Subtilisin variants

Fig. 5.11. Phosphonylating inhibitor derivatives used to select subtilisin variants [64],...

Directed evolution has also been very effective for increasing enzyme activity in organic solvents 14> For example, the serine protease subtilisin can catalyze specific peptide syntheses and transesterification reactions, but organic solvents are required to drive the reaction towards synthesis. Sequential rounds of error-prone PCR and visual screening yielded a subtilisin variant with twelve amino acid substitutions that was 471 times more active than wild-type in 60% dimethylforma-mide (DMF)[145- 22° this enzyme is much more effective for peptide and polymer synthesis. 

Enzymes are highly specific catalysts. The nature of this specificity is believed to result from structural and electrostatic complementarity between the enzyme and its substrate. The serine protease, subtilisin, is being extensively studied as a model system to explore the effects of single amino acid substitutions on its structure and function Q). The gene for Bacillus amyloliquefaciens subtilisin has been expressed and secreted in B. subtilis 12).A site-directed mutagenesis scheme, cassette mutagenesis ( ), has been used to produce a series of subtilisin variants that are more resistant to oxidants (4), and have altered stability ( ), specificity, and specific activity. 

Table II. Kinetic Constants for Specific Subtilisin Variants at Codon 166...

Hydrophilic polymeric support materials provide alternatives to silica for the preparation of HlC sorbents. These materials are more stable to high-pH conditions used as part of the clean in place (CIP) regimes [46]. Representative examples of HIC sorbents are contained in Table 3, and many other in-house laboratory variants can be identified in the scientific literature. From the list of commercialized HIC sorbents found in Table 3, it is apparent that the majority of preparative HIC sorbents can be obtained in particle size ranges (typically 20-65 pm), pore size distributions (typically = 600-1000 A), and surface chemistries suitable for large-scale applications with proteins and peptides. The TSK PS copolymer materials or the Pharmacia cross-linked Superose/agarose range of HIC sorbents are well-known examples in industry. Illustrative of the various uses of silica- and polymer-based HIC sorbents are the examples of avian lysozymes [144], human pituitary prolactin [275], recombinant subtilisin variants [145], and other globular proteins [276-278],... 

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). 

Figure 3. pH profiles of wild type and two variant subtilisins. Activity was assayed with synthetic substrates as described (2) at the indicated pH. 

Figure 4. Oxidative stability of subtilisins. Purified wild type or variant subtilisin was mixed with 0.3% H2O2 in O.IM Tris buffer (pH 8.6). At the times indicated aliquots were removed and tested for remaining enzymatic activity (2).

Rheinnecker, M., Eder, J., Pandey, P. S. Fersht, A. R. (1994). Variants of subtilisin BPN with altered specificity profiles. Biochemistry, 33, 221-5. 

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). 

Oryza sativa (rice) (Poaceae)[seed] Rice a-Amylase-Subtilisin Inhibitor (RAS1) (176 aa 19 kDa 4 Cys 2 S-S) (minor variants also) Subtilisin (A67-A68) ( also inhibits a-Amylase) [389]... 

Vigna (Pliaseolus) angularis (adzuki bean) (Fabaceae)[seed] ASI-I (92 aa 11 kDa 0 Cys) processing variant ASI-II (N-terminal 19 aa missing) Subtilisin (A68-D69) [479]... 

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... 

A product-capture strategy was also used to select improved subtiligases. Subtiligase is a double mutant of subtilisine that catalyses the ligation of peptides. A library of >109 variants involving 25 residues of the active site was constructed on the phage-displayed enzyme. Variants that ligated a biotinylated peptide on their own ex-... 

A second example involves different evolved subtiligases. Subtilisins are proteolytic enzymes sutiligases are mutants that catalyzes the ligation of peptides. Evolved variants of subtiligase that ligate a biotinylated peptide onto their own extended N-termini were selectively captured, as shown in Figure 8.9. The reaction of the biotin-labeled peptide substrate with the evolved enzyme variant results in a... 

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