Production of Galactose Oxidase Inside the Fusarium fujikuroi Species Complex and Recombinant Expression and Characterization of the Galactose Oxidase GaoA Protein from Fusarium subglutinans.

Galactose oxidase catalyzes a two-electron oxidation, mainly from the C6 hydroxyl group of D-galactose, with the concomitant reduction of water to hydrogen peroxide. This enzyme is secreted by Fusarium species and has several biotechnological applications. In this study, a screening of galactose oxidase production among species of the Fusarium fujikuroi species complex demonstrated Fusarium subglutinans to be the main producer. The truncated F. subglutinans gaoA gene coding for the mature galactose oxidase was expressed from the prokaryotic vector pTrcHis2B in the E. coli Rosetta™ (DE3) strain. The purified recombinant enzyme presented temperature and pH optima of 30 °C and 7.0, respectively, KM of 132.6 ± 18.18 mM, Vmax of 3.2 ± 0.18 µmol of H2O2/min, kcat of 12,243 s-1, and a catalytic efficiency (kcat/KM) of 9.2 × 104 M-1 s-1. In the presence of 50% glycerol, the enzyme showed a T50 of 59.77 °C and was stable for several hours at pH 8.0 and 4 °C. Besides D-(+)-galactose, the purified enzyme also acted against D-(+)-raffinose, α-D-(+)-melibiose, and methyl-α-D-galactopyranoside, and was strongly inhibited by SDS. Although the F. subglutinans gaoA gene was successfully expressed in E. coli, its endogenous transcription was not confirmed by RT-PCR.

Changes in soymilk during fermentation with kefir culture: oligosaccharides hydrolysis and isoflavone aglycone production.

The objective of this study was to evaluate the changes in oligosaccharides and isoflavone aglycone content in soymilk during fermentation with commercial kefir culture. Soymilk was fermented with kefir culture at 25 °C for 30 h. The counts of lactic acid bacteria, Lactococcus lactis, Leuconostoc sp and yeasts; measurements of pH, acidity, α-galactosidase and ß-glucosidase activity, sugar and isoflavone contents were performed at the intervals of time. In the fermented soymilk, the lactic acid bacteria counts increased from 7.6 log to 9.1 CFU g(-1), pH reached to 4.9 and lactic acid reached 0.34 g 100 g(- 1). The α-galactosidase was produced (0.016 AU g(-1)) with 100% raffinose and 92% stachyose hydrolysis being observed after the depletion of galactose, glucose and sucrose. Kefir culture produced ß-glucosidase (0.0164 AU g(-1)), resulting in 100% bioconversion of glycitin and daidzin and 89% bioconversion of genistin into the corresponding aglycones. The fermented soymilk presented 1.67 µmol g(-1) of daidzein, 0.28 µmol g(-1) of glicitein and 1.67 µmol g (-1) of genistein.

Reduction of soybean meal non-starch polysaccharides and α-galactosides by solid-state fermentation using cellulolytic bacteria obtained from different environments.

Soybean meal (SBM) is an important protein source in animal feed. However, the levels of SBM inclusion are restricted in some animal species by the presence of antinutritional factors (ANFs), including non-starch polysaccharides (NSPs) and α-galactosides (GOSs). The aim of this study was to reduce the soybean meal NSPs and GOSs by solid-state fermentation (SSF) using a combination of cellulolytic bacteria isolated from different environments (termites, earthworms, corn silage and bovine ruminal content). To analyse the key enzymatic activities, the isolates were grown in minimal media containing NSPs extracted from SBM. The selected bacterial strains belonged to the genera Streptomyces, Cohnella and Cellulosimicrobium. SSF resulted in a reduction of nearly 24% in the total NSPs, 83% of stachyose and 69% of raffinose and an increase in the protein content. These results suggest that cellulolytic bacteria-based SSF processing facilitates SBM nutritional improvement. In addition, the use of fermented SBM in animal diets can be recommended.

Glass transition and time-dependent crystallization behavior of dehydration bioprotectant sugars.

It has been suggested that the crystallization of a sugar hydrate can provide additional desiccation by removing water from the amorphous phase, thereby increasing the glass transition temperature (T(g)). However, present experiments demonstrated that in single sugar systems, if relative humidity is enough for sugar crystallization, the amorphous phase will have a short life. In the conditions of the present experiments, more than 75% of amorphous phase crystallized in less than one month. The good performance of sugars that form hydrated crystals (trehalose and raffinose) as bioprotectants in dehydrated systems is related to the high amount of water needed to form crystals, but not to the decreased water content or increased T(g) of the amorphous phase. The latter effect is only temporary, and presumably shorter than the expected shelf life of pharmaceuticals or food ingredients, and is related to thermodynamic reasons: if there is enough water for the crystal to form, it will readily form.

The effect of rewarming media composition on the ammonia detoxification ability of cold preserved rat hepatocytes.

We examined how different media composition of rewarming solutions affected ammonium detoxification function, urea synthesis and the viability of hepatocytes after 72 hs of cold storage in UW solution. Freshly isolated rat hepatocytes were incubated at 37 C in a cell culture medium (MEM-E) with 3 mM glycine, 5 mM fructose and 2.5 mM adenosine (group 1) and in Krebs-Heinseleit buffer with 3 mM glycine, 5 mM fructose, 2 mM ornithine, 10 mM lactate and adenosine, that was used in two different concentrations: 2.5 mM (group 2) and 10 mM (group 3). We found that freshly isolated cells produced ammonium in group 1 and 2 but the cells were able to diminish ammonium extracellular concentration in group 3. Urea synthesis and ammonium extracellular concentration in group 1 was higher than in group 2. As a result of this observations, we used the Krebs-Heinseleit solution with addition of 10 mM adenosine to determinate the effect of hypothermic preservation on ammonium detoxification and urea synthesis ability of cells. In conclusion the addition of 2.5 mM adenosine into the rewarming medium interfered with the detection of ammonium detoxification of hepatic cells.

Characterization of alpha-galactosidases from germinating soybean seed and their use for hydrolysis of oligosaccharides.

Raffinose oligosaccharides (RO) are the major factors responsible for flatulence following ingestion of soybean derived products. Removal of RO from seeds or soymilk would then have a positive impact on the acceptance of soy-based foods. Enzymic hydrolysis of the RO is accomplished by alpha-galactosidase. While the content of RO decreases during seed germination, the activity of alpha-galactosidase increases substantially. Two alpha-galactosidases were isolated from germinating seeds by partition in an aqueous two-phase system followed by ion-exchange and affinity chromatography. One of the enzyme preparations (P1) showed a single protein with M(r) of 33 kDa, and the second (P2) had two proteins with M(r) of 31 and 33 kDa. Maximal activities against the synthetic substrate rho-nitrophenyl-alpha-D-galactopyranoside (rhoNPGal) were detected at pH 5.0-5.5 and 45-50 degrees C. Both enzymes were fairly stable at 40 degrees C, but lost most of their activities after 30 min at 50 degrees C. The K(m) values for hydrolysis of rhoNPGal by the P1 and P2 enzymes were 1.55 and 0.76 mM, respectively. The K(m) values determined for hydrolysis of raffinose and melibiose by the P2 enzyme were 5.53 and 5.34 mM, respectively and galactose was a competitive inhibitor (K(i)=0.65 mM). To different extents, both enzymes were sensitive to inhibition by galactose, melibiose, CuSO(4), and SDS. Sucrose and beta-mercaptoethanol showed discrete inhibitory effects on both enzymes.

Clinical impact of histidine-ketoglutarate-tryptophan (HTK) cardioplegic solution on the perioperative period in open heart surgery patients.

BACKGROUND: Ischemia-reperfusion injury during open heart surgery related to unsuccessful myocardial protection may increase morbidity or mortality. We analyze the clinical outcome after cardiac surgery with a cardioplegic solution based on intracellular components added with histidine-ketoglutarate-tryptophan. METHODS: Thirty patients programmed for elective open heart surgery were randomized into two groups. In group I (n = 15), myocardial protection was carried out with Bretschenider solution (HTK), and in group II (n = 15) with conventional crystalloid cardioplegia. The incidence of arrhythmias, inotropic support requirement, and length-of-stay in the intensive care unit were evaluated. RESULTS: During reperfusion, there was no difference in incidence of arrhythmias; however, in the postoperative period group I had a lower incidence of arrhythmias (p = 0.001). Inotropic support (p = 0.003) and length-of-stay in the intensive care unit (p = 0.037) were lower in group I. There were no deaths in either group. CONCLUSIONS: It was concluded that myocardial protection with Bretschneider solution effectively decreases incidence of arrhythmias, inotropic support, and length-of-stay in the intensive care unit.