Subtilisin chemical modification

G. DeSantis, X. Shang, and J. B. Jones, Toward tailoring the specificity of the SI pocket of subtilisin B. lentus chemical modification of mutant enzymes as a... 

D Haring, P Schreier. Novel biocatalysts by chemical modification of known enzymes cross-linked microcrystals of semi-synthetic peroxidase seleno-subtilisin. Angew Chem Int Ed 37 2471-2473, 1998. 

Plettner E., DeSantis G., Stabile M. and Jones J. B. (1999) Modulation of esterase and amidase activity of subtilisin Bacillus lentus by chemical modification of cysteine mutants. J. Am. Chem. Soc. 121, 4977-4981. 

Scheme 5.1.6. Controlled site-selective modification of subtilisin by a combined site-directed mutagenesis chemical modification approach.

Combining site-directed mutagenesis strategies with chemical modification is a popular tool in both enzyme engineering and mechanistic studies. This has often been applied to the subtilisin from Bacillus lentus (SBL), or savinase. Subtilisins are... 

Subtilisins are a family of serine proteases, the most important members of which are subtilisin Carlsberg (from Bacillus licheniformis) and subtilisin BPN (from Bacillus amyloliquefaciens)luoK Both enzymes are alkaline proteases with a pH optimum of 6-9. Because of their industrial importance, both subtilisin Carlsberg and subtilisin BPP have been studied intensively and are produced on a large scale. The crystal structures of both subtilisins have been determined1821. Directed evolution and site-directed mutagenesis and chemical modification of subtilisin were carried out in order to influence the stability, activity and enantioselectivity of the enzyme, in particular in organic solvents11111. As in the case of other enzymes,... 

The term enzyme engineering [11] (see Fig. 1) describes a range of techniques from deliberate chemical modification to remodeling a wild-type enzyme by gene technology. Subti-ligase , a mutant of subtilisin BNP , was prepared by Jackson et al. [12] by protein design and used in a further total synthesis of RNase... 

Chemically Modified Enzymes. Besides varying the physicochemical properties of enzymes (such as their solubility), the catalytic properties of an enzyme can be fundamentally altered by chemical modification of the chemical operator in the active site. This teclmique leads to semisynthetic enzymes, which often do not have much in conunon with their natural ancestors [492, 493]. Early efforts in this directiOTi focussed on the modification of nucleophilic OH- or SH-residues in Ser-or Cys-hydrolases, such as subtihsin or papain, respectively. For example, the Ser-hydroxy group within the active site of subtilisin was converted to its selenium... 

Svendsen 1. Chemical modifications of the subtilisins with special reference to the binding of large substrates a review. Carlsberg Res Commun 1976 41 237-291. 

Classical approaches to chemical enzyme modification, however, often suffer from lack of regio-selectivity, which can yield heterogeneous and irreproducible enzyme mixtures. For example, preparation of methyl-chymotrypsin, -subtilisin or -trypsin using methyl sulfonate reagents, originally used to methylate the histidine of the... 

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