Table subtilisin

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).

Later on the crucial role played by the solvent was enlightened in the protease-catalyzed resolution of racemic amines [26]. As shown in Table 1.3, the ratio of the initial rates of acylation of the (S)- and the (Ji)-enantiomers or racemic a-methyl-benzylamine (9) varied from nearly 1 in toluene to 7.7 in 3-methyl-3-pentanol. Similarly, the same authors found a significant solvent effect for the subtilisin-catalyzed transesterification of racemic 1-phenylethanol (10) using vinyl butyrate as acyl donor (Table 1.4 [27]). 

The lipase-catalyzed DKRs provide only (/ )-products to obtain (5 )-products, we needed a complementary (5 )-stereoselective enzyme. A survey of (5 )-selective enzymes compatible to use in DKR at room temperature revealed that subtilisin is a worthy candidate, but its commercial form was not applicable to DKR due to its low enzyme activity and instability. However, we succeeded in enhancing its activity by treating it with a surfactant before use. At room temperature DKR with subtilisin and ruthenium catalyst 5, trifluoroethyl butanoate was employed as an acylating agent and the (5 )-products were obtained in good yields and high optical purities (Table 3)P... 

The (5 )-selective DKR of alcohols with subtilisin was also possible in ionic liquid at room temperature (Table 14). " In this case, the cymene-ruthenium complex 3 was used as the racemization catalyst. In general, the optical purities of (5 )-esters were lower than those of (R)-esters described in Table 5. 

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. 

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. 

Table II is a partial list of known Bacillus genes which have an effect upon subtilisin production. Henner and coworkers (14) placed beta-galactosidase under the control of the subtilisin promoter. By using promoter deletions, they were able to identify regions of the promoter which are regulated by sacU, sacQ (present designation degU and degQ respectively, see Table II) and hpr.

Table II. Genetic Loci of Bacillus subtilis Which Regulate Subtilisin Production...

Members of both structural families of serine proteinases, the trypsinlike and the subtilisin-like, have been found to bind Ca " (references in Tables 1 and II). The role of Ca " in all of these proteolytic enzymes appears to be one of stabilization of structure and/or maintenance of... 

This method was employed for transesterification reactions with both a-chymotrypsin and subtilisin Carlsberg with a variety of H+/Na+ buffers [53]. With both enzymes (which differ widely in secondary and tertiary structures) and two polar solvents, acetonitrile and THF, the activating effect of the solid-state buffer was clearly evident (Table 3.3). The observation that a variety of buffer pairs show success in activating two dissimilar enzymes in synthetically useful solvents makes this method for activation promising and novel. 

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). 

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

Data are adapted from compilations of Dure et at. (1989). O, high osmoticum (PEG or salt) D, desiccation C, cold W, wounding H, heat , untested or unknown. Sd, seed St, stem Pase, protease ASI, a-amylase/subtilisin inhibitor WGA, wheat germ agglutinin RNP, ribonuclear protein not organ specific. Table updated from Skriver Mundy (1990). 

Table 12.4 Correlation of the enantioselectivity E of subtilisin in the transtesterification of racemic 1-phenylethanol in various organic solvents, and the dielectric constants eofthe solvents (Fritzpatrick, 1992).

When switching from water to an organic solvent, or switching between organic solvents, the substrate specificity can change. In the example of the standard reaction, transesterification of N-acetyl-i-phenylalanine ethyl ester with n-propanol by Subtilisin Carlsberg, which has been mentioned several times in this chapter already, the relative specificity between the rather hydrophobic phenylalanine compound and its more hydrophilic analog N-acetyl-L-serine ethyl ester varies with the solvent (Table 12.8) (Wescott, 1993). 

Table 2.1 List of the chemical shifts of [M]SSI in free and complexed state with subtilisin BPN (40° C, pD=7.3). Complexation shift (c)=shift (b) - shift (a)...

Three-dimensional structures of four subtilisin-type enzymes, subtilisin BPN, 36373 subtilisin Carlsberg,37,383 thermitase,39,403 and proteinase K,40,413 are known, but that of aqualysin I has not yet been determined. The Ca atoms of the known structures were superimposed to obtain maximal overlap of the backbone structures, and large parts of all four structures overlap very well (Fig. 12.3) 423 On the basis of such analyses, structurally equivalent core residues (194 residues) are identified, and higher sequence identity was found to correspond to a closer overlap of mainchain atoms in the core (Table 12.1).423... 

As for aqualysin I, its high sequence identity in the core residues with the four proteases (Table 12.1) suggests that the three-dimensional structure of aqualysin I is similar to those of the proteases. The similarity to subtilisins and proteinase K is probably higher than to thermitase. 

Thermostability of subtilisin E is increased by the introduction of the disulfide bond (Table 12.3). The half-life of the disulfide mutant is 2-3 times longer than that of the wild-type enzyme at 45°-60°C. On the other hand, those of the single-cysteine mutants are... 

Table 12.2 Kinetic constants of wild-type, Cys61-Cys98 disulfide, and single-cysteine mutant subtilisin Esl3)...

Table 12.3 Effect of the disulfide bond introduced in subtilisin E on the...

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