Purification and Characterization of the alpha-Glucosidase Produced by Thermophilic Fungus Thermoascus aurantiacus CBMAI 756

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Purificação e caracterização bioquímica da α-glicosidase termoestável produzida por Thermoascus aurantiacus CBMAI 756

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Metabolismo di oligosaccaridi prebiotici in Bifidobacterium per il potenziale sviluppo di nuovi prodotti alimentari funzionali

The growth and the metabolism of Bifidobacterium adolescentis MB 239 fermenting GOS, lactose, galactose, and glucose were investigated. An unstructerd unsegregated model for growth of B. adolescentis MB 239 in batch cultures was developed and kinetic parameters were calculated with a Matlab algorithm. Galactose was the best carbon source; lactose and GOS led to lower growth rate and cellular yield, but glucose was the poorest carbon source. Lactate, acetate and ethanol yields allowed calculation of the carbon fluxes toward fermentation products. Similar distribution between 3- and 2-carbon products was observed on all the carbohydrates (45 and 55%, respectively), but ethanol production was higher on glucose than on GOS, lactose and galactose, in decreasing order. Based on the stoichiometry of the fructose 6-phosphate shunt and on the carbon distribution among the products, ATP yield was calculated on the different carbohydrates. ATP yield was the highest on galactose, while it was 5, 8, and 25% lower on lactose, GOS, and glucose, respectively. Therefore, a correspondance among ethanol production, low ATP yields, and low biomass production was established demonstrating that carbohydrate preferences may result from different sorting of carbon fluxes through the fermentative pathway. During GOS fermentation, stringent selectivity based on the degree of polymerization was exhibited, since lactose and the trisaccharide were first to be consumed, and a delay was observed until longer oligosaccharides were utilized. Throughout the growth on both lactose and GOS, galactose accumulated in the cultural broth, suggesting that β-(1-4) galactosides can be hydrolysed before they are taken up. The physiology of Bifidobacterium adolescentis MB 239 toward xylooligosaccharides (XOS) was also studied and our attention was focused on an extracellular glycosyl-hydrolase (β-Xylosidase) expressed by a culture of B. adolescentis grown on XOS as sole carbon source. The extracellular enzyme was purified from the the supernatant, which was dialyzed and concentrated by ultrafiltration. A two steps purification protocol was developed: the sample was loaded on a Mono-Q anion exchange chromatography and then, the active fractions were pooled and β-Xylosidase was purified by gel filtration chromatography on a Superdex-75. The enzyme was characterized in many aspects. β- Xylosidase was an homo-tetramer of 160 kDa as native molecular mass; it was a termostable enzyme with an optimum of temperature at 53 °C and an optimum of pH of 6.0. The kinetics parameter were calculated: km = 4.36 mM, Vmax = 0.93 mM/min. The substrate specificity with different di-, oligo- and polysaccharides was tested. The reactions were carried out overnight at pH 7 and at the optimum of temperature and the carbohydrates hydrolysis were analyzed by thin layer chromatography (TLC). Only glycosyl-hydrolase activities on XOS and on xylan were detected, whereas sucrose, lactose, cellobiose, maltose and raffinose were not hydrolyzed. It’s clearly shown that β-Xylosidase activity was higher than the Xylanase one. These studies on the carbohydrate preference of a strain of Bifidobacterium underlined the importance of the affinity between probiotics and prebiotics. On the basis of this concept, together with Barilla G&R f.lli SpA, we studied the possibility to develop a functional food containing a synbiotic. Three probiotic strains Lactobacillus plantarum BAR 10, Streptococcus thermophilus BAR 20, and Bifidobacterium lactis BAR 30 were studied to assess their suitability for utilization in synbiotic products on the basis of antioxidative activity, glutathione production, acid and bile tolerance, carbohydrates fermentation and viability in food matrices. Bile and human gastric juice resistance was tested in vitro to estimate the transit tolerance in the upper gastrointestinal tract. B. lactis and L. plantarum were more acid tolerant than S. thermophilus. All the strains resisted to bile. The growth kinetics on 13 prebiotic carbohydrates were determined. Galactooligosaccharides and fructo-oligosaccharides were successfully utilized by all the strains and could be considered the most appropriate prebiotics to be used in effective synbiotic formulations. The vitality of the three strains inoculated in different food matrices and maintained at room temperature was studied. The best survival of Lactobacillus plantarum BAR 10, Streptococcus thermophilus BAR 20, and Bifidobacterium lactis BAR 30 was found in food chocolate matrices. Then an in vivo clinical trial was carried out for 20 healthy volunteers. The increase in faecal bifidobacteria and lactobacilli populations and the efficacy of the pre-prototype was promising for the future develop of potential commercial products.

Substrate recognition and catalysis by the Holliday junction resolving enzyme Hje

Two archaeal Holliday junction resolving enzymes, Holliday junction cleavage (Hjc) and Holliday junction endonuclease (Hje), have been characterized. Both are members of a nuclease superfamily that includes the type II restriction enzymes, although their DNA cleaving activity is highly specific for four-way junction structure and not nucleic acid sequence. Despite 28% sequence identity, Hje and Hjc cleave junctions with distinct cutting patterns—they cut different strands of a four-way junction, at different distances from the junction centre. We report the high-resolution crystal structure of Hje from Sulfolobus solfataricus. The structure provides a basis to explain the differences in substrate specificity of Hje and Hjc, which result from changes in dimer organization, and suggests a viral origin for the Hje gene. Structural and biochemical data support the modelling of an Hje:DNA junction complex, highlighting a flexible loop that interacts intimately with the junction centre. A highly conserved serine residue on this loop is shown to be essential for the enzyme's activity, suggesting a novel variation of the nuclease active site. The loop may act as a conformational switch, ensuring that the active site is completed only on binding a four-way junction, thus explaining the exquisite specificity of these enzymes.

The FAM deubiquitylating enzyme localizes to multiple points of protein trafficking in epithelia, where it associates with E-cadherin and β-catenin

Ubiquitylation is a necessary step in the endocytosis and lysosomal trafficking of many plasma membrane proteins and can also influence protein trafficking in the biosynthetic pathway. Although a molecular understanding of ubiquitylation in these processes is beginning to emerge, very little is known about the role deubiquitylation may play. Fat Facets in mouse (FAM) is substrate-specific deubiquitylating enzyme highly expressed in epithelia where it interacts with its substrate, β-catenin. Here we show, in the polarized intestinal epithelial cell line T84, FAM localized to multiple points of protein trafficking. FAM interacted with β-catenin and E-cadherin in T84 cells but only in subconfluent cultures. FAM extensively colocalized with β-catenin in cytoplasmic puncta but not at sites of cell-cell contact as well as immunoprecipitating with β-catenin and E-cadherin from a higher molecular weight complex (~500 kDa). At confluence FAM neither colocalized with, nor immunoprecipitated, β-catenin or E-cadherin, which were predominantly in a larger molecular weight complex (~2 MDa) at the cell surface. Overexpression of FAM in MCF-7 epithelial cells resulted in increased β-catenin levels, which localized to the plasma membrane. Expression of E-cadherin in L-cell fibroblasts resulted in the relocalization of FAM from the Golgi to cytoplasmic puncta. These data strongly suggest that FAM associates with E-cadherin and β-catenin during trafficking to the plasma membrane.

Aromatic l-amino acid decarboxylase : histochemical localization in rat kidney and lack of effect of dietary potassium or sodium loading on enzyme distribution

Utilizing a mono-specific antiserum produced in rabbits to hog kidney aromatic L-amino acid decarboxylase (AADC), the enzyme was localized in rat kidney by immunoperoxidase staining. AADC was located predominantly in the proximal convoluted tubules; there was also weak staining in the distal convoluted tubules and collecting ducts. An increase in dietary potassium or sodium intake produced no change in density or distribution of AADC staining in kidney. An assay of AADC enzyme activity showed no difference in cortex or medulla with chronic potassium loading. A change in distribution or activity of renal AADC does not explain the postulated dopaminergic modulation of renal function that occurs with potassium or sodium loading.

Effects of soluble dietary cellulose on specific growth rate, survival and digestive enzyme activities in three freshwater crayfish (Cherax) species

Three native freshwater crayfish Cherax species are farmed in Australia namely; Redclaw (Cherax quadricarinatus), Marron (C. tenuimanus), and Yabby (C. destructor). Lack of appropriate data on specific nutrient requirements for each of these species, however, has constrained development of specific formulated diets and hence current use of over-formulated feeds or expensive marine shrimp feeds, limit their profitability. A number of studies have investigated nutritional requirements in redclaw that have focused on replacing expensive fish meal in formulated feeds with non-protein, less expensive substitutes including plant based ingredients. Confirmation that freshwater crayfish possess endogenous cellulase genes, suggests their potential ability to utilize complex carbohydrates like cellulose as nutrient sources in their diet. To date, studies have been limited to only C. quadricarinatus and C. destructor and no studies have compared the relative ability of each species to utilize soluble cellulose in their diets. Individual feeding trials of late-juveniles of each species were conducted separately in an automated recirculating culture system over 12 week cycles. Animals were fed either a test diet (TD) that contained 20% soluble cellulose or a reference diet (RD) substituted with the same amount of corn starch. Water temperature, conductivity and pH were maintained at constant and optimum levels for each species. Animals were fed at 3% of their body weight twice daily and wet body weight was recorded bi-weekly. At the end of experiment, all animals were harvested, measured and midgut gland extracts assayed for alpha-amylase, total protease and cellulase activity levels. After the trial period, redclaw fed with RD showed significantly higher (p<0.05) specific growth rate (SGR) compare with animals fed the TD while SGR of marron and yabby fed the two diets were not significantly different (p<0.05). Cellulase expression levels in redclaw were not significantly different between diets. Marron and yabby showed significantly higher cellulase activity when fed the RD. Amylase and protease activity in all three species were significantly higher in the animals fed with RD (Table 1). These results indicate that test animals of all species can utilize starch better than dietary soluble cellulose in their diet and inclusion of 20% soluble cellulose in diets does not appear to have any significant negative effect on their growth rate but survival was impacted in C. quadricarinatus while not in C. tenuimanus or C. destructor.

Effects of soluble dietary cellulose on specific growth rate, survival and digestive enzyme activities in three freshwater crayfish (Cherax) species

The current study evaluated the effect of soluble dietary cellulose on growth, survival and digestive enzyme activity in three endemic, Australian freshwater crayfish species (redclaw: Cherax quadricarinatus, marron: C. tenuimanus, yabby: C. destructor). Separate individual feeding trials were conducted for late-stage juveniles from each species in an automated recirculating freshwater, culture system. Animals were fed either a test diet (TD) that contained 20% soluble cellulose or a reference diet (RD) substituted with the same amount of corn starch, over a 12 week period. Redclaw fed with RD showed significantly higher (p<0.05) specific growth rates (SGR) compared with animals fed the TD, while SGR of marron and yabby fed the two diets were not significantly different. Expressed cellulase activity levels in redclaw were not significantly different between diets. Marron and yabby showed significantly higher cellulase activity when fed the RD (p<0.05). Amylase and protease activity in all three species were significantly higher in the animals fed with RD (p<0.05). These results indicate that test animals of all three species appear to utilize starch more efficiently than soluble dietary cellulose in their diet. The inclusion of 20% soluble cellulose in diets did not appear, however, to have a significant negative effect on growth rates.

Effect of pretreatment on saccharification of sugarcane bagasse by complex and simple enzyme mixtures

Saccharification of sugarcane bagasse pretreated at the pilot-scale with different processes (in combination with steam-explosion) was evaluated. Maximum glucan conversion with Celluclast 1.5 L (15–25 FPU/g glucan) was in the following order: glycerol/HCl > HCl > H2SO4 > NaOH, with the glycerol system achieving ∼100% conversion. Surprisingly, the NaOH substrate achieved optimum saccharification with only 8 FPU/g glucan. Glucan conversions (3.6–6%) obtained with mixtures of endo-1,4-β-glucanase (EG) and β-glucosidase (βG) for the NaOH substrate were 2–6 times that of acid substrates. However, glucan conversions (15–60%) obtained with mixtures of cellobiohydrolase (CBH I) and βG on acidified glycerol substrate were 10–30% higher than those obtained for NaOH and acid substrates. The susceptibility of the substrates to enzymatic saccharification was explained by their physical and chemical attributes. Acidified glycerol pretreatment offers the opportunity to simplify the complexity of enzyme mixtures required for saccharification of lignocellulosics.