Trypsin trimethylsilanol

Protease-Catalyzed Condensation Study. Based on the exceptional activity of trypsin and a-chymotrypsin from bovine pancreas (Table I), protease enzymes were identified as target catalysts. Consequently, a series of proteases (i.e. serine, cysteine, aspartic, and metallo) were selected in order to screen their ability to catalyze siloxane condensation with trimethylsilanol. The reactions... 

Trypsin preferentially catalyzed the condensation of trimethylsilanol under mild conditions (87% HMDS). Subs tial condensation of trimethylsilanol was not observed in the negative control, non-specific protein (i.e. BSA, y-globulins), small molecule (i.e. CaCl2, imidazole, N-methylimidazole), and polypeptide (i.e. poly-L-lysine) reactions in comparison to the raw material (< 1% HMDS). Based on an inqjurity study with small molecule inhibitors (23), the exceptional activity of trypsin and a-chymotrypsin observed in the original enzyme-catalyzed condensation study was soley due to a tryptic impurity. The tertiary... 

Figure 5. Turnover numbers of the trypsin-catalyzed hydrolysis of trimethylethoxysilane and condensation of trimethylsilanol at 10 C.

Inhibition Study. A proteinaceous inhibition study was conducted to study the role of the enzymatic active site in the hydrolysis and condensation of trimethylethoxysilane. Prior to reaction, trypsin was independently inhibited with an excess amount of the Bowman-Birk inhibitor (34) (4 1 BBI to trypsin mole ratio) and die Popcorn inhibitor (35) (2 1 PCI to trypsin mole ratio) in stirred neutral media for two hours. Based on standard enzymatic activity assays (36), trypsin was fully inhibited by the BBI (98%) and PCI (91%). The reactions were formulated with an 1000 1 trimethylethoxysilane to trypsin mole ratio and conducted at 25°C for three hours. The reaction products were isolated and quantitatively analyzed by GC (Table II). Although the treated enzymes were observed to catalyze the hydrolysis of trimethylethoxysilane, the condensation of trimethylsilanol was conqiletely inhibited in conq>arison to the control reactions. Notably, the rate of hydrolysis decreased in the presence of the BBI- and PCI-inhibited trypsin. Following thermal denaturation, tiie activity of trypsin was comparable to the proteinaceous inhibition experiments. Based on a standard enzymatic activity assay (36), the relative decrease in the rate of silanol condensation correlated with the enhanced stability of trypsin at higher protein concentrations (25). Consequently, it appears that non-specific interactions with trypsin including the active site promoted the hydrolysis of trimethylethoxysilane. Therefore, the active site of trypsin was determined to selectively catalyze the in vitro condensation of trimethylsilanol imder mild conditions. 

Based on the estimated solubility of trimethylsilanol in water (42.56 mg/mL) (32), the concentration of trimethylsilanol ( 160 mg/mL) saturated the aqueous medium and created a two-phase reaction mixture. Since proteases will only interact with water-soluble substrates (31), the trypsin-catalyzed condensation of trimethylsilanol was postulated to occur in the aqueous phase. Although the condensation reaction was conducted in water, the enzyme-catalyzed reaction was promoted by the phase separation of the product. The immiscibility of the product, hexamethyldisiloxane, changed the equilibrium (37) and promoted the condensation reaction in the presence of water. Since the aqueous medium was saturated with trimethylsilanol, the reactant would continue to enter the aqueous phase due to the dynamic equilibrium of the condensation reaction. In addition, the hydrolysis or reverse reaction would be severely hindered due to the immiscibility of the disiloxane product in the aqueous phase. 

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