Production of Galacto-oligosaccharides by the β-Galactosidase from Kluyveromyces lactis : comparative analysis of permeabilized cells versus soluble enzyme

The transgalactosylation activity of Kluyveromyces lactis cells was studied in detail. Cells were permeabilized with ethanol and further lyophilized to facilitate the transit of substrates and products. The resulting biocatalyst was assayed for the synthesis of galacto-oligosaccharides (GOS) and compared with two soluble β-galactosidases from K. lactis (Lactozym 3000 L HP G and Maxilact LGX 5000). Using 400 g/L lactose, the maximum GOS yield, measured by HPAEC-PAD analysis, was 177 g/L (44% w/w of total carbohydrates). The major products synthesized were the disaccharides 6-galactobiose [Gal-β(1→6)-Gal] and allolactose [Gal-β(1→6)-Glc], as well as the trisaccharide 6-galactosyl-lactose [Gal-β(1→6)-Gal-β(1→4)-Glc], which was characterized by MS and 2D NMR. Structural characterization of another synthesized disaccharide, Gal-β(1→3)-Glc, was carried out. GOS yield obtained with soluble β-galactosidases was slightly lower (160 g/L for Lactozym 3000 L HP G and 154 g/L for Maxilact LGX 5000); however, the typical profile with a maximum GOS concentration followed by partial hydrolysis of the newly formed oligosaccharides was not observed with the soluble enzymes. Results were correlated with the higher stability of β-galactosidase when permeabilized whole cells were used.     

Hydrolysis of cellobiose by β-glucosidase in the presence of soil minerals - Interactions at solid-liquid interfaces and effects on enzyme activity levels

Extracellular enzymatic activities in soils are essential for the cycling of organic matter. These activities take place in multiphase environments where solid phases profoundly affect biocatalytic activities. Aspergillus niger is ubiquitous in soils; its β-glucosidase plays an important role in the degradation of cellulose, and therefore in the global carbon cycle and in the turnover of soil organic matter. However, the information on the interactions of this protein with soil minerals is very limited, and even less is known about their consequences for the hydrolysis of the natural substrate cellobiose. We therefore characterised the sorptive interactions of this enzyme with the soil minerals montmorillonite, kaolinite and goethite and quantified the resulting changes in the hydrolysis rate of cellobiose. Fractions of adsorbed protein, and the resulting catalytic activity loss, were lower for montmorillonite than for kaolinite and goethite at given experimental conditions; adsorption was 9.7 ± 7.3% for montmorillonite, 70.3 ± 3.1% for kaolinite and 71.4 ± 1.8% for goethite, respectively. Adsorption of the protein to the minerals caused a total decrease in the catalytic activity of 18.8 ± 3.4% for kaolinite and 17.9 ± 4.7% for goethite whereas it was not significant for montmorillonite. The average catalytic activity lost by the pool of adsorbed molecules was 26.8% for kaolinite and 25.0% for goethite. Both the amount of adsorbed protein and the resulting loss of catalytic activity were found to be independent of the specific surface areas yet were influenced by the electrical properties of the mineral surfaces. Under the experimental conditions, montmorillonite and kaolinite are negatively charged whereas goethite is positively charged. However, because of the adsorption of phosphate anions from the buffer, a charge reversal took place at the surface of goethite. This was confirmed by zeta (ζ)-potential measurements in phosphate buffer, revealing negative values for all the tested minerals. Indeed goethite interacted with the enzyme as a negatively charged surface: the amount of adsorbed protein and the resulting catalytic activity loss were very similar to those of kaolinite. Our results show that, even if an important fraction of β-glucosidase is adsorbed to the minerals, the catalytic activity is largely retained. We suggest that this strong activity retention in presence of soil minerals results from a selective pressure on A. niger, which benefits from the activity of the adsorbed, and thus stabilized, enzyme pool.     

Application of “Four-Plane Model” to the Adsorption of K+, NO3 -, and SO4 2- from a Mixed Solution of KNO3 and K2SO4 on Andisols

To evaluate the interaction between anionic species when they were adsorbed on variable charge soils, we measured the adsorption of NO₃ ^- and SO₄ ^2- on. an Andisol equilibrated with a mixture of KNO₃ and K₂SO₄ solutions. The amount of NO₃ ^- adsorption declined with the increase in the concentration of SO₄ ^2-. On the other hand, the amount of SO₄ ^2- adsorption increased with the increase in the concentration of NO₃ ^-. These phenomena can not be explained by the simple “exchange theory” or the “Three-plane model” unless surface complexation is associated with the changes in the electrical potential of the surface. Therefore, in order to explain the phenomena by using the “Four-plane model” with surface complexation, a new equation was derived for the Diffuse Double Layer (DDL) charge from the “main equation of DDL theory” (equation of electrical potential in DDL) instead of the “Gouy-Chapman equation,” which can be used only for single symmetric electrolytes but nor for a mixture. By introducing the new equation to the computer program for analyzing the Four-plane model, the interaction between anionic species adsorbed on Andisols could be explained well. It was considered that SO₄ ^2- acted as a regulator for the ionic concentration, osmotic pressure and pH of the soil solution, and consequently, these factors affected the vertical distribution and mobility of anionic species and also the mobility of counter-ions in Andisols.     

Free Radical Scavenging Activity of 4-(3,4-Dihydroxybenzoyloxymethyl)phenyl-O-β-d-glucopyranoside from Origanum vulgare and Its Protection against Oxidative Damage

4-(3,4-Dihydroxybenzoyloxymethyl)phenyl-O-β-d-glucopyranoside (DBPG), a polyphenolic glycoside, isolated from Origanum vulgare has shown 1,1-diphenyl-2-picrylhydrazyl (DPPH(?))-scavenging capacity in previous work. This study demonstrated that DBPG exhibits antioxidant activity by a series of DPPH(?), 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS(?+)), and superoxide anion radical (O(2)(?-)) radical-scavenging assays. The inhibition of lipid peroxidation (LP) by DBPG exceeded that by l-ascorbic acid (AA) in a liposome model system. Adding DBPG to mouse liver and brain tissue inhibited the formation of thiobarbituric acid reactive substances (TBARS) to a greater extent than did trolox. In the oxygen stress test, BNLCL2 and HaCaT cells pretreated with DBPG showed increased activities of glutathione peroxidase (GPx), perhaps as a result of reduction of the production of reactive oxygen species (ROS). These findings proved that DBPG had antioxidant activities and a cytoprotective effect in hepatocytes and keratinocytes, suggesting that DBPG may be a useful food and cosmetic additive.     

Molecular Weight Distribution of (1→3)(1→4)-β-Glucan Affects Pasting Properties of Flour from Oat Lines with High and Typical Amounts of β-Glucan

Experimental (N979-5-2-4 and IA95111) and traditional oat lines (Jim and Paul) with average %β-glucan of 7.5, 7.8, 4.9, and 4.4%, respectively, were grown in 2002, 2003, and 2004. Molecular weight (MW) distributions of the β-glucans were examined for potential variations among growing years and for relationships with pasting properties measured by Rapid ViscoAnalyser under three separate conditions: 1) in silver nitrate (SN) solution to inactivate enzymes; 2) hydrolyzed by α-amylase to eliminate the effect of starch; and 3) treated with lichenase to remove β-glucan. The β-glucan was extracted by a process involving multiple precipitation and dialysis steps, and the MW distributions were determined by HPLC. %β-Glucan in N979-5-2-4 and IA95111 lines were consistently and significantly greater (P < 0.05) than in Jim and Paul lines during three growing years. The contribution of β-glucan to peak viscosity on the RVA was substantially greater than that of the starch for all three years. The molecular number average and peak MW of β-glucan from N979-5-2-4 and IA95111 were greater than these values for Jim and Paul, and values were consistent among years. The MW of extracted β-glucan was associated with pasting properties after amylase hydrolysis, but not after treatment with lichenase or in SN solution.     

Synthesis, analytical features, and biological relevance of 5-(3',4'-dihydroxyphenyl)-γ-valerolactone, a microbial metabolite derived from the catabolism of dietary flavan-3-ols

The physiological significance of 5-(3',4'-dihydroxyphenyl)-γ-valerolactone, an important metabolite derived from the catabolism of flavan-3-ols by gut microbiota, has been often overlooked due to the lack of the commercial standard. In the present work, this metabolite has been chemically synthesized, and its analytical parameters and antioxidant capacity have been determined in comparison to other chemical analogues [isomer 3-(3',4'-dihydroxyphenyl)-δ-valerolactone and γ-valerolactone] and other structurally related compounds [(+)-catechin, (-)-epicatechin, and 3-(3,4-dihydroxyphenyl)-propionic acid]. The synthesized compound was also used to perform a targeted analysis in samples collected during the in vitro fermentation of a grape seed flavan-3-ol extract with human fecal microbiota from three healthy volunteers. The time-course formation of 5-(3',4'-dihydroxyphenyl)-γ-valerolactone revealed large interindividual differences among volunteers, with concentrations ranging from 3.31 to 77.54 μM at 10 h of fermentation. These results are further discussed in view of the scarce reports quantifying 5-(3',4'-dihydroxyphenyl)-γ-valerolactone in in vitro fermentation studies, and pharmacokinetic and intervention studies.     

PO 4 3? Removal by and Permeability of Industrial Byproducts and Minerals: Granulated Blast Furnace Slag, Cement Kiln Dust, Coconut Shell Activated Carbon, Silica Sand, and Zeolite

Excess PO ₄ ^3? from agricultural subsurface drainage and runoff degrades the overall water quality of the receiving surface waters in a cumulatively damaging process known as eutrophication. In the past 25 years, PO ₄ ^3? removal by industrial byproducts and minerals has received considerable attention because these materials are both abundant and inexpensive. In this study, the saturated falling-head hydraulic conductivity and phosphate removal capability of granulated blast furnace slag (GBFS), cement kiln dust (CKD), zeolite, silica sand, and coconut shell activated carbon (CS-AC) were assessed. GBFS, zeolite, silica sand, CS-AC, and 5:95% and 10:90% CKD/sand blends all exhibited hydraulic conductivities ??0.001 cm/s. GBFS and the CKD/sand blends exhibited >98% PO ₄ ^3? removal while CS-AC removed 70?C79% of initial PO ₄ ^3? concentrations. In contrast, silica sand and zeolite removed 21?C58% of PO ₄ ^3? . The phosphate removal data for each material was modeled against the Langmuir, Freundlich, Temkin, Dubinin-Radushkevich, and Frumkin sorption isotherms to yield insight into possible removal mechanisms. Overall, GBFS, CKD, zeolite, silica sand, and CS-AC were sufficiently permeable and removed significant amounts of PO ₄ ^3? and should be considered for use in treatment of agricultural effluent.     

Transport across Caco-2 cell monolayer and sensitivity to hydrolysis of two anxiolytic peptides from αs1-casein, α-casozepine, and αs1-casein-f91-97: effect of bile salts

α-Casozepine and f91-97, peptides from α(s1)-casein, display anxiolytic activity in rats and may have to cross the intestinal epithelium to exert this central effect. We evaluated their resistance to hydrolysis by the peptidases of Caco-2 cells and their ability to cross the cell monolayer. To mimic physiological conditions, two preparations of bile salts were used in noncytotoxic concentrations: porcine bile extract and an equimolar mixture of taurocholate, cholate, and deoxycholate. The presence and composition of bile salts appeared to modulate the peptidase activities of the Caco-2 cells involved (i) in the hydrolysis of α-casozepine, leading to much higher formation of fragments f91-99, f91-98, and f91-97, and (ii) in the hydrolysis of f91-97, leading to lower degradation of this peptide. Transport of α-casozepine across Caco-2 monolayer increased significantly, in the presence of bile extract, and of fragment f91-97, in the presence of bile salts.     

Influence of cadmium on the metabolic quotient, <Emphasis Type="SmallCaps">l</Emphasis>- : <Emphasis Type="SmallCaps">d</Emphasis>-glutamic acid respiration ratio and enzyme activity : microbial biomass ratio under laboratory conditions

 This study was carried out to investigate the effect of very high cadmium concentrations (50 and 500 μg Cd g^–1 soil) on some biochemical and microbiological measurements under laboratory conditions involving daily soil samplings. The data for both DTPA- and water-soluble Cd showed two distinctive patterns during soil incubation; from 0 to 4 days, values were about 50–500 and 1–100 μg g^–1 dry weight soil, whereas they decreased markedly after 7 days. Both daily respiration and the ATP content but not the microbial biomass C determined by the fumigation–extraction method were lowered by high DTPA- and water-soluble Cd concentrations. Dehydrogenase and phosphatase activities as well as both enzyme activity : microbial biomass ratios were decreased by the high DTPA- and water-soluble Cd concentrations. In the first 2 days of incubation, the metabolic quotient (qCO₂) was also decreased by the highest values of available Cd. The early (after 6 h) mineralization of l- but not d-glutamic acid to CO₂ was inhibited during the 0–4 day incubation period by the highest Cd concentration. Possibly the l-enantiomer was used by a larger fraction of soil microorganisms than the d-enantiomer or, if they were used by the same fraction of soil microorganisms, the d-enantiomer was mineralized at a lower rate. The l- : d-glutamic acid respiration ratio was decreased by the high available Cd content because under polluted conditions soil microorganisms probably discriminated less between the two stereoisomers of glutamic acid. Received: 13 July 1999     

Book reviews,Hunting the Wild Potato in the South American Andes: Memories of the British Empire Potato Collecting Expedition to South America 1938– 1939. J. [John ‘Jack’] G. [Gregory] Hawkes.Botanical and Experimental Garden,University of Nijmegen,The Netherlands 2004. 224 pp.ISBN 90-9018021-4.

Book Review

Book reviews, Hunting the Wild Potato in the South American Andes: Memories of the British Empire Potato Collecting Expedition to South America 1938– 1939. J. [John ‘Jack’] G. [Gregory] Hawkes.Botanical and Experimental Garden, University of Nijmegen, The Netherlands 2004. 224 pp.ISBN 90-9018021-4.