A novel fibrinolytic protease from Streptomyces sp. CS684

A fibrinolytic protease (FP84) was purified from Streptomyces sp. CS684, with the aim of isolating economically viable enzyme from a microbial source. SDS-PAGE and fibrin zymography of the purified enzyme showed a single protein band of approximately 35 kDa. Maximal activity was at 45 °C and pH 7–8, and the enzyme was stable between pH 6 and 9 and below 40 °C. It exhibited fibrinolytic activity, which is stronger than that of plasmin. FP84 hydrolyzed Bβ-chains of fibrinogen, but did not cleave Aα- and γ-chains. K_m, V_max and K_cat values for azocasein were 4.2 mg ml⁻¹, 305.8 μg min⁻¹ mg⁻¹ and 188.7 s⁻¹, respectively. The activity was suppressed by Co²⁺, Zn²⁺, Cu²⁺ and Fe²⁺, but slightly enhanced by Ca²⁺ and Mg⁺². Additionally, the activity was slightly inhibited by aprotinin and PMSF, but significantly inhibited by pefabloc, EDTA and EGTA. The first 15 amino acids of N-terminal sequence were GTQENPPSSGLDDID. They are highly similar to those of serine proteases from various Streptomyces strains, but different with known fibrinolytic enzymes. These results suggest that FP84 is a novel serine metalloprotease with potential application in thrombolytic therapy.     

Carbon flux from decomposing wood and its dependency on temperature,wood N2 fixation rate,moisture and fungal composition in a Norway spruce forest

Globally 40–70 Pg of carbon (C) are stored in coarse woody debris on the forest floor. Climate change may reduce the function of this stock as a C sink in the future due to increasing temperature. However, current knowledge on the drivers of wood decomposition is inadequate for detailed predictions. To define the factors that control wood respiration rate of Norway spruce and to produce a model that adequately describes the decomposition process of this species as a function of time, we used an unprecedentedly diverse analytical approach, which included measurements of respiration, fungal community sequencing, N₂ fixation rate, nifH copy number, ¹⁴C‐dating as well as N%, δ¹³C and C% values of wood. Our results suggest that climate change will accelerate C flux from deadwood in boreal conditions, due to the observed strong temperature dependency of deadwood respiration. At the research site, the annual C flux from deadwood would increase by 27% from the current 117 g C/kg wood with the projected climate warming (RCP4.5). The second most important control on respiration rate was the stage of wood decomposition; at early stages of decomposition low nitrogen content and low wood moisture limited fungal activity while reduced wood resource quality decreased the respiration rate at the final stages of decomposition. Wood decomposition process was best described by a Sigmoidal model, where after 116 years of wood decomposition mass loss of 95% was reached. Our results on deadwood decomposition are important for C budget calculations in ecosystem and climate change models. We observed for the first time that the temperature dependency of N₂ fixation, which has a major role at providing N for wood‐inhabiting fungi, was not constant but varied between wood density classes due to source supply and wood quality. This has significant consequences on projecting N₂ fixation rates for deadwood in changing climate.     

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.     

A comparative study of the use of inorganic carbon resources by Chara aspera and Potamogeton pectinatus

We studied the potential role of dissolved inorganic carbon (DIC) in determining vegetation dominance of Potamogeton pectinatus L. and Chara aspera Deth. ex Willd. by monitoring the seasonal dynamics of DIC in a shallow lake and comparing the use of DIC of the two species. The HCO₃⁻-concentration in summer dropped from 2.5 to <0.5 mM with seasonally increasing Chara biomass, whereas outside the vegetation concentrations remained at 2.5 mM. Inside Potamogeton spp. vegetation DIC decreased from 2.5 to ca. 0.75 mM HCO₃⁻. A growth experiment showed ash-free biomass for P. pectinatus was nearly two times as high as for C. aspera at 3 mM HCO₃⁻, but almost two times lower at 0.5 mM than at 3.0. In a separate experiment, P. pectinatus precultured at a relatively low HCO₃⁻-level had a lower net photosynthetic rate (P_max, 0.1 mmol O₂ g⁻¹ DW h⁻¹) than C. aspera (P_max, 0.1 mmol O₂ g⁻¹ DW h⁻¹) over the range of HCO₃⁻-concentrations tested (P_max, 0.14 mmol O₂ g⁻¹ DW h⁻¹). In response to CO₂ no significant differences between the compensation points (P. pectinatus, 28 mM; C. aspera 66 mM), were observed, but the photosynthetic rate increased faster than for C. aspera than for P. pectinatus. Under field conditions, the use of CO₂ is not important since inside vegetation CO₂-concentrations were below 10 μM, and thus, not available for photosynthesis of either species during the main part of the growth season. It is suggested that C. aspera may be a better competitor for HCO₃⁻ than P. pectinatus in conditions with a low HCO₃⁻ supply. As HCO₃⁻ is a strong limiting factor for growth inside the vegetation and probably the only carbon source available, the superior ability of C. aspera to use HCO₃⁻ may be an important factor explaining its present dominance in Veluwemeer.     

Purification and biochemical characterization of two extracellular peroxidases from Phanerochaete chrysosporium responsible for lignin biodegradation

Two extracellular peroxidases from Phanerochaete chrysosporium, namely a lignin peroxidase (LiP) and manganese peroxidase (MnP), were purified simultaneously by applying successively, ultrafiltration, ion-exchange and gel filtration chromatography. LiP and MnP have a molecular mass of 36 and 45 kDa, respectively. The optimal pHs for LiP and MnP activities were 3.0 and 4.5, respectively. Both peroxidases showed maximal activity at 30 °C and moderate thermostability. MnP activity was strongly inhibited by Fe²⁺, Zn²⁺, Mg²⁺ and Hg²⁺, and enhanced by Mn²⁺, Ca²⁺ and Cu²⁺. LiP activity was enhanced by Ca²⁺, Na⁺ and Co²⁺ and it was inhibited in the presence of K⁺, Hg⁺, Fe²⁺, Mg²⁺ and high concentrations of Cu²⁺ and Zn²⁺. The K_m and V_max for LiP toward veratryl alcohol as a substrate were 0.10 mM and 15.2 U mg⁻¹, respectively and for MnP toward Mn²⁺, they were respectively 0.03 mM and 25.5 U mg⁻¹. The two peroxidases were also able to break down rice lignin in a small-scale solid state treatment system. Data suggest these two peroxidases may be considered as potential candidates for the development of enzyme-based technologies for lignin degradation.     

Production of ethanol and xylanolytic enzymes by yeasts inhabiting rotting wood isolated in sugarcane bagasse hydrolysate

Yeasts associated with rotting wood from four Atlantic Rain forest sites in Brazil were investigated using a culture medium based on sugarcane bagasse hydrolysate. A total of 330 yeast strains were isolated. Pichia manshurica, Candida pseudolambica, and Wickerhamomyces sp. 3 were the most frequently isolated species. Fourteen novel species were obtained in this study. All isolates were tested for their ability to ferment d-xylose and to produce xylanases. In the fermentation assays using d-xylose (30 g L⁻¹), the main ethanol producers were Scheffersomyces stipitis (14.08 g L⁻¹), Scheffersomyces sp. (7.94 g L⁻¹) and Spathaspora boniae (7.16 g L⁻¹). Sc. stipitis showed the highest ethanol yield (0.42 g g⁻¹) and the highest productivity (0.39 g L⁻¹h⁻¹). The fermentation results using hemicellulosic hydrolysate showed that Sc. stipitis was the best ethanol producer, achieving a yield of 0.32 g g⁻¹, while Sp. boniae and Scheffersomyces sp. were excellent xylitol producers. The best xylanase-producing yeasts at 50 °C belonged to the species Su. xylanicola (0.487 U mg⁻¹) and Saitozyma podzolica (0.384 U mg⁻¹). The results showed that rotting wood collected from the Atlantic Rainforest is a valuable source of yeasts able to grow in sugarcane bagasse hydrolysate, including species with promising biotechnological properties.     

Influence of daylight surface aggregation behavior on nutrient cycling during a Karenia brevis (Davis) G. Hansen & Ø. Moestrup bloom: Migration to the surface as a nutrient acquisition strategy

The toxic HAB dinoflagellate Karenia brevis (Davis) G. Hansen & Ø. Moestrup (formerly Gymnodinium breve) exhibits a migratory pattern atypical of dinoflagellates: cells concentrate in a narrow (∼0–5 cm) band at the water surface during daylight hours due to phototactic and negative geotactic responses, then disperse downward at night via non-tactic, random swimming. The hypothesis that this daylight surface aggregation behavior significantly influences bacterial and algal productivity and nutrient cycling within blooms was tested during a large, high biomass (chlorophyll a >19 μg L⁻¹) K. brevis bloom in October of 2001 by examining the effects of this surface layer aggregation on inorganic and organic nutrient concentrations, cellular nitrogen uptake, primary and bacterial productivity and the stable isotopic signature (δ¹⁵N, δ¹³C) of particulate material. During daylight hours, concentrations of K. brevis and chlorophyll a in the 0–5 cm surface layer were enhanced by 131% (±241%) and 32.1% (±86.1%) respectively compared with an integrated water sample collection over a 0–1 m depth. Inorganic (NH₄, NO₃₊₂, PO₄, SiO₄) and organic (DOP, DON) nutrient concentrations were also elevated within the surface layer as was both bacterial and primary productivity. Uptake of nitrogen (NH₄⁺, NO₃⁻, urea, dissolved primary amines, glutamine and alanine) compounds by K. brevis was greatest in the surface layer for all compounds tested, with the greatest enhancement evident in urea uptake rates, from 0.08 × 10⁻⁵ ng N K. brevis cell⁻¹ h⁻¹ to 3.1 × 10⁻⁵ ng N K. brevis cell⁻¹ h⁻¹. These data suggests that this surface aggregation layer is not only an area of concentrated cells within K. brevis blooms, but also an area of increased biological activity and nutrient cycling, especially of nitrogen. Additionally, the classic dinoflagellate migration paradigm of a downward migration for access to elevated NO₃⁻ concentrations during the dark period may not apply to certain dinoflagellates such as K. brevis in oligotrophic nearshore areas with no significant nitricline. For these dinoflagellates, concentration within a narrow surface layer in blooms during daylight hours may enhance nutrient supply through biological cycling and photochemical nutrient regeneration.     

Reevaluation of the rate constants for the reaction of hypochlorous acid (HOCl) with cysteine,methionine, and peptide derivatives using a new competition kinetic approach

Activated white cells use oxidants generated by the heme enzyme myeloperoxidase to kill invading pathogens. This enzyme utilizes H₂O₂ and Cl⁻, Br⁻, or SCN⁻ to generate the oxidants HOCl, HOBr, and HOSCN, respectively. Whereas controlled production of these species is vital in maintaining good health, their uncontrolled or inappropriate formation (as occurs at sites of inflammation) can cause host tissue damage that has been associated with multiple inflammatory pathologies including cardiovascular diseases and cancer. Previous studies have reported that sulfur-containing species are major targets for HOCl but as the reactions are fast the only physiologically relevant kinetic data available have been extrapolated from data measured at high pH (>10). In this study these values have been determined at pH 7.4 using a newly developed competition kinetic approach that employs a fluorescently tagged methionine derivative as the competitive substrate (k(HOCl + Fmoc-Met), 1.5×10⁸ M⁻¹ s⁻¹). This assay was validated using the known k(HOCl + NADH) value and has allowed revised k values for the reactions of HOCl with Cys, N-acetylcysteine, and glutathione to be determined as 3.6×10⁸, 2.9×10⁷, and 1.24×10⁸ M⁻¹ s⁻¹, respectively. Similar experiments with methionine derivatives yielded k values of 3.4×10⁷ M⁻¹ s⁻¹ for Met and 1.7×10⁸ M⁻¹ s⁻¹ for N-acetylmethionine. The k values determined here for the reaction of HOCl with thiols are up to 10-fold higher than those previously determined and further emphasize the critical importance of reactions of HOCl with thiol targets in biological systems.     

Isolation and divalent-metal activation of a β-xylosidase,RUM630-BX

The gene encoding RUM630-BX, a β-xylosidase/arabinofuranosidase, was identified from activity-based screening of a cow rumen metagenomic library. The recombinant enzyme is activated as much as 14-fold (k_cat) by divalent metals Mg²⁺, Mn²⁺ and Co²⁺ but not by Ca²⁺, Ni²⁺, and Zn²⁺. Activation of RUM630-BX by Mg²⁺ (t_0.5 144 s) is slowed two-fold by prior incubation with substrate, consistent with the X-ray structure of closely related xylosidase RS223-BX that shows the divalent-metal activator is at the back of the active-site pocket so that bound substrate could block its entrance. The enzyme is considerably more active on natural substrates than artificial substrates, with activity (k_cat/K_m) of 299 s⁻¹ mM⁻¹ on xylotetraose being the highest reported.     

Highly thermostable carbonic anhydrase from Persephonella marina EX-H1: Its expression and characterization for CO2-sequestration applications

The codon-optimized carbonic anhydrase gene of Persephonella marina EX-H1 (PMCA) was expressed and characterized. The gene with the signal peptide removed, PMCA(sp−), resulted in the production of approximately five times more purified protein than from the intact gene PMCA using an Escherichia coli expression system. PMCA(sp−) is formed as homo-dimer complex. PMCA(sp−) has a wide pH tolerance (optimum pH 7.5) and a high thermostability even at 100 °C (88 min of thermal deactivation half-life). The melting temperature for PMCA(sp−) was 84.5 °C. The apparent k_cat and K_m values for CO₂ hydration were 3.2 × 10⁵ s⁻¹ and 10.8 mM. The activity of the PMCA(sp−) enzyme was enhanced by Zn²⁺, Co²⁺, and Mg²⁺, but was strongly inhibited by Cu²⁺, Fe³⁺, Al³⁺, Pb²⁺, Ag⁺, and Hg²⁺. PMCA(sp−) readily catalyzed the hydration of CO₂, precipitating CaCO₃ as calcite in the presence of Ca²⁺.     

Nitrogen uptake kinetics of Prymnesium parvum (Haptophyte)

The uptake rates of different nitrogen (N) forms (NO₃⁻, urea, and the amino acids glycine and glutamic acid) by N-deficient, laboratory-grown cells of the mixotrophic haptophyte, Prymnesium parvum, were measured and the preference by the cells for the different forms determined. Cellular N uptake rates (ρ_cell, fmol N cell⁻¹ h⁻¹) were measured using ¹⁵N-labeled N substrates. P. parvum showed high preference for the tested amino acids, in particular glutamic acid, over urea and NO₃⁻ under the culture nutrient conditions. However, extrapolating these rates to Baltic Seawater summer conditions, P. parvum would be expected to show higher uptake rates of NO₃⁻ and the amino acids relative to urea because of the difference in average concentrations of these substrates. A high uptake rate of glutamic acid at low substrate concentrations suggests that this substrate is likely used through extracellular enzymes. Nitrate, urea and glycine, on the other hand, showed a non-saturating uptake over the tested substrate concentration (1–40 μM-N for NO₃⁻ and urea, 0.5–10 μM-N for glycine), indicating slower membrane-transport rates for these substrates.     

Apogamic induction of haploid plasmodia in a myxomycete,Didymium iridis

Interisolate crosses between haploid (mean DNA = 0.32) CR 5-5 (A²) myxamoebae and polyploid (mean DNA = 1.80) CR 2–25 (A⁵) myxamoebae of the myxomycete Didymium iridis result in plasmodia that have the haploid (mean DNA = 0.32) DNA content rather than the predicted polyploid value. F₁ clones possess the mating type allele of the CR 5-5 clone only, and they also have the same mean DNA content as CR 5-5 myxamoebae. Crosses between these F₁ clones and CR 2–25 myxamoebae again resulted in the production of haploid plasmodia. Hence, the polyploid CR 2–25 clone appears to induce the CR 5-5 clone to produce plasmodia without involving itself in nuclear fusion.     

Uptake and inhibition kinetics of nitrogen in Microcystis aeruginosa: Results from cultures and field assemblages collected in the San Francisco Bay Delta,CA

The nitrogen (N) uptake kinetic parameters for Microcystis field assemblages collected from the San Francisco Bay Delta (Delta) in 2012 and non-toxic and toxic laboratory culture strains of M. aeruginosa were assessed. The ¹⁵N tracer technique was used to investigate uptake of ammonium (NH₄⁺), nitrate (NO₃⁻), urea and glutamic acid over short-term incubations (0.5–1 h), and to study inhibition of NO₃⁻, NH₄⁺ and urea uptake by NH₄⁺, NO₃⁻ and NH₄⁺, respectively. This study demonstrates that Delta Microcystis can utilize different forms of inorganic and organic N, with the greatest capacity for NH₄⁺ uptake and the least for glutamic acid uptake, although N uptake did not always follow the classic Michaelis–Menten hyperbolic relationship at substrate concentrations up to 67 μmol N L⁻¹. Current ambient N concentrations in the Delta may be at sub-saturating levels for N uptake, indicating that if N loading (especially NH₄⁺) were to increase, Delta Microcystis assemblages have the potential for increased N uptake rates. Delta Microcystis had the highest specific affinity, α, for NH₄⁺ and the lowest for NO₃⁻. In culture, N uptake by non-toxic and toxic M. aeruginosa strains was much higher than from the field, but followed similar N utilization trends to those in the field. Neither strain showed severe inhibition of NO₃⁻ uptake by NH₄⁺ or inhibition of NH₄⁺ uptake on NO₃⁻, but both strains showed some inhibition of urea uptake by NH₄⁺.     

Degradation of polychlorinated biphenyls (PCBs) by four bacterial isolates obtained from the PCB-contaminated soil and PCB-contaminated sediment

We investigated the PCB-degrading abilities of four bacterial strains isolated from long-term PCB-contaminated soil (Alcaligenes xylosoxidans and Pseudomonas stutzeri) and sediments (Ochrobactrum anthropi and Pseudomonas veronii) that were co-metabolically grown on glucose plus biphenyl which is an inducer of the PCB catabolic pathway. The aim of study was to determine the respective contribution of biomass increase and expression of degrading enzymes on the PCB degrading abilities of each isolate. Growth on 5 g l⁻¹ glucose alone resulted in the highest stimulation of the growth of bacterial strains, whereas grown on 10 mg l⁻¹, 100 mg l⁻¹, 1 g l⁻¹, or 5 g l⁻¹ biphenyl did not effected the bacterial growth. None of the strains used in this study was able to grow on PCBs as the sole carbon source. Cells grown on glucose exhibited enhanced degradation ability due to an increased biomass. Addition of biphenyl at concentrations of 1 or 5 g l⁻¹ did not increase total PCB degradation, but stimulated the degradation of highly chlorinated congeners for some of the strains. The degradation of di- and tri-chlorobiphenyls was significantly lower for cells grown on 5 g l⁻¹ biphenyl independently on glucose addition. The highest degradation of the PCBs was obtained for A. xylosoxidans grown in the presence of glucose. Thus A. xylosoxidans appears to be the most promising among the four bacterial isolates for the purpose of bioremediation.     

A new fungal peroxidase with alkaline-tolerant,chloride-enhancing activity and dye decolorization capacity

A new fungal peroxidase (Pspd) from Perenniporia subacida was purified by ammonium sulfate precipitation, DEAE-cellulose DE52 anionic exchange and Sepharose GL-6B chromatography, resulting in a high specific activity of 9.138 U mg⁻¹, 3.622-fold higher than that of crude enzyme at the same level. Polyacrylamide gel electrophoresis and UV–vis adsorption spectrum analysis showed that the purified enzyme is a heme-containing monomer with a molecular mass of 43.0 kDa. Optimal peroxidase activity was obtained at pH 5.5 and 30 °C when using 100.0 mM n-propanol as substrate, and under these conditions, the catalytic efficiency (k_cat/K_m) is 1.57 s⁻¹ μM⁻¹. Pspd was inhibited by l-cysteine, dithiothreitol, EDTA and sodium azide, but stimulated by Mn²⁺, Na⁺, Mg²⁺ and K⁺. The enzyme is stable over a broad pH range of 7.0–8.5 after incubation for 72 h, which indicated that the enzyme is lasting alkaline-tolerant. It was worth noting that the chloride at relatively low concentrations can enhance the peroxidase activity, with concomitant increase in substrate affinity. Additionally, Pspd performed high decolorization capability toward structurally various dyes and the capability was independent of the oxidizing mediators, with 75.31% of Neutral Red (50.0 mg L⁻¹) being decolorized by 1.5 U mL⁻¹ pure enzyme after incubation for 72 h. These properties demonstrated that Pspd has potentials for textile dyes decolorization applications.     

Purification and characterization of four key enzymes from a feather-degrading Bacillus subtilis from the gut of tarantula Chilobrachys guangxiensis

A feather-degrading bacterium was isolated from the gut of the tarantula Chilobrachys guangxiensis, and was classified as Bacillus subtilis (named Bacillus subtilis CH-1) according to both the phenotypic characteristics and 16S rRNA profile. The improved culture conditions for feather-degrading were 10.0 g l⁻¹ mannitol, 10.0 g l⁻¹ tryptone, 0.1 g l⁻¹ MgCl₂, 0.4 g l⁻¹ KH₂PO₄, 0.3 g l⁻¹ K₂HPO₄, 0.5 g l⁻¹ NaCl, and 2.0 g l⁻¹ intact feather, with pH 8.5 and 37 °C. In the optimized medium, the intact black feather was completely degraded by Bacillus subtilis CH-1 in 24 h. Furthermore, four kinds of enzymes which include extracellular protease Vpr, peptidase T, γ-glutamyl transpeptidase and glyoxalmethylglyoxal reductase were identified as having principal roles. Simultaneously, the relationship between the disulfide bond reducing activity (DRT) and the keratinase activity (KT) in B. subtilis CH-1 fermentation system was discussed. This is the first report for a feather-degrading enteric bacterium from tarantula. The identification of the enzymes shines a light on further understanding the molecular mechanism of feather-degrading by microbes.     

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.     

Effects of flooding on photosynthesis and root respiration in saltcedar (Tamarix ramosissima), an invasive riparian shrub

The introduced shrub Tamarix ramosissima invades riparian zones, but loses competitiveness under flooding. Metabolic effects of flooding could be important for T. ramosissima, but have not been previously investigated. Photosynthesis rates, stomatal conductance, internal (intercellular) CO₂, transpiration, and root alcohol dehydrogenase (ADH) activity were compared in T. ramosissima across soil types and under drained and flooded conditions in a greenhouse. Photosynthesis at 1500 μmol quanta m⁻² s⁻¹ (A₁₅₀₀) in flooded plants ranged from 2.3 to 6.2 μmol CO₂ m⁻² s⁻¹ during the first week, but A₁₅₀₀ increased to 6.4–12.7 μmol CO₂ m⁻² s⁻¹ by the third week of flooding. Stomatal conductance (g_s) at 1500 μmol quanta m⁻² s⁻¹ also decreased initially during flooding, where g_s was 0.018 to 0.099 mol H₂O m⁻² s⁻¹ during the first week, but g_s increased to 0.113–0.248 mol H₂O m⁻² s⁻¹ by the third week of flooding. However, photosynthesis in flooded plants was reduced by non-stomatal limitations, and subsequent increases indicate metabolic acclimation to flooding. Root ADH activities were higher in flooded plants compared to drained plants, indicating oxygen stress. Lower photosynthesis and greater oxygen stress could account for the susceptibility of T. ramosissima at the onset of flooding. Soil type had no effect on photosynthesis or on root ADH activity. In the field, stomatal conductance, leaf water potential, transpiration, and leaf δ¹³C were compared between T. ramosissima and other flooded species. T. ramosissima had lower stomatal conductance and water potential compared to Populus deltoides and Phragmites australis. Differences in physiological responses for T. ramosissima could become important for ecological concerns.     

Metabolite secretion,Fe3+-reducing activity and wood degradation by the white-rot fungus Trametes versicolor ATCC 20869

Trametes versicolor is a promising white-rot fungus for the biological pretreatment of lignocellulosic biomass. In the present work, T. versicolor ATCC 20869 was grown on Pinus taeda wood chips under solid-state fermentation conditions to examine the wood-degrading mechanisms employed by this fungus. Samples that were subjected to fungal pretreatment for one-, two- and four-week periods were investigated. The average mass loss ranged from 5 % to 8 % (m m⁻¹). The polysaccharides were preferentially degraded: hemicellulose and glucan losses reached 13.4 % and 6.9 % (m m⁻¹) after four weeks of cultivation, respectively. Crude enzyme extracts were obtained and assayed using specific substrates and their enzymatic activities were measured. Xylanases were the predominant enzymes, while cellobiohydrolase activities were marginally detected. Endoglucanase activity, β-glucosidase activity, and wood glucan losses increased up to the second week of biodegradation and remained constant after that time. Although no lignin-degrading enzyme activity was detected, the lignin loss reached 7.5 % (m m⁻¹). Soluble oxalic acid was detected in trace quantities. After the first week of biodegradation, the Fe³⁺-reducing activity steadily increased with time, but the activity levels were always lower than those observed in the undecayed wood. The progressive wood polymer degradation appeared related to the secretion of hydrolytic enzymes, as well as to Fe³⁺-reducing activity, which was restored in the cultures after the first week of biodegradation.     

The effects of light intensity on the growth of Japanese Gambierdiscus spp. (Dinophyceae)

Marine toxic dinoflagellates of the genus Gambierdiscus are the causative agents of ciguatera fish poisoning (CFP), a form of seafood poisoning that is widespread in tropical, subtropical and temperate regions worldwide. The distributions of Gambierdiscus australes, Gambierdiscus scabrosus and two phylotypes of Gambierdiscus spp. type 2 and type 3 have been reported for the waters surrounding the main island of Japan. To explore the bloom dynamics and the vertical distribution of these Japanese species and phylotypes of Gambierdiscus, the effects of light intensity on their growth were tested, using a photoirradiation-culture system. The relationship between the observed growth rates and light intensity conditions for the four species/phylotypes were formulated at R > 0.92 (p < 0.01) using regression analysis and photosynthesis-light intensity (P-L) model. Based on this equation, the optimum light intensity (L_max) and the semi-optimum light intensity range (L_s-opt) that resulted in the maximum growth rate (μ_max) and ≥80% μ _max values of the four species/phylotypes, respectively, were as follows: (1) the L_max and L_s-opt of G. australes were 208 μmol photons m⁻² s⁻¹ and 91–422 μmol photons m⁻² s⁻¹, respectively; (2) those of G. scabrosus were 252 and 120–421 μmol photons m⁻² s⁻¹, respectively; (3) those of Gambierdiscus sp. type 2 were 192 and 75–430 μmol photons m⁻² s⁻¹, respectively; and (4) those of Gambierdiscus sp. type 3 were ≥427 and 73–427 μmol photons m⁻² s⁻¹, respectively. All four Gambierdiscus species/phylotypes required approximately 10 μmol photons m⁻² s⁻¹ to maintain growth. The light intensities in coastal waters at a site in Tosa Bay were measured vertically at 1 m intervals once per season. The relationships between the observed light intensity and depth were formulated using Beer’s Law. Based on these equations, the range of the attenuation coefficients at Tosa Bay site was determined to be 0.058–0.119 m⁻¹. The values 1700 μmol photons m⁻² s⁻¹, 500 μmol photons m⁻² s⁻¹, and 200 μmol photons m⁻² s⁻¹ were substituted into the equations to estimate the vertical profiles of light intensity at sunny midday, cloudy midday and rainy midday, respectively. Based on the regression equations coupled with the empirically determined attenuation coefficients for each of the four seasons, the ranges of the projected depths of L_max and L_s-opt for the four Gambierdiscus species/phylotypes under sunny midday conditions, cloudy midday conditions, and rainy midday conditions were 12–38 m and 12–54 m, 1–16 m and 1–33 m, and 0 m and 0–16 m, respectively. These results suggest that light intensity plays an important role in the bloom dynamics and vertical distribution of Gambierdiscus species/phylotypes in Japanese coastal waters.