Subtilisin Carlsberg peptides

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). 

M Meldal, K Breddam. Anthranilamide and nitrotyrosine as a donor-acceptor pair in internally quenched fluorescent substrates for endopeptidases multicolumn peptide synthesis of enzyme substrates for subtilisin Carlsberg and pepsin. Anal Biochem 195 141-147, 1991. 

The alkaline serine protease of Bacillus licheniformis, also known as Subtilisin Carlsberg, is the preferred protease in most nonionic and anionic detergents. It attacks many peptide bonds and easily dissolves proteins. It may be used at temperatures up to 65°C, and its pH optimum is close to 9.0, the pH normally used in washing fluids. 

A very straightforward approach in route C (Fig. 2) would have been the direct enzyme-catalyzed peptide formation (cf. Chen et al. [18]) by enantioselective aminolysis of diester 9 with (S)-tert-leucine methylamide 13 or even racemic 13. This would combine three synthetic objectives the resolution of (rac)-9, the resolution of (roc)-13 and the coupling step. In orientating experiments monoester 10 was tested as a model substrate. It was contacted with an equal amount of (S)-amine 13 in the presence and absence of an organic solvent. Solid or liquid subtilisin Carlsberg preparations (Alcalase 2.0 T or Alcalase 2.5 L, respectively) were used as the catalyst. Only with the liquid enzyme preparation was the formation of minor amounts of one of two possible diastereoisomeric peptides observed [19], whereas most of the ester was hydrolyzed to the acid. Likewise, a few selected lipases also provided negative results. 

M. Meldal, I. Svendsen. Direct visualization of enzyme inhibitors using a portion mixing inhibitor library containing a quenched fluorogcnic peptide substrate. 1 Inhibitors for subtilisin Carlsberg. J. Chem. Soc., Perkin Trans. 1 1995, 1591-1596. 

Subtilisins can catalyze a variety of C-terminal modification reactions [11-13] (Figure 15.5). Quaedflieg and coworkers investigated the use of subtilisin Carlsberg in anhydrous solvents for converting Cbz-protected amino adds and Cbz dipeptides to methyl or ethyl esters. They also studied the conversion of peptide f-butyl esters, which are readily obtained by solid-phase peptide S5mfiiesis, to methyl, ethyl, or benzyl esters [11]. 

Among many other peptide splitting enzymes such as (bacterial) subtilisin and thermolysin, (vegetable) papain, ficin and bromelain, (mammalian) cathepsin and others, the yeast enzyme carboxypeptidase Y finally deserves special mention. The enzyme is an exopeptidase, like carboxypeptidase A i.e. it catalyzes, rather unspecifically, the hydrolytic fission of the carboxy-terminal a-amino acids from a peptide chain. J.T. Johansen and his associates at the Carlsberg laboratory in Copenhagen showed about 10 years ago that CPD-Y is an effective catalyst of peptide bond synthesis [36]. 

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