Peptidase Activity of Proteinivorax Bacteria and Their Possible Ecological Role in the Microbial Communities of Tanatar Soda Lakes (Altai Krai,Russia)

Microbiology - Peptidase activity of alkaliphilic anaerobic proteolytic Proteinivorax bacteria, P. tanatarense Z-910T and P. hydrogeniformans Z-710T, isolated from the Tanatar system of hypersaline...     

Midgut proteinases of the cockroachNauphoeta cinerea

The study of properties of proteolytic enzymes in midgut of imago of the cockroachNauphoeta cinerea Oliv. Has been carried out. It is shown that the total proteolytic activity of digestive proteases, measured with azocasein as substrate, is maximal at pH 11.5 both in the anterior and in the posterior parts of the midgut. The predominant part of this activity (67%) was present in the posterior part. Fractionation of preparation from the posterior part on a column with Sephadex G-50 and subsequent analysis of the activity in the obtained fractions using specificp-nitroanilide substrates and effects of activators and inhibitors of active center have allowed revealing three types of activity of serine proteinases and one cysteine proteinase. No activity of aspartic and metalloproteinases were detected. Among serine proteinases, one trypsin-like, one unusual SHdependent serine, one chymotrypsin-like, and not less than two enzymes hydrolyzing specific substrate of subtilisin were established. The fractionation of the preparation from the anterior part has allowed revealing only three proteinases that were similar by their properties to cysteine, SHdependent serine, and chymotrypsin-like ones in the posterior part of midgut. Their activity was lower in the anterior, than in the posterior part of the midgut. The probable causes of the low proteolytic activity in the anterior part of the midgut are discussed.     

Extracellular proteinases of filamentous fungi as potential markers of phytopathogenesis

he presence of proteins in the culture liquid of filamentous fungi under study was found to induce the secretion of proteinases. The inhibitory analysis of the major extracellular proteinases of the saprotrophic fungus Trichoderma harzianum and the phytopathogenic fungus Alternaria alternata showed that they both belong to the group of serine proteinases. The substrate specificity of these proteinases and their sensitivity to inhibitors suggest that the enzyme of T. harzianum is a subtilisin-like proteinase and the enzyme of A. alternata is a trypsin-like proteinase. This difference between the proteinases may reflect the physiological difference between their producers (saprotroph and phytopathogen).     

Effect of Antioxidant BHT on the Proteolytic Apparatus of Aging Coleoptiles of Wheat Seedlings Grown in Light

The dynamics of changes in total proteolytic activity and activities of various groups of proteases in the coleoptiles of 3- to 12-day-old wheat seedlings grown in light with and without antioxidant BHT (2,6-di-tert-butyl-4-methylphenol) was studied. It was established that the specialized proteases that easily hydrolyze specific synthetic substrates and the enzymes actively hydrolyzing histone H1 dominate in young coleoptiles of 3- to 4-day-old seedlings. Proteases that degrade equally well the majority of the studied substrates are accumulated in the cells of old coleoptiles of 11- to 12-day-old seedlings. Under the effect of BHT, the plants grown in the light (in comparison with etiolated seedlings) demonstrated a somewhat changed dynamics of proteolytic activity in young coleoptiles and the disappearance of proteases active toward histone H1. An inhibitory analysis revealed a relative domination of cysteine proteases in young coleoptiles at the initial development stage of seedlings, whereas the fraction of serine proteases markedly increased in old coleoptiles. We presume that the revealed quantitative and qualitative changes in the proteolytic apparatus of the coleoptile cells induced by BHT may be largely responsible for the retardant and geroprotective effect of this antioxidant in plants.     

Extracellular proteolytic enzymes of microscopic fungi from thermal springs of the Barguzin Valley (Northern Baikal region)

Production of extracellular proteolytic enzymes was studied in thermophilic fungi Paecelomyces variotii and Aspergillus carneus, isolated from thermal springs of the Barguzin Valley. Protease synthesis in these fungi requires protein in the ambient medium. The composition of the enzymes secreted by A. carneus depends on the kind of carbohydrate present in the medium. The proteinase of this fungus digests synthetic substrates and gelatin (optimum pH 7.7). It belongs to neutral serine proteases. Extracellular P. variotii proteases digest gelatin (optimum pH 9.7–10.4). According to inhibitor analysis data, they can be classified as alkaline metalloproteinases and serine proteinases.     

Extracellular proteases of mycelial fungi as participants of pathogenic process

The interest in proteases secreted by mycelial fungi is due to several reasons of which one of the most important is their involvement in the initiation and development of the pathogenic process. A comparison of saprophytic and phytopathogenic mycelial fungi revealed one characteristic feature, namely, the appearance of a new trypsin-like activity in phytopathogens that is absent in saprophytes. To clear up the question of whether the degree of pathogenicity of a fungus is related to the activity of secreted trypsin-like protease, several species of Fusarium of various pathogenicity were compared. In two species, F. sporotrichioides (which causes ear fusariosis of rye) and F. heterosporum (the causative agent of root rot in wheat), a clear correlation between the activity and pathogenicity was revealed: the more pathogenetic F. sporotrichioides exhibited a higher extracellular trypsin-like activity than the less pathogenetic species F. heterosporum. Thus, the presence of trypsin-like activity in a saprotroph-pathogen pair may be an indicator of the pathogenicity of a fungus; in some cases, the value of this activity may indicate the degree of its pathogenicity. This suggests that trypsin-like proteases specific to phytopathogens are directly involved in the pathogenetic process, probably, through interaction with the “sentry” protein or the product of the resistance gene.     

Isolation and Primary Structure of Trypsin from the King Crab Paralithodes camtschaticus

Trypsin from hepatopancreas of the crab Paralithodes camtschaticuswas isolated in homogeneous state by successive ion-exchange chromatography on DEAE-Sephadex, affinity chromatography on Agarose modified with peptide ligands from trypsin hydrolysate of salmin, and ion-exchange chromatography on a Mono Q column. The total yield of the protein was 64%. Its N-terminal amino acid sequence was determined (IVGGTEVTPG-). A sample of amplified total cDNA of hepatopancreas of king crab was obtained. A cDNA fragment containing the complete coding part of the gene was isolated on the basis of the known N-terminal amino acid sequence of the mature form of the trypsin. The polypeptide chain of the proenzyme consists of 266 aa. The mature trypsin involves 237 aa, which corresponds to its molecular mass of 24.8 kDa. A comparison of the amino acid sequence of the king crab trypsin with those of trypsins from other species of crustaceans demonstrated their high structural homology. The trypsin from the shrimp Penaeus vannamei appeared to be closest in primary structure to that of the king crab (65% identity).     

Secreted protease of the entomopathogenic fungus <Emphasis Type="Italic">Cordyceps militaris</Emphasis>. II. Enzyme properties and adsorption on the insect cuticle

Electrophoretically pure preparation of a secreted subtilisin-like protease was isolated from the culture liquid of an entomopathogenic fungus C. militaris strain KP-97-2. The enzyme had an optimum activity at 45°C and pH 7–9. Data were collected on the substrate specificity of the enzyme and its stability at different values of pH and temperature. Adsorption and desorption of the extracellular protease on the cuticle of Nauphoeta cinerea was studied in function of pH.     

Manganese-dependent ribonucleotide reductase ofPropionibacterium freudenreichii subsp.shermanii: Partial purification, characterization, and role in DNA biosynthesis

LikeLactobacillus leichmanii, Rhizobium meliloti, andEuglena gracilis, P. freudenreichii implicates cobalamin in DNA anabolism via adenosylcobalamin-dependent ribonucleotide reductase. However, in the absence of corrinoids,P. freudenreichii is able to synthesize DNA with the involvement of an alternative ribonucleotide reductase, which is independent of adenosylcobalamin. This enzyme is localized in both the cytoplasm (80% of activity) and the cytoplasmic membrane (20% of activity), being loosely bound to the latter. Experiments with partially purified ribonucleotide reductase isolated from extracts of corrinoid-deficient cells showed that manganese specifically stimulates this enzyme and that it is composed of two protein components, a feature that is typical of all metal-containing reductases activated by molecular oxygen. Low concentrations of manganese ions enhanced DNA synthesis in corrinoid-deficient manganese-limited cells. This effect was prevented by the addition of 80 mM hydroxyurea, a specific inhibitor of metal-containing aerobic ribonucleotide reductases. It was concluded that, in adenosylcobalamin-deficientP. freudenreichii cells, DNA synthesis is provided with deoxyribosyl precursors through the functioning of manganese-dependent aerobic ribonucleotide reductase composed of two subunits.     

Secreted protease of an entomopathogenic fungus <Emphasis Type="Italic">Cordyceps militaris</Emphasis>. I. Selection of medium components and development of purification procedure

Extracellular serine proteases from the culture liquids of two strains of an entomopathogenic fungus Cordyceps militaris were isolated and characterized. Their activity was demonstrated to depend on the composition of the culture medium. Electrophoretically homogeneous preparations of the secreted enzymes were obtained by affinity chromatography on bacitracin-silochrome and gel filtration on Superdex G-75. Molecular weight of the proteins was 24 kDa.