Subtilisin Carlsberg and

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... 

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. 

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... 

Table 3.4 Effect of KCI as a salt matrix on subtilisin Carlsberg and chymotrypsin in anhydrous hexanew [88].

Solvent polarity is known to affect catalytic activity, yet consistent correlations between activity and solvent dielectric (e) have not been observed [12,102]. However, a striking correlation was found between the catalytic efficiency of salt-activated subtilisin Carlsberg and the mobility of water molecules (as determined using NMR relaxation techniques) associated with the enzyme in solvents of varying polarities (Figure 3.11) [103]. As the solvent polarity increased, the water mobility of the enzyme increased, yet the catalytic activity of the enzyme decreased. This is consistent with previous EPR and molecular dynamics (MD) studies, which indicated that enzyme flexibility increases with increasing solvent dielectric [104]. 

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 enzymes used were Alcalase(R) 0.6 L, a liquid, food-grade preparation of subtilisin Carlsberg, and Neutrase(R) 0.5 L, a liquid, food-grade preparation of a B. subtilis neutral protease. Both enzymes are commercially available from Novo Industri A/S (7 ). All other reagents were analytical grade laboratory chemicals. ... 

Of particular interest is the study of the biological mechanisms associated with enzyme stereoselectivity and enantioselectivity. For example, MD simulations have been successful in explaining the different affinities of trypsin and acetylcholinesterase to the diastereomers of soman inhibitors [154] and the ability of subtilisin Carlsberg and a-chymotrypsin to discriminate between R-and S- configurations of chiral aldehyde inhibitors [155, 156]. 

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,... 

Kamat et al. found a pressure effect on the stereoselectivity of transesterifications in supercritical fluoroform [64]. The stereoselectivity of subtilisin Carlsberg and Aspergillus proteases increased, while the activity decreased with increasing fluoroform pressure from 66 to 352 bar. They conclude that the stereoselectivity of these proteases increases because fluoroform becomes more hydrophilic as the pressure rises. This finding is in accordance with a previously presented hypothesis for substrate binding in enzymes. 

Figure 14.4.3.2. The dependence of (A) subtilisin Carlsberg and (B) a-chymotrypsin substrate specificity for substrates 1 and 2 on the ratio of their Raoult s law activity coefficients. For the stractures of the substrates 1 and 2, and the solvents a through m in (A) and a to g in (B), refer to Ref 16. [Adapted, by permission, Ifom C.R. Wescott and A.M. Klibanov, Biotechnol. Bioeng., 56, 343(1997)].

C. A. McPhalen and M. N. G. James, Biochemistry, 27, 6582 (1988). Structural Comparison of Two Serine Proteinase-Protein Inhibitor Complexes Eglin-c-Subtilisin Carlsberg and CI-2-Subtilisin Novo. 

As organic solvents are not the natural environment of enzymes, it has been proposed that they may induce changes in the three-dimensional structure of enzymes. The methods developed to study protein structures are difficult to apply to solid enzymes suspended in nonaqueous environments. However, it was possible to show that the structure of subtilisin Carlsberg and y-chymotrypsin is little affected by the passage to organic media (42,43). 

We can take a closer look at the protease reaction. The use of different proteases gives somewhat different results. For exanq)le, three proteases (Thermolysin, subtilisin Carlsberg, and Alcalase ) all react with cationic guar. However, in an aqueous reaction, Alcalase was found to be the most reactive (Table 2). Up to 95% of the protein could be removed, and there was no significant change in its physical properties. A bonus was a 50% decrease in the turbidity of the guar solution after treatment. 

Fig. 7. Prediction of secondary a-mm and 3-d] structure of subtilisin BPN , subtilisin Carlsberg and thermitase by 4 independent methods by Scheraga, Nagano and Robson cited in [17]. The final prediction (common) is represented by the agreement of at least three of the four predictions.

Products from the ( -selective DKR of secondary alcohols with subtilisin Carlsberg and 5. 

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