РЕЗЮМЕПредставена е схема по пречистването на вътреклстъчната протеиназа Е на флавиногенната гъба Eremothecium ashbyii. Изследвани са свойствата на изолирания ензим—рН-оптимум, температурен оптимум, рН-стабилност, субстратна специфичност, инхибитори и активатори, влиянието на ензима in vitro на активността на рибофлавинкиназа и ГТФ-циклохидролаза—ензими, участвуващи в началните и крайните етапи на флавиногенезата.

o C without any adverse effect on protease activity may prove a protective mechanism to temperature stress. These results clearly demonstrated stability and activity of these serine protease isozymes at high temperature and alkalinity which is advantageous for various industrial applications.

Two proteolytic enzyme systems were observed in the culture filtrates of Rhizopus oligosporus. One has an optimum pH at 3.0; the other, at 5.5. Both enzyme systems have maximum activities at 50–55 °C and are fairly stable at pH 3.0–6.0. Maximum production of the enzymes occurred after 72 to 96 hours of incubation and then it decreased rapidly. Higher proteolytic activity was noted in the culture filtrates of the organism grown in wheat flour medium than in soybean flour. Data suggest that formation of the enzyme systems appears to be inhibited by soybean extracts.

Two experiments were conducted to study the effect of methionine- and lysine-supplemented raw and hydrothermically processed full-fat soybean as a dietary protein source on growth performance, digestibility, and digestive enzyme activity in grey mullet, Mugil cephalus (mean BW 0.55 g), and milkfish, Chanos chanos (mean BW 3.83 g). Eight isocaloric (gross energy about 18.27 kj g−1) and isonitrogenous (crude protein about 40%) diets (1–4 = raw soybean based; 5–8 = processed full-fat soybean based) were formulated. A fishmeal-based diet was used as the reference/control diet. Growth performance, nutrient retention, feed conversion ratio (FCR), and protein digestibility in fishes correlated well with the trypsin inhibitor activity levels of the diets. Carcass phosphorous levels were significantly (P < 0.05) higher in fish fed the fishmeal control diet. Irrespective of the fish species, significantly (P < 0.05) lower levels of total ammonia excretion and reactive phosphate production (mg kg−1 BW d−1) were recorded where the fish were fed processed full-fat soybean diets compared with the fish fed fishmeal or raw soybean diets. Among these diets, the best growth performance and the lowest levels of metabolic waste were obtained in fishes fed the methionine- and lysine-supplemented processed soybean-based diets containing no fishmeal.

This work focuses on the adsorption of Bromelain in expanded bed conditions, such as the adsorption kinetics parameters. The adsorption kinetics parameters showed that after 40 minutes equilibrium was achieved and maximum adsorption capacity was 6.11 U per resin mL. However, the maximum adsorption capacity was only determined by measuring the adsorption isotherm. Only by the Langmuir model the maximum adsorption capacity, Qm, and dissociation constant, kd, values could be estimated as 9.18 U/mL and 0.591, respectively, at 25 oC and 0.1 mol/L phosphate buffer pH 7.5. A column made of glass with an inner diameter of 1 cm was used for the expanded bed adsorption (EBA). The residence time was reduced 10 fold by increasing the expansion degree 2.5 times; nonetheless, the plate number (N) value was reduced only 2 fold. After adsorption, the bromelain was eluted in packed bed mode, with a downward flow. The purification factor was about 13 fold and the total protein was reduced 4 fold. EBA showed to be feasible for purification of bromelain.

Thirty adult Angelwing clams, Pholas orientalis, (89.78 ± 6.60 g wet weight; 118.13 ± 2.92 mm standard length) were acclimated in the laboratory for 7 d, randomly segregated into 3 groups which were exposed to different photoperiod regimes (24 h light, 12 h light 12 h dark, 24 h dark). After 7 d, the clams were sacrificed and their crystalline styles excised and the digestive enzymes α-amylase, CMcellulase, agarase, laminarinase and protease were assayed. All the digestive enzymes, amylase, cellulase, agarase, laminarinase and protease exhibited significantly the highest activities when the clams were exposed to 24 h darkness for 7 d and those exposed to continuous artificial light or to equal light and dark hours exhibited lower activities but were not significantly different from each other.

The thermal inactivation of broad specificity proteases such as thermolysin and subtilisin is initiated by partial unfolding processes that render the enzyme susceptible to autolysis. Previous studies have revealed that a surface-located region in the N-terminal domain of the thermolysin-like protease produced by Bacillus stearothermophilus is crucial for thermal stability. In this region a disulfide bridge between residues 8 and 60 was designed by molecular modelling, and the corresponding single and double cysteine mutants were constructed. The disulfide bridge was spontaneously formed in vivo and resulted in a drastic stabilization of the enzyme. This stabilization presents one of the very few examples of successful stabilization of a broad specificity protease by a designed disulfide bond. We propose that the success of the present stabilization strategy is the result of the localization and mutation of an area of the molecule involved in the partial unfolding processes that determine thermal stability.

The susceptibility of exocrine and serum immunoglobulins and antibodies to proteolytic degradation was assessed. Colostral and duodenal fluid exocrine 11S IgA, monomeric serum IgA, and IgG were digested with trypsin, chymotrypsin, or duodenal fluid. Exocrine IgA was more resistant to digestion than were the serum immunoglobulins. Under conditions of the experiments, most of colostral IgA retained its 11S quaternary structure, including the secretory piece; the portion degraded was reduced almost entirely to peptides. The superior resistance of exocrine IgA was also demonstrated by digestion of serum IgG and nasal exocrine IgA diphtheria antitoxins with trypsin or duodenal fluid. Selective precipitation of trypsin-digested antitoxins with antibodies to heavy chains, light chains, or secretory piece revealed that the differences in susceptibility to digestion were due to differences in lability of the Fc portions of the IgA and IgG antibody molecules. The Fc portions of IgG antibody molecules were degraded or cleaved from the Fab units of the molecules, whereas the Fc-like portions of IgA antibody molecules remained associated with their Fab-like units and the secretory piece. On the other hand, trypsin treatment did not affect the antigen binding ability of the Fab parts of either the exocrine IgA or IgG antibodies. The Fc-like portions of exocrine IgA may be protected from tryptic degradation by the quaternary structure of the 11S molecules, which includes a dimer of 7S IgA subunits and the secretory piece.

The serum concentrations of α1-antitrypsin and α2-macroglobulin were determined immunochemicaHy in 50 cases with α1-antitrypsin deficiency. The mean α1-antitrypsin concentration was found to be 14.5 per cent and the mean α2-macroglobulin concentration 134 per cent of the normal concentration. No significant difference could be demonstrated between subjects with and without chronic lung disease. The trypsin inhibiting capacity of the inter-α trypsin inhibitor was estimated in two serum pools from subjects with and without pulmonary disease after separation of the serum inhibitors by gel filtration. It was found to be normal in both.

The rice green semi looper, Naranga aenescens Moore (Lepidoptera: Noctuidae) causes severe damage to rice fields in Eastern Asia and Middle East. We demonstrate that two types of serine proteases are active in the midgut of the third instar larvae of N. aenescens, but trypsin-like proteases are considerably more active than chymotrypsin-like proteases. To develop better control strategies, purification and biochemical characterization of a major trypsin-like digestive protease from the midgut of the third instar larvae of N. aenescens was achieved by gel filtration and anion exchange chromatography. After the final purification step, the enzyme was purified 9.62-fold with a recovery of 16.1% and a specific activity of 4.12 U/mg protein and a molecular mass of approximately 88.5 kDa. Biochemical characterization indicated that the purified protease had highest activity at pH 10 and 30°C and was stable for up to 6 h under those conditions. Divalent cations, especially Ca2+, Mg2+, and Cu2+, increased the enzyme activity and synthetic inhibitors that target trypsin-like activity caused a significant reduction in caseinolytic activity. These data may be used to develop inhibitors that decrease the damage of N. aenescens to rice cultivars in the field.

The removal of a complex substrate bovine serum albumin (BSA) by activated sludge was examined with respect to the initial level of substrate adsorbed, the rate of substrate removal, and the induction of exoprotease (located both on the sludge matrix and in the cell‐free supernatant). The relationship of these various processes, which together make up the overall removal process, was investigated with respect to the level of substrate present expressed both per unit volume and per unit biomass. A higher degree of correlation was observed when the substrate per unit biomass (S/B) ratio was used, thus providing some basis for empirical models of the activated sludge system based on the food‐to‐microorganisms (F/M) ratio. The interaction of sludge concentration and substrate concentration, plus the fact that there is not a clear distinction, as in pure culture, between substrate and microorganisms, makes the use of models devised for pure culture questionable.

The radiation yeilds of unfolding (Gconf) determined by the method of tryptophan fluorescence coincide with the radiation yields of proteolytic inactivation (Gin) for chymotrypsin-like (CT-like) enzymes on irradiation in air, both in solution and in the dry state with futher dissolution at pH7. It can be supposed that the unfolding is the main process determining the proteolytic gamma-inactivation of CT-like enzymes. It was also shown that the transition of chymotrypsin and trypsin gamma-irradiated at acid pH to neutral pH is an additional action, leading to unfolding of part of the molecules.

The properties and agglutination activity of acutolysin C, a hemorrhagic metalloproteinase obtained from Agkistrodon acutus venom, were studied herein. Acutolysin C is a basic glycoprotein consisting of a single polypeptide chain with a molecular weight of 23.1 kDa and pI 8.7, containing one Zn2+ and one Ca2+ per molecule. It possesses caseinolytic, weak lethal (LD50 = 7.6 mg/kg) and weak hemorrhagic (MHD = 12.0 μg) activities, but does not present fibrinolytic, fibrinogenolytic, arginine esterase and phospholipase A2 actions. In addition, it revealed agglutination activity on some animal lymphocytes, including five species of mammals, six of birds, three of reptiles and one of amphibians, but had no effect on lymphocytes from two species of reptiles, one amphibian and nine species of fish. It had no effects on the erythrocytes and platelets of all 26 animal species tested. Both leucoagglutination and caseinolytic activities were inhibited by EDTA; while cysteine, 2-mercaptoethanol, 1,4-dithiothreitol, glutathione, serum against acutolysin C and serum against homologous snake venom as well as glucose, sucrose, mannose, lactose and galactose had no effects on inhibition. The lowest concentration of acutolysin C that induced mouse lymphocyte agglutination was 2.5 μg/mL. Acutolysin C is an interesting substance since it is the first member of the hemorrhagin family to be shown to have leucoagglutination activity.

The production of a clotting factor (peptocoagulase) by bovine clinical isolates of Peptococcus indolicus and its nature were investigated. Extracellular peptocoagulase was demonstrated in culture filtrates of 93% and cell associated with washed cell suspensions of 100% of the 75 isolates tested. Both citrated and heparinized plasma were clotted. Crude peptocoagulase was nondialyzable, precipitated with (NH4)2SO4 at 40% saturation, somewhat resistant to heating at both neutral and acid pH, and chloroform insoluble. Culture filtrate did not contain proteolytic activity with albumin and casein, as substrates and no esterase activity was detected with tosylarginine and benzoylarginine methyl esters as substrates. The clotting reaction required peptocoagulase, prothrombin, and fibrinogen. The activation of prothrombin appeared to involve a stoichiometric reaction with peptocoagulase, possibly by formation of a stable complex.

The production of a clotting factor (peptocoagulase) by bovine clinical isolates of Peptococcus indolicus and its nature were investigated. Extracellular peptocoagulase was demonstrated in culture filtrates of 93% and cell associated with washed cell suspensions of 100% of the 75 isolates tested. Both citrated and heparinized plasma were clotted. Crude peptocoagulase was nondialyzable, precipitated with (NH4)2SO4 at 40% saturation, somewhat resistant to heating at both neutral and acid pH, and chloroform insoluble. Culture filtrate did not contain proteolytic activity with albumin and casein, as substrates and no esterase activity was detected with tosylarginine and benzoylarginine methyl esters as substrates. The clotting reaction required peptocoagulase, prothrombin, and fibrinogen. The activation of prothrombin appeared to involve a stoichiometric reaction with peptocoagulase, possibly by formation of a stable complex.

The partitioning of protease expressed by Penicillium fellutanum from the Brazilian savanna in a novel inexpensive and stable aqueous two‐phase system (ATPS) composed of poly(ethylene glycol) (PEG) and sodium polyacrylate (NaPA) was studied in this work using factorial design. The ATPS is formed by mixing both polymers with a salt (NaCl) and fermented broth of P. fellutanum. The effects of molar mass (2,000, 4,000, and 6,000 g⋅mol−1) and concentration (6, 8, and 10 wt%) of PEG and that of NaPA concentration (6, 8, and 10 wt%) on protease partitioning (K) at 25 °C were studied. A two‐level factorial design (23) was implemented. The effect of Na2SO4 concentration (5, 10, and 15 wt%) on the reextraction of the enzyme was also analyzed. The partition coefficient K ranged from 77.51 to 1.21, indicating the versatility of the method. The reextraction was achieved with the addition of 5% Na2SO4, allowing the partitioning of the protease to the upper phase, whereas total proteins were directed to the bottom phase. The results of partitioning using the PEG/NaPA/NaCl system and that of the subsequent reextraction with Na2SO4 suggest that this method can be used to purify proteases from fermented broth of P. fellutanum.

The objective of the present study investigated on the effects Chamomile seeds exposure to magnetic fields on seed germination, early growth and enzyme activity, under laboratory conditions. Chamomile seeds (dry and wet) were studied after treating with different intensities of magnetic field (0, 25, 50 and 75 mT) and exposure time (0, 30 and 60 min). There were significant effects among treatments for germination percentage, root length, seedling dry mass and seedling vigor index in Chamomile seeds under different magnetic intensity. Magnetic field treatments were not affected to shoot length. In the germinating seeds enzyme activities of α-amylase, dehydrogenase and protease were significantly higher in treated seeds in contrast to controls. The results of this research indicated that, among various combinations of magnetic field (MF) strength and duration, best results were obtained with MF of 50 mT for 60 min exposure in wet seeds of Chamomile.

The noradrenaline-sensitive receptor for catecholamine may be blocked by 2-halogenoalkylamine compounds (of which dibenamine was the first). This process is demonstrable as an antagonism to some of the actions of noradrenaline which is at first surmountable, and partially reversible in vitro-i.e., competitive-but progresses with increasing concentration of the blocking drug or duration of exposure of the tissue to that drug to an insurmountable antagonism which is irreversible in vitro and of variable but frequently prolonged duration in vivo (Graham, 1962, p. 139, Table 4.4, compounds Nos. 1, 3 and 5; also Table I of present paper). Dibenamine occupies these receptors slowly but firmly, 2-bromo-N-ethyl-N-(a-naphthylmethyl)ethylamine (SY28) more quickly but less firmly, and N,N-dimethyl-2-bromo-2-phenethylamine hydrobromide (compound L2; Graham & James, 1961) quickest but least firmly. The insurmountable and prolonged block has been attributed to inactivation of the receptor, involving alkylation (Harvey & Nickerson, 1954). It is postulated here that subsequent regeneration in vivo is the result of an enzymatic process, the speed and extent of which will vary with the firmness of the alkylation of the receptor substance, which is a function of the physicochemical properties of the blocking molecule. A candidate for the enzymatic role is trypsin; should this prove to be so, consideration of the relation of this enzyme, the 2-halogenoalkylamine, the agonist and the tissue may be expected to throw some light on the attributes of the receptor.

The lysis of dead Bacterium coli by concentrates prepared from filtrates of a Streptomyces sp. is probably due to proteolysis. In any event the lysis can be accounted for by proteolysis, even if the action of other bacteriolytic enzymes cannot be excluded. The streptomyces preparation contains at least two proteolytic enzymes, one digesting casein and the other digesting protein(s) present in Bact. coli. The substrates of the autolytic enzymes of Bact. coli are probably different from those of the streptomyces enzymes.

The lethal and enzymatic activities of venom from Naja sumatrana (Equatorial spitting cobra) were determined and compared to venoms from three other Southeast Asian cobras (Naja sputatrix, Naja siamensis and Naja kaouthia). All four venoms exhibited the common characteristic enzymatic activities of Asiatic cobra venoms: low protease, phosphodiesterase, alkaline phosphomonoesterase and L-amino acid oxidase activities, moderately high acetylcholinesterase and hyaluronidase activities and high phospholipase A2. Fractionation of N. sumatrana venom by Resource® S cation exchange chromatography (GE Healthcare, USA) yielded nine major protein peaks, with all except the acidic protein peak being lethal to mice. Most of the protein peaks exhibit enzymatic activities, and L-amino acid oxidase, alkaline phosphomonoesterase, acetylcholinesterase, 5'-nucleotidase and hyaluronidase exist in multiple forms. Comparison of the Resource® S chromatograms of the four cobra venoms clearly indicates that the protein composition of N. sumatrana venom is distinct from venoms of the other two spitting cobras, N. sputatrix (Javan spitting cobra) and N. siamensis (Indochinese spitting cobra). The results support the revised systematics of the Asiatic cobra based on multivariate analysis of morphological characters. The three spitting cobra venoms exhibit two common features: the presence of basic, potentially pharmacologically active phospholipases A2 and a high content of polypeptide cardiotoxin, suggesting that the pathophysiological actions of the three spitting cobra venoms may be similar.