反应详情

The cellobiase was prepared as follows: 1 mL of the crude cellobiase was mixed with 4.0 mL of a solution of 50 mM sodium acetate at pH 5.0. The sodium acetate solution acts as a buffer, ensuring that the proper pH level is maintained. The cellobiose was added to protect the enzyme's catalytic site from any possible inactivation by glutaraldehyde. Two aliquots, each consisting of 2.5 mL of this solution, were then filtered through a PD-10 column, and the filtered cellobiase (total volume 7.0 mL) was used for gel entrapment as described below. One gram of bone gelatin and 0.2567 grams of cellobiose were dissolved in 44 mL of a solution of 50 mM sodium acetate at pH 5.0. The final cellobiose concentration was 15 mM. One mL of glutaraldehyde and 5.0 mL of the filtered cellobiase were added to this solution, and the mixture was incubated at 23° C. for 1 hour. This was done to covalently cross-link the cellobiase, a protein, to gelatin, which is also a protein. The solution was then dialyzed at 4° C. for 20 hours to remove excess glutaraldehyde and essentially all of the cellobiose. 25 mL of the dialyzed solution was then placed in a mixing apparatus. The activity of the filtered cellobiase was determined to be approximately 170 units/mL, where 1 unit is defined as that amount of enzyme required to produce 1 μmol glucose/minute from 10 mM cellobiose at 40° C. in 50 mM sodium acetate at pH 5.0. Two grams of propylene glycol alginate, 14.5 grams of bone gelatin, and 75 mL of 50 mM sodium acetate at pH 5.0 were added to the mixture. The 2% (w/v) propylene glycol alginate used in the manufacture of the gel beads is important for maintaining structural integrity prior to their being soaked in glutaraldehyde. Propylene glycol alginate and bone gelatin are not inhibitors of cellobiase activity. The mixture was stirred at 40° C. for 15-30 minutes until everything was dissolved, resulting in the formation of a viscous solution. The viscous solution was then transferred to a gel bead production apparatus and kept at 40° C. Gel beads were formed by using 20 lbs/in. of gas pressure (helium or nitrogen) to force the viscous liquid through the needle as discrete droplets which fell into 500 mL of mineral oil on the surface of 200 mL of ice cold 50 mM sodium acetate at pH 5.0. One hundred mL of gel beads, 2 mm in diameter were formed. The gel beads were removed from the oil/aqueous interface and stored for 20 hours at 4° C. in a 0.5% (w/v) solution of glutaraldehyde at pH 5.0. They were then washed with 4×500 mL of distilled water and 3×200 mL of 50 mM sodium acetate at pH 5.0, and stored in the sodium acetate solution at pH 5.0. Enzymatic activity was monitored during the procedures involved in the entrapment of cellobiase within the gel beads, and the results are shown in Table 1.

1. customThe cellobiase was prepared
2. temperatureis maintained
3. filtrationwere then filtered through a PD-10 column
4. dissolutionOne gram of bone gelatin and 0.2567 grams of cellobiose were dissolved in 44 mL of a solution of 50 mM sodium acetate at pH 5.0
5. additionOne mL of glutaraldehyde and 5.0 mL of the filtered cellobiase were added to this solution
6. customThe solution was then dialyzed at 4° C. for 20 hours
7. customto remove excess glutaraldehyde