Schistosoma mansoni cercarial elastase (SmCE): differences in immunogenic properties of native and recombinant forms

SUMMARYThe Schistosoma mansoni cercarial elastase (SmCE) has previously been shown to be poorly immunogenic in mice. However, a minority of mice were able to produce antibodies against SmCE after multiple immunizations with crude preparations containing the enzyme. These mice were partially protected against challenge infections of S. mansoni. In the present study, we show that in contrast to the poor immunogenicity of the enzymatically active native form of SmCE derived from a crude preparation (cercarial transformation fluid), immunization of CBA/Ca mice with two enzymatically inactive forms, namely purified native SmCE or a recombinant SmCE fused to recombinant Schistosoma japonicum glutathione S-transferase (rSmCE-SjGST), after adsorption onto aluminum hydroxide adjuvant, induced specific anti-SmCE immunoglobulin G (IgG) in all mice within 2 weeks of the second immunization. The IgG antibody response to rSmCE-SjGST was mainly of the IgG1 subclass. These results suggest that inactive forms of the antigen could be used to obtain the optimum immunogenic effects as a vaccine candidate against schistosomiasis. Mice immunized with the rSmCE-SjGST on alum had smaller mean worm burdens and lower tissue egg counts when compared with adjuvant alone- and recombinant SjGST-injected controls. The native SmCE was antigenically cross-reactive with homologous enzymes of Schistosoma haematobium and Schistosoma margrebowiei.

Production of Biosurfactants By Arthrobacter Sp . N3 , a Hydrocarbon Degrading Bacterium

Different screening methods, such as emulsification capacity and oil spreading assays, hydrocarbon overlay agar and modified drop collapse methods were used to detect biosurfactant production by hydrocarbon degrading Arthrobacter sp N3 strain. It was indicated that oil spreading assay was the most reliable method to detect biosurfactant production. To investigate biosurfactant production, batch cultivation of Arthrobacter sp N3 was carried out in a fermenter with complex nutrient medium supplemented by sunflower oil as a carbon source. The highest oil displacement activity was achieved when Arthrobacter sp N3 strain was cultivated in two stages (with aeration for cell production and without aeration for biosurfactant synthesis). Then, two forms of the biosurfactant (crude preparation and partially purified biosurfactant) were recovered from the culture liquid. Furthermore, the biosurfactant produced by Arthrobacter sp N3 strain was analyzed by thin layer chromatography and it was estimated that even a few compounds have surface activity. The effect of temperature and pH on biosurfactant activity was also studied. It was observed that no appreciable changes in biosurfactant activity occurred at temperature and pH values ranges of 4–125 ºC and 5–10, respectively.

Characterization of a NewProvidenciasp. Strain X1 Producing Multiple Xylanases on Wheat Bran

Providenciasp. strain X1 showing the highest xylanase activity among six bacterial isolates was isolated from saw-dust decomposing site. Strain X1 produced cellulase-free extracellular xylanase, which was higher in wheat bran medium than in xylan medium, when cultivated at pH 8.0 and 35°C. Zymogram analysis of crude preparation of enzymes obtained while growing on wheat bran and birchwood xylan revealed the presence of seven and two distinct xylanases with estimated molecular weight of 33; 35; 40; 48; 60; 75; and 95 kDa and 33 and 44 kDa, respectively. The crude xylanases were produced on wheat bran medium and showed optimum activity at pH 9.0 and 60°C. The thermotolerance studies showed activity retention of 100% and 85% at 40°C and 60°C after 30 min preincubation at pH 9.0. It was tolerant to lignin, ferulic acid, syringic acid, and guaiacol and retained 90% activity after ethanol treatment. The enzyme preparation was also tolerant to methanol and acetone and showed good activity retention in the presence of metal ions such as Fe2+, Mg2+, Zn2+, and Ca2+. The crude enzyme preparation was classified as endoxylanase based on the product pattern of xylan hydrolysis. Pretreatment of kraft pulp with crude xylanases for 3 h at 60°C led to a decrease in kappa number by 28.5%. The properties of present xylanases make them potentially useful for industrial applications.

Isolation of Candida rugosa lipase isoforms

Convenient source of lipases for science and industry is yeast Candida rugosa. Crude preparation of Candida rugosa lipase (CRL) consists of several extracellular lipases. Isoenzyme profile depends on the culture or fermentation conditions. All isoforms are coded by lip pseudogene family; they are monomers of 534 amino acids and molecular weight of about 60 kDa. They share the same catalytic mechanism and interfacial mode of activation. Isoenzymes differ in isoelectric points, post-translational modifications, substrate specificity and hydrophobicity. The presence of different lipase isoforms and other substances (i. e. inhibitors) in crude preparation leads to lack of their productivity in biocatalytic reactions. Purification of specific isoform improves its overall performance and stability. This paper provides an overview of different methods for purification of CRL isoenzymes up to date, their advantages and disadvantages.

Substrate specifity and inhibitors of polyphenol oxidase in aspect of darkening of fresh and frozen mushrooms (Agaricus bisporus (Lange) Sing.)

Activity of mushroom polyphenol oxidase (PPO) toward 6 substrates and inhibitory effect of cysteine, 2-mercaptoethanol, benzoic acid and sodium metabisulphite were determined. The o-diphenols which appeared to be the best substrates were: catechin, DOPA (L-3,4-dihydro-xyphenylalanine) and chlorogcnic acid. Affinity of PPO crude preparation substrates to enzyme, expressed as inverse value of Michaelis constant was lower then affinity of catechol. Inhibitory effect depended on specifity of inhibitors and their concentration. Electrophoretic patterns of PPO of mushrooms reveals slow and fast moving 4 isoforms when DOPA was used as a substrate, 2 bands for catechin and chlorogenic acid while only one band showed activity toward tyrosine and p-cresol.

Partitioning of cellulolytic activity in the polyethylene glycol/dextran two-phase systems

This study is concerned with the partitioning of cellulolytic activity in the polyethylene glycol/dextran two-phase systems. In the system of 10% (w/w) polyethylene glycol 1500/5% (w/w) dextran 500,000/80% (w/w) crude enzyme at the pH 5, 100%, yield of cellulolytic activity from Penicillium sp. in the top phase was achieved in a single extraction step. Addition of KH2PO4 to this system at a concentration of 15 mmol/L improved the purification factor in the top phase for cellulolytic activity from crude preparation to a value of 2.6, although it had an adverse effect on the yield in the same phase.

SELECTED PROTEOLYTIC ACTIVITY BY EXTRACTS OF DERMESTID LARVAE

The larvae of the Beetle Dermestes maculatus De Geer can subsist on a diet consisting largely of protein. Studies have been undertaken to investigate the nature of proteolytic enzymes. A water extract of the larvae yielded a crude preparation that hydrolyzes gelatin, bide powder, hemoglobin substrate, benzoyl-DL-arginine p-nitroamilide, and glutaryl-L-phenylalanine p-nitroanilide. Enzyme activity was found in a non-dialyzable, heat- and acid0labile portion of the extract yielded two fractions with high specific activity towards gelatin. These are precipitated between 40% to 60% saturation of ammonium sulfate and 60% to 80% saturation. The higher specific activity was observed in the 40%-60% fraction. These results suggest that the larvae of these dermestids contain proteolytic enzymes with actions similar to mammalian trypsin and chymotrypsin. The results also suggest that other proteolytic enzymes may be present as well.