Subtilisins characterization

The serine proteases are the most extensively studied class of enzymes. These enzymes are characterized by the presence of a unique serine amino acid. Two major evolutionary families are presented in this class. The bacterial protease subtilisin and the trypsin family, which includes the enzymes trypsin, chymotrypsin, elastase as well as thrombin, plasmin, and others involved in a diverse range of cellular functions including digestion, blood clotting, hormone production, and complement activation. The trypsin family catalyzes the reaction ... 

A practical enzymatic procedure using alcalase as biocatalyst has been developed for the synthesis of hydrophilic peptides.Alcalase is an industrial alkaline protease from Bacillus licheniformis produced by Novozymes that has been used as a detergent and for silk degumming. The major enzyme component of alcalase is the serine protease subtilisin Carlsberg, which is one of the fully characterized bacterial proteases. Alcalase has better stability and activity in polar organic solvents, such as alcohols, acetonitrile, dimethylformamide, etc., than other proteases. In addition, alcalase has wide specificity and both l- and o-amino acids that are accepted as nucleophiles at the p-1 subsite. Therefore, alcalase is a suitable biocatalyst to catalyse peptide bond formation in organic solvents under kinetic control without any racemization of the amino acids (Scheme 5.1). 

Subtilisins are a class of related serine endo proteases produced by members of the Bacillus genus. The B.amvloliauefaciens subtilisin (BPN ) is well-characterized with regard to its DNA sequence 4 protein sequence (5), X-ray crystal structure (6) and kinetic properties (7). With this wealth of information available, BPN was chosen as the model enzyme for our recombinant approach. 

St Leger et al. (1987b) characterized two subtilisin-like proteases (chymoelastases) and three tiypsin-like proteases from M. anisopliae. A subtilisin-like protease (Prl pl=10.3, Mw=25 kDa) and a trypsin-like protease (Pr2 pl=4.42, Mw=28.5 kDa) were purified to homogeneity. Inhibition studies have revealed that both enzymes possess essential Ser and His residues in the active site. Prl exhibited higher activity to locust cuticle than Pr2 and it showed activity to elastin as well. 

The purification and characterization of BmSI-7 and BmSI-6, two subtilisin inhibitors from Boophilus microplus (BmSI) was reported by Sasaki et al. (2008). The inhibitors were found to exhibit significant inhibition on the activity of purified Prl proteases from M. anisopliae. 

Narinx, E., Baise, E., and Gerday, C. (1997). Subtilisin from psychrophilic antarctic bacteria characterization and site-directed mutagenesis of residues possibly involved in the adaptation to cold. Protein Engineering, 10, 1271—1279. 

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... 

Figure 3 Cysteine protease and subtilisin-like protease pathways for proneuropeptide processing. Distinct cysteine protease and subtilisin-like protease pathways have been demonstrated for pro-neuropeptide processing. Recent studies have identified secretory vesicle cathepsin L as an important processing enzyme for the production of the endogenous enkephalin opioid peptide. Preference of cathepsin L to cleave at the NH2-terminal side of dibasic residue processing sites yields peptide intermediates with NH2-terminal residues, which are removed by Arg/Lys aminopeptidase. The well-established subtilisin-like protease pathway involves several prohormone convertases (PC). PC1/3 and PC2 have been characterized as neuroendocrine processing proteases. The PC enzymes preferentially cleave at the COOH-terminal side of dibasic processing sites, which results in peptide intermediates with basic residue extensions at their COOH-termini that are removed by carboxypeptidase E/H.

Spectroscopic methods can be utilized to correlate changes in secondary structure with reduction in activity. Subtilisin (pH 7.8) solubilized in co-solvent solutions of glycerol, TFE or DMSO was characterized by FTIR (Griebenow and Klibanov, 1997 Xu et ak, 1997). The structural information was then compared with the activity data (Table 5). Solutions in glycerol or water exhibited similar ratios of a-helix and (3-sheet content, both correlating with good activity. TFE demonstrated an increase in a-helicity, and DMSO showed a decrease in both a-helix and (3-sheet content, where both DMSO and TFE solutions showed little activity. 

Other enzymes that are not homologs of chymotrypsin have been found to contain very similar active sites. As noted in Chapter 6, the presence of very active sites in these different protein families is a consequence of convergent evolution. Subtilisin, a protease in bacteria such as Bacillus amyloliq-uejadens, is a particularly well characterized example. The active site of this enzyme includes both the catalytic triad and thcoxyanion hole. However, one NH groups that forms the oxyanion hole comes from the side chain of an asparagine residue rather than from the peptide backbone (Figure 9.14). 

M. G. Griitter, W. MSrki, and H. P. Walliser. Crystals of the complex between recombinant N-acetyleglin c and subtilisin. A preliminary characterization. J. Biol. Chem. 260 11436(1985). 

Once enzymes have been modified, they are crystallized and their spatial structure is determined by x-ray crystallography to characterize the structural effects of the specific modifications. Molecular modeling helps to identify which positions in the molecule can be further modified by protein engineering to get a desired effect (specificity and affinity for a substrate, stability at a given pH and temperature). This approach has been used to alter the substrate specificity of subtilisin proteases, to broaden both their pH-activity and pH-stability profiles, and to increase their bleach stability [57]. 

In addition to the trypsin inhibitors are very interesting inhibitors of chymotrypsin and subtilisin, which are the most heterogeneous structure and variability of all species of wheat and corn and barley, which are controlled by fifth chromosome, while the wheat and rye - I homoeologous group of chromosomes of different genomes. In diploid wheat, a number of species (T, timopheevii) identified the protein components capable of inhibiting both subtilisin and chymotrypsin, which are characterized by low variability (Konarev et al., 2004). 

Applied Biochemistry and Microbiology, Vol. 44, No. 3, pp. 233-241, ISSN 0003-6838 Mundy, J., Hejgaard, J., Svendsen, I. (1984). Characterization of a bifundional wheat inhibitor of endogenous alpha-amylase and subtilisin. FEBS, Vol. 167, pp. 210-214, ISSN 1742-4658... 

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