Isolation of a seed coagulant Moringa oleifera lectin

In this work hemagglutinating activity (HA) was investigated in distinct Moringa oleifera tissue extracts. A new lectin from seeds (cMoL) was purified and characterized; hemagglutinating and coagulating activities were evaluated. HA was detected in 0.15 M NaCl extracts from flowers and rachis inflorescence (5%, w/v), seeds, leaves, fundamental tissue of stem and steam bark (10%, w/v). cMoL isolated after saline extraction and guar gel column chromatography was active at pH range 4.0–9.0 agglutinating erythrocytes from rabbit and human blood types. Extracts of tissues and cMoL activities were carbohydrate inhibited; azocasein and asialofetuin abolished cMoL HA. The lectin was thermostable at 100 °C during 7 h. Polyacrylamide gel electrophoresis under reduced conditions revealed a main polypeptide band of 26.5 kDa; native basic cMoL was detected as a unique band. Seed lectin preparations and cMoL showed coagulant activity, similar to aluminium sulphate, the coagulant most widely used in water treatment.     

Screening for hydroxynitrile lyase activity in non-commercialised plants

Hydroxynitrile lyases are used for the synthesis of enantiomerically pure cyanohydrins which are of great importance in the pharmaceutical and fine chemical industries. In this study, the hydroxynitrile lyase activity of 100 plants from 40 families was investigated, first by screening for cyanogenic activity, followed by a hydroxynitrile lyase activity assay. Of the 100 plants, four were found to be cyanogenic and exhibited specific hydroxynitrile lyase activity: Adenia sp. (0.44 U/mg), Adenia firingalavensis (2.88 U/mg), Adenia fruticosa (1.99 U/mg) and, Adenia pechuelii (2.35 U/mg), all from the family Passifloraceae. This is the first report of hydroxynitrile lyase activity in these plants.     

Transformation of maltose into prebiotic isomaltooligosaccharides by a novel α-glucosidase from Xantophyllomyces dendrorhous

The transglycosylation activity of a novel α-glucosidase from the basidiomycetous yeast Xanthophyllomyces dendrorhous (formerly Phaffia rhodozyma) was studied using maltose as glucosyl donor. The enzyme synthesized oligosaccharides with α-(1 → 2), α-(1 → 4) and α-(1 → 6) bonds. Using 200 g/l maltose, the yield of oligosaccharides was 53.8 g/l, with prebiotic oligosaccharides containing at least one α-(1 → 6) linkage (panose, 6-O-α-glucosyl-maltotriose and 6-O-α-isomaltosyl-maltose) being the major products (47.1 g/l). The transglycosylatying yield was 3.6 times higher than the observed with the α-glucosidase from Saccharomyces cerevisiae (53.8 vs. 14.7 g/l). Moreover, when increasing the maltose concentration up to 525 g/l, the maximum production of tri- and tetrasaccharides reached 167.1 g/l, without altering the percentage of oligosaccharides in the mixture. Compared with other microbial α-glucosidases in which the main transglycosylation product is a disaccharide, the enzyme from X. dendrorhous yields a final product enriched in trisaccharides and tetrasaccharides.     

Prevalencia de hiperandrogenismo y síndrome de ovario poliquístico en transexuales de mujer a hombre

IntroductionPrevalence of hyperandrogenism (HA), including the polycystic ovary syndrome (PCOS), in female-to-male transsexuals (FMT) is high. This has been related to metabolic syndrome (MS), which appears to increase cardiovascular morbidity and mortality throughout cross-sex hormone (CSH) therapy.ObjectivesTo assess the prevalence of HA and PCOS in FMT patients before the start of CSH therapy, and their association to MS and its components, insulin resistance (IR) and other cardiovascular risk (CVR) factors.Materials and methodsSeventy-seven FMTs underwent clinical and biochemical assessment for HA before the start of CSH therapy. CVR, IR, and other MS parameters were also assessed.ResultsPrevalence of HA was 49.4% (73.7% were cases of PCOS [Rotterdam criteria]), and prevalence of PCOS in the overall sample was 36.4%. Prevalence of MS was 38.4% and 51.7% according to ATP-III and IDF criteria respectively). MS (according to ATP-III and IDF criteria respectively) was found in 36.8% and 57.9% as compared to 25.6% and 41% of patients with and without HA respectively (p < 0.0001 and P < 0.01 respectively). Of total patients, 54.5% had normal weight (body mass index [BMI] 18.5-24.9 kg.m^-2), 26% were overweight (BMI 25-29.9 kg.m^-2), and 19.5% were obese (BMI ≥ 30 kg.m^-2). After adjusting for BMI, the comparison of hormonal, metabolic, and anthropometric parameters showed statistically significant differences in plasma glucose, HOMA-IR, and abdominal circumference (P < 0.001 for all), as well as HDL cholesterol (HDL) (P = 0.033), but not in total testosterone or calculated free testosterone levels. In the total sample, 27.3% had HDL levels less than 50 mg/dL.ConclusionsOverall HA, and PCOS in particular, are highly prevalent in FMTs. HA and PCOS are related to early development of SM, IR, and other CVR factors with unknown consequences in adulthood.     

Structural characterization of natural ideal 6-O-sulfated agarose from red alga Gloiopeltis furcata

A charge and size uniform polysaccharide GW2M was extracted with cold water from red alga Gloiopeltis furcata and purified by strong anion ion-exchange and gel permeation chromatography. Its chemical structure was identified by methylation, ¹H–¹H COSY, ¹H–¹³C HMQC and ¹H–¹³C HMBC techniques. The experimental data showed that GW2M was composed of galactose (40.3%), 3,6-anhydro-galactose (34.1%) and sulfate (24.8%) with an average molecular mass of 20.6 kDa. The results proved GW2M was a linear repeating sequence of alternating (1 → 3)-linked 6-O-sulfated-β-d-galactose (G6S) and (1 → 4)-linked 3,6-anhydro-α-l-galactose (A) which made it to be an ideal 6-O-sulfated-agarose. The sequences of serial oligosaccharides prepared by mild acid and reductive acid hydrolysis from GW2M were confirmed using electrospray collision induced dissociation tandem mass spectrometry (ES-CID-MS/MS) technique.     

Purification and characterization of a Na+/K+ dependent alginate lyase from turban shell gut Vibrio sp. YKW-34

An alginate lyase with high specific enzyme activity was purified from Vibrio sp. YKW-34, which was newly isolated from turban shell gut. The alginate lyase was purified by in order of ion exchange, hydrophobic and gel filtration chromatographies to homogeneity with a recovery of 7% and a fold of 25. This alginate lyase was composed of a single polypeptide chain with molecular mass of 60 kDa and isoelectric point of 5.5–5.7. The optimal pH and temperature for alginate lyase activity were pH 7.0 and 40 °C, respectively. The alginate lyase was stable over pH 7.0–10.0 and at temperature below 50 °C. The alginate lyase had substrate specificity for both poly-guluronate and poly-mannuronate units. The k_cat/K_m value for alginate (heterotype) was 1.7 × 10⁶ s⁻¹ M⁻¹. The enzyme activity was completely lost by dialysis and restored by addition of Na⁺ or K⁺. The optimal activity exhibited in 0.1 M of Na⁺ or K⁺. This enzyme was resistant to denaturing reagents (SDS and urea), reducing reagents (β-mercaptoethanol and DTT) and chelating reagents (EGTA and EDTA).     

Selective and stable production of physiologically active chitosan oligosaccharides using an enzymatic membrane bioreactor

We investigated the production of chitosan oligosaccharides by continuous hydrolysis of chitosan in an enzyme membrane bioreactor, with the goal of improving the yield of physiologically active oligosaccharides (pentamers and hexamers) and achieving operational stability. The bioreactor was a continuous-flow stirred-tank reactor equipped with an ultrafiltration membrane with a molecular weight cut-off of 2000 Da, and the hydrolysis was accomplished with chitosanase from Bacillus pumilus. After optimization of the reaction parameters, such as the amount of enzyme, the yield of the target oligosaccharides produced in the membrane bioreactor with free chitosanase reached 52% on the basis of the fed concentration of chitosan. An immobilized chitosanase prepared by the multipoint attachment method was used to improve the operational stability of the membrane bioreactor. Under the optimized conditions, pentameric and hexameric chitosan oligosaccharides were steadily produced at 2.3 g/L (46% yield) for a month. The half-life of the productivity of the reactor was estimated to be 50 d under the conditions examined.     

Hyaluronic acid production and molecular weight improvement by redirection of carbon flux towards its biosynthesis pathway

Hyaluronic acid (HA) production in Streptococcus zooepidemicus competes for the carbon source along with biomass formation, lactate formation (via glycolysis) and pentose phosphate pathway (PPP). In our studies, increase in HA molecular weight was observed by redirecting the carbon flux towards HA biosynthesis pathway by partially inhibiting the glycolytic pathway. Batch bioreactor (1.2 L) studies showed that with the addition of 25 μM sodium iodoacetate, 5 g/L tryptophan and 10 g/L pyruvate, which are glycolytic inhibitors, HA molecular weight increased to 3.2, 3.2 and 3.1 MDa respectively compared to control run (2.4 MDa). Yield coefficients Y_HA/S and Y_LA/S showed inverse relationship, indicating competition for glucose between HA and lactic acid formation. Addition of 5 g/L glutamine along with 25 μM sodium iodoacetate also increased the HA concentration to 5.0 g/L from 2.0 g/L in control run. Metabolic flux analysis studies show that concentration and molecular weight of HA is increased by decreasing carbon flux towards glycolysis and PPP and increasing carbon flux towards HA precursor formation. It was observed that specific growth rate of the cells correlated positively to the specific HA production rate and negatively to the molecular weight of HA produced. Addition of antioxidant tannic acid also increased molecular weight to 3.0 MDa.     

Structures of xyloglucans in primary cell walls of gymnosperms,monilophytes (ferns sensu lato) and lycophytes

Little is known about the structures of the xyloglucans in the primary cell walls of vascular plants (tracheophytes) other than angiosperms. Xyloglucan structures were examined in 13 species of gymnosperms, 13 species of monilophytes (ferns sensu lato), and two species of lycophytes. Wall preparations were obtained, extracted with 6 M sodium hydroxide, and the extracts treated with a xyloglucan-specific endo-(1 → 4)-β-glucanase preparation. The oligosaccharides released were analysed by matrix-assisted laser-desorption ionisation time-of-flight mass spectrometry and by high-performance anion-exchange chromatography. The xyloglucan oligosaccharide profiles from the gymnosperm walls were similar to those from the walls of most eudicotyledons and non-commelinid monocotyledons, indicating that the xyloglucans were fucogalactoxyloglucans, containing the fucosylated units XXFG and XLFG. The xyloglucan oligosaccharide profiles for six of the monilophyte species were similar to those of the gymnosperms, indicating they were also fucogalactoxyloglucans. Phylogenetically, these monilophyte species were from both basal and more derived orders. However, the profiles for the other monilophyte species showed various significant differences, including additional oligosaccharides. In three of the species, these additional oligosaccharides contained arabinosyl residues which were most abundant in the profile of Equisetum hyemale. The two species of lycophytes examined, Selaginella kraussiana and Lycopodium cernuum, had quite different xyloglucan oligosaccharide profiles, but neither were fucogalactoxyloglucans. The S. kraussiana profile had abundant oligosaccharides containing arabinosyl residues. The L. cernuum profile indicated the xyloglucan had a very complex structure.     

SEC determination of cross-link efficiency in hyaluronan fillers

The cross-link efficiency in hyaluronan fillers was determined by means of triple detector (RI, LALS-RALS and a Differential Viscometer) GPC/SEC in aqueous buffer. The low water solubility of HA cross-linked with BDDE (HBC) was overcome with an alkaline hydrolysis step. The kinetics of linear HA hydrolysis was investigated at different NaOH concentrations (0.25–0.5–1 M), initial polymer Mws (200–700–1000–1200–1600 kDa) and polymer concentrations (0.1–0.3–0.5 mg/ml). As expected for first-order kinetics, the apparent hydrolysis constant (k_h) was independent of polymer concentration and initial Mws. The k_h was found to be linearly dependent on the NaOH concentration, suggesting a random polymer degradation. A similar behavior was observed for HBC polymers, synthesized with 5–7.5–10–14–18% mol/mol of BDDE for HA repeat unit. The degree of crosslinking was obtained using the Zimm–Stockmayer equation for random, tri-functional polydisperse polymers. The contraction factor (g) was determined after an accurate experimental measurement of the structure factor (?). Comparative studies were performed by ¹H NMR spectroscopy and rheological measurements. Interestingly, the number of effective cross-links found is very low compared to the total BDDE linked; i.e. in a HBC sample only 0.04% of total BDDE linked (equal to 4.6% by ¹H NMR analysis) gives an effective cross-link.     

Effect of blast furnace dust on the degradation of chlorinated organic and endocrine disrupting compounds

This research was to study the effect of blast furnace dust (BFD) as a new reactive material for the degradation of chlorinated organic (COCs) and endocrine disrupting compounds (EDCs). When 100 g/L of BFD was used, the effective degradation of tetrachloroethylene (PCE) was obtained. The cis-DCE (0.93 mM) was dechlorinated to below detection limit within 120 h of reaction. Among various COCs and EDCs, they were degraded at least 90% except for sodium perchlorate. The metabolites of 4-tert-octylphenol (4-t-OP) were identified as 2,4,4-trimethyl-2-pentanol, hydroquinone and 2-tert-octylhydroquinone, respectively. The effective degradation of PCE and 4-t-OP was occurred at range of pH 4–7. A total of 100% of cis-DCE and 86% of 4-t-OP were degraded in fed-batch experiments after 264 h. A solution of highly enriched bacteria completely insolubilized 2.14 mM of Zn eluted from BFD within 168 h of culture. These researches will provide more information related to the application for other contaminated water and wastewater treatments.     

Culture filtrate of root endophytic fungus Piriformospora indica promotes the growth and lignan production of Linum album hairy root cultures

The effect of addition of autoclaved and filter-sterilized culture filtrate of Piriformospora indica (a root endophytic fungus) to the growing Linum album hairy root cultures on growth and lignan production was investigated. The addition resulted in a significant enhancement in lignan production and growth. The podophyllotoxin and 6-methoxypodophyllotoxin (the lignans) concentrations were maximally improved by 3.8 times (233.8 mg/L) and 4.4 times (131.9 mg/L) in comparison to control cultures, respectively, upon addition of 3.0% (v/v) filter-sterilized culture filtrate of P. indica to the hairy root cultures of L. album for exposure time of 48 h. This increase in the lignan content also coincided with the increase in phenylalanine ammonia lyase activity, which was 3.1-fold (371.4 μkat/kg protein) higher compared to control cultures under the same conditions. The maximal increase in hairy root biomass was, however, obtained under different conditions; it was enhanced by 1.4 times (21.8 g/L) in comparison to control cultures, when 2% (v/v) filter-sterilized culture filtrate was in contact with L. album cultures for 96 h.     

Detoxification of aflatoxin B1 by an aqueous extract from leaves of Adhatoda vasica Nees

The effectiveness of aqueous extracts of various medicinal plants in detoxification of aflatoxin B1 (AFB1) was tested in vitro by thin-layer chromatography and enzyme-linked immunosorbent assay (ELISA). Among the different plant extracts, the leaf extract of Vasaka (Adhatoda vasica Nees) showed the maximum degradation of AFB1 (≥98%) after incubation for 24 h at 37 °C. The aflatoxin detoxifying activity of the A. vasica leaf extract was significantly reduced by heating to 100 °C for 10 min or autoclaving at 121 °C for 20 min. Dialysis had no effect on aflatoxin detoxifying ability of A. vasica extract and the dialyzed extract showed similar level of detoxification of AFB1 as that of the untreated extract. A time course study of aflatoxin detoxification by A. vasica extract showed that 69% of the toxin was degraded within 6 h and ≥95% degradation was observed after 24 h of incubation. Detoxification of AFB1 by A. vasica extract was further confirmed by liquid chromatography–mass spectrometry (LC–MS) analysis. Phytochemical analysis revealed the presence of alkaloids in methanolic extract of A. vasica leaves. A partially purified alkaloid from A. vasica leaves by preparative TLC exhibited strong AFB1 detoxification activity.     

Characterization and high-level expression of a metagenome-derived alkaline pectate lyase in recombinant Escherichia coli

Alkaline pectate lyases (PLs) play an important role in mild and eco-friendly bioscouring pretreatment processes in the textile industry. However, to date, only a few PLs can be applied in industrial-scale production, and many of them exhibit high production cost, low activity, and/or do not meet the treatment requirements. In this study, an alkaline PL gene was cloned from the metagenomic DNA of alkaline environment soils. The gene pelB consisted of 1263 nucleotides and encoded a mature protein (PelB) of 399 amino acids, which was expressed in Escherichia coli. The maximum catalytic activity of the enzyme exhibited a bimodal distribution at pH 8.1 and 9.8 and an optimal temperature of 55 °C. The K_m and V_max values of PelB were 1.78 g/L and 1084.8 μmol/(L min) at 45 °C, respectively. Substrate specificity analysis demonstrated the high cleavage capability of PelB on a broad range of substrates of natural methylated pectin. Based on the degradation products, PelB was considered to be an endo-acting lyase. Using high-cell-density cultivation in 7-L bioreactor, the highest PL activity (1816.2 U/mL) was achieved. Thus, the recombinant PelB, with promising properties for use in bioscouring in the textile pretreatment process, should be a potential enzyme for industrial applications.     

A simple noninvasive index to predict significant liver fibrosis in patients with advanced schistosomiasis japonica

BackgroundSchistosoma japonicum causes marked liver fibrosis, while lethal syndromes present in advanced schistosomiasis patients. Its management depends on the degree of fibrosis present.Patients and methodsFifty-two patients were recruited to assess the diagnostic value of bio-markers in patients with advanced schistosomiasis japonica. Fibrosis was assessed in liver biopsies using METAVIR system. The correlation between conventional parameters and significant fibrosis (F2-F4) was assessed using univariate analysis and logistic regression. The method of area under receiver operating characteristic curves (AUROCs) was used as a measurement of diagnostic efficacy.ResultsWhite blood cell counts, platelet counts and albumin (all P < 0.05) were significantly lower, while prothrombin time, international normalized ratio (INR), hyaluronic acid (HA), IV collagen and ultrasound fibrosis scores (all P < 0.01) were significantly elevated in F2-F4 patients compared with F0-F1 patients. HA and INR were identified as independent predictors by multivariate analysis (P = 0.023 and P = 0.013, respectively). Of the routine laboratory tests for the diagnosis of significant fibrosis, HA gave the best AUROC of 0.875 (95% confidence interval (CI): 0.701–0.997). We constructed a new simple index (INR × HA/100) to discriminate between F2-F4 patients and F0-F1 patients. It showed the highest AUROC of 0.921 (95% CI: 0.828-1.000), and had better diagnostic values than APRI and FIB-4.ConclusionHA and INR were reliable markers for differentiating significant liver fibrosis in patients with advanced schistosomiasis japonica. And the new simple index can easily predict significant liver fibrosis with a high degree of accuracy.     

Partial purification of extracellular exo-inulinase from Ulocladium atrum

Eight fungal species were cultivated on the Czapek liquid medium and a good starting extracellular and intracellular exo-inulinase were selected. Extracellular inulinase from Ulocladium atrum was prepared in the presence of 1% inulin source and 0.2% sodium nitrate as the best carbon and nitrogen sources. Incubation for the U. atrum was increased till it reached its maximum (36 U/ml) at the sixth day of incubation at 30 °C which was the best temperature for the production of exo-inulinase. Effect of all metal ions inhibited inulase production by U. atrum. Exo-inulinase was purified by using ammonium sulfate precipitation, ion exchange chromatography on DEAE-cellulose. Three active inulinase forms INI, INII and INIII were resolved, each for DEAE cellulose. The specific activity of INI was 1915 U/mg protein which represented 2.65-fold purification over the crude extract with 42.8% recovery pooling of INI placed on CM cellulose chromatography and INI was resolved into INIa, INIb and INIc. The specific activity of INIa was 2479.2 U/mg protein which represented 3.43-fold purification over the crude extract with 24.2% recovery.     

Covalent immobilization of hydroperoxide lyase on chitosan hybrid hydrogels and production of C6 aldehydes by immobilized enzyme

Five kinds of spacer arm attached chitosan hybrid hydrogels were tested for the possibility of being used as carriers for the immobilization of hydroperoxide lyase from amaranthus tricolor leaves. The 1,6-hexamethylenediamine attached chitosan-κ-carrageenan with biomimetic hydrophobic surface was proved to be the most suitable carrier. A maximum activity of 7.49 ± 0.19 U/g and a yield of 95% were obtained under optimized coupling condition. Meanwhile, the affinity between enzyme and substrates was not reduced after immobilization, as evidenced by the fact that the K_m value of hydroperoxide lyase decreased from 108.6 to 79.97 μM for 13-hydroperoxy-linoleic-acid and almost unchanged for 13-hydroperoxy-linolenic-acid. Furthermore, the thermal, operational and storage stabilities of HPL were significantly improved after immobilization. Using the immobilized enzyme as the catalyst, the yield of 2(E)-hexenal and hexanal reached 1374.8 ± 51.8 mg/L and 1987.9 ± 67.9 mg/L, respectively, and the amount of immobilized enzyme needed in the reaction mixture was much lower than its free counterpart.     

An extremely alkaline novel chitinase from Streptomyces sp. CS495

An extremely alkaline chitinase from Streptomyces sp. CS495 was isolated from a Korean soil sample, purified by single-step chromatography, and biochemically characterized. The extracellular chitinase was purified 7.0 fold with a 33.9% yield by Sepharose Cl-6B column. The molecular mass of the enzyme (Ch495) was approximately 41 kDa. Ch495 was found to be stable over a broad pH range (5–12.5) and to 50 °C and have an optimum temperature of 60 °C. Ch495 had K_m and V_max values of 1.34 ± 2.9 mg/mL and 889 ± 3.6 mmol/min, respectively using different concentrations of colloidal chitin. N-terminal sequence of Ch495 was APREKINLLYFLGYF. HPLC and TLC analysis of Ch495 shows the production of produced N-acetyl d-glucosamine (GlcNAc) as minor and diacetylchitobiose (GlcNAc)₂ as major products. Ch495 shows antifungal activity against Fusarium solani and Aspergillus brasiliensis which can be used for the biological control of fungus. As being simple in purification, extreme alkalophilic, stable in broad range of pH, ability to produce oligosaccharides, and antifungal activity shows that Ch495 has potential applications in industries as for chitooligosaccharides production used as medical prebiotics or/and for the biological control of plant pathogens in agriculture.     

Lowering induction temperature for enhanced production of polygalacturonate lyase in recombinant Pichia pastoris

Various effects of temperature on heterologous alkaline polygalacturonate lyase produced in recombinant Pichia pastoris were investigated. The results indicated that PGL activity could be improved significantly by decreasing the cultivation temperature. It was reached 931 U/mL with temperature lowered to 22 °C at the beginning of induction phase, which were 2.1-fold and 2.9-fold increase compared to that at 30 and 26 °C. The mechanisms behind the temperature effect on recombinant PGL production may be ascribed to poor cell viability, decrease of intracellular adenosine phosphate levels, of AOX activity but increase of extracellular proteases activities. Our study demonstrated that cultivation at lower temperatures resulted in higher cell viability, significant improvement of PGL stability and an increase intracellular AOX activity, but a lower activity of released host proteases which possibly caused the degradation of recombinant PGL. In addition, the evidence of higher intracellular adenosine phosphate levels but lower energy charge level was provided at a lower temperature induction.     

Metabolic engineering of Saccharomyces cerevisiae to improve 1-hexadecanol production

Fatty alcohols are important components of a vast array of surfactants, lubricants, detergents, pharmaceuticals and cosmetics. We have engineered Saccharomyces cerevisiae to produce 1-hexadecanol by expressing a fatty acyl-CoA reductase (FAR) from barn owl (Tyto alba). In order to improve fatty alcohol production, we have manipulated both the structural genes and the regulatory genes in yeast lipid metabolism. The acetyl-CoA carboxylase gene (ACC1) was over-expressed, which improved 1-hexadecanol production by 56% (from 45 mg/L to 71 mg/L). Knocking out the negative regulator of the INO1 gene in phospholipid metabolism, RPD3, further enhanced 1-hexadecanol production by 98% (from 71 mg/L to 140 mg/L). The cytosolic acetyl-CoA supply was next engineered by expressing a heterologous ATP-dependent citrate lyase, which increased the production of 1-hexadecanol by an additional 136% (from 140 mg/L to 330 mg/L). Through fed-batch fermentation using resting cells, over 1.1 g/L 1-hexadecanol can be produced in glucose minimal medium, which represents the highest titer reported in yeast to date.