It was shown that acetylated dipeptides, Ac-D-Phe-D-Phe-OH, Ac-L-Phe-L-Phe-OH, Ac-D-Phe-L-Phe-OH, and Ac-L-Phe-D-Phe-OH, are formed during D-phenylalanine racemization. The overall content of these dipeptides in the reaction mixture ranged from 40 to 60% depending on the reaction conditions. We concluded that, like alpha-aminoisobutyric acid, phenylalanine is prone to polymerization under racemization conditions.

Buffalo chymosin was isolated from abomasum mucosa extract of buffalo calves by affinity chromatography on gramicidin S-agarose followed by ion exchange chromatography on gamma-aminopropylsilochrom. Its molecular weight, 36 +/- 1 kDa, is similar to that of bovine calf chymosin. The N-terminal sequence Gly-Glu-Val-Ala-Ser-Val-Pro- coincides with that of bovine enzyme, whereas some differences were found in the amino acid composition of these enzymes. Buffalo and bovine enzyme possess similar but not identical structures. General proteolytic and milk-clotting activities of buffalo chymosin are also similar to those of bovine proteinase. pH-Optimum of its activity against hemoglobin lies at pH 4.0, somewhat higher than that for bovine chymosin, which indicates subtle differences in the functional properties of two enzymes.

Chymotrypsin hydrolyzate of the intracellular serine protease was separated by ion-exchange chromatography on a sulphocationite resine followed by HPLC to yield fifty one individual peptides. Their sequences, corresponding in total to 381 amino acid residues, were determined by the manual Edman procedure.

Chymotrypsin hydrolyzate of the intracellular serine protease was separated by ion-exchange chromatography on a sulphocationite resin followed by HPLC to yield fifty one individual peptides. Their sequences, corresponding in total to 381 amino acid residues, were determined by the manual Edman procedure.

Stepwise application of affinity chromatography on bacitracin-silochrome, gel filtration on Acrylex P-10, rechromatography on bacitracin-Sepharose 4B and gel filtration on Sephadex G-15, a homogeneous metalloproteinase (M(r) = 35,000 Da) has been isolated from the cultural filtrate of B. megaterium strain 599. The amino acid composition and N-terminal sequence (20 amino acids) of the enzyme have been determined. The proteinase is not inhibited by diisopropyl-fluorophosphate, is inhibited by o-phenanthroline, EDTA, and Zn2+, and is activated by Co2+. The enzyme has a peak activity at 60-65 degrees C. The maximum of the enzymatic activity after hydrolysis of synthetic substrates is at pH 6.5-7.0. The enzyme is stable at pH 7.0-9.0 and retains its stability at 45-60 C for several hours. In acid media the enzyme undergoes irreversible inactivation. The dependence of kcat/Km on pH points to the involvement of an ionogenic group with pKa 7.5 in the catalytic act, most probably of the imidazole group of histidine. The metalloproteinase hydrolyzes synthetic peptide substrates at the bonds formed by the amino groups of hydrophobic amino acids-Phe, Leu, Ile and Val.

A homogenous metalloproteinase with molecular weight 35 kD was isolated from the culture medium of Bacillus megaterium strain 599 by stepwise application of affinity chromatography on bacitracin-silochrom, gel filtration on Acrylex P-10, rechromatography on bacitracin-Sepharose 4B, and gel filtration on Sephadex G-25 The amino acid composition and N-temminal sequence (20 amino acids) were determined The proteinase is not inhibited by diisopropyl fluorophosphate. It is suppressed by o-phenanthroline, EDTA, and Zn2+ but activated by Co2+. The enzyme shows peak activity at 60-65 degrees C. The activity for synthetic substrates is maximal at pH 6.5-7.0. The enzyme is stable at pH 7.0-9.0 and retains its stability at 45-60 degrees C for several hours. In acid media the enzyme is irreversibly inactivated The pH dependence of k(cat)/K-m suggests the involvement of an anionic group with pK(a) 7.5 (probably the imidazole group of histidine) in catalysis. The metalloproteinase hydrolyses synthetic peptide substrates at the bonds formed by amino groups of hydrophobic amino acids (Phe, Leu, Ile, Val).

Kinetic constants for the hydrolysis of the series of p-nitroanilide peptide substrates catalyzed by subtilisin from Bacillus subtilis strain 72 have been determined. The series of N-protected p-nitroanilides of the Z-A2-A1-pNA, Z-A3-A2-A1-pNA, Z-A4-A3-A2-A1-pNA types (Z-, benzyloxycarbonyl-1; -pNA, p-nitroanilide; A1-An, amino acid residues of the L-configuration) have been used. Subsite S1 reveals a preference for hydrophobic amino acid residues, i.e., leucine and phenylalanine. A preference for Leu over Phe at this position is manifested at the catalytic step, but not during the binding process. The beta-branched (Val, Ile) and the basic (Arg) amino acid residues cannot interact with the S1 subsite and the hydrolysis of the corresponding peptides occurs exclusively at the A2-A1 bond. If S1/A1 interactions are weak (Ala, Nva, Nle), the amino acid residue A1 can interact with subsites S1 and S'1 resulting in the hydrolysis at two bonds (A1-pNA and A2-A1). The data obtained suggests that the S'1 subsite is of broad selectivity. Subsite S2 reveals a preference for small amino acid residues. At pH 5.5-9 and below 50 degrees C, the subtilisin study does not lose its activity. At higher temperatures a rapid thermoinactivation occurs. Substrate binding stabilizes the enzyme. The temperature dependences of the kinetic and thermodynamic parameters suggest that the enzyme exists in two, i.e., 'cold' and 'hot' forms. At 22 degrees C the 'cold' form turns into the 'hot' one possibly owing to a conformational change. The enzyme-substrate complex does not exhibit such behavior and exists in only one form in the whole temperature range studied. The activity of an uncomplexed enzyme is controlled by a group of pKa = 7.2 +/- 0.1, which probably belongs to the histidine imidazole.

The intracellular peptidase of Brevibacterium E531, a lysine-producing bacterial species, was purified 6500-fold by chromatography on DEAE-cellulose and the affinity adsorbent H-Thr(But)-Phe-Pro-hexamethylene-diamine-Sepharose 4B and by gel filtration on Sephadex G-200. The enzyme displayed the maximum activity towards proline p-nitroanilide at pH 7.7-7.9 and readily split glycine, alanine and proline from di-, tri- and tetrapeptides but did not practically hydrolyze oligopeptides of a greater chain length. The enzyme was not inhibited by complexons (EDTA, 8-oxiquinoline and 1.10-phenanthroline). The peptidase was not activated by divalent metal ions and was inhibited by Zn2+; Cd2+, Hg2+ and Cu2+. Data brom gel filtration on Sephadex G-200 suggest that the molecular mass of the enzyme is no less than 250 kDa. In the presence of sodium dodecyl sulfate the molecular mass of the enzyme is 43 kDa, which is suggestive of the presence of a quaternary structure. One peculiarity of the enzyme is its activation by alkaline metal halogenides and sodium nitrate which reaches a maximum at the 0.05-0.1 M concentration of the salts.

The properties of new killer toxins secreted by the yeasts Williopsis subsufficiens and Williopsis beijerinckii were studied. The toxins have wide spectra of activity against yeasts of different taxonomic groups, including pathogenic yeasts of the genus Candida. The two toxins had different spectra of activity, and these spectra were also different from those of toxins produced by other species of the genus Williopsis. Both killer toxins were active over the pH range 3.0 to 8.0, and were thermostable. The molecular weights of the toxins of W. subsufficiens and W. beijerinckii were 15,800 and 12,600 Da respectively, and their isoelectric points were in the range 4.0-4.5.

A comparative study of the hydrolysis of various p-nitroanilide substrates (Z-A2-A1-pNA, Z-A3-A2-A1-pNA, and Z-A4-A3-A2-A1-pNA, where A1-An are various amino acid residues, Z is the benzoyloxycarbonylic group and pNA is the p-nitroanilide group), catalyzed by serine proteinase from Bacillus subtilis strain 72, was carried out. It was found that depending on the substrate structure, the hydrolysis may involve both the peptide-p-nitroaniline and the amino acid-amino acid bonds. A kinetic analysis of substrate hydrolysis occurring simultaneously at these two bonds was carried out. The physico-chemical meaning of the kinetic parameters of the given scheme was determined. The quantitative estimation of the enzyme specificity with respect to both hydrolyzing bonds can be found by using the parameters calculated during the analysis of the kinetic curve of p-nitroaniline production. It was found that according to their specificity the amino acid residues at position A1 can be arranged in the following order: L-Leu greater than P-Phe greater than L-Ile greater than L-Ala. The beta-branched amino acid residues, L-Val and L-Ile, do not bind to subsite S1. If these residues occupy position A1, the substrate splitting occurs exclusively between residues A1 and A2. The tetrapeptide N-protected p-nitroanilide substrates are also hydrolyzed at this bond. Partial hydrolysis of the amino acid-amino acid bond between residues A1 and A2 occurs in two cases: i) when residue A1 is loosely bound to subsite S1 and/or, ii) when residue A2 is firmly bound to subsite S1.

Using amino acid analysis, the ability of destabilize to hydrolyze the epsilon-(gamma-Glu)-Lys isopeptide bond was demonstrated. Incubation of the epsilon-(gamma-Glu)-Lys isopeptide with the enzyme was accompanied by a decrease of the amount of the isopeptide and an increase of equimolar amounts of lysine and glutamic acid. Complete hydrolysis of the isopeptide was observed after 96 hour incubation with destabilize. It was supposed that the isopeptide is a less specific substrate for destabilize compared to L-gamma-Glu-pNA.

Extracellular beta-galactosidase from P. canescens culture medium was purified by ion-exchange chromatography on DEAE and CM-Sepharose CL-6B and gel filtration. The enzyme active form was shown to be a monomer with a molecular weight of about 120 kDa; the isoelectric point is 6.7 and the sedimentation coefficient is 6.5. In terms of physico-chemical and catalytic properties, the purified enzyme is similar to beta-galactosidases of other fungi of genus Penicillium. The amino acid composition and the NH2-terminal sequence of 24 residues non-homologous to the corresponding sequences of bacterial and yeast beta-galactosidases were determined.

Serine proteinase and metalloproteinase of Asp. oryzae, extracellular metalloproteinase of L. pneumophila and chymotrypsin-like proteinase of S. rutgersensis can hydrolyze pepsinogen by converting it into pepsin (pH 5.0, 37 degrees C). The localization of the site of hydrolysis depends on the nature of the enzyme: serine proteinase from Asp. oryzae induces the synthesis of a mixture of 60% pepsin, 25% leucyl-pepsin and 15% alanyl-leucyl-pepsin; metalloproteinase of Asp. oryzae converts pepsinogen only into leucyl-pepsin, while metalloproteinase of L. pneumophila yields a mixture of 33% pepsin, 53% leucyl-pepsin and 14% alanyl-leucyl-pepsin. Thus, the region of the activating pepsinogen peptide--Ala 42P-Ile 1 bond--seems to the most probable site for hydrolysis by exogenous proteinases. This site contains a Leu 44P-Ile 1 bond which is subjected to intermolecular hydrolysis during autocatalytic activation of pepsinogen. The experimental results emphasize the importance of the intermolecular pathway of pepsinogen activation.

Extracellular carboxypeptidase was isolated from culture filtrates of Str. spheroides strain 35, using affinity chromatography on bacitracin-silochrome, bacitracin-Sepharose and CABS-Sepharose. The electrophoretically homogenous enzyme was obtained with a 44% yield and 4160-fold purification. The enzyme-molecular weight is 33,000 Da; pI is 4.7. The amino acid composition of carboxypeptidase is as follows: Asp43, Thr30, Ser35, Glu33, Pro30, Gly47-50, Ala38, 1/2 Cys5-6, Val16, Met2, Ile11, Leu15, Tyr8, Phe10, Lys10, His6, Arg9. The enzyme shows an activity optimum at pH 7.5 is stable at pH 6-8, is completely inhibited with EDTA and can be reactivated by Ca2+. The carboxypeptidase from Str. spheroides strain 35 has a dual substrate specificity, i. e., it splits N-substituted di-, three- and tetrapeptides having both neutral and basic amino acids at the C-ends similar to mammalian carboxypeptidases A and B. The enzyme belongs to the family of metallocarboxypeptidases; its properties are very similar to those of carboxypeptidase S from Str. griseus K-1 and of carboxypeptidase T from Thermoactinomyces sp.