Subtilisin enzymatic hydrolysis

Se-enriched yeast Enzymatic hydrolysis (protease XIV and subtilisin) with an ultrasonic probe HPLC-ICP-MS... 

The resolution of ( )-Z/77 7 o-mcthylphcnidate (10) free base by enantioselective enzymatic hydrolysis was first reported by us (Novartis) (Scheme 9).[ ] a-Chy-motrypsin and subtilisin carlsberg exhibited selectivity towards the hydrolysis of the (2/ ,2 / )-enantiomer. 

Two-step enzymatic hydrolysis with a non-specific protease (subtilisin) separation on a Spherisorb 5 ODS/AMINO column using two different chromatographic conditions, namely phosphate buffers at pH 2.8 and 6.0 as mobile phases on-line ICP-MS... 

Modification of Ultrafiltered versus Acid Precipitated Soy Protein. When the retentate obtained from the ultrafiltration of soybean extract is subjected to an enzymatic hydrolysis as described earlier (2) for acid precipitated protein, a hydrolysis curve (DH versus time) may be drawn. A comparison of such hydrolysis curves is shown in Fig. 2 for acid precipitated soy protein isolate and ultrafiltered soy protein isolate. The curves are drawn on the basis of the same hydrolysis parameters. The enzyme used is the microbial alkaline protease subtilisin Carlsberg (ALCALASE ). 

Considerable attention has also been given to enantioselective enzymatic hydrolysis of esters of a-amino acids. This is of particular importance as a means of preparing enantiopure samples of unusual (non-proteinaceous) a-amino acids. The readily available proteases a-chymotrypsin (from bovine pancreas) and subtilisin (from Bacillus lichenformis) selectively hydrolyze the L-esters, leaving D-esters unreacted. These enzymatic hydrolysis reactions can be applied to V-protected amino acid esters, such as those containing r-Boc and Cbz protecting groups. 

One of the most desired transformations is a hydrolysis of above substances resulting in free lysergic acid, the substrate for many semisynthetic preparations. Chemical hydrolysis of its derivatives gives low yields (50-65%). Fermentative production of lysergic acid is somehow complicated. Enzymatic hydrolysis of peptide alkaloids is still impracticable. Common proteolytic enzymes (papain, subtilisin, chymotrypsin, termolysin) do not attack peptidic bond in ergokryptine... 

Early reports on the effects of the choice of solvent on enzymatic enantioselectivity showed that substantial changes may be observed. For the transesterification reaction of sec-phenethyl alcohol with vinyl butyrate catalyzed by subtilisin Carlsberg, a 20-fold increase in the E-value was reported when the medium was changed from acetonitrile to dioxane [59]. Similar changes were recorded for the prochiral selectivity of Pseudomonas sp. lipase in the hydrolysis of 2-substituted... 

Carbadox-related residues can also be monitored by conversion to quinoxa-line-2-carboxylic acid. This conversion as well as its liberation are accomplished by alkaline hydrolysis of tissue, followed by isolation of the analyte from indigenous hydrolysis products through extraction and cleanup procedures (419-422). Enzymatic digestion of swine tissues with subtilisin A, which liberates quinoxa-line-2-carboxylic acid from macromolecules, has also been reported (423). 

The authors of the studies cited above found that the enzymes used during sample preparation could not completely hydrolyze sample proteins into amino acids. This is partly understandable as both proteinase K and subtilisin are known to show hydrolyzing preference for specific residues of proteins. Accordingly, they do not necessarily arrive at total hydrolysis. At the same time, neither pronase E nor protease XIV could always provide 100 percent extraction of Se from the samples (see Table 19.1). Therefore, alternative methods should be developed in the field of sample preparation to replace enzymatic methods. 

Enzymatic Kinetic Resolution of N-Boc-Amino Add-Thioesters Coupled with Base-catalyzed Racemization Recently, a new method leading to the preparation of a number of aryl-glycines of the L-configuration has been published. The method is based on the hydrolysis of N-Boc-amino acid thioesters 15 catalyzed by an industrial preparation of the protease subtilisin (Scheme 13.16) [43]. 

The 6 -monobutanoate of methyl p-lactoside was prepared by subtilisin-catalyzed ester transfer from 2,2,2-trichloroethyl butanoate. Mesitoylation gave the hexaester which was converted to the 6 -fluoride (see Chapter 8). 2,3,6,3, 4 -Penta-0-acetylsucrose, the precursor of sucralose, was available from the octaacetate by selective hydrolysis first with alcalase, then with AP-6 lipase. The main by-product, the 2,3,4,3, 4 -pentaacetate, was readily converted to the required isomer by acetyl migration on treatment with phosphate buffer. An enzymatic acylation of cyclodextrin is referred to in Chapter 3. 

In enzymatic cleavage from the microspheres either with subtilisin or lipase, the release of the substrate was greater than 60%. Impressively, the release of azo-BSA from the microspheres catalyzed by lipase was greater than 90%. However, nonspecific hydrolysis of azo-BSA by lipase was detected, probably attributed to the similarities between the ester and amide bonds. Also, both reactions were performed in closed systems, contrary to the biological... 

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