °C and NaF added to induce gelation.  With this method methyl, mercapto, and vinyl surface modifications were introduced to mesoporous silica gels. 

Although there are many reports using TEOS to produce amino-functionalized silica gels (Lee, Kim et al. 2001; Bois, Bonhomme et al. 2003; Yokoi, Yoshitake et al. 2004), only one reference has been found for a co-condensation process which uses sodium silicate (Shah, Kim et al. 2004).  They report a maximum of 50 mol% functionalization of available sites with 3-aminopropyltrimethoxysilane.

While tremendous advances have been made in the fabrication of composites with a high surface area, high functional loading, and controlled morphology, the immobilization of biological specie on these composites has not seen a significant improvement compared to materials of the past.  Efficient immobilization of biologics requires not only a matrix with designed properties, but also the proper protocols and processing conditions.  Design of such a system requires a proper understanding of the underlying process and an ability to control it.