Selection and characterization of mannanase-producing bacteria useful for the formation of prebiotic manno-oligosaccharides from copra meal

In order to select bacterial strains effectively secreting mannanase activity for the production of prebiotic mannooligosaccharides, a two-step screening procedure was performed. Enriched cultures from isolation medium containing copra meal were primary screened on an isolation agar medium containing 1% locust bean gum (LBG), which resulted in 48 mannanase-producing bacterial isolates with significant clearing zones on the mannan-containing agar. However, only nine isolates showed appreciable mannanase activities against copra meal in their culture supernatants (0.054–0.185 U/mg of protein) as determined in a standard assay based on the detection of reducing sugars released from this substrate. The isolates CW2-3 and ST1-1 displayed the highest activity against LBG and copra meal, respectively. Copra mannan hydrolysates that were obtained by using crude mannanase from these nine isolates were further used for a secondary screening towards a growth-enhancing activity on Lactobacillus reuteri and inhibitory activity against Escherichia coli as well as Salmonella Enteritidis, resulting in 0.09–2.15 log CFU/ml enhancing activity and low inhibitory activity of 0.46–1.78 log CFU/ml as well as 0.37–1.72 log CFU/ml, respectively. The hydrolysate of CW2-3 mannanase showed the highest enhancing activity of 2.15 log CFU/ml while isolate ST1-1 was most effective with respect to growth inhibition against E. coli E010 and S. Enteritidis S003 with 0.76 and 1.61 log CFU/ml, respectively. Based on morphological, physical, biochemical and genetics properties, isolates CW2-3 and ST1-1 were identified as Klebsiella oxytoca and Acinetobacter sp., respectively. Crude mannanase activity from these two strains was characterized preliminarily. The pH optima of mannanase activity from Klebsiella oxytoca CW2-3 and Acinetobacter sp. ST1-1 were 7 and 6, respectively. The enzymes were stable at 4°C over a pH range of 3–6 and 3–10, respectively.     

Innate immune response and disease resistance in Carassius auratus by triherbal solvent extracts

This study reports the effect of aqueous, ethanol and methanol triherbal solvent extract from Azadirachta indica, Ocimum sanctum and Curcuma longa on innate immune mechanisms such as phagocytosis activity, respiratory burst activity, alternative complement activity and lysozyme activity and disease resistance in goldfish (Carassius auratus) against Aeromonas hydrophila. Fish were intraperitoneally injected with different doses of 0, 5, 50 and 100 mg kg⁻¹ body weight of each triherbal solvent extracts. The functional immunity in terms of percentage mortality and Relative Percent Survival (RPS) and innate immune response was assessed on week 1, 2 and 4 by challenging with live A. hydrophila (1 × 10⁷ cells ml⁻¹). All the chosen innate immune parameters were enhanced in the ethanol and methanol triherbal solvent extract treatment after week 2. However, the aqueous triherbal extract was enhanced only after week 4. The ethanol and methanol triherbal solvent extracts administration preceding the challenge with live A. hydrophila decreased the percentage mortality in the experimental groups with the consequence increase in RPS values. The study indicates that all the doses of ethanol or methanol triberbal solvent extracts could be positively influence the immune response and protect the heath status of goldfish against A. hydrophila infection.     

Characterization of Esterases in a Brazilian Population of Zaprionus Indianus (Diptera: Drosophilidae)

The aim of this study was to characterize esterases in Zaprionus indianus, a drosophilid recently introduced into Brazil. A further aim was study the variation of activity of esterases in the presence of inhibitors and their expression according to sex, sexual activity and age of individual flies. Polymorphisms were detected in two esterase loci (Est-2 and Est-3) and monomorphisms in four others (Est-1, Est-4, Est-5 and Est-6). Biochemical tests using α- and β-naphthyl acetate and the inhibitors malathion, eserine sulphate and PMSF allowed us to classify EST-2 and EST-5 as β-esterases, both carboxyl-esterases, and EST-1, EST-3, EST-4 and EST-6 as α-esterases. EST-1 and EST-3 were classified as carboxyl-esterases and EST-4 and EST-6 as cholinesterases. EST-5 activity was more pronounced in males and EST-2 was restricted to them or to recently copulated females. EST-4, rarely detected, was not characterized. Based on their biochemical characteristics possible roles for these enzymes are suggested.     

Antifungal substances produced by Penicillium oxalicum strain PY-1—potential antibiotics against plant pathogenic fungi

This paper reports the isolation from soil of Penicillium strain PY-1 with strong antagonistic activity against plant pathogenic fungi. On the basis of its morphological characteristics and the sequence of the ITS region, strain PY-1 was identified as P. oxalicum. Strain PY-1 produces antifungal substances that suppress the mycelial growth of Sclerotinia sclerotiorum and many other plant pathogenic fungi tested; the highest antagonistic activity was detected at 72 h when cultured in a 250-ml flask containing 80 ml potato dextrose broth. Compared with carbendazim, the relative activity of the antifungal substances produced by strain PY-1 was approximately 4 μg active ingredient (a.i.) per milliliter. The antifungal substances were extracted with ethyl acetate and further separated by high-performance liquid chromatography (HPLC); at least two active components were discovered. The ability to control plant disease with strain PY-1 was confirmed with S. sclerotiorum, a widespread pathogenic fungus that attacks rapeseed (Brassica napus) and other plants. Spores (10⁶ or 10⁷ ml⁻¹) and filtrate (tenfold diluted or undiluted) of strain PY-1 could significantly suppress infection and/or the extent of infection by S. sclerotiorum of plants at seven-true-leaves stage. The potential of strain PY-1 for identifying new antibiotics to control fungal disease and for biological control of plant disease, for example oilseed rape stem rot, is discussed.     

Screening of chitin deacetylase from Mucoralean strains (Zygomycetes) and its relationship to cell growth rate

Chitin deacetylase (CDA) is an enzyme that catalyzes the hydrolysis of acetamine groups of N-acetyl-d-glucosamine in chitin, converting it to chitosan in fungal cell walls. In the present study, the activity in batch culture of CDA from six Mucoralean strains, two of them wild type, isolated from dung of herbivores of Northeast Brazil, was screened. Among the strains tested, Cunninghamella bertholletiae IFM 46114 showed a high intracellular enzyme activity of 0.075 U/mg protein after 5 days of culture, and a wild-type strain of Mucor circinelloides showed a high intracellular enzyme activity of 0.060 U/mg protein, with only 2 days of culture, using N-acetylchitopentaose as substrate. This enzyme showed optimal activity at pH 4.5 in 25 mM glutamate-sodium buffer at 50°C, and was stable over 1 h preincubation at the same temperature. The kinetic parameters of CDA did not follow Michaelis-Menten kinetics, but rather Hill affinity distribution, showing probable allosteric behavior. The apparent K_HILL and V_max of CDA were 288±34 nmol/l and 0.08±0.01 U mg protein^–1 min^–1, respectively, using N-acetylchitopentaose as substrate at pH 4.5 at 50°C.     

The effect of nitrogen and sucrose concentrations on the growth of Eucomis autumnalis (Mill.) Chitt. plantlets in vitro, and on subsequent anti-inflammatory activity in extracts prepared from the plantlets

Large amounts of anti-inflammatory activity are present in extractsprepared from Eucomis plants. Extracts prepared from in vitroplantlets grown on a modified Murashige and Skoog medium supplementedwith 1 mg &ell^–1 NAA and 1 mg &ell^–1 BA, were tested intwo cyclooxygenase assays (COX-1 and COX-2). Ethanol extracts showedhigh levels of COX-1 and COX-2 inhibitory activity, with a COX-2/COX-1inhibition ratio of 1.1. Further experimental work aimed to determine thefactors affecting the accumulation of anti-inflammatory compounds inin vitro plantlets. High concentrations of sucrose (40 g &a,p;ell^–1) inthe culture medium significantly increased the number of shoots initiated,but had no effect on the subsequent anti-inflammatory activity. Lowconcentrations of sucrose (10 g &ell^–1) led to a significantdecrease in COX-1 inhibition. Changig the amount of nitrogen in the medium(but not the ratio of nitrate to ammonium ions) had no significant effect onthe COX-1 inhibitory activity of the extracts.     

Heterologous expression of Fusarium oxysporum tomatinase in Saccharomyces cerevisiae increases its resistance to saponins and improves ethanol production during the fermentation of Agave tequilana Weber var. azul and Agave salmiana must

This paper describes the effect of the heterologous expression of tomatinase from Fusarium oxysporum f. sp lycopersici in Saccharomyces cerevisiae. The gene FoTom1 under the control of the S. cerevisiae phosphoglycerate kinase (PGK1) promoter was cloned into pYES2. S. cerevisiae strain Y45 was transformed with this vector and URA3 transformant strains were selected for resistance to α-tomatine. Two transformants were randomly selected for further study (designated Y45-1 and Y45-2). Control strain Y45 was inhibited at 50 μM α-tomatine, in contrast, transformants Y45-1 and Y45-2 did not show inhibition at 200 μM. Tomatinase activity was detected by HPLC monitoring tomatine disappearance and tomatidine appearance in the supernatants of culture medium. Maximum tomatinase activity was observed in the transformants after 6 h, remaining constant during the following 24 h. No tomatinase activity was detected in the parental strain. Moreover, the transformants were able to grow and produce ethanol in a mix of Agave tequilana Weber var. azul and Agave salmiana must, contrary to the Y45 strain which was unable to grow and ferment under these conditions.     

A comparative study on the antimutagenic properties of aqueous extracts of Aspalathus linearis (rooibos), different Cyclopia spp. (honeybush) and Camellia sinensis teas

Antimutagenic activity of aqueous extracts of the South African herbal teas, Aspalathus linearis (rooibos) and Cyclopia spp. (honeybush) was compared with that of Camellia sinensis (black, oolong and green) teas in the Salmonella mutagenicity assay using aflatoxin B₁ (AFB₁) and 2-acetylaminofluorene (2-AAF) as mutagens. The present study presents the first investigation on antimutagenic properties of C. subternata, C. genistoides and C. sessiliflora. The herbal teas demonstrated protection against both mutagens in the presence of metabolic activation, with the exception of “unfermented” (green/unoxidised) C. genistoides against 2-AAF, which either protected or enhanced mutagenesis depending on the concentration. Antimutagenic activity of “fermented” (oxidised) rooibos was significantly (P < 0.05) less than that of Camellia sinensis teas against AFB₁, while for 2-AAF it was less (P < 0.05) than that of black tea and similar (P > 0.05) to that of oolong and green teas. Antimutagenic activity of unfermented C. intermedia and C. subternata exhibited a similar protection as fermented rooibos against AFB₁. Against 2-AAF, fermented rooibos exhibited similar protective properties than unfermented C. intermedia and C. sessiliflora. Unfermented rooibos was less effective than the C. sinensis teas and fermented rooibos, but had similar (P > 0.05) antimutagenicity to that of fermented C. sessiliflora against AFB₁ and fermented C. subternata against 2-AAF. Fermented C. intermedia and C. genistoides exhibited the lowest protective effect against 2-AAF, while fermented C. intermedia exhibited the lowest protection when utilising AFB₁ as mutagen. Aspalathin and mangiferin, major polyphenols in rooibos and Cyclopia spp., respectively, exhibited weak to moderate protective effects when compared to the major green tea catechin, (−)epigallocatechin gallate (EGCG). Antimutagenic activity of selected herbal tea phenolic compounds indicated that they contribute towards (i) observed antimutagenic activity of the aqueous extracts against both mutagens and (ii) enhancement of the mutagenicity of 2-AAF by unfermented C. genistoides. Antimutagenic activity of the South African herbal teas was mutagen-specific, affected by fermentation and plant material, presumably due to changes and variation in phenolic composition.     

Transesterification activity of a novel lipase from Acinetobacter venetianus RAG-1

Transesterification activity and the industrial potential of a novel lipase prepared from Acinetobacter ventiatus RAG-1 were evaluated. Purified lipase samples were dialyzed against pH 9.0 buffer in a single optimization step prior to lyophilization. The enzyme and organic phase were pre-equilibrated (separately) to the same thermodynamic water activities (a _w) ranging from a _w 0.33 to 0.97. Production of 1-octyl butyrate by lipase-catalyzed transesterification of vinyl butyrate with 1-octanol in hexane was monitored by gas chromatography. Production of 1-octyl butyrate and initial rate of reaction depended on water activity. Product synthesis and rate of transesterification increased sharply with increase from a _w 0.33 to 0.55. Highest product concentration (218 mM) and rate of reaction (18.7 μmol h⁻¹ · 10 μg protein) were measured at a _w 0.86. Transesterification activity in hexane represented 32% of comparable hydrolytic activity in aqueous buffer.     

Pullulanases of alkaline and broad pH range from a newly isolated alkalophilicBacillus sp. S-1 and aMicrococcus sp. Y-1

Summary Two highly alkalophilic bacteria, and potent producers of alkaline pullulanase, were isolated from Korean soils. The two isolates, identified asBacillus sp. S-1 andMicrococcus sp. Y-1, grow on starch under alkaline conditions and effectively secrete extracellular pullulanases. The two isolates were extremely alkalophilic since bacterial growth and enzyme production occurred at pH values ranging from pH 6.0 to 12.0 forMicrococcus sp. Y-1 and pH 6.0 to 10.0 forBacillus sp. S-1. Both strains secrete enzymes that possess amylolytic and pullulanolytic acitivities. Extracellular crude enzymes of both isolates gave maltotriose as the major product formed from soluble starch and pullulan hydrolysis. Compared to other alkalophilic microbes such asMicrococcus sp. (0.57 units ml^–1),Bacillus sp. KSM-1876 (0.56 units ml^–1) andBacillus No. 202-1 (1.89 units ml^–1) these isolates secreted extremely high concentrations (7.0 units ml^–1 forBacillus sp. S-1 and 7.6 units ml^–1 forMicrococcus sp. Y-1) of pullulanases in batch culture. The pullulanase activities from both strains were mostly found in the culture medium (85–90%). The extracellular enzymes of both bacteria were alkalophilic and moderately thermoactive; optimal activity was detected at pH 8.0–10.0 and between 50 and 60°C. Even at pH 12.0, 65% of original Y-1 pullulanase activity and 10% of S-1 pullulanase activity remained. The two newly isolated strains had broad pH ranges and moderate thermostability for their enzyme activities. These result strongly indicate that these new bacterial isolates have potential as producers of pullulanases for use in the starch industry.     

Cloning, heterologous gene expression and biochemical characterization of the alpha-1,3-glucanase from the filamentous fungus Penicillium purpurogenum

There has been much recent interest in α-1,3-glucanases (mutanases) as they have the potential to be used in the treatment of dental caries. Mutanases have been reported in a number of bacteria, yeast and fungi but remain a relatively uncharacterised family of enzymes. In this study we heterologously expressed the mutanase gene from the filamentous fungus Penicillium purpurogenum to enable further characterization of its enzymatic activity. The mutanase cDNA was cloned and expressed in the methylotrophic yeast Pichia pastoris. The molecular mass of the secreted protein was about 102 kDa. The recombinant enzyme hydrolyzed mutan with a specific activity of 3.9 U/mg of protein. The recombinant enzyme was specific for mutan and could not cleave a variety of other polysaccharides demonstrating a specificity for α-1,3-glucosidic linkages. The pH and temperature optima were pH 4.6 and 45 °C, respectively. Synthetic compounds were also tested as substrates to assess whether the P. purpurogenum mutanase has an exo- or endo-type mechanism of hydrolysis. The results suggest an endo-hydrolytic mode of action. The type of mechanism was confirmed since mutanase activity was not suppressed in the presence of inhibitors of exo-type enzymes.     

Inhibition of hepatic lipase by m-aminophenylboronate application of phenylboronate affinity chromatography for purification of human postheparin plasma lipases

Phenylboronates are competitive inhibitors of serine hydrolases including lipases. We studied the effect of m-aminophenylboronate on triglyceride-hydrolyzing activity of hepatic lipase (EC 3.1.1.3). m-Aminophenylbo ronate inhibited hepatic lipase activity with a K₁ value of 55 μM. Furthermore, m-aminophenylboronate protected hepatic lipase activity from inhibition by di-isopropyl fluorophosphate, an irreversible active site inhibitor of serine hydrolases. Inhibition of hepatic lipase activity by m-aminophenylboronate was pH-dependent. The inhibition was maximal at pH 7.5, while at pH 10 it was almost non-existent. These data were used to develop a purification procedure for postheparin plasma hepatic lipase and lipoprotein lipase. The method is a combination of m-aminophenylboronate and heparin-Sepharose affinity chromatographies. Hepatic lipase was purified to homogeneity as analyzed on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The specific activity of purified hepatic lipase was 5.46 mmol free fatty acids h⁻¹ mg⁻¹ protein with a total purification factor of 14 400 and a final recovery of approximately 20%. The recovery of hepatic lipase activity in m-aminophenylboronate affinity chromatography step was 95%. The purified lipoprotein lipase was a homogeneous protein with a specific activity of 8.27 mmol free fatty acids h⁻¹ mg⁻¹ The purification factor was 23 400 and the final recovery approximately 20%. The recovery of lipoprotein lipase activity in the m-aminophenylboronate affinity chromatography step was 87%. The phenylboronate affinity chromatography step can be used for purification of serine hydrolases which interact with boronates.     

Codon optimization of <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> β-1,3-1,4-glucanase gene and its expression in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

β-1,3-1,4-glucanase (EC3.2.1.73) as an important industrial enzyme has been widely used in the brewing and animal feed additive industry. To improve expression efficiency of recombinant β-1,3-1,4-glucanase from Bacillus licheniformis EGW039(CGMCC 0635) in methylotrophic yeast Pichia pastoris GS115, the DNA sequence encoding β-1,3-1,4-glucanase was designed and synthesized based on the codon bias of P. pastoris, the codons encoding 96 amino acids were optimized, in which a total of 102 nucleotides were changed, the G+C ratio was simultaneously increased from 43.6 to 45.5%. At shaking flask level, β-1,3-1,4-glucanase activity is 67.9 and 52.3 U ml⁻¹ with barley β-glucan and lichenan as substrate, respectively. At laboratory fermentor level, the secreted protein concentration is approximately 250 mg l⁻¹. The β-1,3-1,4-glucanase activity is 333.7 and 256.7 U ml⁻¹ with barley β-glucan and lichenan as substrate, respectively; however, no activity of this enzyme on cellulose is observed. Compared to the nonoptimized control, expression level of the optimized β-1,3-1,4-glucanase based on preferred codons in P. pastoris shown a 10-fold higher level. The codon-optimized enzyme was approximately 53.8% of the total secreted protein. The optimal acidity and temperature of this recombinant enzyme were pH 6.0 and 45°C, respectively.     

Evaluation of antiprotozoal and antimycobacterial activities of the resin glycosides and the other metabolites of Scrophularia cryptophila

Resin glycosides are secondary metabolites exclusive to the convolvulaceous plants. In this study, crypthophilic acids A–C (1–3), the first resin glycosides occurring in another family (Scrophulariaceae), and the other constituents of Scrophularia cryptophila were examined for in vitro antiprotozoal and antimycobacterial potentials. Except for crypthophilic acid B (2), all tested compounds exhibited growth-inhibitory effect against Trypanosoma brucei rhodesiense, with l-tryptophan (6) and buddlejasaponin III (7) being the most potent ones (IC₅₀'s 4.1 and 9.7 μg/ml). In contrast, the activity towards Trypanosoma cruzi was poor, and only crypthophilic acid C (3), 6 and 7 were trypanocidal at concentrations above 40 μg/ml. With the exception of 2 and 6, all compounds were active against Leishmania donovani. Harpagide (4) and 3 emerged as the best leishmanicidal agents (IC₅₀'s 2.0 and 5.8 μg/ml). Only compounds 3, 6 and 7 showed antimalarial activity against Plasmodium falciparum with IC₅₀ values of 4.2, 16.6 and 22.4 μg/ml. Overall the best and broadest spectrum activity was presented by compounds 3 and 7, as they inhibited all four parasitic protozoa. None of the isolates had significant activity against Mycobacterium tuberculosis (MICs >100 μg/ml) or were toxic towards mammalian (L6) cells. This is the first report of antiprotozoal activity for natural resin glycosides, as well as for harpagide (4), acetylharpagide (5), tryptophan (6) and buddlejasaponin III (7).     

Development and characterization of a potent producer of penicillin G amidase by mutagenization

Summary The penicillin G amidase (PGA) activity of a parent strain of E. coli (PCSIR-102) was enhanced by chemical mutagenization with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). After screening and optimization, a penicillinase deficient mutant (MNNG-37) was isolated and found effective for the production of penicillin G amidase as compared to the parent strain of E. coli (PCSIR-102). Penicillin G amidase activity of MNNG-37 appeared during an early stage of growth, whereas PCSIR-102 did not exhibit PGA activity due to the presence of penicillinase enzyme which inhibits the activity of enzyme PGA. However, MNNG-37 gave a three-fold increase in enzyme activity (231 IU mg⁻¹) as compared to PCSIR-102 (77 IU mg⁻¹) in medium containing 0.15 and 0.1% concentrations of phenylacetic acid, respectively which was added after 6 h of cultivation. The difference in K _m values of the enzyme produced by parent strain PCSIR-102 (0.26 mM) and mutant strain MNNG-37 (0.20 mM) is significant (1.3-fold increase in K _m value) which may show the superiority of the latter in terms of better enzyme properties.     

Production of the <Emphasis Type="Italic">Aspergillus aculeatus</Emphasis> endo-1,4-β-mannanase in <Emphasis Type="Italic">A. niger</Emphasis>

The β-mannanase gene (man1) from Aspergillus aculeatus MRC11624 (Izuka) was patented for application in the coffee industry. For production of the enzyme, the gene was originally cloned and expressed in Saccharomyces cerevisiae. However the level of production was found to be economically unfeasible. Here we report a 13-fold increase in enzyme production through the successful expression of β-mannanase of Aspergillus aculeatus MRC11624 in Aspergillus niger under control of the A. niger glyceraldehyde-3-phosphate dehydrogenase promoter (gpd _P) and the A. awamori glucoamylase terminator (glaA_T). The effect of medium composition on mannanase production was evaluated, and it was found that the glucose concentration and the organic nitrogen source had an effect on both the volumetric enzyme activity and the specific enzyme activity. The highest mannanase activity levels of 16,596 nkat ml⁻¹ and 574 nkat mg⁻¹ dcw were obtained for A. niger D15[man1] when cultivated in a process-viable medium containing corn steep liquor as the organic nitrogen source and high glucose concentrations.     

Effect of lectins from Opuntia ficus indica cladodes and Moringa oleifera seeds on survival of Nasutitermes corniger

Biodegradation by termites is a serious problem for wood and crop industries worldwide, and new environmentally friendly alternatives for termite control have been developed. This work investigated the effects of crude and purified preparations containing lectins from Opuntia ficus indica cladodes (OfiL) and Moringa oleifera seeds (WSMoL and cMoL) on Nasutitermes corniger workers and soldiers. Purified OfiL was more active than cladode extracts, showing a stronger termiticidal activity against workers (LC₅₀ of 0.116 mg ml⁻¹) than against soldiers. OfiL was active against soldiers only at 1.5 mg ml⁻¹. All preparations containing WSMoL and cMoL were active only at concentrations of 1.0 and 1.5 mg ml⁻¹. The tested preparations did not exert repellent activity against N. corniger. OfiL was able to kill workers and therefore is potentially a new tool for N. corniger control; as a consequence, this lectin could disturb organization, structure, and maintenance of termite colonies.     

Cytotoxic and antioxidant triterpene saponins from Butyrospermum parkii (Sapotaceae)

Three new triterpenoid saponins, elucidated as 3-O-β-d-glucopyranosyloleanolic acid 28-O-β-d-xylopyranosyl-(1→4)-α-l-rhamnopyranosyl-(1→2)-β-d-xylopyranoside (parkioside A, 1), 3-O-[β-d-apifuranosyl-(1→3)-β-d-glucopyranosyl]oleanolic acid 28-O-[β-d-apifuranosyl-(1→3)-β-d-xylopyranosyl-(1→4)-[α-l-rhamnopyranosyl-(1→3)]-α-l-rhamnopyranosyl-(1→2)β-d-xylopyranoside (parkioside B, 2) and 3-O-β-d-glucuronopyranosyl-16α-hydroxyprotobassic acid 28-O-α-l-rhamnopyranosyl-(1→3)-β-d-xylopyranosyl-(1→4)-α-l-rhamnopyranosyl-(1→2)-β-d-xylopyranoside (parkioside C, 3), were isolated from the n-BuOH extract of the root bark of Butyrospermum parkii, along with the known 3-O-β-d-glucopyranosyloleanolic acid (androseptoside A). The structures of the isolated compounds were established on the basis of chemical and spectroscopic methods, mainly 1D and 2D NMR data and mass spectrometry. The new compounds were tested for both radical scavenging and cytotoxic activities. Compound 2 showed cytotoxic activity against A375 and T98G cell lines, with IC₅₀ values of 2.74 and 2.93 μM, respectively. Furthermore, it showed an antioxidant activity comparable to that of Trolox or butylated hydroxytoluene (BHT), used as controls, against 2,2-diphenyl-1-picryl hydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), oxygen and nitric oxide radicals.     

Inhibitory effect of extracts from Brazilian medicinal plants on the adhesion of <Emphasis Type="Italic">Candida albicans</Emphasis> to buccal epithelial cells

Plants are known to produce a plethora of secondary metabolites which are recognized as a useful source of new drugs or drug leads. Extracts and fractions of Schinus terebinthifolius Raddi (Anacardiaceae), Piper regnellii C.D.C. (Piperaceae), Rumex acetosa L. (Polygonaceae), and Punica granatum L. (Punicaceae) were assessed for their antifungal activity against eight clinical isolates of C. albicans. They were also evaluated for their effect on the adhesion of these C. albicans isolates to buccal epithelial cells (BECs). The ethyl acetate fraction from the leaves of S. terebinthifolius showed promising activity, inhibiting the growth of three C. albicans isolates at 7.8 μg ml⁻¹ and significantly inhibiting their adhesion to BEC at 15 μg ml⁻¹ . In addition, this fraction did not show cytotoxic activity against murine macrophages. The results show the potential of the plant extracts studied as a source of new antifungal compounds. Further studies are necessary for isolation and characterization of the active compounds of these plants.     

High level expression of a recombinant phospholipase C from <Emphasis Type="Italic">Bacillus cereus</Emphasis> in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Twenty-two Bacillus cereus strains were screened for phospholipase C (PLC, EC 3.1.4.3) activity using p-nitrophenyl phosphorylcholine as a substrate. Two strains (B. cereus SBUG 318 and SBUG 516) showed high activity at elevated temperatures (>70°C) at acidic pH (pH 3.5–6) and were selected for cloning and functional expression using Bacillus subtilis. The genes were amplified from B. cereus DNA using primers based on a known PLC sequence and cloned into the expression vector pMSE3 followed by transformation into B. subtilis WB800. On the amino acid level, one protein (PLC318) was identical to a PLC described from B. cereus, whereas PLC516 contained an amino acid substitution (E173D). PLC production using the recombinant strains was performed by an acetoin-controlled expression system. For PLC516, 13.7 U g⁻¹ wet cell weight was determined in the culture supernatant after 30 h cultivation time. Three purification steps resulted in pure PLC516 with a specific activity of 13,190 U mg⁻¹ protein.