Proteases subtilisin

Husum et al. found that the hydrolytic activities of P-galactosidase from E. coli and the protease subtilisin in a 50 % aqueous solution of the water-miscible ionic liquid [BMIM][Bp4] were comparable to those in 50 % aqueous solutions of ethanol or acetonitrile (Entry 9) [37]. 

The serine proteases are the most extensively studied class of enzymes. These enzymes are characterized by the presence of a unique serine amino acid. Two major evolutionary families are presented in this class. The bacterial protease subtilisin and the trypsin family, which includes the enzymes trypsin, chymotrypsin, elastase as well as thrombin, plasmin, and others involved in a diverse range of cellular functions including digestion, blood clotting, hormone production, and complement activation. The trypsin family catalyzes the reaction ... 

Table 1.3 Influence ofthe organic solvent on the enantioselectivity of the protease subtilisin in the kinetic resolution ofthe racemic amine (9) (expressed as the ratio ofthe initial rate of acylation of the pure enatiomers, Vs/vr).

Scheme 4 6 -0-Acyl-lactose (acetyl, propionyl, butyryl) derivatives prepared via the selective enzymatic acylation of lactose by the protease subtilisin were used as acceptors for enzymatic transglycosylations catalyzed by a-D-galactosidase from Talaromyces flavus, forming iso-globotriose a-Gal(l -> 3)-p-Gal-(l -> 4).92 (/) Protease N, pyridine, 37 °C (ii) a-galactosidase from Talaromyces flavus.

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. 

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). 

In order to prove enzyme engineering feasibility, it was important to develop a model system. One of the prime considerations for any model would be the commercial potential of the model. Table I lists the major commercial enzymes and the market size in US dollars (5). The alkaline proteases (subtilisins) are clearly the major single class of enzymes in commercial use today, representing 25% of the total enzyme market of 600 million. The primary use of subtilisins is as additives in laundry detergents to aid in the removal of proteinaceous stains from cloth. 

The application of CPO, HRP and CiP is limited to sterically unencumbered substrates and all these peroxidases produce the same absolute configuration of the chiral hydroperoxide. To overcome this limitation, the semisynthetic enzyme selenosubtilisin, a mimic for glutathione peroxidase, with the peptide framework of the serine protease subtilisin was developed by Bell and Hilvert. This semisynthetic peroxidase catalyzes the reduction of hydrogen peroxide and hydroperoxides in the presence of 5-mercapto-2-nitrobenzoic acid. It was utilized by Adam and coworkers and Schreier and coworkers for the kinetic resolution of racemic hydroperoxides (equation 17) . The results obtained were very promising. 

Recently, improved hydrophilicity and dyeability with acid and disperse dyes of nylon 6 fibres after treatment with protease (subtilisin) was reported [31]. Similarly, various proteases were used for surface hydrolysis of nylon 6,6 fibres, leading to... 

Khmelnitsky et al. were the first to observe the activating effects salt showed on enzymes in the nonaqueous environment [88]. As shown in Figure 3.7, the transesterification activity of the serine protease subtilisin Carlsberg in anhydrous solvents is strongly dependent on the KC1 content in a lyophilized enzyme preparation and increases sharply as the salt content is increased. This increase in activity was determined to be a result primarily of an increase in kcat and not a decrease in Km, as shown in (Table 3.4). 

As mentioned earlier (Section 4.2.1.1), empirical rules for the enantioselectivity of hydrolases have been developed. It is important to keep in mind that these rules do not work for all substrates. Most rules are based on pockets, which indicate how the steric bulk of the substituents in the substrate fit into the environment of the active site. Thus, such rules have been suggested for pig liver esterase(PLE) [66], the protease subtilisin [66-68], and certain lipases [69-71]. For secondary alcohols, most lipases follow the simple rule of Kazlauskas, which was developed for Pseudomonas cepacia, and which is depicted in Figure 4.4 [72]. This model implies that the fast-reacting enantiomers binds to the active site as described in Figure 4.4, whereas the slowly reacting one is not able to achieve a comfortable fit, because it will require the large substituent L to fit into the smaller pocket. In contrast to lipases, subtilisin displays opposite enantioselectivity toward secondary alcohols [68]. 

The above-described acylation of the sugar moiety of the nucleotide adenosine (3) [2] has been followed by a series of papers reporting on the chemoselec-tive enzymatic modification of natural compounds carrying both hydroxyl and amino groups. In addition to the extensive work on nucleosides developed by Gotor and coworkers [8], the biocatalyzed esterification of the hydroxylated alkaloids castanospermine (4) and 1-deoxynojirimidn (5) should be mentioned. Both compounds were selectively acylated at their C-6 and/or C-2 OH by the protease subtilisin, despite the presence of a potentially more reactive amino functions [9]. 

The enantiopreference of the protease subtilisin in the acylalion of chiral alcohols is known to be opposite to that observed with lipases, providing for access to both enantiomers with DKR, depending on the enzyme used [137, 138, 139]. Acylation using 2,2,2-trifluoroethyl butyrate as the acyl donor was combined with in situ racemization, affording the corresponding esters in high yield and [135]. 

An early discovery by Frederick Richards that turned out to be useful was that the protein could be cleaved between residues 20 and 21 by the bacterial serine protease, subtilisin. The resulting two polypeptides were separated and purified. They were enzymatically inactive individually, but regained the activity of the native enzyme when they were recombined. This work shows that strong, nonco-valent interactions occur that can hold protein chains together even when one of the peptide links is cut. It also makes it possible to modify specific amino acid residues of the two polypeptide chains independently and to explore how each residue contributes to the reassembly of the protein and the recovery of enzymatic activity. 

A number of steroids have been regioselectively acylated in a similar manner (99,104). Chromobacterium viscosum lipase esterifies 5a-androstane-3p,17p-diol [571-20-0] (75) with 2,2,2-trifluoroethyl butyrate in acetone with high selectivity. The lipase acylates exclusively the hydroxy group in the 3-position giving the 3p-(monobutyryl ester) of (75) in 83% yield. In contrast, bacillus subtilis protease (subtilisin) displays a marked preference for the C-17 hydroxyl. Candida cylindracea lipase (CCL) suspended in anhydrous benzene regioselectively acylates the 3a-hydroxyl group of several bile acid derivatives (104). 

A considerably simpler approach in the context of a biocatalytic pathway was reported by Sidler et al. (Scheme 4.16). Here, the methyl ester 45 could be hydrolyzed selectively by the protease subtilisin (lipases and esterases were unreactive), allowing hydrolysis of the unwanted (R)-enantiomer. The desired (S)-45 was recovered from the solution in 80-90% chemical yield (98% ee) and was further manipulated into (S) L-771,668 [191]. 

Besides these rather complex coenzyme-dependent enzymes, the none-coenzyme requiring protease subtilisin is the most extensively mutated enzyme. The substrate specificity of the enzyme as well as its dependence on pH and its stability were altered by site-directed mutagenesis [72-78]. As the knowledge about exact details of the structure and active site of the enzyme is essential for the application of this method, progress in this field is difficult to achieve. Site-directed mutagenesis as a means of catalyst improvements will be used only after extensive application of conventional optimization procedures. 

Little was done in the area of cross-linked enzyme crystals over the next 10 years. In 1977, the kinetic properties of CLCs of the protease subtilisin were reported by Tuchsen and Ottesen [3], They reported that cross-linked enzyme crystals of subtilisin were highly effective catalysts with increased thermal stability and increased stability toward acid compared to the soluble enzyme. They further reported that the CLCs of subtilisin showed essentially no autodigestion at 30°C. Like Quiocho and Richards before them, Tuchsen and Ottesen found... 

Researchers at the biotech company EntreMed, Inc., have recently prepared and tested 2-phthalimidino-glutaric acid analogs of thalidomide and found them to be potent inhibitors of tumor metastasis [28], The key to the success of their synthesis was a resolution via enantioselective ester hydrolysis catalyzed by ChiroCLEC -BL, the CLC form of the protease subtilisin. The authors were able to isolate both enantiomers of the desired product with good optical purity (95% ee) (see Fig. 7). 

Kano, H., Taguchi, S., and Momose, H. (1997). Cold adaptation of a mesophilic serine protease, subtilisin, by in vitro random mutagenesis. Appl. Microbiol. Biotechnol, 47, 46-51. 

In the catalytic mechanism of the serine protease subtilisin, the tetrahedral intermediate is believed to be stabilized by a hydrogen bond to the side chain of Asn 155. Replacement of Asn 155 with Gly left the substrate binding unaffected, but inhibited the catalytic step, confirming the proposed mechanism. 

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