Javier D. Breccia

Polyethyleneimine–protein interactions and implications on protein stability

Protein stability assessment of seven model proteins in the presence of low molecular weight polyethyleneimine (PEI, MW 2000 Da) was performed. Thermodynamic stability, monitored by circular dichroism (CD) spectroscopy, showed that the polymer did not have a major effect on the melting temperature (T(m)) of the basic proteins - muscle lactate dehydrogenase (LDH), ribonuclease A, lysozyme and cutinase, while for the acidic ones - human growth hormone, human serum albumin and heart lactate dehydrogenase - there was a shift in T(m) towards lower temperatures. The secondary structures of the basic proteins were essentially the same, with none or a slight increase in the CD spectra, in presence of the polymer. In the case of the acidic proteins, the CD spectra were diminished mostly due to phase separation. Assuming a homogeneous distribution of the net charge on the protein surface a quantitative inverse relationship was established between surface charge density of the acidic proteins and the PEI(2000) concentration required for maximum flocculation. Despite lowering the thermal stability of acidic proteins, PEI(2000) was seen to protect heart LDH at an increasing oxidative stress.

Isolation and molecular characterization of Shewanella sp. G5, a producer of cold-active β -D-glucosidases

β ‐Glucosidase is a highly desired glycosidase, especially for hydrolysis of glycoconjugated precursors in musts and wines for the release of active aromatic compounds. A Shewanella sp. G5 strain was isolated from the intestinal content of benthonic organism (Munida subrrugosa) from different coastal areas of the Beagle Channel, Tierra del Fuego (Argentina). This marine bacterium was able to grow at a temperature range between 4 to 20 °C using different β ‐glycoside substrates, such as cellobiose, as carbon source. In this work, the Shewanella sp. G5 strain exhibited high β ‐glucosidase activity on plate at low temperature (4 and 20 °C). Two genes encoding different cold‐active β ‐glucosidases were amplified and sequenced and the nucleotide sequences were submitted to the GenBank. 16S rDNA and gyrB gene sequences were used for the molecular characterization of Shewanella sp. G5. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Clearance of Tritrichomonas foetus in experimentally infected heifers protected with vaccines based on killed-T. foetus with different adjuvants

Tritrichomonas foetus is a flagellated protozoan that causes a sexually transmitted disease in cattle. Trichomonosis is characterized by early abortions, subfertility and a significant decrease in productivity. Vaccine preparations containing whole T. foetus can reduce the time of residence of the pathogen in the host cervix after experimental infection. Here, T. foetus vaccines prepared with different adjuvants were tested, in parallel with a commercial vaccine, for their efficacy to clear the infection. The median time for clearance of infection was 69days in non-immunized animals, 55days in animals treated with aluminum hydroxide, 41days with oil-in-water or saponin based vaccines or with a commercial vaccine and 27days in animals treated with saponin plus aluminum hydroxide. A slight increase in the risk of T. foetus clearance from the genital tract was found with the saponin based vaccine (hazard ratio, 2.52; 95% confidence interval, 1.03-6.17) or the commercial vaccine (hazard ratio, 2.61; 95% confidence interval, 1.07-6.38). A significant increase in the risk of T. foetus clearance was found with the combination of saponin plus aluminum hydroxide based vaccine (hazard ratio, 5.12; 95% confidence interval, 2.04-12.83).

Screening and quantification of the enzymatic deglycosylation of the plant flavonoid rutin by UV–visible spectrometry

The enzymatic deglycosylation of the plant flavonoid rutin (quercetin-3-O-(6-O-α-l-rhamnopyranosyl-β-d-glucopyranoside) is usually assessed by means of high performance liquid chromatography (HPLC). We have developed a spectrophotometric method for the quantification of the released quercetin. After the enzymatic reaction, quercetin is extracted with ethyl acetate, and subsequently oxidized under basic conditions. The absorbance of quercetin autooxidation products at 320nm was correlated with the quercetin concentration by linear regression (molar extinction coefficient 23.2 (±0.3)×103M-1cm-1). With this method, rutin-deglycosylation activity in buckwheat flour and a commercial naringinase was measured, and showed no significant differences with the results obtained by HPLC. The convenience of this method resides on the enzymatic activity quantification using the natural substrate by UV-visible spectrometry. Moreover, the simplicity and speed of analysis allows its application for a large number of samples.

Experimental infection of rabbits (Oryctolagus cuniculus) with Brucella suis biovar 1 isolated from wild hares (Lepus europaeus)

Brucella suis biovar 1 is the causative agent of brucellosis in several domestic and wild animals and it is a common agent of human brucellosis. European hares (Lepus europaeus) have been shown to be infected by B. suis biovar 1 and the transmission to other animals has been suggested. In this work, experimental rabbits (Cuniculus orictolagus) were infected with B. suis biovar 1 isolated from wild hares. Infected rabbits showed high serological response in 2 weeks after discharge and typical granulomatous lesions (2mm diameter) were found in liver, spleen and kidneys after 50 days. B. suis biovar 1 was cultured from the lesion of the organs mentioned above as well as from urine, placenta and fetuses. These data suggest that hares are a potential source for horizontal transmission of B. suis biovar 1 to other mammalians.

Quantitative relationship between maximum growth rates and the intracellular pattern of α-esterase and β-esterase activity of leguminous infecting bacteria.

Sixteen strains belonging to three families of the Rhizobiales order (Bradyrhizobiaceae, Phyllobacteriaceae and Rhizobiaceae) were evaluated according their specific growth rates (micro) and the activity of intracellular alpha-esterase and beta-esterase isoenzymes. The average esterase activity of 48 isoenzymes assayed belonging to five strains with low (micro(max) = 0.08-0.12 h(-1)), four medium (micro(max) = 0.13-0.22 h(-1)) and seven high (micro(max) = 0.24-0.28 h(-1)) growth rate values were 22.1 +/- 4.3; 8.7 +/- 2.2 and 3.9 +/-1.7 U g(-1) respectively. An inversely proportional relationship between the activity of the whole pattern of esterases and micro(max) was found. Our results illustrate a feature of intracellular esterases, ascribable in a variety of cellular functions, which might be related to characteristics micro(max) of legume infecting bacteria.

Modelling of an Industrial Biotransformation Process for Tryptophan Production

Abstract For an industrial biotransformation process a simple mathematical model has been developed. During the process indole and serine is converted to tryptophan using the enzyme tryptophan synthase, which is supplied in E. coli cells. The tryptophan production has been described by a first order kinetic with respect to serine and the relative cell concentration as well as zero order kinetics with respect to indole. The 10.000 L reactor, in which the production is performed, is described as an ideal stirred tank reactor. The activity of the cell suspension, i. e. the activity of the tryptophan synthase, which was considered in one of the model parameter could be determined from fluorescence measurements (fluorescence intensity at 350 run excitation and 470 nm emission wavelength) using a linear regression model. The model prediction corresponds very well with off-line measurement

Enzyme-mediated transglycosylation of rutinose (6-O-α-l-rhamnosyl-d-glucose) to phenolic compounds by a diglycosidase from Acremonium sp. DSM 24697: Phenolic-Rutinoside Synthesis

The structure of the carbohydrate moiety of a natural phenolic glycoside can have a significant effect on the molecular interactions and physicochemical and pharmacokinetic properties of the entire compound, which may include anti‐inflammatory and anticancer activities. The enzyme 6‐O‐α‐rhamnosyl‐β‐glucosidase (EC 3.2.1.168) has the capacity to transfer the rutinosyl moiety (6‐O‐α‐l‐rhamnopyranosyl‐β‐d‐glucopyranose) from 7‐O‐rutinosylated flavonoids to hydroxylated organic compounds. This transglycosylation reaction was optimized using hydroquinone (HQ) and hesperidin as rutinose acceptor and donor, respectively. Since HQ undergoes oxidation in a neutral to alkaline aqueous environment, the transglycosylation process was carried out at pH values ≤6.0. The structure of 4‐hydroxyphenyl‐β‐rutinoside was confirmed by NMR, that is, a single glycosylated product with a free hydroxyl group was formed. The highest yield of 4‐hydroxyphenyl‐β‐rutinoside (38%, regarding hesperidin) was achieved in a 2‐h process at pH 5.0 and 30 °C, with 36 mM OH‐acceptor and 5% (v/v) cosolvent. Under the same conditions, the enzyme synthesized glycoconjugates of various phenolic compounds (phloroglucinol, resorcinol, pyrogallol, catechol), with yields between 12% and 28% and an apparent direct linear relationship between the yield and the pKa value of the aglycon. This work is a contribution to the development of convenient and sustainable processes for the glycosylation of small phenolic compounds.