543 research outputs found

    Whole-Cell Bioconversion of Renewable Biomasses-Related Aromatics to cis,cis-Muconic Acid

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    Lignin and wheat bran represent renewable feedstocks for generation of useful and value-added compounds such as vanillin (a popular flavoring agent) and cis,cis-muconic acid (ccMA, a building block for the synthesis of plastic materials). In the present work, we report on the setup of an efficient and green process for producing such valuable compounds based on (a) the optimization of the extraction procedures for vanillin from lignin and ferulic acid from wheat bran and (b) the genetic engineering of an Escherichia coli strain with up to three plasmids differing in copy numbers to modulate the expression of up to seven recombinant enzymes. In detail, we used two sequential reactions catalyzed by the decarboxylase Fdc and the dioxygenase Ado to convert wheat bran-derived ferulic acid into vanillin: nature-identical vanillin was produced in one pot with a >85% yield in 20 h. Next, the dehydrogenase LigV, the demethylase VanAB, the decarboxylase AroY, and the dioxygenase C12O converted lignin-derived vanillin into ccMA with a >95% conversion yield and a productivity of 4.2 mg of ccMA/g of Kraft lignin in 30 min. Finally, when the optimized E. coli strain expressing all the abovementioned enzymes was used, ccMA was produced with a >95% conversion yield starting from ferulic acid in 10 h following product isolation, corresponding to 0.73 g of ccMA/g of ferulic acid, 1.4 g of ccMA/L, and 2.2 g of ccMA/g of wheat bran biomass. The optimized whole-cell system represents a sustainable and cost-competitive process for producing high value-added products from renewable resources

    Practical use of blactchford score

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    Aim: To retrospectively evaluate the use of proton pump inhibitor infusions. Methods: A retrospective chart review was conducted of all patients who received an 80 mg bolus of esomeprazole follow by an infusion at 8 mg/hr. An analysis was performed to determine if a Blatchford Score of \u3e 5 was a predictor of upper gastrointestinal ulcers. Results: 300 patients received high dose esomeprazole over a 15 month period. 32% had an ulcer identified on endoscopy. Gastritis and esophagitis were the second most common diagnosis accounting for 16% of patients. A Blatchford Score of \u3e5 as a predictor of upper gastrointestinal ulcers had a sensitivity of 86.5% with a specificity of 32%. Conclusion: Utilizing the Blatchford Score to predict patients that have an upper gastrointestinal ulcer does not appear to be effective in clinical practice

    Antibacterial Properties of D-Amino Acid Oxidase: Impact on the Food Industry

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    Food quality is also related to safety and prevention of spoilage. Biological antimicrobial agents represent suitable alternatives to clinical preservatives in food industry to increase both safety and stability of aliments. Here, we focused on the enzyme D-amino acid oxidase (DAAO) from the yeast Rhodotorula gracilis, a well-studied protein for biotechnological use based on its stability, high activity, and easy recombinant production. DAAO catalyzes the O-2-dependent oxidative deamination of D-enantiomer of amino acids generating alpha -keto acids, ammonia, and hydrogen peroxide. DAAO shows antibacterial activity on both Gram-positive and Gram-negative bacteria in the presence of D-alanine when tested on plates and reduced by half their growth when tested on liquid cultures. Control experiments performed with alternative amino acid-specific flavoenzymes (able or not to generate H2O2 acting on amino acids), a DAAO inactive variant, catalase (H2O2 scavenger), and L-amino acids instead of D-alanine identified H2O2 as the antibacterial agent. DAAO showed a good ability to decrease the bacterial growth on various food stuffs: e.g., 10-fold less colonies were formed on grated cheese incubated for 16 h at 37 degrees C when a tiny amount (0.01 mg corresponding to 1.2 units) of DAAO was added. No exogenous D-amino acids were added since DAAO used the ones naturally occurring or the ones generated during ripening. Notably, simultaneously to H2O2 generation, DAAO also acts as O-2-scavenger thus further hampering food deterioration

    Modulating D-amino acid oxidase substrate specificity: production of an enzyme for analytical determination of all D-amino acids by directed evolution

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    Recent research on the flavoenzyme D-amino acid oxidase from Rhodotorula gracilis (RgDAAO) has revealed new, intriguing properties of this catalyst and offers novel biotechnological applications. Among them, the reaction of RgDAAO has been exploited in the analytical determination of the D-amino acid content in biological samples. However, because the enzyme does not oxidize acidic D-amino acids, it cannot be used to detect the total amount of D-amino acids. We now present the results obtained using a random mutagenesis approach to produce RgDAAO mutants with a broader substrate specificity. The libraries of RgDAAO mutants were generated by error-prone PCR, expressed in BL21(DE3)pLysS Escherichia coli cells and screened for their ability to oxidize different substrates by means of an activity assay. Five random mutants that have a 'modified' substrate specificity, more useful for the analytical determination of the entire content of D-amino acids than wild-type RgDAAO, have been isolated. With the only exception of Y223 and G199, none of the effective amino acid substitutions lie in segments predicted to interact directly with the bound substrate. The substitutions appear to cluster on the protein surface: it would not have been possible to predict that these substitutions would enhance DAAO activity. We can only conclude that these substitutions synergistically generate small structural changes that affect the dynamics and/or stability of the protein in a way that enhances substrate binding or subsequently catalytic turnover

    Treatment of malignant hypercalcaemia with aminohexane bisphosphonate (neridronate).

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    Twenty patients with hypercalcaemia due to malignancy, which persisted following rehydration, were treated with the bisphosphonate, aminohexane bisphosphonate (AHBP), which is structurally similar to pamidronate. The treatment given was a single infusion of 125 mg of AHBP in 500 ml of normal saline infused over 4 h. Serum and urine biochemistry were measured before and after treatment. Acute toxicity was evaluated with particular attention to gastrointestinal symptoms, acute-phase reaction and change in renal function, as judged by serum creatinine. The infusion of AHBP induced a rapid fall apparent by day 3 (P < 0.001), with a nadir at day 7. The serum calcium remained lower at days 14 and 28 than at day 0, but the numbers followed up were low (n = 5 and n = 4). In all 20 patients there was a fall in serum calcium after treatment, and in 13 (65%) normocalcaemia was achieved. Failure to respond completely to AHBP appeared to be associated with a renal mechanism of hypercalcaemia. Treatment was associated with a significant decrease in fasting urinary calcium excretion (P < 0.05). There was no change in white cell count or renal function following AHBP and only two cases of mild pyrexia after infusion. We conclude that aminohexane bisphosphonate is an effective agent in the treatment of tumour-induced hypercalcaemia, with rapid onset of effect and low toxicity

    Commercial hybrid yield of green asparagus (Asparagus officinalis L. var. altilis) processed in two spear-lengths, during the stable productivity stage, in the Province of Buenos Aires, Argentina

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    The perennial crop asparagus reaches maximum yield in its fifth season, exhibits marked genotype-environment interaction and requires productivity evaluation of its hybrids. To determine the behavior of green asparagus genotypes, a randomized complete block design trial (begun 16/11/11, density 23810 pl ha-1, area 1690 m2) was carried out in Azul (36°48’ S, lat. 59°51’ W, long.) within the framework of the ISHS’s Fourth International Asparagus Cultivar Trial, in which the following hybrids were harvested between 5/8/17 and 26/10/17 and evaluated for agronomic performance: ‘Italo’, ‘Vittorio’, ‘Eros’, ‘Ercole’, ‘Giove’, ‘Franco’, ‘Chino’, ‘Early-California’, ‘UC-157’, ‘Patrón’, ‘NJ-1189’, ‘NJ-1123’ and ‘NJ-1192’. Their response to pre-harvest diammonium phosphate (DAP) fertilisation was also evaluated (100 kg ha-1 vs. control). Harvested spears were cut, conditioned to two lengths (long 22 cm, short 17 cm), weighed, counted, washed and calibrated. The following characters were evaluated: total fresh commercial productivity (TFP) and that of long and short commercial spears (CFP-L and CFP-S); total commercial spear number (TSN) and that of long and short commercial spears (CSN-L and CSN-S); mean spear weight (MSW); calibre distribution (CD): (J: Jumbo; XL: Extra-Large; L: Large; M: Medium; S: Small and A: Asparagine). ANOVA-LSD test P≥0.05 was employed. DAP fertilisation raised yield by 3-10%, though not significantly. The following mean values were obtained: TFP: 8678.46 kg ha-1; TSN: 440298 spears ha-1; MSW: 20 g; CFP-L: 3109; CFP-S: 1945 kg ha-1; CSN-L: 165857 and CSN-S: 130467 spears ha-1. The following hybrids performed well: for CFP-L: ‘Vittorio’: 4379(a); ‘Franco’: 4033(ab); ‘NJ-1123’: 3849(abc); for CFP-C: ‘NJ-1123’: 2726(a); ‘Ercole’: 2483(ab); ‘Vittorio’: 2325(abc); for CSN-L: ‘NJ-1123’: 225260(a); ‘Franco’: 214161(a); ‘Vittorio’: 204358(ab); ‘Giove’: 200229(abc); for CSN-C: ‘NJ-1123’: 2725(a); ‘Ercole’: 2482(ab); ‘Vittorio’: 2325(abc); for CD: J: ‘UC-157’: 12767(a); ‘Eros’: 9882(ab); ‘NJ-1123’: 9005(abc); for XL: ‘NJ-1123’: 41124(a); ‘Franco’: 38958(ab); ‘NJ-1192’: 37644(ab); for L: ‘Ercole’: 85083(a); ‘NJ-1123’: 84700(a); ‘Franco’: 83872(a); ‘Italo’: 83095(a); for M: ‘NJ-1123’: 118198(a); ‘Franco’: 116369(a); ‘Vittorio’: 109645(ab); ‘Ercole’: 105984(ab); for S: ‘Giove’: 102748(a); ‘NJ-1123’: 80530(ab). In summary, ‘NJ-1123’ would be chosen for spear number productivity and ‘Vittorio’ and ‘Franco’ for yield.Fil: Castagnino, Ana Maria. Cresca-faa-unicen; Argentina. Asaho; Argentina. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Agronomía. Centro Regional de Estudios Sistémico de Cadenas Agroalimentarias; ArgentinaFil: Diaz, K. E.. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Agronomía. Centro Regional de Estudios Sistémico de Cadenas Agroalimentarias; ArgentinaFil: Rosini, M. B.. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Agronomía. Centro Regional de Estudios Sistémico de Cadenas Agroalimentarias; ArgentinaFil: Guisolis, Andrea Paola. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Agronomía. Centro Regional de Estudios Sistémico de Cadenas Agroalimentarias; ArgentinaFil: Dussi, M. C.. Universidad Nacional del Comahue; ArgentinaFil: Rogers, William John. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Agronomía. Centro Regional de Estudios Sistémico de Cadenas Agroalimentarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones en Biodiversidad y Biotecnología; Argentin
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