Subtilisin-type serine protease

Aqualysin I Belongs to a Family of Subtilisin-type Serine Proteases... 

In the family of subtilisin-type serine proteases, primary sequences of about 40 enzymes are known.423 Among them, no subtilisin produced by Bacillus species has cysteine residues. Aqualysin I (four cysteine residues per molecule),163 proteinase K (five residues),173 and thermitase (one residue)223 are cysteine-containing enzymes (Fig. 12.2). 

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. 

EndopepUdases (proteinoses) catalyse the hydrolysis of bonds within the peptide chain, forming variously sized cleavage peptides. They can be further subdivided into acidic, neutral and basic endopeptidases. Neutral and basic types can each be divided into Serine proteases (see) and thiol proteinases (see Thiol enzymes). Examples of animal endopeptidases are Pepsin (see). Rennet enzyme (see), Ttypsin (see), Elastase (see). Thrombin (see), Plasmin (see) and Renin (see). For examples of plant and bacterial endopeptidases, see Papain, Subtilisin, Bromelain. Endopeptidases have also been isolated from yeast and fungi. 

Subtilisin (EC 3.4.21.4) an extracellular, single chain, alkaline serine protease from Bacillus subtilis and related species. S. are known from four different species of Bacillus S. Carlsberg (274 amino acid residues, M, 27,277), S. BPN (275 amino acid residues, M, 27,537), S. Novo (identical with S.BPN ) and S. amylosacchariticus (275 amino acid residues, M, 27671). The observed sequence differences between different S. represent conservative substitutions and are limited to the surface amino acids. Like the pancreatic proteinases, S. has catalytic Ser22i, His64 and Asnjj residues, but it is structurally very different from the other serine proteases, e. g. the active center of S. is -Thr-Ser-Met-, whereas that of the pancreatic enzymes is -Asp-Ser-Gly- pancreatic enzymes contain 4- disulfide bridges, whereas S. contains none S. contains 31 % a-helical structure and 3 spatially separated domains, whereas the pancreatic enzymes have 10-20% a-helical structure and a high content of p-structures in both types, the active center is a substrate cleft. S. also have a broader substrate specificity than the pancreatic enzymes. This is a notable example of the convergent evolution of catalytic activity in two structurally completely different classes of proteins. S. is used in the structural elucidation... 

When A -nucleophiles such as ammonia, amines or hydrazine are subjected to acyl-transfer reactions, the corresponding Af-acyl derivatives - amides or hydrazides - are formed through interception of the acyl-enzyme intermediate by the iV-nucleophile (Schemes 2.1 and 3.21) [262]. Due to the pronounced difference in nucleophiU-city of the amine (or hydrazine) as compared to the leaving alcohol (R -OH) (Scheme 3.21), aminolysis reactions can be regarded as quasi-irreversible. Any type of serine hydrolase which forms an acyl-enzyme intermediate (esterases, lipases, and most proteases) is able to catalyze these reactirms. Among them, proteases such as subtilisin and peniciUin acylase and lipases from Candida antarctica and Pseudomonas sp. have been used most often. 

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