Two-Stage Approach to Biopolymer Entrapment

The first belief in the possibility of enzyme stabilization on a silica matrix was stated by Dickey in 1955, but he did not give experimental evidence, only mentioning that his experiments were unsuccessful [65]. A sol-gel procedure for enzyme immobilization in silica was first developed by Johnson and Whateley in 1971 [66]. The entrapped trypsin retained about 34 % of its tryptic activity observed in solution before the encapsulation. Furthermore, the enzyme was not released from the silica matrix by washing, demonstrating the increased stability and working pH range. Unfortunately, the article did not attract attention, although their method contained all the details that may be found in the present-day common approach. This was probably due to its publication in a colloid journal that was not read by biochemists. 

The next publication appeared in 1984. Venton et al. [67] entrapped antiprogesterone antiserum into a sol-gel derived silica. It retained about 56 % of progesterone binding capacity. The authors mentioned the considerable promise of the sol-gel technique for biopolymer immobilization, but their study was not further continued. 

Interest in sol-gel processing was awakened by the work of Avnir et al. in 1990 who performed successful experiments with such enzymes as [1-glucosidasc, alkaline phosphatase, chitinase and aspartase [68]. This gave impetus to their own systematic study of the entrapment of biopolymers in a silica matrix as well as those of other teams [69-79]. The results have been summarized and discussed in numerous review articles (see, e.g., Refs. [41—43,45—49,51,80—85]). 

The immobilization procedure performed in two stages allows one to exclude the detrimental effect of acid on the entrapped proteins [44,71,86]. It was demonstrated [87] by the example of a set of oxidases that their activity was retained if the entrapment was carried out at a pH as close to their isoelectric point (pi) as possible. Because the pi value of most enzymes is in the neutral region, the two-stage procedure favors the retention of their functionality. Therefore, the pH shift to the optimal region provides a means of extending the sol-gel entrapment to a wide range of enzymes [45,71]. 

The two-stage procedure eliminates the detrimental effect of acid, but an alcohol, which is produced in the course of precursor hydrolysis Equation (2), remains in solution. This can cause unfolding of the biomacromolecule and denaturation of 

---