Analysis of Maillard reaction precursors and secondary metabolites in Chilean potatoes and neoformed contaminants during frying.

Southern Chile native potatoes are an interesting raw material to produce novel snacks like colored potato chips. These novel products should be comprehensively evaluated for the presence of undesirable compounds such as acrylamide, 5-hydroxymethylfurfural and furan, the main neoformed contaminants in starchy rich fried foods. This study evaluated the neoformed contaminant levels and oil content on chips made from eleven Chilean potato accessions and compared them with commercial samples. The neoformed contaminant contents were related to Maillard reaction precursor levels (reducing sugars and asparagine) and secondary metabolites (phenolic compounds and carotenoids). Neoformed contaminants correlated well among them and were weakly correlated with reducing sugars and asparagine. Acrylamide level in native potato chips ranged from 738.2 to 1998.6 µg kg-1 while from 592.6 to 2390.5 µg kg-1 in commercial samples. Thus, there is need to implement neoformed contaminant mitigation strategies at different steps of the production chain of colored potato chips.

Chemical composition and sensory profiling of coffees treated with asparaginase to decrease acrylamide formation during roasting.

Acrylamide is an amide formed in the Maillard reaction, with asparagine as the primary amino acid precursor. The intake of large amounts of acrylamide has induced genotoxic and carcinogenic effects in hormone-sensitive tissues of animals. The enzime asparaginase is one of the most effective methods for lowering the formation of acrylamide in foods such as potatoes. However, the reported sensory outcomes for coffee have been unsatisfactory so far. This study aimed to produce coffees with reduced levels of acrylamide by treating them with asparaginase while retaining their original sensory and bioactive profiles. Three raw samples of Coffea arabica, including two specialty coffees, and one of Coffea canephora were treated with 1000, 2000, and 3000 ASNU of the enzyme. Asparagine and bioactive compounds (chlorogenic acids-CGA, caffeine, and trigonelline) were quantified in raw and roasted beans by HPLC and LC-MS, while the determination of acrylamide and volatile organic compounds was performed in roasted beans by CG-MS. Soluble solids, titratable acidity, and pH were also determined. Professional cupping by Q-graders and consumer sensory tests were also conducted. Results were analyzed by ANOVA-Fisher, MFA, PCA and Cluster analyses, with significance levels set at p ≤ 0.05. Steam treatment alone decreased acrylamide content by 18.4%, on average, and 6.1% in medium roasted arabica and canefora coffees. Average reductions of 32.5-56.0% in acrylamide formation were observed in medium roasted arabica beans when 1000-3000 ASNU were applied. In the canefora sample, 59.4-60.7% reductions were observed. However, steam treatment primarily caused 17.1-26.7% reduction of total CGA and lactones in medium roasted arabica samples and 13.9-22.0% in canefora sample, while changes in trigonelline, caffeine, and other evaluated chemical parameters, including the volatile profiles were minimal. Increasing enzyme loads slightly elevated acidity. The only sensory changes observed by Q-graders and or consumers in treated samples were a modest increase in acidity when 3000 ASNU was used in the sample with lower acidity, loss of mild off-notes in control samples, and increased perception of sensory descriptors. The former was selected given the similarity in chemical outcomes among beans treated with 2000 and 3000 ASNU loads.

Identifying the origin of acrylamide in Peruvian panela production to inform strategies for its reduction.

Maximum levels of acrylamide have been set by the European Commission (EU) 2017/2158 for several food products due to its carcinogenic properties. Although not regulated yet, European buyers are requesting maximum levels of 0.8 mg kg-1 in artisanal panela (raw cane sugar) from northern Peru. Panela in this area is produced by 600 small holder farmers and exportation guarantees a respectable price in an area with a high index of poverty. The objective here was to determine the cause of high acrylamide concentrations in panela to inform cost effective minimisation strategies. We monitored panela production from field to final product to understand the scale of the problem, identify the cause of acrylamide formation, as well as the effect of storage on its concentration. We also determined the utility of rapid kits for asparagine quantification. Our results indicate that high acrylamide levels are a widespread problem (85% of samples analysed) and there was a correlation between acrylamide and asparagine of R2 = 0.58 (p < 0.001), but not with any post-harvest processing variable. We estimate that with a concentration of asparagine of <0.1 g l-1 in sugarcane juice, the threshold set by buyers for acrylamide can be met. Potential solutions to reduce asparagine include varietal selection, improved agronomic practices and the use of asparaginase during panela production. However, any proposed measure should be applicable in the context of the rural Peru. Additionally, we confirm the utility of rapid and low-cost kits for measuring asparagine. This pioneering study provides a baseline for effective management for acrylamide minimization in panela.

Technological Prospecting: Mapping Patents on L-asparaginases from Extremophilic Microorganisms.

BACKGROUND: L-asparaginase (L-ASNase, L-asparagine amidohydrolase, E.C.3.5.1.1) is an enzyme with wide therapeutic applicability. Currently, the commercialized L-ASNase comes from mesophilic organisms, presenting low specificity to the substrate and limitations regarding thermostability and active pH range. Such factors prevent the maximum performance of the enzyme in different applications. Therefore, extremophilic organisms may represent important candidates for obtaining amidohydrolases with particular characteristics desired by the biotechnological market. OBJECTIVES: The present study aims to carry out a technological prospecting of patents related to the L-asparaginases derived from extremophilic organisms, contributing to pave the way for further rational investigation and application of such enzymes. METHODS: This patent literature review used six patents databases: The LENS, WIPO, EPO, USPTO, Patent Inspiration, and INPI. RESULTS: It was analyzed 2860 patents, and 14 were selected according to combinations of descriptors and study criteria. Approximately 57.14% of the patents refer to enzymes obtained from archaea, especially from the speciesPyrococcus yayanosii (35.71% of the totality). CONCLUSION: The present prospective study has singular relevance since there are no recent patent reviews for L-asparaginases, especially produced by extremophilic microorganisms. Although such enzymes have well-defined applications, corroborated by the patents compiled in this review, the most recent studies allude to new uses, such as the treatment of infections. The characterization of the catalytic profiles allows us to infer that there are potential sources still unexplored. Hence, the search for new L-ASNases with different characteristics will continue to grow in the coming years and, possibly, ramifications of the technological routes will be witnessed.

Effect of Fe (III) on L-asparagine monohydrate investigated under low- and high-temperature conditions.

Raman spectra of Fe-doped L-asparagine monohydrate (LAM:Fe) crystal were studied under several temperatures varying from 17 to 490 K. The effect of Fe (III) ion on the stability of the crystal in changing temperature through the vibrational spectra was discussed. The behavior of inter and intra-molecular vibration modes has indicated two phase transitions and an amorphous transformation. These effects were also clarified by X-ray powder diffraction measurements which corroborate very well the Raman data. In addition, we have determinated the lattice parameters of all phases and verified that under low temperature conditions the crystal undergoes a conformational transition whereas under high temperatures its structure transforms from the orthorhombic (P212121-space group) to the monoclinic (P21-space group) symmetry and, after this process, it goes to an amorphous phase due to the start of the decomposition. Finally, differential scanning calorimetry analysis was utilized as complementary technique to investigate the structural stability of LAM:Fe and results are in a good agreement with the Raman and the X-ray diffraction data.

Insertion of carbon nanotubes in Langmuir-Blodgett films of stearic acid and asparaginase enhancing the catalytic performance.

In this paper, carbon nanotubes (CNT) were adsorbed on stearic acid (SA) Langmuir monolayers to serve as matrices for the incorporation of asparaginase. The interaction between the components at the air-water interface was evaluated by surface pressure-area isotherms, surface potential-area isotherms, polarization-modulation reflection absorption infrared spectroscopy (PM-IRRAS), and Brewster angle microscopy (BAM). The enzyme expanded the monolayers and changed the thermodynamic and electrical properties of the SA-CNT monolayers, as detected with the isotherms. PM-IRRAS spectra showed that the enzyme keeps its secondary structure when adsorbed at the monolayers and also alters the morphology of the air-water interface, as identified with BAM. The hybrid floating films were transferred to solid supports through the Langmuir-Blodgett (LB) technique, and the cotransfer of the enzyme was confirmed with fluorescence spectroscopy. The catalytic activity of asparaginase in the LB films was studied with UV-vis spectroscopy, which showed that the presence of CNT in the enzyme-lipid LB film not only tuned the catalytic activity, but also helped conserve its enzyme activity after weeks, showing higher persisting values of activity. UV-vis spectroscopy also showed that the catalytic activity is dependent basically on the enzyme molecules present on the surface of the LB films since multilayer films did not provide a proportional increase of enzyme activity. These results are related to the synergism between the compounds on the active layer, leading to a molecular architecture that allowed the adequate molecular accommodation of the analyte with the catalytic sites of the enzyme, which also preserved the asparaginase activity. This work then demonstrates the feasibility of employing LB films composed of fatty acids, CNT, and enzymes as devices for biosensing applications.

Effect of temperature on the formation of acrylamide in cocoa beans during drying treatment: An experimental and computational study.

The aim of this work was to determine the effect of temperature on the formation of acrylamide in cocoa beans during drying treatment by an experimental and computational study, in order to assess the presence of this neoformed compound from postharvest stage. The computational study was conducted on the reaction between fructose, glyoxal from glucose, and on asparagine at the M06-2X/6-31+G(d,p) level, under cocoa bean drying conditions at 323.15 to 343.15 K. The proposed reaction for acrylamide formation consisted of seven steps, which required to progress a via cyclic transition state of the four members. In addition, step III (decarboxylation) was considered to be the rate-determining step. Glucose followed an E1-like elimination and fructose exhibited an E1cb-like elimination. Computational model showed that the reaction of acrylamide formation was favored by fructose rather than glucose. The content of reducing sugars, asparagine and acrylamide in fermented and dried cocoa from two subregions of Antioquia-Colombia, as well as roasted cocoa, were evaluated by UHPLC-C-CAD and UHPLC-QqQ. The concentrations of monosaccharides measured at the end of the fermentation and drying process of cocoa nibs showed greater decreases in the levels of fructose as compared to glucose, supporting the main model hypothesis. Acrylamide formation only occurred in Bajo Cauca due to the presence of both precursors and fast drying time (72 h). Finally, it was possible to find the conditions to which acrylamide can be formed from the drying process and not only from roasting, information that can be used for future control strategies.

Peach Fruit Development: A Comparative Proteomic Study Between Endocarp and Mesocarp at Very Early Stages Underpins the Main Differential Biochemical Processes Between These Tissues.

Peach (Prunus persica) is an important economically temperate fruit. The development follows double sigmoid curve with four phases (S1-S4). We centered our work in the early development. In addition to S1, we studied the very early stage (E) characterized by the lag zone of the exponential growing phase S1, and the second stage (S2) when the pit starts hardening. "Dixiland" peach fruit were collected at 9 (E), 29 (S1), and 53 (S2) days after flowering (DAF) and endocarp and mesocarp were separated. There was a pronounced decrease in total protein content along development in both tissues. Quantitative proteomic allowed the identification of changes in protein profiles across development and revealed the main biochemical pathways sustaining tissue differentiation. Protein metabolism was the category most represented among differentially proteins in all tissues and stages. The decrease in protein synthesis machinery observed during development would be responsible of the protein fall, rather than a proteolytic process; and reduced protein synthesis during early development would reroute cell resources to lignin biosynthesis. These changes were accompanied by net decrease in total amino acids in E1-S1 and increase in S1-S2 transitions. Amino acid profiling, showed Asn parallels this trend. Concerted changes in Asn and in enzymes involved in its metabolism reveal that increased synthesis and decreased catabolism of Asn may conduct to an Asn increase during very early development and that the ß-Cyano-Alanine synthase/ß-Cyano-Alanine hydratase could be the pathway for Asn synthesis in "Dixiland" peach fruit. Additionally, photosynthetic machinery decays during early development in mesocarp and endocarp. Proteins related to photosynthesis are found to a higher extent in mesocarp than in endocarp. We conclude mesocarpic photosynthesis is possible to occur early on the development, first providing both carbon and reductive power and latter only reductive power. Together with proteomic, histological tests and anatomical analysis help to provide information about changes and differences in cells and cell-walls in both tissues. Collectively, this work represents the first approach in building protein databases during peach fruit development focusing on endocarp and mesocarp tissues and provides novel insights into the biology of peach fruit development preceding pit hardening.

In-solution behavior and protective potential of asparagine synthetase A from Trypanosoma cruzi.

l-Asparagine synthetase (AS) acts in asparagine formation and can be classified into two families: AS-A or AS-B. AS-A is mainly found in prokaryotes and can synthetize asparagine from ammonia. Distinct from other eukaryotes, Trypanosoma cruzi produces an AS-A. AS-A from Trypanosoma cruzi (Tc-AS-A) differs from prokaryotic AS-A due to its ability to catalyze asparagine synthesis using both glutamine and ammonia as nitrogen sources. Regarding these peculiarities, this work uses several biophysical techniques to provide data concerning the Tc-AS-A in-solution behavior. Tc-AS-A was produced as a recombinant and purified by three chromatography steps. Circular dichroism, dynamic light scattering, and analytical size exclusion chromatography showed that Tc-AS-A has the same fold and quaternary arrangement of prokaryotic AS-A. Despite the tendency of protein to aggregate, stable dimers were obtained when solubilization occurred at pH ≤ 7.0. We also demonstrate the protective efficacy against T. cruzi infection in mice immunized with Tc-AS-A. Our results indicate that immunization with Tc-AS-A might confer partial protection to infective forms of T. cruzi in this particular model.

Avaliação da resistência de formas mutantes da enzima L-asparaginase a proteases séricas humanas / Evaluation of L-asparaginase mutant forms resistance to human serum proteases

O tratamento para a Leucemia Linfoblástica Aguda LLA utiliza, entre outros fármacos, a enzima L-asparaginase (ASNase) proveniente da bactéria Escherichia coli. Reações imunológicas estão entre os problemas do tratamento com ASNase, e a formação de anticorpos contra essa proteína pode impedir o sucesso no tratamento. Duas cisteíno proteases lisossomais estão relacionadas com a degradação de ASNase nos seres humanos, a Catepsina B (CTSB) e Asparagina Endopeptidase (AEP). Em estudos prévios do nosso grupo obteve-se mutantes de ASNase resistentes a degradação por CTSB e/ou AEP in vitro. Nesse trabalho avaliamos essas mutantes quanto a sua citotoxicidade em linhagens celulares de leucemia e conduzimos estudos in vivo, aplicando as proteoformas de ASNases em camundongos Balb C para avaliar a atividade asparaginase sérica das enzimas ao longo do tempo, bem como obter informações sobre a formação de anticorpos contra essas proteoformas. Nos ensaios de citotoxicidade, duas das proteoformas testadas tiveram efeito citotóxico semelhante a forma selvagem, enquanto uma outra proteoforma tem a citotoxicidade sensivelmente reduzida. Já nos ensaios in vivo, uma proteoforma demonstrou meia vida sérica maior da atividade asparaginásica, e duas proteoformas causaram reduzida formação de anticorpos. Juntos, esses resultados colaboram para a obtenção de uma nova geração de ASNases com melhor biodisponibilidade, e efeitos adversos reduzidos, gerando a possibilidade de menores doses e frequência de aplicações

The Treatment for Acute Lymphoblastic Leukemia (ALL) includes the biopharmaceutical L-asparaginase (ASNase) from Escherichia coli. Immunological reactions are among the problems of treatment using ASNase, and the antibodies formation protein may prevent success in treatment. Lysosomal cysteine proteases are related to ASNase degradation, Cathepsin B (CTSB) and Asparagine Endopeptidase (AEP). In previous studies, ASNase mutants resistant to CTSB and / or AEP degradation in vitro were obtained. In this work, mutants were evaluated in cytotoxicity in ALL cell lines and, in vivo studies, applying doses of the wild and mutant ASNases in Balb C mice to evaluate serum asparaginase activity of the enzymes over time, as well as to obtain information on the formation of antibodies against these proteoforms. Regarding to the cytotoxicity, two proteoforms among the tested had similar cytotoxicity than the wild-type. While another proteoform had the cytotoxicity severely reduced. One proteoform have demonstrated greater serum half-life of asparaginase activity, while two other mutants caused reduced antibody formation. Together, these results collaborate to obtain a new generation of ASNases with increased bioavailability and reduced side effects, generating the possibility of lower doses and frequency of applications

Trade-off between soluble protein production and nutritional storage in Bromeliaceae.

BACKGROUND AND AIMS: Bromeliads are able to occupy some of the most nutrient-poor environments especially because they possess absorptive leaf trichomes, leaves organized in rosettes, distinct photosynthetic pathways [C3, Crassulacean acid metabolism (CAM) or facultative C3-CAM], and may present an epiphytic habit. The more derived features related to these traits are described for the Tillandsioideae subfamily. In this context, the aims of this study were to evaluate how terrestrial predators contribute to the nutrition and performance of bromeliad species, subfamilies and ecophysiological types, whether these species differ in their ecophysiological traits and whether the physiological outcomes are consistent among subfamilies and types (e.g. presence/absence of tank, soil/tank/atmosphere source of nutrients, trichomes/roots access to nutrients). METHODS: Isotopic (15N-enriched predator faeces) and physiological methods (analyses of plant protein, amino acids, growth, leaf mass per area and total N incorporated) in greenhouse experiments were used to investigate the ecophysiological contrasts between Tillandsioideae and Bromelioideae, and among ecophysiological types when a predatory anuran contributes to their nutrition. KEY RESULTS: It was observed that Bromelioideae had higher concentrations of soluble protein and only one species grew more (Ananas bracteatus), while Tillandsioideae showed higher concentrations of total amino acids, asparagine and did not grow. The ecophysiological types that showed similar protein contents also had similar growth. Additionally, an ordination analysis showed that the subfamilies and ecophysiological types were discrepant considering the results of the total nitrogen incorporated from predators, soluble protein and asparagine concentrations, relative growth rate and leaf mass per area. CONCLUSIONS: Bromeliad subfamilies showed a trade-off between two strategies: Tillandsioideae stored nitrogen into amino acids possibly for transamination reactions during nutritional stress and did not grow, whereas Bromelioideae used nitrogen for soluble protein production for immediate utilization, possibly for fast growth. These results highlight that Bromeliaceae evolution may be directly associated with the ability to stock nutrients.

Klebsiella pneumoniae Asparagine tDNAs Are Integration Hotspots for Different Genomic Islands Encoding Microcin E492 Production Determinants and Other Putative Virulence Factors Present in Hypervirulent Strains.

Due to the developing of multi-resistant and invasive hypervirulent strains, Klebsiella pneumoniae has become one of the most urgent bacterial pathogen threats in the last years. Genomic comparison of a growing number of sequenced isolates has allowed the identification of putative virulence factors, proposed to be acquirable mainly through horizontal gene transfer. In particular, those related with synthesizing the antibacterial peptide microcin E492 (MccE492) and salmochelin siderophores were found to be highly prevalent among hypervirulent strains. The determinants for the production of both molecules were first reported as part of a 13-kbp segment of K. pneumoniae RYC492 chromosome, and were cloned and characterized in E. coli. However, the genomic context of this segment in K. pneumoniae remained uncharacterized. In this work, we provided experimental and bioinformatics evidence indicating that the MccE492 cluster is part of a highly conserved 23-kbp genomic island (GI) named GIE492, that was integrated in a specific asparagine-tRNA gene (asn-tDNA) and was found in a high proportion of isolates from liver abscesses sampled around the world. This element resulted to be unstable and its excision frequency increased after treating bacteria with mitomycin C and upon the overexpression of the island-encoded integrase. Besides the MccE492 genetic cluster, it invariably included an integrase-coding gene, at least seven protein-coding genes of unknown function, and a putative transfer origin that possibly allows this GI to be mobilized through conjugation. In addition, we analyzed the asn-tDNA loci of all the available K. pneumoniae assembled chromosomes to evaluate them as GI-integration sites. Remarkably, 73% of the strains harbored at least one GI integrated in one of the four asn-tDNA present in this species, confirming them as integration hotspots. Each of these tDNAs was occupied with different frequencies, although they were 100% identical. Also, we identified a total of 47 asn-tDNA-associated GIs that were classified into 12 groups of homology differing in theencoded functionalities but sharing with GIE492 a conserved recombination module and potentially its mobility features. Most of these GIs encoded factors with proven or potential role in pathogenesis, constituting a major reservoir of virulence factors in this species.

Optimization of asparaginase production from Zymomonas mobilis by continuous fermentation / Otimização da produção de asparaginase de Zymomonas mobilis por fermentação contínua

A asparaginase é uma enzima usada em tratamento clínico como agente quimioterapêutico e em tecnologia de alimentos na prevenção de formação de acrilamida em alimentos fritos e assados. Asparaginase é industrialmente produzida por micro-organismos, principalmente bactérias gram negativas. Zymomonas mobilis é uma bactéria gram negativa que utiliza glicose, frutose e sacarose como fonte de carbono e é conhecida por sua eficiência para produzir etanol, sorbitol, levana, ácido glicônico, e mais recentemente, tem despertado interesse no uso desse micro-organismo na produção de asparaginase. Este trabalho teve como objetivo otimizar a produção de asparaginase de Z. mobilis por fermentação contínua, pelo uso do delineamento experimental e da metodologia da superfície de resposta, testando as variáveis: sacarose, extrato de levedura e asparagina. A condição ótima alcançada, com produção de 117,45 UI L-1 foi na taxa de diluição 0,20 h-1, utilizando 0,5 g L-1 de extrato de levedura, 20 g L-1 de sacarose e 1,3 g L-1 de asparagina. Observou-se que a relação carbono:nitrogênio (1:0,025) exerceu forte influência na resposta da atividade de asparaginase. A utilização de Z. mobilis por fermentação contínua demonstrou ser uma alternativa promissora na produção biotecnológica da asparaginase.

Asparaginase is an enzyme used in clinical treatments as a chemotherapeutic agent and in food technology to prevent acrylamide formation in fried and baked foods. Asparaginase is industrially produced by microorganisms, mainly gram-negative bacteria. Zymomonas mobilis is a Gram-negative bacterium that utilizes glucose, fructose and sucrose as carbon source and has been known for its efficiency in producing ethanol, sorbitol, levan, gluconic acid and has recently aroused interest for asparaginase production. Current assay optimizes the production of Z. mobilis asparaginase by continuous fermentation using response surface experimental design and methodology. The studied variables comprised sucrose, yeast extract and asparagine. Optimized condition obtained 117.45 IU L-1 with dilution rate 0.20 h-1, yeast extract 0.5 g L-1, sucrose 20 g L-1 and asparagine 1.3 g L-1. Moreover, carbon:nitrogen ratio (1:0.025) strongly affected the response of asparaginase activity. The use of Z. mobilis by continuous fermentation has proved to be a promising alternative for the biotechnological production of asparaginase.

Flooding of the root system in soybean: biochemical and molecular aspects of N metabolism in the nodule during stress and recovery.

Nitrogen fixation of the nodule of soybean is highly sensitive to oxygen deficiency such as provoked by waterlogging of the root system. This study aimed to evaluate the effects of flooding on N metabolism in nodules of soybean. Flooding resulted in a marked decrease of asparagine (the most abundant amino acid) and a concomitant accumulation of γ-aminobutyric acid (GABA). Flooding also resulted in a strong reduction of the incorporation of (15)N2 in amino acids. Nodule amino acids labelled before flooding rapidly lost (15)N during flooding, except for GABA, which initially increased and declined slowly thereafter. Both nitrogenase activity and the expression of nifH and nifD genes were strongly decreased on flooding. Expression of the asparagine synthetase genes SAS1 and SAS2 was reduced, especially the former. Expression of genes encoding the enzyme glutamic acid decarboxylase (GAD1, GAD4, GAD5) was also strongly suppressed except for GAD2 which increased. Almost all changes observed during flooding were reversible after draining. Possible changes in asparagine and GABA metabolism that may explain the marked fluctuations of these amino acids during flooding are discussed. It is suggested that the accumulation of GABA has a storage role during flooding stress.

Optimization of asparaginase production from Zymomonas mobilis by continuous fermentation / Otimização da produção de asparaginase de Zymomonas mobilis por fermentação contínua

Asparaginase is an enzyme used in clinical treatments as a chemotherapeutic agent and in food technology to prevent acrylamide formation in fried and baked foods. Asparaginase is industrially produced by microorganisms, mainly gram-negative bacteria. Zymomonas mobilis is a Gram-negative bacterium that utilizes glucose, fructose and sucrose as carbon source and has been known for its efficiency in producing ethanol, sorbitol, levan, gluconic acid and has recently aroused interest for asparaginase production. Current assay optimizes the production of Z. mobilis asparaginase by continuous fermentation using response surface experimental design and methodology. The studied variables comprised sucrose, yeast extract and asparagine. Optimized condition obtained 117.45 IU L-1 with dilution rate 0.20 h-1, yeast extract 0.5 g L-1, sucrose 20 g L-1 and asparagine 1.3 g L-1. Moreover, carbon:nitrogen ratio (1:0.025) strongly affected the response of asparaginase activity. The use of Z. mobilis by continuous fermentation has proved to be a promising alternative for the biotechnological production of asparaginase.(AU)

A asparaginase é uma enzima usada em tratamento clínico como agente quimioterapêutico e em tecnologia de alimentos na prevenção de formação de acrilamida em alimentos fritos e assados. Asparaginase é industrialmente produzida por micro-organismos, principalmente bactérias gram negativas. Zymomonas mobilis é uma bactéria gram negativa que utiliza glicose, frutose e sacarose como fonte de carbono e é conhecida por sua eficiência para produzir etanol, sorbitol, levana, ácido glicônico, e mais recentemente, tem despertado interesse no uso desse micro-organismo na produção de asparaginase. Este trabalho teve como objetivo otimizar a produção de asparaginase de Z. mobilis por fermentação contínua, pelo uso do delineamento experimental e da metodologia da superfície de resposta, testando as variáveis: sacarose, extrato de levedura e asparagina. A condição ótima alcançada, com produção de 117,45 UI L-1 foi na taxa de diluição 0,20 h-1, utilizando 0,5 g L-1 de extrato de levedura, 20 g L-1 de sacarose e 1,3 g L-1 de asparagina. Observou-se que a relação carbono:nitrogênio (1:0,025) exerceu forte influência na resposta da atividade de asparaginase. A utilização de Z. mobilis por fermentação contínua demonstrou ser uma alternativa promissora na produção biotecnológica da asparaginase.(AU)

Thermodynamic study of asparagine and glycyl-asparagine using computational methods

This work aimed to develop an ab initio procedure for accurately calculating pKa values and applied it to study the acidity of asparagine and glycyl-asparagine. DFT methods with B3LYP composed by 6-31+G(d) basis set were applied for calculating the acidic dissociation constant of asparagine and glycyl-asparagine. The formation of intermolecular hydrogen bonds between the available species and water was analyzed using Tomasi,s method. Results showed that in alkaline solutions, the cation, anion and neutral species of asparagine and glycyl-asparagine were solvated with one, two, three and four molecules of water, respectively. There was an excellent similarity between the experimentally attained pKa values and the theoretically ones in this work.

Effect of salt on the growth and metabolism of Glycine max

Soybean plants cultivated with 50, 100 and 200 mM of NaCl, revealed that root growth was less affected by salinity than shoots. Salinity led to a reduction in leaf area and an increase in water content of the roots. These factors could contribute to the adaptation of the plant, improving its hydration. Although nitrate and free amino acid levels were reduced by salt treatment in roots, protein content of leaves was not altered. Salinity led to alterations in xylem amino acid composition, with increases in Ser, Ala, Gaba and Pro and a decrease in Asn. Similar changes were seen for Asn and Ser in roots together with a much stronger increase in Gaba. It is suggested that the decline in Asn reflects its conversion to Ala and Gaba (via Glu) in the roots while the increase in Pro and Gaba could be related to the adaptation of the plant to salinity.

Xylem sap nitrogen compounds of some Crotalaria species

Thirteen species of Crotalaria were analysed for nitrogen compounds in the xylem root bleeding sap. Amino acids were the main form of organic nitrogen found, but only traces of ureides were present. Of the four species analysed for amino acid composition, asparagine was found to be the major amino acid, accounting for over 68% of the nitrogen transported. No striking deviations from this general pattern was found between species, between vegetative and floral stages of development, or between nodulated and non-nodulated plants. It was concluded that the Crotalaria species studied here have an asparagine-based nitrogen metabolism, consistent with many other non-ureide-producing legume species.

Treze espécies de Crotalaria foram analisadas quanto aos compostos nitrogenados presentes na seiva do xilema. Os aminoácidos foram os principais compostos nitrogenados encontrados, e apenas traços de ureídeos estavam presentes. Uma análise da composição de aminoácidos realizada para quatro espécies revelou que a asparagina é o aminoácido predominante, representando mais de 68% do nitrogênio transportado. Nenhum desvio marcante deste padrão foi encontrado entre espécies, entre plantas noduladas e não-noduladas e nem entre estádios florais e vegetativos. Chegou-se a conclusão de que as espécies aqui estudadas possuem metabolismo de nitrogênio baseado na asparagina, concordando com dados da literatura para muitas outras espécies de leguminosas que pertencem à categoria de não-produtoras de ureídeos.

Xylem sap nitrogen compounds of some Crotalaria species

Thirteen species of Crotalaria were analysed for nitrogen compounds in the xylem root bleeding sap. Amino acids were the main form of organic nitrogen found, but only traces of ureides were present. Of the four species analysed for amino acid composition, asparagine was found to be the major amino acid, accounting for over 68% of the nitrogen transported. No striking deviations from this general pattern was found between species, between vegetative and floral stages of development, or between nodulated and non-nodulated plants. It was concluded that the Crotalaria species studied here have an asparagine-based nitrogen metabolism, consistent with many other non-ureide-producing legume species.

Treze espécies de Crotalaria foram analisadas quanto aos compostos nitrogenados presentes na seiva do xilema. Os aminoácidos foram os principais compostos nitrogenados encontrados, e apenas traços de ureídeos estavam presentes. Uma análise da composição de aminoácidos realizada para quatro espécies revelou que a asparagina é o aminoácido predominante, representando mais de 68% do nitrogênio transportado. Nenhum desvio marcante deste padrão foi encontrado entre espécies, entre plantas noduladas e não-noduladas e nem entre estádios florais e vegetativos. Chegou-se a conclusão de que as espécies aqui estudadas possuem metabolismo de nitrogênio baseado na asparagina, concordando com dados da literatura para muitas outras espécies de leguminosas que pertencem à categoria de não-produtoras de ureídeos.