Staphylococcus cohnii HFUTY-08: a novel acid urease-producing strain

Urea in alcoholic beverages is a precursor of ethyl carbamate (EC), which is carcinogenic. At present, removal of urea by acid urease is considered to be the most effective method. In this study, a strain with higher acid urease production was screened and the enzyme activity was 1.12 U/mL. The strain was identified as Staphylococcus cohnii via a phylogenetic analysis of its 16S rDNA gene sequence, its morphological characteristics, and its physiological and biochemical properties, named as Staphylococcus cohnii HFUTY-08. Optimum culture conditions were determined through a single-factor test and an orthogonal test, with results as follows: glucose concentration 30 g/L, peptone concentration 15 g/L, initial pH 5, and an optimal inoculation amounts of 5%. Under these conditions, the activity of acid urease produced by strain Staphylococcus cohnii HFUTY-08 was 1.78 U/ml. Besides, the crude enzyme was purified to electrophoretic homogeneity by ion exchange chromatography and gel filtration chromatography. The molecular weight of the enzyme was estimated to be 295 kDa and the structural features of the enzyme were defined as (αβγ)₃. Finally, the preliminary study on the removal of urea by acid urease in Chinese rice wine (CRW) showed that the enzyme could remove about 75% urea within 72 h at 37 °C, which effectively prevented EC production.     

Immobilization of soybean (Glycine max) urease on alginate and chitosan beads showing improved stability: Analytical applications

The soybean (Glycine max) urease was immobilized on alginate and chitosan beads and various parameters were optimized and compared. The best immobilization obtained were 77% and 54% for chitosan and alginate, respectively. A 2% chitosan solution (w/v) was used to form beads in 1N KOH. The beads were activated with 1% glutaraldehyde and 0.5 mg protein was immobilized per ml of chitosan gel for optimum results. The activation and coupling time were 6 h and 12 h, respectively. Further, alginate and soluble urease were mixed to form beads and final concentrations of alginate and protein in beads were 3.5% (w/v) and 0.5 mg/5 ml gel. From steady-state kinetics, the optimum temperature for urease was 65 °C (soluble), 75 °C (chitosan) and 80 °C (alginate). The activation energies were found to be 3.68 kcal mol⁻¹, 5.02 kcal mol⁻¹, 6.45 kcal mol⁻¹ for the soluble, chitosan- and alginate-immobilized ureases, respectively. With time-dependent thermal inactivation studies, the immobilized urease showed improved stability at 75 °C and the t_1/2 of decay in urease activity was 12 min, 43 min and 58 min for soluble, alginate and chitosan, respectively. The optimum pH of urease was 7, 6.2 and 7.9 for soluble, alginate and chitosan, respectively. A significant change in K_m value was noticed for alginate-immobilized urease (5.88 mM), almost twice that of soluble urease (2.70 mM), while chitosan showed little change (3.92 mM). The values of V_max for alginate-, chitosan-immobilized ureases and soluble urease were 2.82 × 10² μmol NH₃ min⁻¹ mg⁻¹ protein, 2.65 × 10² μmol NH₃ min⁻¹ mg⁻¹ protein and 2.85 × 10² μmol NH₃ min⁻¹ mg⁻¹ protein, respectively. By contrast, reusability studies showed that chitosan–urease beads can be used almost 14 times with only 20% loss in original activity while alginate–urease beads lost 45% of activity after same number of uses. Immobilized urease showed improved stability when stored at 4 °C and t_1/2 of urease was found to be 19 days, 80 days and 121 days, respectively for soluble, alginate and chitosan ureases. The immobilized urease was used to estimate the blood urea in clinical samples. The results obtained with the immobilized urease were quite similar to those obtained with the autoanalyzer^®. The immobilization studies have a potential role in haemodialysis machines.     

Genetic microheterogeneity and phenotypic variation of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> arginase in clinical isolates

Background  

Clinical isolates of the gastric pathogen Helicobacter pylori display a high level of genetic macro- and microheterogeneity, featuring a panmictic, rather than clonal structure. The ability of H. pylori to survive the stomach acid is due, in part, to the arginase-urease enzyme system. Arginase (RocF) hydrolyzes L-arginine to L-ornithine and urea, and urease hydrolyzes urea to carbon dioxide and ammonium, which can neutralize acid.     

Effects of acetic acid, ethanol, and SO2 on the removal of volatile acidity from acidic wines by two Saccharomyces cerevisiae commercial strains

Herein, we report the influence of different combinations of initial concentration of acetic acid and ethanol on the removal of acetic acid from acidic wines by two commercial Saccharomyces cerevisiae strains S26 and S29. Both strains reduced the volatile acidity of an acidic wine (1.0 g l⁻¹ acetic acid and 11% (v/v) ethanol) by 78% and 48%, respectively. Acetic acid removal by strains S26 and S29 was associated with a decrease in ethanol concentration of 0.7 and 1.2% (v/v), respectively. Strain S26 revealed better removal efficiency due to its higher tolerance to stress factors imposed by acidic wines. Sulfur dioxide (SO₂) in the concentration range 95–170 mg l⁻¹ inhibits the ability of both strains to reduce the volatile acidity of the acidic wine used under our experimental conditions. Therefore, deacidification should be carried out either in wines stabilized by filtration or in wines with SO₂ concentrations up to 70 mg l⁻¹. Deacidification of wines with the better performing strain S26 was associated with changes in the concentration of volatile compounds. The most pronounced increase was observed for isoamyl acetate (banana) and ethyl hexanoate (apple, pineapple), with an 18- and 25-fold increment, respectively, to values above the detection threshold. The acetaldehyde concentration of the deacidified wine was 2.3 times higher, and may have a detrimental effect on the wine aroma. Moreover, deacidification led to increased fatty acids concentration, but still within the range of values described for spontaneous fermentations, and with apparently no negative impact on the organoleptical properties.     

N-substituted aminomethanephosphonic and aminomethane-<Emphasis Type="Italic">P</Emphasis>-methylphosphinic acids as inhibitors of ureases

Small unextended molecules based on the diamidophosphate structure with a covalent carbon-to-phosphorus bond to improve hydrolytic stability were developed as a novel group of inhibitors to control microbial urea decomposition. Applying a structure-based inhibitor design approach using available crystal structures of bacterial urease, N-substituted derivatives of aminomethylphosphonic and P-methyl-aminomethylphosphinic acids were designed and synthesized. In inhibition studies using urease from Bacillus pasteurii and Canavalia ensiformis, the N,N-dimethyl derivatives of both lead structures were most effective with dissociation constants in the low micromolar range (K _i  = 13 ± 0.8 and 0.62 ± 0.09 μM, respectively). Whole-cell studies on a ureolytic strain of Proteus mirabilis showed the high efficiency of N,N-dimethyl and N-methyl derivatives of aminomethane-P-methylphosphinic acids for urease inhibition in pathogenic bacteria. The high hydrolytic stability of selected inhibitors was confirmed over a period of 30 days using NMR technique.     

Evaluation of the acetaldehyde production and degradation potential of 26 enological <Emphasis Type="Italic">Saccharomyces</Emphasis> and non-<Emphasis Type="Italic">Saccharomyces</Emphasis> yeast strains in a resting cell model system

Acetaldehyde is relevant for wine aroma, wine color, and microbiological stability. Yeast are known to play a crucial role in production and utilization of acetaldehyde during fermentations but comparative quantitative data are scarce. This research evaluated the acetaldehyde metabolism of 26 yeast strains, including commercial Saccharomyces and non-Saccharomyces, in a reproducible resting cell model system. Acetaldehyde kinetics and peak values were highly genus, species, and strain dependent. Peak acetaldehyde values varied from 2.2 to 189.4 mg l⁻¹ and correlated well (r ² = 0.92) with the acetaldehyde production yield coefficients that ranged from 0.4 to 42 mg acetaldehyde per g of glucose in absence of SO₂. S. pombe showed the highest acetaldehyde production yield coefficients and peak values. All other non-Saccharomyces species produced significantly less acetaldehyde than the S. cerevisiae strains and were less affected by SO₂ additions. All yeast strains could degrade acetaldehyde as sole substrate, but the acetaldehyde degradation rates did not correlate with acetaldehyde peak values or acetaldehyde production yield coefficients in incubations with glucose as sole substrate.     

A novel and sensitive resonance scattering assay for detection of urea in serum coupled urease catalytic reaction and NH4 + associated particle reaction

In the pH 6.6 Na₂HPO₄–NaH₂PO₄ buffer solutions and in the presence of urease catalyst, urea can be decomposed to form NH₄ ⁺. The NH₄ ⁺ reacted with sodium tetraphenyl boron (NaTPB) to form the association particles that exhibited a resonance scattering (RS) peak at 474 nm. When the urea concentration increased, NH₄ ⁺ increased, and RS intensity at 474 nm enhanced linearly. Under the chosen conditions, the increased RS intensity (ΔI _474 nm) had a linear response to the urea concentration in the range of 0.125–15 μM, with a detection limit of 0.058 μM urea, and a regression equation of ΔI _474 nm = 31.6C + 2.1, a correlation coefficient of 0.9986. This catalytic RS method was applied for the detection of urea in human serum sample, with good selectivity and sensitivity, and the results were consistent with the reference method.     

The role of Azolla cover in improving the nitrogen use efficiency of lowland rice

The development of management techniques to improve the poor N use efficiency by lowland rice (Oryza sativa L.) and reduce the high N losses has been an important focus of agronomic research. The potential of an Azolla cover in combination with urea was assessed under field conditions in Laguna, Philippines. Two on-station field experiments were established in the 1998–1999 dry season and eight on-farm experiments per season were carried out in the 2000–2001 wet and dry seasons. Treatment combinations consisting of N levels applied alone or combined with Azolla were evaluated with respect to floodwater chemistry, ¹⁵N recovery, crop growth, and grain yield. A full Azolla cover on the floodwater surface at the time of urea application prevented the rapid and large increase in floodwater pH and floodwater temperature. As a consequence, the partial pressure of ammonia (NH₃), which is an indicator of potential NH₃ volatilization, was significantly depressed. ¹⁵N recovery was higher in plots covered with Azolla where the total ¹⁵N recovery ranged between 77 and 99%, and the aboveground (grain and straw) recovery by rice ranged between 32 and 61%. The tiller count in Azolla-covered plots was significantly increased by 50% more than the uncovered plots at all urea levels. Consequently, the grain yield was likewise improved. Grain yields from the 16 on-farm trials increased by as much as 40% at lower N rates (40 and 50 kg N ha^–1) and by as much as 29% at higher N rates (80 and 100 kg N ha^–1). In addition, response of rice to treatments with lower N rates with an Azolla cover was comparable to that obtained with the higher N rates without a cover. Thus, using Azolla as a surface cover in combination with urea can be an alternative management practice worth considering as a means to reduce NH₃ volatilization losses and improve N use efficiency.     

Amperometric electrode for determination of urea using electrodeposited rhodium and immobilized urease

An amperometric biosensor was developed for determination of urea using electrodeposited rhodium on a polymer membrane and immobilized urease. The urease catalyzes the hydrolysis of urea to NH₄⁺ and HCO₃⁻ ions and the liberated ammonia is catalytically and electrochemically oxidized by rhodium present in the rhodinized membrane on the Pt working electrode. Three types of rhodinized polymer membranes were prepared by varying the number of electrodeposition cycles: membrane 1 with 10 deposition cycles, membrane 2 with 40 cycles and membrane 3 with 60 cycles. The morphologies of the rhodinized membranes were investigated by scanning electron microscopy and the results showed that the deposition of rhodium was like flowers with cornices-like centers. The influence of the amount of electrodeposited rhodium over the electrode sensitivity to different concentrations of ammonia was examined initially based on the cyclic voltammetric curves using the three rhodium modified electrodes. The obtained results convincingly show that electrode with rhodinized membrane 1, which contain the lowest amount of electrodeposited rhodium is the most active and sensitive regarding ammonia. It was found that the anodic oxidation peak of ammonia to nitrogen occurs at 0.60 V. In order to study the performance of urease amperometric sensor for the determination of urea, experiments at constant potential (0.60 V) were performed. The current–time experiments were carried out with urease rhodinized membrane 1 (10 cycles). The amperometric response increased linearly up to 1.75 mM urea. The detection limit was 0.05 mM. The urea biosensor exhibited a high sensitivity of 1.85 μA mM⁻¹ cm⁻² with a response time 15 s. The Michaelis–Menten constant K_m for the urea biosensor was calculated to be 6.5 mM, indicating that the immobilized enzyme featured a high affinity to urea. The urea sensor showed a good reproducibility and stability. Both components rhodium and urease contribute to the decreasing of the production cost of biosensor by avoiding the use of a second enzyme.     

Expression of an Acid Urease with Urethanase Activity in <Emphasis Type="Italic">E. coli</Emphasis> and Analysis of Urease Gene

Urea in alcoholic beverage is a precursor of ethyl carbamate (EC), which is carcinogenic. Enzymatic elimination of urea has attracted much research interest. Acid urease with good tolerance toward ethanol and acid is ideal enzyme for such applications. In the present work, the structural genes of urease from Providencia rettgeri JN-B815, ureABC were efficiently expressed in E. coli BL21(DE3) in an active form (apourease) exhibiting both urease and urethanase (hydrolyze EC) activities. The specific activities of the purified apourease were comparatively low, which were 2.1 U/mg for urease and 0.6 U/mg for urethanase, respectively. However, apourease exhibited good resistance toward ethanol and acidic conditions. The relative activities of urease and urethanase remained over 80% in the buffers within pH 4–7. And the recoveries of both urease and urethanase activities were more than 50% in 5–25% ethanol solution. Apourease was utilized to eliminate urea in wine, and the residual urea in model wine was less than 50% after treatment with apourease for 30 h. Then 3D structure of UreC was predicted, and it was docked with urea and EC, respectively. The docking result revealed that three hydrogen bonds were formed between urea and amino acid residues in the active site of urease, whereas only one hydrogen bond can be formed between EC and the active center. Moreover, EC exhibited greater steric hindrance than urea when combined with the active site. Due to the low specific activities of apourease, both structural genes and accessory genes of urease were co-expressed in E. coli BL21(DE3). The holoenzyme was expressed as inclusion body. After renaturation and purification, the specific activities of urease and urethanase reached 10.7 and 3.8 U/mg, which were 5.62-fold and 6.33-fold of those of apourease, respectively. Therefore, accessory subunits of urease play an important role in enhancing urease and urethanase activities.     

Responses of morphological, physiological, and biochemical parameters in Euglena gracilis to 7-days exposure to two commonly used fertilizers DAP and urea

Diammonium phosphate (DAP) and urea are commonly used fertilizers throughout the world. The effects of these fertilizers on the freshwater flagellate Euglena gracilis was studied after 7 days of growth using morphological, physiological and biochemical parameters as end points. NOEC and EC₅₀ values for various parameters like cell density, motility, velocity, cell shape, gravitaxis, chlorophyll a, b and total carotenoids were calculated. NOEC and EC₅₀ values of DAP varied from 0.5 to 2.5 g L⁻¹ and 3.14 to 5.96 g L⁻¹, respectively, for different parameters. NOEC and EC₅₀ values for urea ranged from 5 to 25 g L⁻¹ and 28 to 44.05 g L⁻¹, respectively, for various parameters. Photosynthetic pigments were found to be more sensitive to both fertilizers as compared to other measured end points. The NOEC and EC₅₀ values obtained for DAP were much lower than those for urea; i.e., DAP showed a stronger inhibitory effect as compared to urea. Application of DAP resulted in an increased concentration of ammonia in Euglena cultures but urea did not. The stronger inhibitory effect of DAP is attributed to release of free ammonia in the culture due to DAP decompostion. No release of ammonia by urea occurred due to the absence of the enzyme urease in E. gracilis.     

Influence of N and Ni supply on nitrogen metabolism and urease activity in rice (Oryza sativa L.)

Nickel is considered to be an essential micronutrient in plants because of its role in the metalloenzyme urease. In order to characterize the metabolic consequences of Ni deprivation, the significance of Ni supply for growth and N metabolism of rice plants grown with either NH4NO3 or urea as sole N source was evaluated. Growth of plants receiving NH4NO3 was not affected by the Ni status, and neither were the activities of arginase and glutamine synthetase. However, urease activity was not detectable in leaves of low-Ni plants, which in conjunction with arginase action, led to the accumulation of urea in plants grown with NH4NO3. Amino acid contents and mineral nutrient status (except Ni) were not affected by the Ni treatment.Urea-grown Ni-deprived plants showed reduced growth and accumulated large amounts of urea owing to the lack of urease activity. These plants were further characterized by low amino acid contents indicating impaired usage of the N supplied. They also exhibited reduced levels of the urea precursor arginine, which is merely attributed to the overall N economy in these plant. When urea-grown plants were supplied with 0.5 mmol m^-3 Ni in the nutrient solution, the dry weight and the amino acid N contents were increased at the expense of the urea contents, indicating efficient use of urea N in Ni-supplemented plants.A critical Ni concentration in the shoot regarding dry matter production of NH4NO3-grown plants could not be deduced, while 25 g Ni kg^-1 DW is certainly inadequate for urea-grown plants. This suggests that the Ni requirement strongly depends on the N source employed.Keywords: Amino acids, ornithine cycle, Ni supply, rice, urea, urease activity.      

重组酸性脲酶对黄酒中尿素和氨基甲酸乙酯的降解应用

氨基甲酸乙酯(Ethyl carbamate,EC)作为一种潜在致癌物质普遍存在于传统发酵食品中。利用酸性脲酶消除EC前体物质尿素是一种具有潜在重要应用价值的策略。本研究在前期成功实现食品级耐乙醇酸性脲酶高效表达制备的基础上,系统研究了重组酸性脲酶对尿素和EC的水解过程。重组酸性脲酶对模拟体系以及黄酒体系中的尿素具有很好的降解能力(60mg/L的尿素在25h内完全被降解),表明该重组酸性脲酶适用于黄酒中尿素的消除。虽然重组酸性脲酶也具有降解EC的催化活性,但在黄酒中添加重组酸性脲酶对EC的浓度无明显影响。进一步研究发现重组酸性脲酶对尿素和EC的Km值分别为0.714 7mmol/L和41.32mmol/L,研究结果为应用定向进化策略改造重组酸性脲酶实现同时水解尿素和EC提供了理论依据。     

The use of urea by Evernia prunastri thalli

Thalli of Evernia prunastri floated on 40 mM urea synthesize urease (EC3.5.1.5) which is, in part, retained in the cells as well as secreted into the external medium. By using [¹⁴C]urea, it has been shown that the ¹⁴CO₂ evolved by the action of urease is mainly incorporated into phenolic compounds. Evernic acid has the highest radioactivity when incubations are carried out in the light. The orsellinate moiety of this molecule contains ten times more radioactivity than the everninic acid moiety. This could be explained by the assumption that orsellinic acid is the first product of cyclisation of the polyketide chain in the biosynthetic pathway.     

Response surface design for the optimization of enzymatic detection of mercury in aqueous solution using immobilized urease from vegetable waste

Soluble and alginate immobilized urease was utilized for detection and quantitation of mercury in aqueous samples. Urease from the seeds of pumpkin, being a vegetable waste, was extracted and purified to apparent homogeneity (sp. activity 353 U/mg protein; A₂₈₀/A₂₆₀ = 1.12) by heat treatment at 48 ± 0.1 °C and gel filtration through Sephadex G-200. Homogeneous enzyme preparation was immobilized in 3.5% alginate leading to 86% immobilization, no leaching of enzyme was found over a period of 15 days at 4 °C. Urease catalyzed urea hydrolysis by soluble and immobilized enzyme revealed a clear dependence on the concentration of Hg²⁺. Inhibition caused by Hg²⁺ was non-competitive (K_i = 1.2 × 10⁻¹ μM for soluble and 1.46 × 10⁻¹ μM for alginate immobilized urease.). Time-dependent inhibition both in presence and in absence of Hg²⁺ ion revealed a biphasic inhibition in activity. For optimization of this process response surface methodology (RSM) was utilized where two-level-two-full factorial (2²) central composite design (CCD) has been employed. The regression equation and analysis of variance (ANOVA) were obtained using MINITAB^® 15 software. Predicted values thus obtained were closed to experimental value indicating suitability of the model. 3D response surface plot, iso-response contour plot and process optimization curve were helpful to predict the results by performing only limited set of experiments.     

Transgenic Rice Plants Expressing a Modified <Emphasis Type="Italic">cry1Ca1</Emphasis> Gene are Resistant to <Emphasis Type="Italic">Spodoptera litura</Emphasis> and <Emphasis Type="Italic">Chilo</Emphasis><Emphasis Type="Italic">suppressalis</Emphasis>

Nucleotide sequence encoding the truncated insecticidal Cry1Ca1 protein from Bacillus thuringiensis was extensively modified based on the codon usage of rice genes. The overall G + C contents of the synthetic cry1Ca1 coding sequence were raised to 65% with an additional bias of enriching for G and C ending codons as preferred by monocots. The synthetic gene was introduced into the Chinese japonica variety, Xiushui 11, by Agrobacterium-mediated transformation. Transgenic rice plants harboring this gene were highly resistant to Chilo suppressalis and Spodoptera litura larvae as revealed by insect bioassays. High levels of Cry1Ca1 protein were obtained in the leaves of transgenic rice, which were effective in achieving 100% mortality of S. litura and C. suppressalis larvae. The levels of Cry1Ca1 expression in the leaves of these transgenic plants were up to 0.34% of the total soluble proteins. The larvae of C. suppressalis and S. litura could consume a maximum of 1.89  and 4.89 mm² of transgenic leaf area whereas the consumption of non-transgenic leaves by these larvae was significantly higher; 58.33 and 61.22 mm², respectively. Analysis of R₁ transgenic plants indicated that the cry1Ca1 was inherited by the progeny plants and provided complete protection against C. suppressalis and S. litura larvae.     

Synthesis, molecular docking and biological evaluation of metronidazole derivatives as potent Helicobacter pylori urease inhibitors

Fourteen metronidazole derivatives (compounds 3a–f and 4b–h) have been synthesized by coupling of metronidazole and salicylic acid derivatives. All of them are reported for the first time. Their chemical structures are characterized by ¹H NMR, MS, and elemental analysis. The inhibitory activities against Helicobacter pylori urease have been investigated in vitro and many compounds have showed promising potential inhibitory activities of H. pylori urease. The effect of compounds 4b (IC₅₀ = 26 μM) and 4g (IC₅₀ = 12 μM) was comparable with that of acetohydroxamic acid, a well known H. pylori urease inhibitor used as a positive control. The experimental values of IC₅₀ showed that inhibitor was potent urease inhibitor. A docking analysis using the autodock 4.0 program could explain the inhibitory activities of compound 4g against H. pylori urease.     

微生物酶法消除黄酒中氨基甲酸乙酯研究进展

氨基甲酸乙酯(Ethyl carbamate,EC)具有致癌性,广泛存在于酒精饮料中。我国的黄酒因EC含量高而带来的食品安全问题越来越受到人们的关注。微生物酶法消除黄酒中的EC具有直接、高效的特性而被深入研究。文中从黄酒中EC的形成机制、酸性脲酶研究现状、氨基甲酸乙酯水解酶研究现状等方面概述了微生物酶法消除黄酒中EC的研究进展及存在的问题。并针对这些问题,提出了寻找新型氨基甲酸乙酯水解酶、Fe~(3+)依赖型双功能酸性脲酶食品级表达与定向进化及双酶并用将尿素和EC一起消除的策略。     

Energy Demands of Nitrogen Supply in Mass Cultivation of Two Commercially Important Microalgal Species, <Emphasis Type="Italic">Chlorella vulgaris</Emphasis> and <Emphasis Type="Italic">Dunaliella tertiolecta</Emphasis>

Mass culture of microalgae is a potential alternative to cultivation of terrestrial crops for bioenergy production. However, microalgae require nitrogen fertiliser in quantities much higher than plants, and this has important consequences for the energy balance of these systems. The effect of nitrogen fertiliser supplied to microalgal bubble-column photobioreactor cultures was investigated using different nitrogen sources (nitrate, urea, ammonium) and culture conditions (air, 12% CO₂). In 20 L cultivations, maximum biomass productivity for Chlorella vulgaris cultivated using nitrate and urea was 0.046 and 0.053 g L⁻¹ day⁻¹, respectively. Maximum biomass productivity for Dunaliella tertiolecta cultivated using nitrate, urea and ammonium was 0.033, 0.038 and 0.038 g L⁻¹ day⁻¹, respectively. In intensive bubble-column photobioreactors using 12% CO₂, maximum productivity reached 0.60 and 0.83 g L⁻¹ day⁻¹ for C. vulgaris and D. tertiolecta, respectively. Recycling of nitrogen within the photobioreactor system via algal exudation of nitrogenous compounds and bacterial activity was identified as a potentially important process. The energetic penalty incurred by supply of artificial nitrogen fertilisers, phosphorus, power and CO₂ to microalgal photobioreactors was investigated, although analysis of all energy burdens from biomass production to usable energy carriers was not conducted. After subtraction of the power, nitrogen and phosphorus energy burdens, maximum net energy ratios for C. vulgaris and D. tertiolecta cultivated in bubble columns were 1.82 and 2.10. Assuming CO₂ was also required from a manufactured source, the net energy ratio decreased to 0.09 and 0.11 for C. vulgaris and D. tertiolecta, so that biomass production in this scenario was unsustainable. Although supply of nitrogen is unlikely to be the most energetically costly factor in sparged photobioreactor designs, it is still a very significant penalty. There is a need to optimise both cultivation strategies and recycling of nitrogen in order to improve performance. Data are supported by measurements including biochemical properties (lipid, protein, heating value) and bacterial number by epifluorescence microscopy.     

Identification and Characterization of Proteins Involved in Rice Urea and Arginine Catabolism

Rice (Oryza sativa) production relies strongly on nitrogen (N) fertilization with urea, but the proteins involved in rice urea metabolism have not yet been characterized. Coding sequences for rice arginase, urease, and the urease accessory proteins D (UreD), F (UreF), and G (UreG) involved in urease activation were identified and cloned. The functionality of urease and the urease accessory proteins was demonstrated by complementing corresponding Arabidopsis (Arabidopsis thaliana) mutants and by multiple transient coexpression of the rice proteins in Nicotiana benthamiana. Secondary structure models of rice (plant) UreD and UreF proteins revealed a possible functional conservation to bacterial orthologs, especially for UreF. Using amino-terminally StrepII-tagged urease accessory proteins, an interaction between rice UreD and urease could be shown. Prokaryotic and eukaryotic urease activation complexes seem conserved despite limited protein sequence conservation for UreF and UreD. In plant metabolism, urea is generated by the arginase reaction. Rice arginase was transiently expressed as a carboxyl-terminally StrepII-tagged fusion protein in N. benthamiana, purified, and biochemically characterized (K_m = 67 mm, k_cat = 490 s⁻¹). The activity depended on the presence of manganese (K_d = 1.3 μm). In physiological experiments, urease and arginase activities were not influenced by the external N source, but sole urea nutrition imbalanced the plant amino acid profile, leading to the accumulation of asparagine and glutamine in the roots. Our data indicate that reduced plant performance with urea as N source is not a direct result of insufficient urea metabolism but may in part be caused by an imbalance of N distribution.Nitrogen (N) availability often limits plant performance in natural ecosystems (Vitousek and Howarth, 1991), causing a selective pressure to optimize the use of N resources. This ecophysiological selection has even led to a reduction of the N content of plant proteins in comparison with animal orthologs (Elser et al., 2006). Because N is a limiting resource, plants do not only require efficient N uptake mechanisms but also possess enzymatic pathways for N remobilization.Arg is the most important single metabolite for N storage in plant seeds. In a survey of 379 plant species, Arg N accounted on average for 17.3% of total seed N (Vanetten et al., 1967). In several rice (Oryza sativa) varieties, values ranging from 16.1% to 17.1% were measured (Mosse et al., 1988). To access the N stored in the guanidinium group of Arg, it must first be hydrolyzed by mitochondrial arginase to Orn and urea. Urea leaves the mitochondria and is hydrolyzed by urease in the cytosol, releasing ammonia, which is reassimilated into amino acids by the combined action of Gln synthetase and Glu synthase.Urea not only originates from Arg breakdown but may also be taken up from the environment by urea transporters (Kojima et al., 2007; Wang et al., 2008). Therefore, urease is involved in N remobilization as well as in primary N assimilation. Plant ureases and arginases are housekeeping enzymes found in many if not all plant species (Witte and Medina-Escobar, 2001; Brownfield et al., 2008). Urease is a nickel metalloenzyme that in Arabidopsis (Arabidopsis thaliana) requires three urease accessory proteins (UAPs; AtUreD, AtUreF, and AtUreG) for activation (Witte et al., 2005a). Studies in bacteria demonstrated that UAPs form a complex with apo-urease and are required for posttranslational Lys carboxylation of apo-urease and the subsequent incorporation of two nickel ions into the active center. After activation, the UAPs dissociate from urease. The exact molecular function of each accessory protein in this process is not yet understood (Carter et al., 2009). Like urease, arginase is a metalloenzyme. It is best activated by manganese (Carvajal et al., 1996; Hwang et al., 2001), not requiring accessory proteins for activation.Urea plays an important role in agriculture because it is the most used N fertilizer worldwide (http://www.fertilizer.org/ifa), intensively employed in Asia for the cultivation of rice. Urea N partly reaches the plant as ammonium or nitrate because the fertilizer is already degraded in the environment by microbial ureases and may then be subject to nitrification. Alternatively, plants are capable of taking up urea from fertilization directly and assimilate its N (Kojima et al., 2007; Wang et al., 2008). Although rice is a major crop plant and rice production is heavily dependent on urea fertilization, the enzymes and the corresponding genes involved in rice urea metabolism have not yet been investigated. In this study, we identified the genes and cloned the corresponding cDNAs coding for rice arginase, urease, and the UAPs UreD, UreF, and UreG. The functionality of the corresponding proteins was demonstrated and biochemical parameters were determined. The general gene and protein structure of plant UreD and UreF were investigated and a direct interaction of rice UreD with apo-urease was discovered, leading to a refinement of the mechanistic view of plant urease activation. In physiological experiments, rice urease and arginase activities showed no significant response to different N-fertilizing regimes, while the amino acid composition in urea-grown plants was strongly imbalanced, indicating that urea N disturbs plant metabolism downstream of N assimilation.