Entrapment and Covalent Linking of Trypsin in Sol-Gel Silica Matrices

• Main Authors: Yu-Chun Chuang, 莊玉君
• Other Authors: Lai-Kwan Chau
• Format: Others
• Language: zh-TW
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/81160225266702047648

碩士 === 國立中正大學 === 化學研究所 === 92 === Abstract This research attempts to prepare mesoporous sol-gel silica to immobilize trypsin, so that large protein molecules can pass through the mesopores and cleavaged by trypsin. However, trypsin may leach out from sol-gel silica. Therefore, immobilization of trypsin was achieved by covalent linking of trypsin with the silica matrix. To prepare mesoporous sol-gel silica materials, three methodologies have been attempted. The first method is entrapment, followed by drying with supercritical carbon dioxide extraction. The results show that the activity of trypsin increases. However, supercritical carbon dioxide extraction cannot extract all water out. As a result, aerogel was not formed. Hence, this method needs to be improved. The second method is by addition of PEG (Polyethylene glycol, MW=300) into silica sol, where trypsin has been linked covalently with the sol-gel precursor ATPES by glutaraldehyde. However, BET results show that this material is not a mesoporous material. Unexpectedly, this method does not decrease the catalytic activity of trypsin. The catalytic activity of trypsin by this method (Km=4.80×10-4M，Kcat=3.64×103 min-1) is higher than that by the entrapment method (Km=1.18×10-3M, Kcat=9.60×102min-1). The last method is to use PEG (MW=8500) as a template to prepare mesoporous sol-gel silica and link trypsin on the surface of the sol-gel silica covalently. Results of this method show that large protein molecules can pass through the mesopore and cleaved by trypsin.