Bacillus subtilis subtilisin from

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... 

Subtilisin (from Bacillus subtilis) [9014-01-1 ] [EC 3.4.21.62]. Purified by affinity chromatography using 4-(4-aminophenylazo)phenylarsonic acid complex to activated CH-Sepharose 4B. [Chandraskaren and Dhai Anal Biochem 150 141 7955]. 

DNA polymerase I has been purified to homogeneity. When the pure enzyme is treated with subtilisin, a proteolytic enzyme from Bacillus subtilis, the polymerase is cleaved into two pieces. The small fragment retains the 5 to 3 nuclease activity, whereas the larger piece, called a Klenow fragment, has both polymerase activity and the 3 to 5 exonuclease activity. The Klenow fragment is sold commercially for use in labeling DNA for use in detecting recombinant DNA. 

Subtilisin (from Bacillus subtilis) [9014-01-1 ] [EC 3.4.21.62]. Purified by affinity... 

The sugar alkaloid colchicoside and its synthetic sulfur analog thiocol-chicoside were acylated with esters of trifluoroethanol and with isopro-penylacetate in pyridine in the presence of the enzyme subtilisin from Bacillus subtilis (132). The 13C-NMR spectra of the ester alkaloids, which... 

The first step in setting up a successful directed evolution protocol is the development of an efficient expression system using an appropriate bacterial host. This is not a trivial task, in particular when overexpression is to be coupled to enzyme secretion. Fortunately, some proteins can easily be overexpressed and secreted by using commercially available systems [27 - 29], a prominent example being subtilisin of Bacillus subtilis [30]. However, many enzymes of interest are not amenable to such systems examples include a variety of different lipases from Pseudomonas species. 

Superior to lipases with regard to activity at higher solvent polarity are other hydrolases such as proteases. For example, subtilisin from Bacillus subtilis can be used to acetylate sucrose in dimethylformamide to the 1-mono-acetylated derivative [13], whereas the chemical acetylation reaction regioselectively leads to the 6-mono-acetylated product [ 14]. Thus, the two procedures nicely complement each other. 

Subtilisin (from Bacillus subtilis) [9014-01-1] Mr 27,000 (sedimentation equilibrinm) [EC 3.4.21.62], This alkaline protease is purified 211-fold by affinity chromatography using 4-(4-aminophenylazo)phenylarsonic acid complex to activated CH-Sepharose 4B. It is inhibited by 2-phenylethane boronic acid, PMSF, 3,4-dichloroisocoumarin, acetone and benzamide. [Chandraskaren Dhar Anal Biochem 150 141 1985, Schomburg Schomburg Springer Handbook of Enzymes 2nd Edn vol 7 p 286 2002.]... 

A further possibility for the enzymatic removal of C-terminal blocking groups is opened up by the application of enzymes which generally display a high esterase/ protease ratio. Such a biocatalyst is the alkaline protease from Bacillus subtilis DY which shows similarities to Subtilisin Carlsberg. For this enzyme the ratio of esterase to protease activity is >105. It selectively removes methyl, ethyl and benzyl esters from a variety of Trt-, Z- and Boc-protected di- and tripeptides and a pentapeptide at pH 8 and 37 °C (Fig. 18-9) (91l... 

The practical utilization of pH during the sorption of neutral proteinase from Bacillus subtilis on Sepharose with attached glycyl-D-phenylalanine (through a spacer 23 atoms long) is shown in Fig. 4.7.5. Walsh et al. [62] demonstrated that between pH 5 and 6.5 neutral proteinase was effectively adsorbed and thus separated both from subtilisin and other proteins present in the culture filtrate. At higher pH values, an effective separation of neutral proteinase from subtilisin does not take place. 

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. 

Wong, S.L. et al (1984) The subtilisin E gene of Bacillus subtilis is transcribed from a sigma 37 promoter in vivo. 

Comparative studies on the primary structures of homologous P. from different species (e.g. hemoglobin from vertebrates, see Homologous proteins) or analogous P. (e. g. subtilisin from Bacillus subtilis and mammalian trypsin) have made a valuable biochemical contribution to questions of divergent and convergent evolution. However, for an explanation of P. function and behavior, especially the mechanism of enzyme action, the primary structure alone is insufficient, and a knowledge of secondary and tertiary structure is needed. 

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... 

Purification of subtilisin DY and isolation of alkaline proteinase from culture medium of Bacillus subtilis. 

Purafect OxP contains an engineered subtilisin gene from Bacillus lentus (substitution of a single amino add at an oxidizable residue near the active site) similar cleaning performance as Purafect G produced by a selected strain of asporogenic Bacillus subtilis. 

Peng, Y, Yang, X. J., Xiao, L., Zhang, Y. Z. Cloning and expression of a fibrinolytic enzyme (subtilisin DFE) gene from Bacillus amyloliquefaciens DC in Bacillus subtilis. Res Microbiol 2004,155, 167-173. 

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