Temperature subtilisin

FIGURE 9.5. The potential surface for the 0"C = 0— 0-C-0" step in amide hydrolysis in solution, where the surface is given in terms of the angle 0 and the distance b. The heavy contour lines are spaced by fi (at room temperature) and can be used conveniently in estimating entropic effects. The figure also shows the regions (cross hatched) where the potential is less than for the corresponding reaction in the active site of subtilisin. 

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. 

Unlike many other enzymes, the subtilisins are fairly stable towards e.g. organic solvents, anionic surfactants, high temperatures and high pH. This makes the subtilisins very suitable as detergent proteases. But despite this fact, stabilization of these protease enzymes in liquid detergents remains a major issue. 

DKR of secondary alcohol is achieved by coupling enzyme-catalyzed resolution with metal-catalyzed racemization. For efficient DKR, these catalyhc reactions must be compatible with each other. In the case of DKR of secondary alcohol with the lipase-ruthenium combinahon, the use of a proper acyl donor (required for enzymatic reaction) is parhcularly crucial because metal catalyst can react with the acyl donor or its deacylated form. Popular vinyl acetate is incompatible with all the ruthenium complexes, while isopropenyl acetate can be used with most monomeric ruthenium complexes. p-Chlorophenyl acetate (PCPA) is the best acyl donor for use with dimeric ruthenium complex 1. On the other hand, reaction temperature is another crucial factor. Many enzymes lose their activities at elevated temperatures. Thus, the racemizahon catalyst should show good catalytic efficiency at room temperature to be combined with these enzymes. One representative example is subtilisin. This enzyme rapidly loses catalytic activities at elevated temperatures and gradually even at ambient temperature. It therefore is compatible with the racemization catalysts 6-9, showing good activities at ambient temperature. In case the racemization catalyst requires an elevated temperature, CALB is the best counterpart. 

When 18-crown-6 was co-lyophilized with a-chymotrypsin, a 470-fold activation was seen over the free enzyme in the transesterification of APEE with 1-propanol in cyclohexane (Scheme 3.2) [96]. There was a low apparent specificity for the size and macrocyclic nature of the crown ether additives, suggesting that, during lyophilization, 18-crown-6 protects the overall native conformation and acts as a lyoprotectant. To examine this global effect, FTIR was used to examine the effect of crown ethers on the secondary structure of enzymes. In one study [98], subtilisin Carlsberg was shown to retain its secondary structure in 1,4-dioxane when lyophi-lized in a 1 1 ratio with 18-crown-6. In addition, examination of FTIR spectra from varying incubation temperatures indicated that an increase in crown ether content in the final enzyme preparation resulted in a decreased denaturation temperature in the solvent, indicating a more flexible protein structure. 

Naturally occurring Upases are (R)-selective for alcohols according to Kazlauskas rule [58, 59]. Thus, DKR of alcohols employing lipases can only be used to transform the racemic alcohol into the (R)-acetate. Serine proteases, a sub-class of hydrolases, are known to catalyze transesterifications similar to those catalyzed by lipases, but, interestingly, often with reversed enantioselectivity. Proteases are less thermostable enzymes, and for this reason only metal complexes that racemize secondary alcohols at ambient temperature can be employed for efficient (S)-selective DKR of sec-alcohols. Ruthenium complexes 2 and 3 have been combined with subtilisin Carlsberg, affording a method for the synthesis of... 

A 5 L, 3-neck round bottom flask equipped with a mechanical stirrer was charged with the N-acetyl-3-(4-thiazolyl)-DL-alanine ethyl ester (210.0 g, 0.87 mol), distilled water (1.6 L), and 1 M aqueous KCI (0.8 L). The homogeneous solution was adjusted to pH 7.0 with 0.1 M NaOH and then was treated with Subtilisin Carlsberg (1.8 g) dissolved in 0.1 M aqueous KCI (25 ml). The reaction mixture was stirred at room temperature with 1.0 M NaOH added as required to maintain the pH at 6.25-7.25. After 4 h, 430 ml of base had been consumed and the reaction was judged to be complete. The reaction mixture was then extracted with chloroform (4x1.5 L), the aqueous phase was carefully acidified to pH 4 with 2 M HCI and then was concentrated under reduced pressure. Residual water was removed by consecutive evaporation... 

With regard to the use of protease in the synthetic mode, the reaction can be carried out using a kinetic or thermodynamic approach. The kinetic approach requires a serine or cysteine protease that forms an acyl-enzyme intermediate, such as trypsin (E.C. 3.4.21.4), a-chymotrypsin (E.C. 3.4.21.1), subtilisin (E.C. 3.4.21.62), or papain (E.C. 3.4.22.2), and the amino donor substrate must be activated as the ester (Scheme 19.27) or amide (not shown). Here the nucleophile R3-NH2 competes with water to form the peptide bond. Besides amines, other nucleophiles such as alcohols or thiols can be used to compete with water to form new esters or thioesters. Reaction conditions such as pH, temperature, and organic solvent modifiers are manipulated to maximize synthesis. Examples of this approach using carboxypeptidase Y (E.C. 3.4.16.5) from baker s yeast have been described.219... 

We chose subtilisin E to test our prediction that directed evolution makes mutations at uncoupled positions (Voigt et al., 2000b). Directed evolution increased the temperature optimum for activity, T pt, of Bacillus subtilis subtilisin E from 59° to 76°C, with eight mutations (Zhao and Arnold, 1999). In an independent study, thirteen mutations improved the activity toward the hydrolysis of su c c i iivI-A 1 a-A1 a-Pro-Phe- >-nitroanilide (s-AAPF-j Na) in the organic solvent dimethylformamide (DMF). The mutants were found by screening 2000 to 5000 clones from... 

Fig. 11. Dependence of activity on temperature for wildtype subtilisin E and the thermostable mutant 5-3H5.

Taguchi et al. (1998) used directed evolution to increase the low-temperature activity of the mesophilic subtilisin BPN. Random muta-... 

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. 

---