Biosynthesis of selenium-containing polysaccharides with antioxidant activity in liquid culture of Hericium erinaceum

The effects of inorganic selenium (sodium selenite) and Selol containing selenitetriglycerides synthesized from sunflower oil on mycelial growth and selenium-containing extracellular (EPS) and intracellular (IPS) polysaccharides production were examined in shake flask cultures of Hericium erinaceum.Unlike sodium selenite which inhibited mycelial growth, Selol increased in biomass production in a dose-dependent manner. Selol also dramatically enhanced EPS formation to 2.25 g/L which is 2.5–fold higher than in the control. Selenium content in EPS and IPS obtained from Selol-enriched medium reached a maximum of 4.89 and 4.69 mg/g, respectively.The in vitro antioxidant activities of polysaccharides were evaluated by reducing power, inhibition of lipid peroxidation, and 1,1-diphenyl-dipicrylhydrazyl radicals scavenging assays. The selenium-containing EPS showed an excellent antioxidant activity correlated well with increasing concentrations.The results suggested that selenium-containing EPS from H. erinaceum submerged culture should be explored as a novel selenium source in dietary supplements, with potent antioxidant properties.     

Stimulatory effects of Coix lacryma-jobi oil on the mycelial growth and metabolites biosynthesis by the submerged culture of Ganoderma lucidum

Effects of Coix lacryma-jobi oil (CLO) addition on the mycelia growth and production of bioactive metabolites, such as triterpenoids, exopolysaccharide (EPS), and intracellular polysaccharide (IPS) in the submerged culture of Ganoderma lucidum were studied. The results showed that when a level of 2% CLO was added at the beginning of culture, the biomass, triterpenoids, EPS, and IPS productions reached a maximum of 10.71 g/L, 92.94 mg/L, 0.33 g/L, and 0.389 g/L, respectively, that were 3.34-fold, 2.76-fold, 2.2-fold, and 2.23-fold compared to that of control. Analysis of fermentation kinetics of G. lucidum suggested that glucose concentration in the culture of CLO-added group decreased more quickly as compared to the control group from day 2 to day 7 of fermentation process, while the triterpenoids and polysaccharides biosynthesis were promoted at the same culture period. However, the culture pH profile was not affected by the addition of CLO. There were no new components in the two types of polysaccharides obtained by the addition of CLO. Enzyme activities analysis indicated CLO or its fatty acids affected the synthesis level of phosphoglucose isomerase and α-phosphoglucomutase at different stage.     

Production of β-fructofuranosidases by Aspergillus niveus using agroindustrial residues as carbon sources: Characterization of an intracellular enzyme accumulated in the presence of glucose

The production of β-fructofuranosidases by Aspergillus niveus, cultivated under submerged fermentation using agroindustrial residues, was investigated. The highest productivity of β-fructofuranosidases was obtained in Khanna medium supplemented with sugar cane bagasse as carbon source. Glucose enhanced the production of the intracellular enzyme, whereas that of the extracellular one was decreased. The intracellular β-fructofuranosidase was a trimeric protein of approximately 141 kDa (gel filtration) with 53.5% carbohydrate content, composed of 57 kDa monomers (SDS-PAGE). The optimum temperature and optimum pH were 60 °C and 4.5, respectively. The purified enzyme showed good thermal stability and exhibited a half-life of 53 min at 60 °C. β-Fructofuranosidase activity was slightly activated by Cu²⁺, Mn²⁺, Mg²⁺, and Na⁺ at 1 mM concentration. The enzyme hydrolyzed sucrose, raffinose, and inulin, with K_d values of 5.78 mM, 5.74 mM, and 1.74 mM, respectively.     

Magnesium uptake of Arabidopsis transporters,AtMRS2-10 and AtMRS2-11, expressed in Escherichia coli mutants: Complementation and growth inhibition by aluminum

Magnesium (Mg^2 +) plays a critical role in many physiological processes. Mg^2 + transport systems in Salmonella have been well documented, but those in Escherichia coli have not been fully elucidated. We examined the effects of corA, mgtA, yhiD and corC gene deletion on Mg^2 + transport in E. coli. We obtained every combination of double, triple and quadruple mutants. The corA and mgtA double mutant required addition of 10 mM Mg^2 + to Luria-Bertani (LB) medium for growth, and the corA, mgtA and yhiD triple mutant TM2 required a higher Mg^2 + concentration. The Mg^2 + requirement of the quadruple mutant was similar to that of TM2. The results demonstrated that either CorA or MgtA is necessary for normal E. coli growth in LB medium and that YhiD plays a role in Mg^2 + transport under high Mg^2 + growth conditions in E. coli. The Arabidopsis Mg^2 + transporters, AtMRS2-10 and AtMRS2-11, were heterologously expressed in TM2 cells. TM2 cells expressing AtMRS2-10 and AtMRS2-11 could grow in LB medium that had been supplemented with 1 mM Mg^2 + and without Mg^2 + supplementation, respectively, and cell growth was inhibited by 2 mM AlCl₃. The results indicated that the growth of TM2 expressing AtMRS2-10 and AtMRS2-11 reflected these AtMRS2 function for Mg^2 + and aluminum. The E. coli TM2 cells are useful for functional analysis of Arabidopsis MRS2 proteins.     

Salt effects on β-glucosidase: pH-profile narrowing

Salts inhibit the activity of sweet almond β-glucosidase. For cations (Cl⁻ salts) the effectiveness follows the series: Cu⁺², Fe⁺² > Zn⁺² > Li⁺ > Ca⁺² > Mg⁺² > Cs⁺ > NH₄⁺ > Rb⁺ > K⁺ > Na⁺ and for anions (Na⁺ salts) the series is: I⁻ > ClO₄⁻ > ⁻SCN > Br⁻ ≈ NO₃⁻ > Cl⁻ ≈ ⁻OAc > F⁻ ≈ SO₄^− 2. The activity of the enzyme, like that of most glycohydrolases, depends on a deprotonated carboxylate (nucleophile) and a protonated carboxylic acid for optimal activity. The resulting pH-profile of k_cat/K_m for the β-glucosidase-catalyzed hydrolysis of p-nitrophenyl glucoside is characterized by a width at half height that is strongly sensitive to the nature and concentration of the salt. Most of the inhibition is due to a shift in the enzymic pK_as and not to an effect on the pH-independent second-order rate constant, (k_cat/K_m)^lim. For example, as the NaCl concentration is increased from 0.01 M to 1.0 M the apparent pK_a1 increases (from 3.7 to 4.9) and the apparent pK_a2 decreases (from 7.2 to 5.9). With p-nitrophenyl glucoside, the value of the pH-independent (k_cat/K_m)^lim (= 9 × 10⁴ M^− 1 s^− 1) is reduced by less than 4% as the NaCl concentration is increased. There is a similar shift in the pK_as when the LiCl concentration is increased to 1.0 M. The results of these salt-induced pK_a shifts rule out a significant contribution of reverse protonation to the catalytic efficiency of the enzyme. At low salt concentration, the fraction of the catalytically active monoprotonated enzyme in the reverse protonated form (i.e., proton on the group with a pK_a of 3.7 and dissociated from the group with a pK_a of 7.2) is very small (≈ 0.03%). At higher salt concentrations, where the two pK_as become closer, the fraction of the monoprotonated enzyme in the reverse protonated form increases over 300-fold. However, there is no increase in the intrinsic reactivity, (k_cat/K_m)^lim, of the monoprotonated species. For other enzymes which may show such salt-induced pK_a shifts, this provides a convenient test for the role of reverse protonation.     

Using Taguchi experimental design methods to optimize trace element composition for enhanced surfactin production by Bacillus subtilis ATCC 21332

In this work, optimizing trace element composition was attempted as a primary strategy to improve surfactin production from Bacillus subtilis ATCC 21332. Statistical experimental design (Taguchi method) was applied for the purpose of identifying optimal trace element composition in the medium. Of the five trace elements examined, Mg²⁺, K⁺, Mn²⁺, and Fe²⁺ were found to be more significant factors affecting surfactin production by the B. subtilis strain. In the absence of Mg²⁺ or K⁺, surfactin yield decreased to 0.4 g/l, which was only 25% of the value obtained from the control run. When Mn²⁺ and Fe²⁺ were both absent, the production yield also dropped to ca. 0.6 g/l, approximately one-third of the control value. However, when only one of the two metal ions (Fe²⁺ or Mn²⁺) was missing, the B. subtilis ATCC 21332 strain was able to remain over 80% of original surfactin productivity, suggesting that some interactive correlations among the selected metal ions may involve. Taguchi method was thus applied to reveal the interactive effects of Mg²⁺, K⁺, Mn²⁺, Fe²⁺ on surfactin production. The results show that interaction of Mg²⁺ and K⁺ reached significant level. By further optimizing Mg²⁺ and K⁺ concentrations in the medium, the surfactin production was boosted to 3.34 g/l, which nearly doubled the yield obtained from the original control.     

Proton production from nitrogen transformation drives stream export of base cations in acid-sensitive forested watersheds

The biogeochemical cycles of nitrogen (N) and base cations (BCs), (i.e., K⁺, Na⁺, Ca²⁺, and Mg²⁺), play critical roles in plant nutrition and ecosystem function. Empirical correlations between large experimental N fertilizer additions to forest ecosystems and increased BCs loss in stream water are well demonstrated, but the mechanisms driving this coupling remain poorly understood. We hypothesized that protons generated through N transformation (PPR_N)—quantified as the balance of NH₄⁺ (H⁺ source) and NO₃⁻ (H⁺ sink) in precipitation versus the stream output will impact BCs loss in acid-sensitive ecosystems. To test this hypothesis, we monitored precipitation input and stream export of inorganic N and BCs for three years in an acid-sensitive forested watershed in a granite area of subtropical China. We found the precipitation input of inorganic N (17.71 kg N ha⁻¹ year⁻¹ with 54% as NH₄⁺–N) was considerably higher than stream exported inorganic N (5.99 kg N ha⁻¹ year⁻¹ with 83% as NO₃⁻–N), making the watershed a net N sink. The stream export of BCs (151, 1518, 851, and 252 mol ha⁻¹ year⁻¹ for K⁺, Na⁺, Ca²⁺, and Mg²⁺, respectively) was positively correlated (r = 0.80, 0.90, 0.84, and 0.84 for K⁺, Na⁺, Ca²⁺, and Mg²⁺ on a monthly scale, respectively, P < 0.001, n = 36) with PPR_N (389 mol ha⁻¹ year⁻¹) over the three years, suggesting that PPR_N drives loss of BCs in the acid-sensitive ecosystem. A global meta-analysis of 15 watershed studies from non-calcareous ecosystems further supports this hypothesis by showing a similarly strong correlation between ∑BCs output and PPR_N (r = 0.89, P < 0.001, n = 15), in spite of the pronounced differences in environmental settings. Collectively, our results suggest that N transformations rather than anions (NO₃⁻ and/or SO₄²⁻) leaching specifically, are an important mediator of BCs loss in acid-senstive ecosystems. Our study provides the first definitive evidence that the chronic N deposition and subsequent transformation within the watershed drive stream export of BCs through proton production in acid-sensitive ecosystems, irrespective of their current relatively high N retention. Our findings suggest the N-transformation-based proton production can be used as an indicator of watershed outflow quality in the acid-sensitive ecosystems.     

Cloning,overexpression, purification,and site-directed mutagenesis of xylitol-2-dehydrogenase from Candida albicans

Xylitol-2-dehydrogenase from Candida albicans was cloned and overexpressed in Escherichia coli. The purified recombinant XDH has an apparent molecular weight of 40 kDa which belongs to the medium chain alcohol dehydrogenase family and exclusively uses NAD⁺ as a cofactor. The recombinant caXDH has a K_M of 8.8 mM and 37.7 μM using the substrate xylitol and NAD⁺, respectively, and its catalytic efficiency is 53,200 min^?1 mM^?1. Following site-directed mutagenesis, one of the engineered caXDHs with six mutations at Ser95Cys, Ser98Cys, Tyr101Cys, Asp206Ala, Ile207Arg, and Phe208Ser shifted its cofactor dependence from NAD⁺ to NADP⁺ in which the K_M and k_cat/K_M towards NADP⁺ are 119 μM and 26,200 min^?1 mM^?1, respectively.     

Fluoroquinolones stimulate the DNA cleavage activity of topoisomerase IV by promoting the binding of Mg2 + to the second metal binding site

BackgroundFluoroquinolones target bacterial type IIA topoisomerases, DNA gyrase and topoisomerase IV (Topo IV). Fluoroquinolones trap a topoisomerase–DNA covalent complex as a topoisomerase–fluoroquinolone–DNA ternary complex and ternary complex formation is critical for their cytotoxicity. A divalent metal ion is required for type IIA topoisomerase-catalyzed strand breakage and religation reactions. Recent studies have suggested that type IIA topoisomerases use two metal ions, one structural and one catalytic, to carry out the strand breakage reaction.MethodsWe conducted a series of DNA cleavage assays to examine the effects of fluoroquinolones and quinazolinediones on Mg^2 +-, Mn^2 +-, or Ca^2 +-supported DNA cleavage activity of Escherichia coli Topo IV.ResultsIn the absence of any drug, 20–30 mM Mg^2 + was required for the maximum levels of the DNA cleavage activity of Topo IV, whereas approximately 1 mM of either Mn^2 + or Ca^2 + was sufficient to support the maximum levels of the DNA cleavage activity of Topo IV. Fluoroquinolones promoted the Topo IV-catalyzed strand breakage reaction at low Mg^2 + concentrations where Topo IV alone could not efficiently cleave DNA.Conclusions and general significanceAt low Mg^2 + concentrations, fluoroquinolones may stimulate the Topo IV-catalyzed strand breakage reaction by promoting Mg^2 + binding to metal binding site B through the structural distortion in DNA. As Mg^2 + concentration increases, fluoroquinolones may inhibit the religation reaction by either stabilizing Mg^2 + at site B or inhibition the binding of Mg^2 + to site A. This study provides a molecular basis of how fluoroquinolones stimulate the Topo IV-catalyzed strand breakage reaction by modulating Mg^2 + binding.     

Fractionation,characterization and antioxidant activity of exopolysaccharides from fermentation broth of a Cordyceps sinensis fungus

Exopolysaccharide (EPS) fractions P_1/5, P_2/5, P₁, P₂ and P₅ were isolated from the fermentation medium of a medicinal fungus Cordyceps sinensis by gradient precipitation with ethanol at 1/5, 2/5, 1, 2, and 5 volume ratios to the liquid medium. P_1/5 and P_2/5 were mainly composed of polysaccharides with negligible protein content and a large molecular or particle size (intrinsic viscosity [η] 2025 mL/g; hydrodynamic radius R_h 905 nm). The fraction attained at a higher ethanol volume ratio had a much higher protein and lower carbohydrate content, and a smaller molecular size. In particular, P₅ was composed of mainly protein with an average molecular weight 16 kDa, [η] 4.3 mL/g and R_h 23.5 nm. The antioxidant activities of EPS fractions showed a significant dependence on the protein content, being negligible of P_1/5 and P_2/5, low to moderate of P₁ and P₂, and very strong of P₅. Gradient ethanol precipitation was proven a simple and workable method for initial fractionation of polysaccharides, proteins and their complexes with different molecular sizes and for further identification of the bioactive components.     

Influence of ionic interactions on essential oil and phenolic diterpene composition of Dalmatian sage (Salvia officinalis L.)

The potential of four essential cations (K⁺, Ca²⁺, Mg²⁺ and Fe²⁺) to alleviate salt toxicity was studied in sage (Salvia officinalis L.) plants grown in pots. Two concentrations of the following chloride salts: KCl, CaCl₂, MgCl₂ and FeCl_3, were used together with 100 mM NaCl to study the effects of these nutrients on plant growth, leaf essential oils (EOs) and phenolic diterpenes composition. The sage plants accumulated Na⁺ in their leaves (includers); this has affected secondary metabolites’ biosynthesis. Treatment with 100 mM NaCl slightly decreased borneol and viridiflorol, while increased manool concentrations. Addition of KCl, CaCl₂ and MgCl₂ increased considerably in a dose-dependent manner the oxygen-containing monoterpenes (1.8-cineole, camphor, β-thujone and borneol) in 100 mM NaCl-treated sage. Whereas, the contents of viridiflorol decreased further with the addition of KCl in 100 mM NaCl-treated sage. Our results suggest that the changes in EOs composition were more related to K⁺ and Ca²⁺ availability than to Na⁺ toxicity. Furthermore, treatment with NaCl decreased by 50% carnosic acid (CA), a potent antioxidant, content in the leaves. K⁺ and Ca²⁺ promoted the accumulation of CA and its methoxylated form (MCA) in the leaves. The concentration of CA was positively correlated with leaf K⁺ (r = 0.56, P = 0.01) and Ca²⁺ (r = 0.44, P = 0.05) contents. It appears that different salt applications in combination with NaCl treatments had a profound effect on EOs and phenolic diterpene composition in sage. Therefore, ionic interactions may be carefully considered in the cultivation of this species to get the desired concentrations of these secondary metabolites in leaf extracts.     

Production of bioactive polysaccharides by Inonotus obliquus under submerged fermentation supplemented with lignocellulosic biomass and their antioxidant activity

The effect of lignocellulose degradation in wheat straw, rice straw, and sugarcane bagasse on the accumulation and antioxidant activity of extra- (EPS) and intracellular polysaccharides (IPS) of Inonotus obliquus under submerged fermentation were first evaluated. The wheat straw, rice straw, and sugarcane bagasse increased the EPS accumulation by 91.4, 78.6, and 74.3 % compared with control, respectively. The EPS and IPS extracts from the three lignocellulose media had significantly higher hydroxyl radical- and 2,2-diphenyl-1-picrylhydrazyl radical-scavenging activity than those from the control medium. Of the three materials, wheat straw was the most effective lignocellulose in enhancing the mycelia growth, accumulation and antioxidant activity of I. obliquus polysaccharides (PS). The carbohydrate and protein content, as well as the monosaccharide compositions of the EPS and IPS extracts, were correlated with sugar compositions and dynamic contents during fermentation of individual lignocellulosic materials. The enhanced accumulation of bioactive PS of cultured I. obliquus supplemented with rice straw, wheat straw, and bagasse was evident.     

Effects of chronic exposure to ammonia concentrations on brain monoamines and ATPases of Nile tilapia (Oreochromis niloticus)

The effects of chronic exposure to total ammonia nitrogen (TAN) concentrations on the brain monoamines and ATPases of Nile tilapia, Oreochromis niloticus fingerlings, were studied. The period of exposure was 70 consecutive days, and the initial weight of the fingerlings was 18 ± 2.1 g. In addition to the control, three treatment groups exposed to 2.5 (low), 5 (medium), and 10 (high) mg TAN L^?1 concentrations were tested. The unionized ammonia nitrogen (NH₃) levels calculated in mg L^?1 were 0.059, 0.185, and 0.575 in aquaria at 26 °C. The brain monoamines were serotonin (5-HT), dopamine (DA), and norepinephrine (NE), as well as their derivatives, 5-hydroxyindoleacetic acid (5-HIAA) and dihydroxyphenylacetic acid (DOPAC). Compared with the controls, the levels of brain monoamines and Na⁺/K⁺- and Ca²⁺-ATPase activities were not significantly altered in fish exposed to low TAN concentration. However, there was a significant decrease in 5-HT, DA, and NE levels, and a significant increase in both serotonergic (5-HIAA/5-HT) and dopaminergic (DOPAC/DA) activities of fish exposed to medium TAN and high TAN concentrations. The activities of brain Na⁺/K⁺- and Ca²⁺-ATPases of fish exposed to medium TAN and high TAN concentrations significantly increased, while Mg²⁺-ATPase did not significantly change compared with that of the controls. The quantity of the detected alterations increased in fish exposed to high TAN concentration.     

Structural basis for the magnesium-dependent activation of transketolase from Chlamydomonas reinhardtii

BackgroundIn photosynthetic organisms, transketolase (TK) is involved in the Calvin-Benson cycle and participates to the regeneration of ribulose-5-phosphate. Previous studies demonstrated that TK catalysis is strictly dependent on thiamine pyrophosphate (TPP) and divalent ions such as Mg^2 +.MethodsTK from the unicellular green alga Chlamydomonas reinhardtii (CrTK) was recombinantly produced and purified to homogeneity. Biochemical properties of the CrTK enzyme were delineated by activity assays and its structural features determined by CD analysis and X-ray crystallography.ResultsCrTK is homodimeric and its catalysis depends on the reconstitution of the holo-enzyme in the presence of both TPP and Mg^2 +. Activity measurements and CD analysis revealed that the formation of fully active holo-CrTK is Mg^2 +-dependent and proceeds with a slow kinetics. The 3D–structure of CrTK without cofactors (CrTK_apo) shows that two portions of the active site are flexible and disordered while they adopt an ordered conformation in the holo-form. Oxidative treatments revealed that Mg^2 + participates in the redox control of CrTK by changing its propensity to be inactivated by oxidation. Indeed, the activity of holo-form is unaffected by oxidation whereas CrTK in the apo-form or reconstituted with the sole TPP show a strong sensitivity to oxidative inactivation.ConclusionThese evidences indicate that Mg^2 + is fundamental to allow gradual conformational arrangements suited for optimal catalysis. Moreover, Mg^2 + is involved in the control of redox sensitivity of CrTK.General significanceThe importance of Mg^2 + in the functionality and redox sensitivity of CrTK is correlated to light-dependent fluctuations of Mg^2 + in chloroplasts.     

Purification and characterization of a thermostable α-galactosidase with transglycosylation activity from Aspergillus parasiticus MTCC-2796

An extracellular thermostable α-galactosidase from Aspergillus parasiticus MTCC-2796 was purified 16.59-fold by precipitation with acetone, followed by sequential column chromatography with DEAE-Sephadex A-50 and Sephadex G-100. The purified enzyme was homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). It was found to be a monomeric protein with a molecular weight of about 67.5 kDa. The purified enzyme showed optimum activity against o-nitrophenyl-α-d-galactopyranoside (oNPG) at pH 5.0 and a temperature of 50 °C. The enzyme was thermostable, showing complete activity even after heating at 65 °C for 30 min. The enzyme showed strict substrate specificity for α-galactosides and hydrolyzed oNPG (K_m = 0.83 mM), melibiose (K_m = 2.48 mM) and raffinose (K_m = 5.83 mM). Among metal ions and reagents tested, Ca²⁺ and K⁺ enhanced the enzymatic activity, but Mg²⁺, Mn²⁺, ethylenediaminetetraacetic acid (EDTA) and 2-mercaptoethanol showed no effect, while Ag⁺, Hg²⁺ and Co²⁺ strongly inhibited the activity of the enzyme. The enzyme catalyzed the transglycosylation reaction for the synthesis of melibiose.     

Fed-batch fermentation of Tuber melanosporum for the hyperproduction of mycelia and bioactive Tuber polysaccharides

For the first time, a fed-batch fermentation process of Tuber melanosporum was developed for the efficient production of bioactive mycelia and Tuber polysaccharides. Each 1.67 g/L of peptone and 8.33 g/L of yeast extract were added on day 3, 6, and 9, respectively, and sucrose was fed to maintain its concentration around 35–5 g/L when its residual level decreased to 10–5 g/L. Then, the maximal biomass, the production of extracellular polysaccharides (EPS) and intracellular polysaccharides (IPS) reached 53.72 ± 2.57 g DW/L, 7.09 ± 0.62 and 4.43 ± 0.21 g/L, respectively. Compared with the batch culture conducted in the enriched medium, the biomass, the production of EPS and IPS were enhanced by 55.8%, 222.3% and 103.2%, respectively. Not only the cell density but also the production of EPS and IPS were the highest ever reported in truffle fermentation, and the biomass was also the highest as ever reported in mushroom fermentation.     

Citric acid production by recycling its wastewater treated with anaerobic digestion and nanofiltration

We have developed an integrated citric acid-methane fermentation process to solve the problem of wastewater pollution in the citric acid industry. Citric acid wastewater was initially treated by anaerobic digestion. After subsequent ultrafiltration and nanofiltration, the anaerobic digestion effluent (ADE) could be recycled as process water for the next fermentation, maintaining excellent citric acid production efficiency while eliminating wastewater discharge and reducing water consumption. Untreated ADE was not suitable for direct recycling. The effects on citric acid fermentation of components in the ADE were investigated. Production was inhibited when Na⁺ and Mg²⁺ concentrations in recycled ADE were >200 mg/L and >40 mg/L, respectively. This problem was resolved by treating the ADE using ultrafiltration and nanofiltration to reduce Na⁺ and Mg²⁺ concentrations to acceptable levels. Our results prove the technical feasibility of cleaner production in the citric acid industry and provide a strategy for management of wastewater in other submerged fermentation industries.     

Production,characterization and application of a keratinase from Chryseobacterium L99 sp. nov.

Keratins are important bioresources for apparels and feedstuffs, but recalcitrant to common enzymes. Now, it is popular and essential to develop keratinolytic enzymes for environmental prevention and improvement of keratin product quality. In the study, the medium optimization, purification, characterization and application of the keratinase from a newly isolated Chryseobacterium L99 sp. nov. were conducted. Exogenous sucrose, malt sugar, glucose, starch, tryptone, Mg²⁺, Zn²⁺, Ca²⁺ and Cu²⁺ could promote the keratinase production, while exogenous urea, NH₄Cl and yeast extract exhibited strong inhibition effects. Response surface methodology predicted a maximum keratinase yield of 213.8 U mL⁻¹, at (g L⁻¹) sucrose 16.8, MgCl₂·6H₂O 1.9, feather keratin 40.0, NaH₂PO₄·2H₂O 6.0 and K₂HPO₄·6H₂O 1.0, where dry cell weight nearly had a minimum 8.58 g L⁻¹. Then, a serine keratinase about 33 kDa was purified, and its optimal activity was acquired at 40 °C and pH 8.0 with K⁺, Zn²⁺or Co²⁺. Compared with Savinase 16 L and transglutaminase, the L99 keratinase could efficient prevent shrinkage and eliminate directional frictional effect of wool, indicating it as a promising prospect in the biotreatment of wool fibres.     

Cerebral vasodilator properties of Danshen and Gegen: A study of their combined efficacy and mechanisms of actions

Danshen and Gegen are two commonly used Chinese herbal medicines for treatment of cardiovascular diseases. The aim of the present study was to elucidate the combination effects of these two herbs on cerebral vascular tone and their underlying mechanisms of actions. Basilar artery rings were obtained from rats and precontracted with U46619. Cumulative administrations of aqueous extracts of Danshen, Gegen, or the two herbs combined (DG; ratio 7:3) produced concentration-dependent relaxation of the artery rings. Statistical analysis on these findings produced a combination index (CI) of 1.041 at ED₅₀, which indicates the two herbs produced additive vasodilator effects when used as a combined decoction. Removal of the endothelium had no effect on the vasodilator properties of Danshen, Gegen, and DG. However, their maximum effects (Imax) were significantly blunted by a K_ATP channel inhibitor glibenclamide, a non-selective K⁺ channel inhibitor tetraethylammonium (TEA), and by a combination of K⁺ channel inhibitors (glibenclamide + TEA + iberiotoxin + 4-aminopyridine + barium chloride). In addition, Danshen, Gegen, and DG produced augmentation of K_ATP currents and inhibited Ca²⁺ influx in vascular smooth muscle cells isolated from rat basilar arteries. Furthermore, these agents inhibited CaCl₂-induced contraction in the artery rings. In conclusion, the present study showed that Danshen and Gegen produced additive vasodilator effects on rat cerebral basilar arteries. These effects were independent of endothelium-derived relaxant factors (EDRF), but required the opening of K_ATP channels and inhibition of Ca²⁺ influx in the vascular smooth muscle cells. It is suspected that the cerebral vasodilator effects of Danshen and Gegen produced either on their own or in combination, can help patients with obstructive cerebrovascular diseases.     

Differential tolerance to combined salinity and O2 deficiency in the halophytic grasses Puccinellia ciliata and Thinopyrum ponticum: The importance of K+ retention in roots

Saline environments of terrestrial halophytes are often prone to waterlogging, yet the effects on halophytes of combined salinity and waterlogging have rarely been studied. Either salinity or hypoxia (low O₂) alone can interfere with K⁺ homeostasis, therefore the combination of salinity or hypoxia is expected to impact significantly on K⁺ retention in roots. We studied mechanisms of tolerance to the interaction of salinity with hypoxia in Puccinellia ciliata and Thinopyrum ponticum, halophytic grasses that differ in waterlogging tolerance. Plants were exposed to aerated and stagnant saline (250 mM NaCl) treatments with low (0.25 mM) and high (4 mM) K⁺ levels; growth, net ion fluxes and tissue ion concentrations were determined. P. ciliata was more tolerant than T. ponticum to stagnant-saline treatment, producing twice the biomass of adventitious roots, which accumulated high levels of Na⁺, and had lower shoot Na⁺. After 24 h of saline hypoxic treatment, MIFE measurements revealed a net uptake of K⁺ (∼40 nmol m⁻² s⁻¹) for P. ciliata, but a net loss of K⁺ (∼20 nmol m⁻² s⁻¹) for the more waterlogging sensitive T. ponticum. NaCl alone induced K⁺ efflux from roots of both species, with channel blocker tests implicating GORK-like channels. P. ciliata had constitutively a more negative root cell membrane potential than T. ponticum (−150 versus −115 mV). Tolerance to salinity and hypoxia in P. ciliata is related to increased production of adventitious roots, regulation of shoot K⁺/Na⁺, and a superior ability to maintain negative membrane potential in root cells, resulting in greater retention of K⁺.