Thermostability of Subtilisin

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... 

Other Attempts to Increase the Thermostability of Subtilisin are Unsuccessful... 

An entirely different property of subtilisin was affected by substituting leucine at the 222 location. Native BPN is extremely sensitive to the presence of oxidation agents, showing rapid inactivation when incubated in the presence of 0.3% H2O2 (Figure 4). The Leu-222 variant, in contrast, was found to be totally stable under the same oxidation conditions. The data clearly show that single amino acid alterations can have dramatic effects upon the activity of the enzyme. Similarly, other changes have been shown to affect catalytic properties, substrate specificities and thermostability (7,2,9). 

As for subtilisin BPN, the first attempt at molecular modeling of disulfide mutants was performed with computer graphics using coordinates from the crystal structure of the enzyme,10-121 but increasing enzyme stability was unsuccessful. We have studied a thermostable subtilisin-type protease, aqualysin I, and the introduction site of a disulfide bond was chosen on structural homology between aqualysin I and subtilisin E. Here we describe a successful study to increase the stability of subtilisin E and others done for subtilisin enzymes. 

As for aqualysin I, the disulfide bond seems to be one of the causes of its thermostability, because the enzyme is unstable in the presence of 2-mercaptoethanol.15) Accordingly, increasing the stability of subtilisin E was attempted by engineering disulfide bonds at the sites corresponding to those of aqualysin I. 

Bryan PN, Rollence ML, Pantoliano MW, Wood J, Finzel BC, Gilliland GL, Howard AJ, Poulos TL (1986) Proteases of enhanced stability characterization of a thermostable variant of subtilisin. Proteins Structure, Function and Genetics. 1 326-334... 

Knowing that peptides and amines confer thermal stability on enzymes from certain thermophilic organisms (47-49) led some workers to examine protein stabilization by antibodies. It was found that the presence of specific polyclonal antibodies stabilize several enzymes (50, 51). In addition, not only did antibodies increjise the thermostability of a-amylase, glucoamylase, and subtilisin, but some stability toward acid denaturation, oxidizing agent, and organic solvent exposure was increased in specific cases (52, 53). 

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... 

Fig. 13. The predicted entropy distribution for subtilisin E as determined by a mean-field treatment of the structural model. When all amino acids are equally allowed at a position, ij = In 20 3.0. The red lines are the positions at which mutations discovered by directed evolution improved the thermostability. The blue lines are for mutations that improved the activity (hydrolysis of a peptide substrate) in aqueous dimethylformamide. The bars indicate the average and standard deviation of the structural entropies.

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

The plot of the stabilities and activities of clones from the first generation S41 random mutant library shows once again that most mutations are detrimental to stability and activity (Fig. 14). However, compared to the esterase library (Fig. 7), there are more mutants with improvements in both properties, suggesting that the two enzymes have different adaptive potentials. This may be due to the relatively poor stability of S41, or it may reflect constraints intrinsic to the three-dimensional structures of the two proteins. Evidence for the former can be found by comparing the results for the first generations of the psychrophilic sub-tilisin S41 and the mesophilic subtilisin E. Screening 864 mutants of S41 yielded nine thermostabilized variants (a hit rate of approximately 1%) (Miyazaki and Arnold, 1999) in contrast, screening 5000 subtilisin E mutants identified five thermostable variants (a hit rate of only 0.1%) (Zhao and Arnold, 1999). 

Fig. 5. Activities of 654 active clones from the shuffled subtilisin library compared to twenty-six parents. Relative activities of each clone in five screens are plotted as concentric circles. Each color represents one of the five screening conditions pH5.5 (orange), pH7.5 (blue), pHIO (dark red), thermostability (yellow), and activity in 35% DMF at pH 7.5 (green). The area of the circle is proportional to the activity in the five assays relative to the best parent in each assay.

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