PARTIAL CHARACTERISATION AND THERAPEUTIC APPLICATION OF PROTEASE FROM A FUNGAL SPECIES

Proteases find wide application in textile, leather, food and pharmaceutical industries. Also this enzyme has considerable therapeutic importance in treating inflammation, digestive and vascular conditions. A protease obtained from a fungal isolate, Aspergillus niger, was tested for clinical application. The microorganism was cultivated on a starch-casein medium. The proteolytic activity was assayed with casein substrate using Folin-Ciocalteus method having tyrosine standard. The enzyme was precipitated using 50% ammonium sulphate. Inhibition studies on EDTA, phenyl methyl sulphonyl fluoride, dithiothreitol and pepstatin A revealed that the enzyme is an acid protease. The precipitated protease exhibited significant dehairing, destaining, declotting activites on Wistar rat model. The enzyme showed prominent anti-inflammatory activity and results were compared using diclofenac sodium.

Recovery of Bacillus licheniformis Alkaline Protease from Supernatant of Fermented Wastewater Sludge Using Ultrafiltration and Its Characterization

Investigation on recovery of alkaline protease from B. licheniformis ATCC 21424 fermented wastewater sludge was carried out by centrifugation and ultrafiltration. Optimization of ultrafiltration parameters (transmembrane pressure (TMP) and feed flux) was carried out with 10 kDa membrane. TMP of 90 kPa and feed flux of 714 L/h/m2 gave highest recovery (83%) of the enzyme from the centrifuged supernatant. The recovered enzyme had given maximum activity at temperature of 60°C and at pH 10. It was stable between pH 8 to 10 and retained 97% activity at 60°C after 180 min of incubation. Enzyme activity was significantly augmented by metal ions like Ca2+ and Mn2+. Protease inhibitors like phenylmethyl sulphonyl fluoride (PMSF) and diisopropyl fluorophosphates (DFPs) completely inhibited the enzyme activity. The partially purified protease showed excellent stability and compatibility with various commercial detergents. The detergent (Sunlight) removed the blood stains effectively along with the enzyme as additive.

Shedaoenase, a Novel Fibrinogenase from the Venom of Agkistrodon shedaoenthesis Zhao

Shedaoenase, a serine protease, was isolated from the venom of Agkistrodon shedaoenthesis Zhao with an apparent molecular mass of 36 kDa. It was purified by affinity chromatography on arginine Sepharose 4B column and anion exchange on Mono Q fast protein liquid chromatography. Shedaoenase preferentially cleaved the Aα-chain of human fibrinogen and slowly digested the Bβ-chain. It also showed arginyl esterase activity using Nα-benzoyl-L-arginine ethyl ester as a substrate, and some synthetic chromogentic substrates, such as Chromozym PL, S-2266, and S-2160, could also be hydrolyzed. The enzyme activity of shedaoenase could be completely inhibited by phenylmethyl sulphonyl fluoride and could be little inhibited by the chelating reagent EDTA. The N-terminal sequence of shedaoenase was determined, and its full-length cDNA encoding a protein of 238 amino acid residues was cloned by reverse transcription-polymerase chain reaction from the total mRNA extracted from the snake venom gland. The deduced primary sequence of shedaoenase shares significant homology with other snake venom serine proteases.

Studies on Porphobilinogen-Deaminase from Saccharomyces cerevisiae

Porphobilinogen-deaminase from Saccharomyces cerevisiae has been isolated and partially purified 80-and 230-fold in the absence or presence of phenylmethylsulphonyl fluoride, respectively.Some properties of the isolated enzyme were studied. Porphyrin formation was linear with time and protein concentration. Optimum pH was about 7.5-7.8. Molecular mass of the protein was 30,000 ± 3000 Dalton when the enzyme was purified in the presence of phenylmethyl­ sulphonyl fluoride. A less active and unstable 20,000 Da molecular mass species was obtained when purification was performed in the absence of the protease inhibitor.Porphobilinogen-deaminase exhibited classical Michaelis-Menten kinetics. The apparent Km for uroporphyrinogen formation was 19 μм; Vmax was 3.6 nmol uroporphyrin/h and the Hill coefficient was n = 1.Also the action of several reagents on the activity was studied. Protective thiol agents had no effect. Heavy metals inhibited both porphyrin formation and porphobilinogen consumption, but known sulphydryl inactivating chemicals inhibit the former without modifying the latter. Ammonium ions had no effect on the activity while hydroxylamine completely inhibited both porphyrin formation and porphobilinogen consumption.

Peptidases from two strains ofPseudomonas fluorescens: partial purification, properties and action on milk

SummaryPseudomonas fluorescensstrains 240 and 32A expressed cell-associated peptidase activity which was shown by subcellular fractionation to be primarily intracellular. Two peptidases were partly purified from strain 32A. One specifically hydrolysedN-α-benzoyl-DL-arginine-4-nitroanilide and was termed endopeptidase and the other hydrolysed L-lysine- and L-leucine-4-nitroanilide and was termed aminopeptidase. The endopeptidase had very low activity on bovine serum albumin compared with that of trypsin and probably was not a proteinase. The endopeptidase had a mol. wt of 33000 and a pH optimum of 8·0. The enzyme was stimulated by Ca2+and Mg2+and inhibited by Co2+, Mn2+, Hg2+, Zn2+and leupeptin. Soya bean trypsin inhibitor and phenylmethane sulphonyl fluoride (PMSF) had no effect on its activity. The aminopeptidase had a mol. wt of 44000 and a pH optimum of 8·0. It was inhibited by all the metal ions mentioned above and by PMSF. Little proteolysis was found when ultra high temperature (UHT) sterilized milk was treated with cell-free extract from strain 32A. It was concluded that the cell-associated peptidases fromPseudomonasstrains normally present in raw milk may not contribute significantly to the deterioration of UHT sterilized milk.

Inhibited thrombins. Interactions with fibrinogen and fibrin

Fibrin-monomer-Sepharose was used to study thrombin binding to fibrin and the role of the enzyme active centre in this interaction. Binding properties of preformed enzyme-inhibitor complexes, as well as inhibition of thrombin already adsorbed to fibrin monomer, were investigated. No apparent difference was found in binding properties of phenylmethanesulphonyl fluoride-, D-Phe-Pro-Arg-CH2Cl- and dansylarginine NN-(3-ethylpentane-1,5-diyl)amide-inhibited thrombins. Also, the elution profile of phenylmethane-sulphonyl fluoride-inhibited thrombin from fibrinogen-Sepharose was identical with that of active thrombin from fibrin-monomer-Sepharose. Thus far the only low-Mr inhibitor that prevents thrombin from binding to fibrin monomer is pyridoxal 5′-phosphate. Preformed hirudin-thrombin complexes do not interact with fibrin. The extent to which the active centre of thrombin associated with fibrin is still accessible to substrates and inhibitors was also studied. Thrombin bound to fibrin hydrolyses a synthetic substrate at the same rate as the free enzyme. Water-soluble low-Mr inhibitors such as D-Phe-Pro-Arg-CH2Cl and dansylarginine NN-(3-ethylpentane-1,5-diyl)amide can readily modify the active centre of the fibrin-associated enzyme, and the active centre is exposed to the degree that displacement of dansylarginine NN-(3-ethylpentane-1,5-diyl)amide by D-Phe-Pro-Arg-CH2Cl is possible without disturbing the binding. Hirudin disrupts the affinity between thrombin and fibrin. These data indicate that the active centre of thrombin associated with fibrin through extended binding is fully exposed and freely accessible. It is possible that extended binding may play a regulatory role in the activation of Factor XIII by thrombin, as well as inactivation of this enzyme by antithrombin III.

Studies on Circulating Plasminogen Activator

A plasminogen activator was prepared in crude form from postvenous occlusion plasma by gel filtration of the plasma on Sephadex G-200 in 0.005 M phosphate buffer (pH 7.5) containing 0.15 M NaCl followed by further gel filtration of the fractions with maximal activator activity on Sephadex G-200 in 0.005 M phosphate buffer containing 1 M NaCl. The preparations were free of plasminogen, α1-antitrypsin, α2-macroglobulin, CI inactivator and antithrombin III as assessed by immunological techniques. The activity of the partially purified plasma activator preparations was markedly more stable at 37° C than the original plasma, was stable at 4° C for several days, and withstood heating at 56° C for 1 hour. The activity was inhibited by exposure to phenylmethyl sulphonyl-fluoride, but not by tosyl-L-lysine chloromethyl ketone or iodoacetamide.

The preparation and reactivity of some potassium Purine-, Quinazoline-, and Tetrahydroquinazoline-sulphonates

The potassium salts of purine-6(and 8)-, 9-methylpurine-6-, quinazoline-2(and 4)-, 5,6,7,8-tetrahydroquinazoline-4-, and 5,6,7,8- tetrahydro-2-methylquinazoline-4-sulphonic acid are prepared by treatment of the corresponding thiones with aqueous potassium permanganate. All the suphonates undergo ready hydrolysis to the corresponding oxo derivatives in acid and also in alkali (except for the first two purines which are stabilized as dianions therein) ; t1/2 values at H0 - 1 (25�) are all less than 8 min; at pH 14 (40�) they vary from 2 to 190 min. Potassium quinazoline-4-sulphonate undergoes hydrazinolysis to 4-hydrazinoquinazoline; quinazoline-2-sulphonyl fluoride (prepared by chlorine oxidation of quinazoline-2-thione in the presence of potassium hydrogen difluoride) reacts with appropriate amines to give 2-diethylamino-, 2-dipropylamino-, and 2-amino- quinazoline; and purine-6-sulphonyl fluoride with hydrazine gives purine-6-sulphonohydrazide or 6-hydrazinopurine, according to conditions.