Trypsin catalyzed condensation

The well known specificity of proteinases implies the use of specific amino acids (amides, esters) as acyl donors and—seldom—specific amino acid (derivatives) as acceptors in enzymatic peptide bond formation, since the same structural features of RCONHR that influence the rate of hydrolytic cleavage are also involved in the synthesis. Accordingly trypsin is well suited to the formation of a new -Arg-X or -Lys-X bond. As an example the transformation of the -Lys-AlaOH terminus of the B-chain of porcine insulin into -LysThrOBu of human insuhn may be mentioned. C-terminal Ala was removed by means of car-boxypeptidase A, trypsin-catalyzed condensation of the des-alanine peptide with threonine tert. butylester gave 73% of the ester of human insulin [33] (see also... 

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. 

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 4. Trypsin-catalyzed hydrolysis and condensation of trimethylethoxysilane at 10° C.

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

Since trypsin catalyzed the formation of siloxane bonds, alternate monofunctional aUcoxysilanes were chosen as substrates to investigate the ability of trypsin to selectively catalyze the in vitro hydrolysis and condensation of organo-hmctional alkoxysilanes under mild conditions. l,l-dimethyl-l-sila-2-oxacyclohexane and phenyldimethylethoxysilane were selected to study the activity of trypsin due to different interactions with the substrates. The two-... 

In comparison to a control reaction, trypsin reportedly did not catalyze the polycondensation of a silicic acid analogue, tetraethoxysilane, in an aqueous medium at pH 6.8 (9). Similarly, trypsin did not hydrolyze or condense tetraethoxysilane in a replicate reaction formulated with a 4 1 monomer to enzyme weight ratio and conducted at 25"C for three hours. 

In review, trypsin was observed to selectively catalyze the hydrolysis and condensation of some organo-functional alkoxysilanes under mild conditions. 

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. 

In a similar manner there occurs the formation of 7-glutamyl peptides by sheep kidney extracts from glutathione and free amino acids. The activity of esters as substrates is shown by methionine isopropyl ester condensing to form methionylmethionine and methionylmethionylmethionine catalyzed by trypsin or by chymotrypsin. A similar condensation has been noted for threonine isopropyl ester. ... 

Followed by these observations, Bassindale et al. [ 19,20] studied the use of various homologous lipase and protease enzymes to catalyze the formation of molecules with a single siloxane bond during the in vitro hydrolysis and condensation of alkoxysilanes under mild reaction conditions. They found that non-specific interactions with trypsin promoted the hydrolysis of alkoxysilanes, while the active site was determined to selectively catalyze the condensation of silan-ols. One interesting observation was that when trypsin from various sources was employed different extents of conversion were observed. Comparatively, the activity of trypsin from a bovine pancreas was greater than the alternate sources of trypsin. Although various sources (e.g., mammalian, fish) of trypsin are similar (e.g., tertiary structure), their selectivity and activity was found to be different due to different optimum pH ranges and/or levels of calcium (an additive). 

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