Carbon and nitrogen metabolism of an eutrophication tolerative macrophyte,Potamogeton crispus,under NH4+ stress and low light availability

Submersed macrophytes in eutrophic lakes often experience high NH₄⁺ concentration and low light availability in the water column. This study found that an NH₄⁺–N concentration of 1 mg L^?1 in the water column apparently caused physiological stress on the macrophyte Potamogeton crispus L. The plants accumulated free amino acids (FAA) and lost soluble carbohydrates (SC) under NH₄⁺ stress. These stressful effects of NH₄⁺ were exacerbated under low light availability. Shading significantly increased NH₄⁺ and FAA contents and dramatically decreased SC and starch contents in the plant shoots. At an NH₄⁺–N concentration of 1 mg L^?1 in the water column, neither growth inhibition nor NH₄⁺ accumulation was observed in the plant tissues of P. crispus under normal light availability. The results showed that 1 mg L^?1 NH₄⁺–N in the water column was not toxic to P. crispus in a short term. To avoid NH₄⁺ toxicity, active NH₄⁺ transportation out of the cell may cost energy and thus result in a decline of carbohydrate. When NH₄⁺ inescapably accumulates in the plant cell, i.e. under NH₄⁺ stress and shading, NH₄⁺ is scavenged by FAA synthesis.     

Thermophilic lipase from Thermomyces lanuginosus: Gene cloning,expression and characterization

An extracellular lipase gene ln1 from thermophilic fungus Thermomyces lanuginosus HSAUP₀₃80006 was cloned through RT-PCR and RACE amplification. Its coding sequence predicted a 292 residues protein with a 17 amino acids signal peptide. The deduced amino acids showed 78.4% similarity to another lipase lgy from T. lanuginosus while shared low similarity with other fungi lipases. Higher frequencies hydrophobic amino acids related to lipase thermal stability, such as Ala, Val, Leu and Gly were observed in this lipase (named LN). The sequence, -Gly-His-Ser-Leu-Gly-, known as a lipase-specific consensus sequence of mould, was also found in LN. High level expression for recombinant lipase was achieved in Pichia pastoris GS115 under the control of strong AOX1 promoter. It was purified to homogeneity through only one step DEAE-Sepharose anion exchange chromatography and got activity of 1328 U/ml. The molecular mass of one single band of this lipase was estimated to be 33 kDa by SDS-PAGE. The enzyme was stable at 60 °C and kept 65% enzyme activity after 30 min incubation at 70 °C. It kept half-activity after incubated for 40 min at 80 °C. The optimum pH for enzyme activity was 9.0 and the lipase was stable from pH 8.0 to 12.0. Lipase activity was enhanced by Ca²⁺ and inhibited by Fe²⁺, Zn²⁺, K⁺, and Ag⁺. The cell-free enzyme hydrolyzed and synthesized esters efficiently, and the synthetic efficiency even reached 81.5%. The physicochemical and catalytic properties of the lipase are extensively investigated for its potential industrial applications.     

Codon optimization,promoter and expression system selection that achieved high-level production of Yarrowia lipolytica lipase in Pichia pastoris

Lipase (EC 3.1.1.3) stands amongst the most important and promising biocatalysts for industrial applications. In this study, in order to realize a high-level expression of the Yarrowia lipolytica lipase gene in Pichia pastoris, we optimized the codon of LIP2 by de novo gene design and synthesis, which significantly improved the lipase expression when compared to the native lip2 gene. We also comparatively analyzed the effects of the promoter types (P_AOX1 and P_FLD1) and the Pichia expression systems, including the newly developed PichiaPink system, on lipase production and obtained the optimal recombinants. Bench-top scale fermentation studies indicated that the recombinant carrying the codon-optimized lipase gene syn-lip under the control of promoter P_AOX1 has a significantly higher lipase production capacity in the fermenter than other types of recombinants. After undergoing methanol inducible expression for 96 h, the wet cell weight of Pichia, the lipase activity and the protein content in the fermentation broth reached their highest values of 262 g/L, 38,500 U/mL and 2.82 g/L, respectively. This study has not only greatly facilitated the bioapplication of lipase in industrial fields but the strategies utilized, such as de novo gene design and synthesis, the comparative analysis among promoters and different generations of Pichia expression systems will also be useful as references for future work in this field.     

The effect of glycerol mixed substrate on the heterologous production of a Rhizopus oryzae lipase in Pichia pastoris system

A recombinant Rhizopus oryzae lipase producing Mut^s Pichia pastoris strain was used as a model organism to study the effect of mixed substrates (glycerol and methanol) on the specific product productivity. Different fed-batch cultivations were performed under three constant specific growth rates (0.02, 0.05 and 0.1 h⁻¹), maintaining a constant methanol concentration of 2 g l⁻¹.At the lowest μ tested (0.02 h⁻¹), the specific productivity was 1.23 and 1.61 fold higher and the specific methanol consumption rate (q_sMeOH) was 3 and 3.5 fold higher than values obtained when μ was 0.05 and 0.1 h⁻¹, respectively. This implies a relation between the q_sMeOH and the specific productivity, yielding higher specific productivities whenever the consumption of methanol is higher. Although glycerol was maintained under limiting conditions in all μ tested, when the relation between the μ_Gly and μ_MeOH was larger than 4, an important decrease on the maximal activity value was observed.Finally, a comparison under the same conditions using glycerol or sorbitol as co-substrates was also performed, obtaining better specific productivity when sorbitol was used. In addition, protease activity was detected when glycerol was used as co-substrate.     

Effects of NH4+ concentration on growth,morphology and NH4+ uptake kinetics of Salvinia natans

Many plants develop toxicity symptoms and have reduced growth rates when supplied with ammonium (NH₄⁺) as the only source of inorganic nitrogen. In the present study, the growth, morphology, NH₄⁺ uptake kinetics and mineral concentrations in the tissues of the free-floating aquatic plant Salvinia natans (water fern) supplied exclusively with NH₄⁺–N at concentrations of 0.25–15 mM were investigated. S. natans grew well, with relative growth rates of c. 0.25 g g^?1 d^?1 at external NH₄⁺ concentrations up to 5 mM, but at higher levels growth was suppressed and the plants had small leaves and short roots with stunted growth. The high-affinity transport system (HATS) that mediate NH₄⁺ uptake at dilute NH₄⁺ levels was downregulated at high NH₄⁺ concentrations with lower velocities of maximum uptake (V_max) and higher half-saturation constants (K_1/2). High NH₄⁺ levels also barely affected the concentrations of mineral cations and anions in the plant tissue. It is concluded that S. natans can be characterized as NH₄⁺-tolerant in line with a number of other species of wetland plants as growth was unaffected at NH₄⁺ concentrations as high as 5 mM and as symptoms of toxicity at higher concentrations were relatively mild. Depolarization of the plasma membrane to the equilibrium potential for NH₄⁺ at high external concentrations may be a mechanism used by the plant to avoid excessive futile transmembrane cycling. S. natans is tolerant to the high NH₄⁺ levels that prevail in domestic and agricultural wastewaters, and the inherent high growth rate and the ease of biomass harvesting make S. natans a primary candidate for use in constructed wetland systems for the treatment of various types of nitrogen-rich wastewaters.     

The effects of NH4+ and NO3− on growth,resource allocation and nitrogen uptake kinetics of Phragmites australis and Glyceria maxima

The effects of NH₄⁺ or NO₃⁻ on growth, resource allocation and nitrogen (N) uptake kinetics of two common helophytes Phragmites australis (Cav.) Trin. ex Steudel and Glyceria maxima (Hartm.) Holmb. were studied in semi steady-state hydroponic cultures. At a steady-state nitrogen availability of 34 μM the growth rate of Phragmites was not affected by the N form (mean RGR = 35.4 mg g⁻¹ d⁻¹), whereas the growth rate of Glyceria was 16% higher in NH₄⁺-N cultures than in NO₃⁻-N cultures (mean = 66.7 and 57.4 mg g⁻¹ d⁻¹ of NH₄⁺ and NO₃⁻ treated plants, respectively). Phragmites and Glyceria had higher S/R ratio in NH₄⁺ cultures than in NO₃⁻ cultures, 123.5 and 129.7%, respectively.Species differed in the nitrogen utilisation. In Glyceria, the relative tissue N content was higher than in Phragmites and was increased in NH₄⁺ treated plants by 16%. The tissue NH₄⁺ concentration (mean = 1.6 μmol g fresh wt⁻¹) was not affected by N treatment, whereas NO₃⁻ contents were higher in NO₃⁻ (mean = 1.5 μmol g fresh wt⁻¹) than in NH₄⁺ (mean = 0.4 μmol g fresh wt⁻¹) treated plants. In Phragmites, NH₄⁺ (mean = 1.6 μmol g fresh wt⁻¹) and NO₃⁻ (mean = 0.2 μmol g fresh wt⁻¹) contents were not affected by the N regime. Species did not differ in NH₄⁺ (mean = 56.5 μmol g⁻¹ root dry wt h⁻¹) and NO₃⁻ (mean = 34.5 μmol g⁻¹ root dry wt h⁻¹) maximum uptake rates (V_max), and V_max for NH₄⁺ uptake was not affected by N treatment. The uptake rate of NO₃⁻ was low in NH₄⁺ treated plants, and an induction phase for NO₃⁻ was observed in NH₄⁺ treated Phragmites but not in Glyceria. Phragmites had low K_m (mean = 4.5 μM) and high affinity (10.3 l g⁻¹ root dry wt h⁻¹) for both ions compared to Glyceria (K_m = 6.3 μM, affinity = 8.0 l g⁻¹ root dry wt h⁻¹). The results showed different plasticity of Phragmites and Glyceria toward N source. The positive response to NH₄⁺-N source may participates in the observed success of Glyceria at NH₄⁺ rich sites, although other factors have to be considered. Higher plasticity of Phragmites toward low nutrient availability may favour this species at oligotrophic sites.     

Uptake of dissolved inorganic and organic nitrogen by the benthic toxic dinoflagellate Ostreopsis cf. ovata

Environmental factors that shape dynamics of benthic toxic blooms are largely unknown. In particular, for the toxic dinoflagellate Ostreopsis cf. ovata, the importance of the availability of nutrients and the contribution of the inorganic and organic pools to growth need to be quantified in marine coastal environments. The present study aimed at characterizing N-uptake of dissolved inorganic and organic sources by O. cf. ovata cells, using the ¹⁵N-labelling technique. Experiments were conducted taking into account potential interactions between nutrient uptake systems as well as variations with the diel cycle. Uptake abilities of O. cf. ovata were parameterized for ammonium (NH₄⁺), nitrate (NO₃⁻) and N-urea, from the estimation of kinetic and inhibition parameters. In the range of 0 to 10 μmol N L⁻¹, kinetic curves showed a clear preference pattern following the ranking NH₄⁺ > NO₃⁻ > N-urea, where the preferential uptake of NH₄⁺ relative to NO₃⁻ was accentuated by an inhibitory effect of NH₄⁺ concentration on NO₃⁻ uptake capabilities. Conversely, under high nutrient concentrations, the preference for NH₄⁺ relative to NO₃⁻ was largely reduced, probably because of the existence of a low-affinity high capacity inducible NO₃⁻ uptake system. Ability to take up nutrients in darkness could not be defined as a competitive advantage for O. cf. ovata. Species competitiveness can also be defined from nutrient uptake kinetic parameters. A strong affinity for NH₄⁺ was observed for O. cf. ovata cells that may partly explain the success of this toxic species during the summer season in the Bay of Villefranche-sur-mer (France).     

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⁺.     

Optimization of culture conditions and properties of lipase from Penicillium camembertii Thom PG-3

The culture medium including nitrogen source, carbon source and metal ions, for lipase from Penicillium camembertii Thom PG-3 was optimized and the optimal medium consisted of soybean meal (fat free) 4%, Jojoba oil 0.5%, (NH₄)₂HPO₄, 0.1% Tween 60, initial pH 6.4 and the inoculation was at 28 °C for 96 h. The lipase activity produced was enhanced 3.9-fold and reached 500 U/ml. The lipase was purified 19.8-fold by pH precipitation, ethanol precipitation and ammonium sulphate precipitation as well as DEAE-cellulose chromatography. The purified lipase showed one polypeptide band in SDS-polyacrylamide gel electrophoreses (SDS-PAGE) with molecular weight 28.18 kDa. The optimal pH and temperature for activity of lipase were 6.4 and 48 °C, respectively, which are higher than those lipases from other penicillium sources. The P. camembertii Thom lipase is 1,3-positional specificity for hydrolysis of triglyceride and hydrolyses plant oil preferentially to animal oil. The lipase can be used in short chain ester synthesis with an esterification degree of 95%.     

Two ways to achieve an anammox influent from real reject water treatment at lab-scale: Partial SBR nitrification and SHARON process

A comparative study to produce the correct influent for Anammox process from anaerobic sludge reject water (700–800 mg NH₄⁺-N L⁻¹) was considered here. The influent for the Anammox process must be composed of NH₄⁺-N and NO₂⁻-N in a ratio 1:1 and therefore only a partial nitrification of ammonium to nitrite is required. The modifications of parameters (temperature, ammonium concentration, pH and solid retention time) allows to achieve this partial nitrification with a final effluent only composed by NH₄⁺-N and NO₂⁻-N at the right stoichiometric ratio. The equal ratio of HCO₃⁻/NH₄⁺ in reject water results in a natural pH decrease when approximately 50% of NH₄⁺ is oxidised. A Sequencing batch reactor (SBR) and a chemostat type of reactor (single-reactor high activity ammonia removal over nitrite (SHARON) process) were studied to obtain the required Anammox influent. At steady state conditions, both systems had a specific conversion rate around 40 mg NH₄⁺-N g⁻¹ volatile suspended solids (VSS) h⁻¹, but in terms of absolute nitrogen removal the SBR conversion was 1.1 kg N day⁻¹ m⁻³, whereas in the SHARON chemostat was 0.35 kg N day⁻¹ m⁻³ due to the different hydraulic retention time (HRT) used. Both systems are compared from operational (including starvation experiments) and kinetic point of view and their advantages/disadvantages are discussed.     

The modification of glucose levels and N source in the Hungate's medium to stimulate the production of fibrolytic enzymes of Anaeromyces sp. YQ3 grown on corn stalks

Hungate's method is a well-accepted protocol for the isolation or incubation of anaerobes with a roll tube technique. The aim of this study was to stimulate fungal enzyme production by optimizing the components of Hungate's medium for the growth of a rumen fungus Anaeromyces sp. YQ3. The organism was grown on corn stalks and incubated for 10 days in defined media with two glucose levels (G⁺, glucose in the Hungate's medium as a glucose control; G^?, glucose removed in a modified Hungate's medium) and four N sources (N1: yeast extract + tryptone + (NH₄)₂SO₄ in Hungate's medium (control); N2: yeast extract + (NH₄)₂SO₄; N3: tryptone + (NH₄)₂SO₄; and N4: tryptone + yeast extract). In the G^? media, the recovered activities of feruloyl esterase (FAE) (P<0.0001), acetyl esterase (AE) (P=0.0065) and xylanase (P<0.0001) were decreased, while the G⁺ media with N1 nitrogen stimulated the production of FAE and xylanase (P<0.0001). The G^? medium with N2 nitrogen increased the recovered activities of carboxymethyl cellulase (P=0.0001) and avicelase (P<0.0001), while the N3 and N4 media increased the recovered activity of AE (P=0.0015). The N4 medium was comparable to the N1 medium in stimulating the amount of recovered xylanase activity. The activities of FAE (P<0.0001), AE (P<0.0001), and xylanase (P<0.0001) showed a time-dependent increase and reached their peaks at day 10, while the avicelase activity peaked at day 8 (P=0.0071). The esterase activities (FAE and AE) were positively correlated with the enzyme activities of xylanase and carboxymethyl cellulase (r > 0.48, P<0.05). After a 10-day incubation, the glucose in the Hungate's media contributed to an increase in organic matter disappearance (P<0.0001) and volatile fatty acid (VFA) concentration (P<0.0001), except for molar acetate proportions. The N4 treatment increased organic matter disappearance and total VFA concentration (P=0.0002). The change in N source did not alter molar proportions of acetate, propionate and valerate, while the N2 treatment increased molar butyrate proportion (P<0.0035), and both N2 and N3 increased the molar proportion of branched chain VFAs (P<0.0041). In summary, the glucose in the Hungate's medium is beneficial for stimulating the production of esterases and xylanase, thereby promoting fungal growth. Amending the N source in Hungate's medium brings about different yields of rumen fungal esterases and polysaccharide hydrolases that have important nutritional impacts on fibre degradation in ruminant animals.     

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.     

Pretreatment of immobilized Candida sp. 99-125 lipase to improve its methanol tolerance for biodiesel production

Candida sp. 99-125 lipase immobilized on textile membrane was pretreated with several methods to improve its activity and methanol tolerance for biodiesel production. Lipase pretreatments with short chain alcohols from n-propyl alcohol to isobutyl alcohol did not have any positive effect on the lipase activity and methanol tolerance. While lipase treated with methanol solutions from 10 to 20% volume concentrations did enhance the enzyme activity and methanol tolerance, and this lipase activation effect did not exist when methanol volume concentration was 40%. 1 mM salt solutions of (NH₄)₂SO₄, CaCl₂, KCl, K₂SO₄ and MgCl₂ pretreatments were the useful tools to improve the lipase activity and methanol tolerance. The reason might be that salts could incorporate with the protein molecular to form a more stable molecular to resist conformation change induced by high methanol concentration. The operational stability of pretreated lipase was improved dramatically for biodiesel production during batch reactions.     

An improved method of lipase preparation incorporating both solvent treatment and immobilization onto matrix

A simple and effective preparation of lipases for use in organic solvents is hereby proposed. Lipases in aqueous solution were treated with isopropanol, immediately followed by immobilization onto a commercially available macroporous resin CRBO2 (crosslinked polystyrene with N-methylglucamine as a functional group). The dual modification of lipases by (1) isopropanol treatment and (2) immobilization improved the activity and stability of lipases more significantly than either of the two treatments alone. The degree of lipase activation was dependent on isopropanol–buffer (v/v) ratio and the source of lipase used. Among the lipases tested, Rhizopus oryzae lipase was more significantly activated. The maximum specific activity of R. oryzae lipase after dual modification was 94.9 mmol h⁻¹ g⁻¹, which was, respectively, 3.3-, 2.5- and 1.5-fold of untreated free, untreated immobilized and treated free lipases. The conformations of the treated and untreated free lipases were investigated by circular dichroism (CD) measurement. Changes in the far- and near-UV CD spectra of lipase indicate that lipase activation is accompanied by changes in secondary and tertiary structures of lipases. The increase in negative molar elipticity at 222 nm suggests that the α-helical content of lipase increase after pretreatment.     

Extracellular cold-active lipase of Microbacterium luteolum isolated from Gangotri glacier,western Himalaya: Isolation,partial purification and characterization

A psychrophilic bacterium producing cold-active lipase upon growth at low temperature was isolated from the soil samples of Gangotri glacier and identified as Microbacterium luteolum. The bacterial strain produced maximum lipase at 15 °C, at a pH of 8.0. Beef extract served as the best organic nitrogen source and ammonium nitrate as inorganic for maximum lipase production. Castor oil served as an inducer and glucose served as an additional carbon source for production of cold-active lipase. Ferric chloride as additional mineral salt in the medium, highly influenced the lipase production with an activity of 8.01 U ml^?1. The cold-active lipase was purified to 35.64-fold by DEAE-cellulose column chromatography. It showed maximum activity at 5 °C and thermostability up to 35 °C. The purified lipase was stable between pH 5 and 9 and the optimal pH for enzymatic hydrolysis was 8.0. Lipase activity was stimulated in presence of all the solvents (5%) tested except with acetonitrile. Lipase activity was inhibited in presence of Mn²⁺, Cu²⁺, and Hg²⁺; whereas Fe⁺, Na⁺ did not have any inhibitory effect on the enzyme activity. The purified lipase was stable in the presence of SDS; however, EDTA and dithiothreitol inhibited enzyme activity. Presence of Ca²⁺ along with inhibitors stabilized lipase activity. The cold active lipase thus exhibiting activity and stability at a low temperature and alkaline pH appears to be practically useful in industrial applications especially in detergent formulations.     

Combined strategies for improving the heterologous expression of an alkaline lipase from Acinetobacter radioresistens CMC-1 in Pichia pastoris

In this study, a series of strategies was developed to enhance the expression of an alkaline lipase from Acinetobacter radioresistens (ARL) in Pichia pastoris. Activity of the lipase from recombinant strain carrying a single copy of codon-optimized ARL gene was 65 U/mL in shake flask culture with p-nitrophenyl caprylate as the substrate. The lipase yield was increased to 104 U/mL by introducing a short N-extension spacer peptide coding for the 10 amino acids (EEAEAEAEPK) between α-factor signal peptide and ARL. The N-terminal extension spacer did not affect the pH or temperature properties of the recombinant ARL. After the multi-copy constructs were identified by Q-PCR assay, a higher lipase activity of 180 U/mL was obtained. Further introduction of the spliced HAC1 gene into multi-copy integrants (>6 copies) extensively enhanced the ARL yield by 30–40%. As a result, the ARL yield reached 1.06 × 10⁴ U/mL in a 10-L scaled-up fed-batch fermenter as well as the lipase showed some better properties compared to that wild one from A. radioresistens.     

Production enhancement of Rhizopus arrhizus lipase by feeding oleic acid

A strategy for Rhizopus arrhizus lipase production enhancement by feeding oleic acid was developed. The oleic acid was proved to have strong inducing effect on lipase production, but high concentration oleic acid could repress lipase production. The decrease rate of oleic acid concentration using peanut oil as initial carbon source was figured out according to the change of oleic acid concentration in the fermentation broth. Our feeding strategy designed based on the decrease rate of oleic acid could avoid the repression of lipase production that is caused by high concentration of oleic acid in the fermenting liquor, and this strategy worked as a new feeding method showing excellent performance. The maximum lipase activity was gained by feeding dilute oleic acid every 12 h starting at 60 h, which maintained the oleic acid concentration around 18 mg/L, and the lipase activity was 31% higher than that of no feeding.     

Purification and characterization of trans-3-(4-methoxyphenyl) glycidic acid methyl ester hydrolyzing lipase from Pseudomonas aeruginosa

Enantiospecific lipase was purified from Pseudomonas aeruginosa MTCC 5113 and it was used for the hydrolysis of (±)-methyl trans-3(4-methoxyphenyl) glycidate, a key intermediate in the synthesis of cardiovascular drug, diltiazem. Enzyme from broth supernatant was precipitated with acetone and purified by anion exchange and gel filtration chromatography. The purified lipase was a homogenous protein having a molecular weight of 59.4 kDa as determined by SDS-PAGE. Isoelectric point was found to be approximately 5.5 after 2D electrophoresis. This organic solvent tolerant enzyme was found to be active in presence of EDTA, Tween-80 and β-mercaptoethanol whereas sodium dodecyl sulphate and dithiothreitol inhibited its activity. The K_m and V_max of the enzyme were 50 mM and 27.1 μmol/min mg, respectively using p-nitrophenyl palmitate as a substrate. The activity of lipase was confirmed by (±)-MPGM hydrolysis and zymography.     

Removal of nutrients from wastewater with Canna indica L. under different vertical-flow constructed wetland conditions

Constructed wetlands are becoming increasingly popular worldwide for removing contaminants from domestic wastewater. This study investigated the removal efficiency of nitrogen (N) and phosphorus (P) from wastewater with the simulated vertical-flow constructed wetlands (VFCWs) under three different substrates (i.e., BFAS or blast furnace artificial slag, CBAS or coal burn artificial slag, and MSAS or midsized sand artificial slag), hydraulic loading rates (i.e., 7, 14, and 21 cm d^?1), and wetland operational periods (0.5, 1, and 2 years) as well as with and without planting Canna indica L. The wastewater was collected from the campus of South China Agricultural University, Guangzhou, China. Results show that the percent removal of total P (TP) and ammonium N (NH₄⁺-N) by the substrates was BFAS > CBAS > MSAS due to the high contents of Ca and Al in substrate BFAS. In contrast, the percent removal of total N (TN) by the substrates was CBAS > MSAS > BFAS due to the complicated nitrification/denitrification processes. The percent removal of nutrients by all of the substrates was TP > NH₄⁺-N > TN. About 10% more TN was removed from the wastewater after planting Canna indica L. A lower hydraulic loading rate or longer hydraulic retention time (HRT) resulted in a higher removal of TP, NH₄⁺-N, and TN because of more contacts and interactions among nutrients, substrates, and roots under the longer HRT. Removal of NO₃^?N from the simulated VFCWs is a complex process. A high concentration of NO₃^?N in the effluent was observed under the high hydraulic loading rate because more NH₄⁺-N and oxygen were available for nitrification and a shorter HRT was unfavorable for denitrification. In general, a longer operational period had a highest removal rate for nutrients in the VFCWs.