Subtilisin-Carlsberg

Enzymatic Resolution of Ethyl Sulfopropionate 2 with Subtilisin Carlsberg 

---

Bode, W., Papamokos, E., Musil, D. The high-resolution X-ray crystal structure of the complex formed between subtilisin Carlsberg and eglin c, an elastase inhibitor from the leech Hirudo medicinalis. Eur. J. Biochem. 166 (1987) 673-692... 

McPhalen, C. A., James, M. N. G. Structural comparison of two serine proteinase-protein inhibitor complexes Eglin-C-Subtilisin Carlsberg and CI-2-subtilisin novo. Biochemistry 27 (1988) 6582-6598... 

CH2=CHCH2CH2C02CH2CN, 3-methyl-3-pentanol, Subtilisin Carlsberg. These conditions were used to resolve a chiral amine (43% yield, 97% ee). ... 

An example that refers to the third method additives can be employed is described below. Markedly enhanced enantioselectivity was reported for P. cepacia lipase and subtilisin Carlsberg with chiral substrates converted to salts by treatment with numerous Bronsted-Lowry adds or bases [63]. This effect was observed in various organic solvents but not in water, where the salts apparently dissociate to regenerate... 

KR was an alcalase subtilisin Carlsberg as the major enzyme component). To avoid racemization of the final product, they employed a mixture of 2-methyl-2-propanol/ H2O (19 1). Under these conditions, the product precipitated during the course of hydrolysis (Figure 4.26). A very similar DKR process was reported two years later by Parmar [52]. 

Several lipases were more efficient than PLE and subtilisin Carlsberg for the desymmetrization of an N-t-butoxycarbonyl (Boc) meso-piperidine diester (Figure 6.13). The (3R)-monoester was converted into optically pure isogalactofagomme, a potent galactosidase inhibitor [60]. 

Scheme 5.24 Subtilisin-Carlsberg-catalyzed hydrolysis of sulfinamides.

However, when subtilisin E was replaced by subtilisin Carlsberg, the hydrolysis of the S-N bond in some A(-acyl arenesulfinamides 34 unexpectedly became the main hydrolytic process giving under the kinetic resolution conditions, in addition to the unreacted substrates, the corresponding sulfinic acids and... 

Our parallel experiments, in which subtilisin Carlsberg was used to promote hydrolysis of A-acetyl-A-benzyl arenesulfinamides, led to exclusive S-N bond breaking. However, the recovered substrates were racemic. Moreover, blank experiments showed that a spontaneous chemical hydrolysis contributed to the process to a much higher degree than that in the cases shown in Ref. 47. Hence, a conclusion was drawn that in our case the hydrolysis proceeded without involvement of the subtilisin active site Kielbasihski, P. Albrycht, M. Mikolajczyk, M. Unpublished results. 

The production process for (S)-phenylalanine as an intermediate in aspartame perpetuates the principle of reracemization of the nondesired enantiomer (Figure 4.5) in a hollow fiber/ liquid membrane reactor. Asymmetric hydrolysis of the racemic phenylalanine isopropylester at pH 7.5 leads to enantiopure phenylalanine applying subtilisin Carlsberg. The unconverted enantiomer is continuously extracted via a supported liquid membrane [31] that is immobilized in a microporous membrane into an aqueous solution of pH 3.5. The desired hydrolysis product is charged at high pH and cannot, therefore, be extracted into the acidic solution [32]. 

Ricks, E.E., Estrada-Vades, M.C., McLean, T.L. and Iacobucci, G.A. (1992) Highly enantioselective hydrolysis of (/ ,Sl-phenylalanine isopropyl ester by subtilisin Carlsberg. Continuous synthesis of (Sl-phenylalanine in a hollow fibre/liquid membrane reactor. Biotechnology Progress, 8, 197-203. 

Kawabata, T.T., Babcock, L.S., and Horn, P.A., Specific IgE and IgGl responses to subtilisin Carlsberg (Alcalase) in mice Development of an intratracheal exposure model, Fundam. Appl. Toxicol., 29, 238, 1996. 

For single-tryptophan proteins there is some correlation between blue-shifted fluorescence emission maximum and phosphorescence lifetime (Table 3.2). Another correlation is that three of the proteins which exhibit phosphorescence, azurin, protease (subtilisin Carlsberg), and ribonuclease Tlt are reported to show resolved fluorescence emission at 77 K. Both blue-shifted emission spectra and resolved spectra are characteristic of indole in a hydrocarbon-like matrix. 

Mugford, P.F., Lait, S.M., Keay, B.A. and Kazlauskas, R.J., Enantiocomplementary enzymatic resolution of the chiral auxiliary c7j,c7j-6-(2,2-dimethylpropanamido)spiro-[4.4]nonan- l-ol and the moleuclar basis for the high enantioselectivity of subtilisin Carlsberg. ChemBioChem, 2004, 5, 980-987. 

A practical enzymatic procedure using alcalase as biocatalyst has been developed for the synthesis of hydrophilic peptides.Alcalase is an industrial alkaline protease from Bacillus licheniformis produced by Novozymes that has been used as a detergent and for silk degumming. The major enzyme component of alcalase is the serine protease subtilisin Carlsberg, which is one of the fully characterized bacterial proteases. Alcalase has better stability and activity in polar organic solvents, such as alcohols, acetonitrile, dimethylformamide, etc., than other proteases. In addition, alcalase has wide specificity and both l- and o-amino acids that are accepted as nucleophiles at the p-1 subsite. Therefore, alcalase is a suitable biocatalyst to catalyse peptide bond formation in organic solvents under kinetic control without any racemization of the amino acids (Scheme 5.1). 

The hbraries of enzyme substrates were obtained by spht-pool synthesis to yield one-bead-one-compound hbraries. The substrate assay was performed with a range of proteolytic enzymes such as subtilisin Carlsberg [26], cruzipain [27], protein disulfide isomerase [28-29], matrix metalloprotease MM P-9 [30], papain [31],... 

Subtilisin Carlsberg 2SEC 1.8 0.136 2 Stabilizing McPhalen etal. (1985), McPhalen and James (1988)... 

Tincture of the dried seed, on agar plate at a concentration of 30 p,L/disc, was inactive on Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. Extract of 10 g plant material in 100 mL ethanol was used b Anticoagulation activity. Serpin BSZx (an inhibitor of trypsin and chemotrypsin) inhibited thrombin, plasma kallikrein, factor Vlla/tissue factor, and factor Xa at heparin-independent association rates. Only factor Xa turned a significant fraction of BSZx over as substrate. Activated protein C and leukocyte elastase were slowly inhibited by BSZx, whereas factor Xlla, urokinase and tissue type plasminogen activator, plasmin and pancreas kallikrein, and elastase were not or only weakly affected. Trypsin from Fusarium was not inhibited, while interaction with subtilisin Carlsberg and Novo was rapid, but most BSZx was cleaved as a substrate L... 

Figure 1.6 Dependence of the substrate specificity of subtilisin Carlsberg on the calculated ratio of the solvent-to-water partition coefficients of N-Ac-Phe-OEt and N-Ac-Ser-OEt in various solvents. Reprinted with permission from [47]. Copyright (1993) American Chemical Society.

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... 

It was found that MpSCD is able to restore dehydration-induced structural perturbations to a certain extent. Results were interpreted to show that enantioselectivity and structural intactness in the various solvents investigated were clearly related. Increased enzyme activity, in contrast, is mainly caused by increased structural flexibility of subtilisin in the solvents by M(iCD. Similar results have been reported for subtilisin Carlsberg [124]. 

---