Helix packing of lactose permease in Escherichia coli studied by site-directed chemical cleavage.

Biotinylated lactose permease from Escherichia coli containing a single-cysteine residue at position 330 (helix X) or at position 147, 148, or 149 (helix V) was purified by avidin-affinity chromatography and derivatized with 5-(alpha-bromoacetamido)-1,10-phenanthroline-copper [OP(Cu)]. Studies with purified, OP(Cu)-labeled Leu-330 --> Cys permease in dodecyl-beta-D-maltopyranoside demonstrate that after incubation in the presence of ascorbate, cleavage products of approximately 19 and 6-8 kDa are observed on immunoblots with anti-C-terminal antibody. Remarkably, the same cleavage products are observed with permease embedded in the native membrane. Comparison with the C-terminal half of the permease expressed independently as a standard indicates that the 19-kDa product results from cleavage near the cytoplasmic end of helix VII, whereas the 6- to 8-kDa fragment probably results from fragmentation near the cytoplasmic end of helix XI. Results are entirely consistent with a tertiary-structure model of the C-terminal half of the permease derived from earlier site-directed fluorescence and site-directed mutagenesis studies. Similar studies with OP(Cu)-labeled Cys-148 permease exhibit cleavage products at approximately 19 kDa and at 15-16 kDa. The larger fragment probably reflects cleavage at a site near the cytoplasmic end of helix VII, whereas the 15- to 16-kDa fragment is consistent with cleavage near the cytoplasmic end of helix VIII. When OP(Cu) is moved 100 degrees to position 149 (Val-149 --> Cys permease), a single product is observed at 19 kDa, suggesting fragmentation at the cytoplasmic end of helix VII. However, when the reagent is moved 100 degrees in the other direction to position 147 (Gly-147 --> Cys permease), cleavage is not observed. The results suggest that helix V is in close proximity to helices VII and VIII with position 148 in the interface between the helices, position 149 facing helix VII, and position 147 facing the lipid bilayer.

Effect of lactose solid-state on the aerosolization performance of drug-carrier mixtures

Lactose, in particular α-lactose monohydrate, is the most used carrier for inhalation. Its surface and solid-state properties are of paramount importance in determining drug aerosolization performance. However, these properties may be altered by processing, such as micronization, thus affecting the product performance and stability. The present research project focused on the study of the effect of lactose solid-state on the aerosolization performance of drug-carrier mixtures, giving particular attention to the impact of micronization on lactose physico-chemical properties. The formation of a fraction of hygroscopic anhydrous α-lactose, rather than amorphous lactose, as a consequence of the mechanical stress stemming from micronization was evidenced by 1H NMR, XRPD and DSC analyses performed on samples of micronized lactose. The development of a new DVS method capable to identify and quantify different forms of α-lactose (hygroscopic anhydrous, stable anhydrous and amorphous), even simultaneously present in the same sample, confirmed the results obtained with the above-mentioned techniques. The influence of lactose solid-state on drug respirability was then evaluated through the preparation and in vitro aerodynamic assessment of ternary and binary mixtures containing two different drugs. In particular, the use, as carriers, of anhydrous forms of α-lactose in place of the conventional α-lactose monohydrate resulted in significantly improved respirability in the case of salbutamol sulphate and poorer performance in the case of budesonide. In an attempt to rationalize the obtained results, IGC was selected as a tool to investigate possible variations in the surface energy of the studied lactose carriers and APIs. A direct correlation between the total surface free energy of lactose carriers and drug respirability was not found. However, salbutamol sulphate and budesonide exhibited different specific surface free energy, to which the difference in the aerosolization performance may be, at least in part, ascribed.

Bioreaction and separation in preparative batch chromatographic columns : the hydrology of lactose to yield glucose,galactose and oligosaccharides

The initial aim of this project was to improve the performance of a chromatographic bioreactor-separator (CBRS). In such a system, a dilute enzyme solution is pumped continuously through a preparative chromatographic column, while pulses of substrate are periodically injected on to the column. Enzymic reaction and separation are therefore performed in a single unit operation. The chromatographic columns used were jacketed glass columns ranging from 1 to 2 metres long with an internal diameter of 1.5 cm. Linking these columns allowed 1, 2, 3 and 4 metre long CBRS systems to be constructed. The hydrolysis of lactose in the presence of β~galactosidase was the reaction of study. From previous work at Aston University, there appeared to be no difficulties in achieving complete lactose hydrolysis in a CBRS. There did, however, appear to be scope for improving the separative performance, so this was adopted as an initial goal. Reducing the particle size of the stationary phase was identified as a way of achieving this improvement. A cation exchange resin was selected which had an average particle size of around half that previously used when studying this reaction. A CBRS system was developed which overcame the operational problems (such as high pressure drop development) associated with use of such a particle size. A significant improvement in separative power was achieved. This was shown by an increase in the number of theoretical plates (N) from about 500 to about 3000 for a 2 metre long CBRS, coupled with higher resolution. A simple experiment with the 1 metre column showed that combined bioreaction and separation was achievable in this system. Having improved the separative performance of the system, the factors affecting enzymic reaction in a CBRS were investigated; including pulse volume and the degree of mixing between enzyme and substrate. The progress of reaction in a CBRS was then studied. This information was related to the interaction of reaction and separation over the reaction zone. The effect of injecting a pulse over a length of time as in CBRS operation was simulated by fed batch experiments. These experiments were performed in parallel with normal batch experiments where the substrate is mixed almost instantly with the enzyme. The batch experiments enabled samples to be taken every minute and revealed that reaction is very rapid. The hydrodynamic characteristics of the two injector configurations used in CBRS construction were studied using Magnetic Resonance Imaging, combined with hydrodynamic calculations. During the optimisation studies, galactooligosaccharides (GOS) were detected as intermediates in the hydrolysis process. GOS are valuable products with potential and existing applications in food manufacture (as nutraceuticals), medicine and drug targeting. The focus of the research was therefore turned to GOS production. A means of controlling reaction to arrest break down of GOS was required. Raising temperature was identified as a possible means of achieving this within a CBRS. Studies were undertaken to optimise the yield of oligosaccharides, culminating in the design, construction and evaluation of a Dithermal Chromatographic Bioreactor-separator.

Lactose malabsorption and nutrition

Lactose needs to be digested by the small intestinal enzyme lactase into its constituent monosaccharides, glucose and galactose, before transport across the epithelial cell membranes. Lactase activity is therefore essential for the development of young mammals, since their sole source of nourishment is their mother's milk. This chapter summarizes what is known about the genetics and evolution of the lactase persistence (LP) trait. Symptoms of lactose intolerance can arise after an individual with hypolactasia has ingested lactose. Many dairy products contain only small amounts of lactose, allowing their consumption by most lactase non-persistent individuals. Bacteria and yeast fermentation convert the lactose in milk into various by-products, reducing the content of lactose. Nutritional epidemiology studies, and the staggering selective advantages that have favoured LP-associated alleles over the last 10,000 years, clearly show that adult milk consumption can be highly beneficial.

Evolution of protein bound Maillard reaction end-products and free Amadori compounds in low lactose milk in presence of fructosamine oxidase I

Thermal treatments and storage influence milk quality, particularly in low lactose milk as the higher concentration of reducing sugars can lead to the increased formation of the Maillard reaction products (MRPs). The control of the Amadori products (APs) formation is the key step to mitigate the Maillard reaction (MR) in milk. The use of fructosamine oxidases, (Faox) provided promising results. In this paper, the effects of Faox I were evaluated by monitoring the concentration of free and bound MRPs in low lactose milk during shelf life. Results showed that the enzyme reduced the formation of protein-bound MRPs down to 79% after six days at 37 °C. Faox I lowered the glycation of almost all the free amino acids resulting effective on basic and polar amino acids. Data here reported corroborate previous findings on the potentiality of Faox enzymes in controlling the early stage of the MR in foods.

Isomérisation du lactose en lactulose en solution modèle de lactose et dans du perméat de lactosérum par électro-activation supportée par échange ionique sur résine

L’isomérisation alcaline du lactose en lactulose a été effectuée électro-chimiquement à l’aide d’un réacteur d’électro-activation en combinaison avec des résines échangeuses d’anions de polystyrène de trois types; à savoir Lewatit VP-OC-1065 faible-acide, Lewatit MP-64 moyenne-acide et Lewatit Monoplus M500 forte-acide. Les paramètres opératoires qui ont fait l’objet de cette étude ont été étudiés sur trois blocs expérimentaux pour optimiser le système. Dans le Premier bloc, les paramètres étudiés sont : (1) ratio lactose-5%(p/v) : résine échangeuse d’anions (1:0.5, 1:1 et 1:2), (2) intensité du champ électrique : 50 mA, 100 mA et 200 mA et (3) type de résines : faible, moyenne et forte. Dans le Deuxième bloc, les paramètres mis à l’étude comprenaient : (1) l’intensité du champ électrique : 300 mA, 450 mA et 550 mA, (2) le débit de la solution traitée : 25 ml / min, 50 ml/ min et 100 ml/min et (3) la surface active de la membrane adjacente au compartiment cathodique : 0.78 cm2, 7.06 cm2 et 18.1 cm2. Le Troisième bloc expérimental a été effectué sur la base de la distance entre la membrane et l’électrode : 3.1 cm, 5.6 cm et 9 cm. Le même modèle expérimental a était également réalisé avec du perméat du lactosérum d’une concentration de 7% (p/v). Les résultats obtenus ont révélé que le meilleur rendement de l’isomérisation du lactose en lactulose était obtenu après 30 minutes d’électroactivation en utilisant une solution modèle de lactose-5% avec une valeur d’environ 20.1%. Les conditions opératoires qui ont permis d’avoir ce taux de conversion sont une intensité du courant de 550 mA, un débit de la solution de 25 ml/min, une surface active de la membrane de 7.06 cm2 et une distance de 9 cm entre la cathode et la membrane qui lui y est adjacente. En utilisant le perméat de lactosérum-7%, un taux de conversion de lactose en lactulose de 8.34% a était obtenu avec une intensité du courant de 200 mA, un débit de 120 ml/min, une surface active de de 18.1cm2 et une distance de 9 cm entre la membrane et l’électrode dans le compartiment cathodique. Les analyses de variance ont indiqué un effet catalytique significatif du type de la résine. En effet, la résine-forte a permis d’avoir les plus hauts rendements de la réaction d’isomérisation par électro-activation. La résistance électrique globale du système d’électroactivation dépendait de la valeur de l’intensité du courant. Le produit final était d’une grande pureté, car il ne présentait que quelques traces de galactose (< 4%).

Reaproveitamento da lactose presente em permeado de soro de leite bovino em pó para estudo da síntese de lactulose

Dissertação composta por 2 artigos.

Conversão da lactose e síntese de galacto-oligossacarídeos: uma abordagem experimental e teórica

O presente trabalho avaliou, na etapa experimental, um processo simultâneo de catálise e fermentação láctica visando obter um iogurte com potenciais características nutracêuticas e, na sua etapa teórica, estabeleceu uma interlocução entre a vivência experimentalista e a teoria da cinética enzimática, no que se refere à conversão da lactose e à síntese de galactooligossacarídeos (GOS). Na abordagem experimental, para um substrato específico, avaliouse biocatálise conduzida simultaneamente à fermentação, defasando a adição da enzima em relação ao início do processo fermentativo. A fermentação foi realizada a partir de cultura láctica liofilizada comercial contendo dois micro-organismos probióticos, Bifidobacterium animalis e Lactobacillus acidophilus, associados aos micro-organismos característicos do iogurte, Lactobacillus bulgaricus e Streptococcus thermophilus. Foi utilizado um preparado enzimático contendo -galactosidases obtidas de duas origens distintas: Kluyveromyces lactis e Aspergillus niger. Foram avaliados os efeitos da concentração da enzima e do tempo de adição da enzima em um planejamento experimental 2 2 . As respostas foram às concentrações, ao final do processo, de lactose, de GOS, de glicose e de galactose e a hidrólise dos galactooligossacarídeos ao longo do tempo. No que se refere à abordagem teórica, o presente trabalho considerou modelos matemáticos de hidrólise de dissacarídeos e conversão da lactose, em que a inibição foi representada a partir do incremento da concentração dos produtos da reação. No que se refere à conversão da lactose e síntese de GOS, o presente trabalho buscou estabelecer um modelo matemático em que a inibição ocorreu por efeito do incremento das concentrações de glicose e de galactose, comparando-o com os modelos conhecidos na literatura. Verificou-se que o desempenho do modelo obtido no presente trabalho foi robusto em relação às premissas estabelecidas. Na comparação com resultados experimentais de conversão enzimática, o modelo mostrou-se capaz de minimizar o erro e de ajustar-se aos dados experimentais.

Purificação e caracterização parcial de uma B-D glicosidase de Artemia franciscana com ação sobre celobiose e lactose

-D-glucosidase (EC 3.2.1.21) is one of the most interesting glycosidases, especially for hydrolysis cellobiose releasing glucose, is last step degradation of cellulose. This function makes the -D-glucosidase is of great interest as a versatile industrial biocatalyst, being critical to various bio-treatment / biorefinery processes, such as bioethanol production. Hen in the report, a -D-glucosidase was extracts from protein extracted of the invertebrate marine Artemia franciscana was purified and characterized with a combination of precipitation with ammonium sulfate (0 - 30%, 30 to 50%, 50 to 80%), the fraction saturated in the range of 30 to 50% (called F-II) was applied in a molecular exclusion chromatography, in Sephacryl S-200, the fractions corresponding to the first peak of activity of -D-glucosidase were gathered and applied in a chromatography of ion exchange in Mono Q; the third peak this protein obtained chromatography, which coincides with the peak of activity of -D-glucosidase was held and applied in a gel filtration chromatography Superose 12 where the first peak protein, which has activity of -D-glucosidase was rechromatography on Superose 12. This enzyme is probably multimerica, consisting of three subunit molecular mass of 52.7 kDa (determined by SDS-PAGE) with native molecular mass of 157 kDa (determined by gel filtration chromatography on Superose 12 under the system FPLC). The enzyme was purified 44.09 times with a recovery of 1.01%. Using up p-nitrophenyl-β-D-glucopiranoside as substrate obtained a Km apparent of 0.229 mM and a Vmax of 1.109 mM.60min-1.mL-1mM. The optimum pH and optimum temperature of catalysis of the synthetic substrate were 5.0 and 45 °C, respectively. The activity of the -D-glucosidase was strongly, inhibited by silver nitrate and N- etylmaleimide, this inhibition indicates the involvement of radical sulfidrila the hydrolysis of synthetic substrate. The -D-glucosidase of Artemia franciscana presented degradativa action on celobiose, lactose and on the synthetic substrate -nitrophenyl-β-D-glucopiranoside indicating potential use of this enzyme in the industry mainly for the production of bioethanol (production of alcohol from the participating cellulose), and production hydrolysate milk (devoid of milk lactose)

Uma Lectina D-lactose específica da esponja marinha Cinachyrella apion: purificação, caracterização e interação com Leishmania chagasi

Four different sponge species were screened using Ouchterlony agarose gel and immunodiffusion tests to identify cross-reactivity with the polyclonal antibody IgG anti-deglicosilated CvL, a lectin from Cliona varians. Crude extract from the sponge Cinachyrella apion showed cross-reactivity and also a strong haemmaglutinating activity towards human erythrocytes of all ABO groups. Thus, it was submitted to acetone fractionation, IgG anti-deglicosilated CvL Sepharose affinity chromatography, and Fast Protein Liquid Chromatography (FPLC-AKTA) gel filtration on a Superose 6 10 300 column to purify a novel lectin. C. apion lectin (CaL) agglutinated all types of human erythrocytes with preference for papainized type A and O erythrocytes. The haemagglutinating activity is independent of Ca2+, Mg2+ and Mn2+ ions, and it was strongly inhibited by the disaccharide D-lactose, up to a minimum concentration of 6.25 mM. CaL molecular mass determined by FPLC-AKTA gel filtration on a Superose 12 10 300 column and SDS gel electrophoresis was approximately 124 kDa, consisting of eight subunits of 15.5 kDa, assembled by hydrophobic interactions. The lectin was relatively heat- and pH-stable. Leishmania chagasi romastigotes were agglutinated by CaL, indicating that lactose receptors could be presented in this parasite stage. These findings are indicative of the physiological defense roles of CaL and its possible use in the antibiosis of pathogenic protozoa

Rheological Properties of a Sodium Bentonite and Lactose Aqueous Dispersion

Aim of this paper is show the viscosity measure of a sodium bentonite-water-lactose mixture and your rheological behaviour. This analysis showed the formation of tridimensional structure type and formation of stratified silicate/lactose, this occurred due to different concentrations of organic products into mixture and due to a difference of rotation during viscosity measument. Formation of networks is a consequence of the attraction between the silicate layers in water-lactose mixture. In the present work aqueous solutions of lactose with concentration of 7%, 5%, 3%, 1% and 0% (wt %) were used.