Advances in the structure and function of chitosanase / 生物工程学报

Chitosanases represent a class of glycoside hydrolases with high catalytic activity on chitosan but nearly no activity on chitin. Chitosanases can convert high molecular weight chitosan into functional chitooligosaccharides with low molecular weight. In recent years, remarkable progress has been made in the research on chitosanases. This review summarizes and discusses its biochemical properties, crystal structures, catalytic mechanisms, and protein engineering, highlighting the preparation of pure chitooligosaccharides by enzymatic hydrolysis. This review may advance the understandings on the mechanism of chitosanases and promote its industrial applications.

Enzymatic preparation of fructooligosaccharides-rich burdock syrup with enhanced antioxidative properties

Background: Burdock (Arctium lappa L.) is a fructan-rich plant with prebiotic potential. The aim of this study was to develop an efficient enzymatic route to prepare fructooligosaccharides (FOS)-rich and highly antioxidative syrup using burdock root as a raw material. Results: Endo-inulinase significantly improved the yield of FOS 2.4-fold while tannase pretreatment further increased the yield of FOS 2.8-fold. Other enzymes, including endo-polygalacturonase, endo-glucanase and endo-xylanase, were able to increase the yield of total soluble sugar by 11.1% (w/w). By this process, a new enzymatic process for burdock syrup was developed and the yield of burdock syrup increased by 25% (w/w), whereas with FOS, total soluble sugars, total soluble protein and total soluble polyphenols were enhanced to 28.8%, 53.3%, 8.9% and 3.3% (w/w), respectively. Additionally, the scavenging abilities of DPPH and hydroxyl radicals, and total antioxidant capacity of the syrup were increased by 23.7%, 51.8% and 35.4%, respectively. Conclusions: Our results could be applied to the development of efficient extraction of valuable products from agricultural materials using enzyme-mediated methods.

Caracterización química y cuantificación de fructooligosacáridos, compuestos fenólicos y actividad antirradical de tubérculos y raíces andinos cultivados en el noroeste de Argentina / Chemical characterization and quantification of fructooligosaccharides, phenolic compounds and antiradical activity of Andean roots and tubers grown in Northwest of Argentina

Existe un creciente interés por consumir alimentos que además de aportar los componentes básicos para una buena nutrición provean otros compuestos benéficos para la salud. El objetivo del trabajo fue determinar la composición química de alimentos autóctonos de la región andina y cuantificar algunos componentes funcionales. Se determinó composición centesimal, contenido de vitamina C y compuestos fenólicos totales, actividad antirradical (DPPH) en cáscara y pulpa, fibra dietaria soluble e insoluble, fructooligosacáridos (FOS), almidón total y resistente (en tubérculos y raíces crudos, hervidos y hervidos y almacenados) de 6 variedades de Ocas (Oxalis tuberosa), 4 clones de Mandiocas (Manihot esculenta Crantz) y Yacón (Smallanthus sonchifolius). Los resultados mostraron mayor cantidad de compuestos bioactivos y actividad antirradical en la cáscara de ocas. En todos los casos el contenido de fibra insoluble fue mayor que la soluble. Las mandiocas tuvieron mayor contenido de almidón total que las raíces y tubérculos andinos. El proceso de ebullición disminuyó el contenido de almidón resistente en ocas y mandiocas pero cuando éstas se almacenaron por 48h a 5°C, el contenido de almidón resistente aumentó nuevamente. El contenido de FOS en ocas fue similar para todas las variedades (Aproximadamente 7%). El principal componente de los carbohidratos del yacón fueron los FOS (8,89%). Se puede concluir que las raíces y tubérculos estudiados, además de aportar nutrientes, contienen compuestos funcionales que les confieren un valor adicional como alimentos útiles para la prevención de algunas enfermedades no trasmisibles.

There is great interest in consuming foods that can provide the nutrients for a good nutrition and other health beneficial compounds. The aim of this work was to determine the chemical composition of native foods of the Andean region and to quantify some functional components. Proximal composition, vitamin C, total phenolic compounds, antiradical activity (DPPH) in peel and pulp, dietary fiber soluble and insoluble, fructooligosaccharides (FOS), total and resistant starch (in tubers and raw roots, boiled and boiled and stored) of 6 varieties of Oca (Oxalis tuberosa), 4 clones of manioc (Manihot esculenta Crantz) and yacon (Smallanthus sonchifolius were determined. The results showed greater amount of bioactive compounds and antiradical activity in the skin of these products. The highest content was found in the oca peel. In all cases, the content of insoluble fiber was greater than the soluble. The manioc had higher total starch than Andean roots and tubers. The boiling process decreased the resistant starch content of ocas and maniocs, but when these are stored for 48 h at 5 ° C, the resistant starch content increased. The FOS content of the ocas was similar for all varieties (7%). The main component of yacon carbohydrates were FOS (8,89%). The maniocs did not contain FOS. It can be concluded that the roots and tubers studied, in addition to provide nutrients, contain functional compounds that confer additional helpful value for preventing no communicable diseases.

Action of phosphorylated mannanoligosaccharides on immune and hematological responses and fecal consistency of dogs experimentally infected with enteropathogenic Escherichia coli strains

The therapeutic action of phosphorylated mannanoligosaccharides (MOS) was investigated regarding its prebiotic activity on enteropathogenic Escherichia coli (EPEC). Diarrhea was induced in dogs by experimental infection with EPEC strains. Then MOS was supplied once a day, in water for 20 days. Immunological (IgA and IgG), hematological (lymphocytes, neutrophils and monocytes) and bacteriological variables (PCR detection of the eae gene of EPEC recovered from stool culture), as well as occurrence of diarrhea were evaluated. All strains caused diarrhea at 24, 48 and 72 h after infection. PCR results indicated that E. coli isolated from stool culture of all infected animals had the eae gene. There was no significant difference among groups as to number of blood cells in the hemogram and IgA and IgG production. MOS was effective in recovering of EPEC-infected dogs since prebiotic-treated animals recovered more rapidly from infection than untreated ones (p < 0.05). This is an important finding since diarrhea causes intense dehydration and nutrient loss. The use of prebiotics for humans and other animals with diarrhea can be an alternative for the treatment and prophylaxis of EPEC infections.

Produccion de beta-glucosidasas por cultivos de bacterias termofilas indigenas del altiplano boliviano / beta-Glucoside production by thermophilic bacteria indigenous the bolivian altiplano culture

Las beta-glucosidasas son enzimas que poseen actividad hidrolitica y transferasa o transglucosidasa. Tienen diversas aplicaciones; en la biosintesis de oligosacaridos, produccion de etanol utilizando residuos agricolas y en la industria de vinos. La aplicacion industrial, sin embargo, requiere estabilidad a temperaturas elevadas, por lo que los microorganismos termofilos tienen gran interes. El proposito de esta investigacion es el de optimizar el medio de cultivo anaerobio de bacterias termofilas, para aumentar la produccion de beta-glucosidasas. Esta enzima es producida por tres aislados bacterianos: FT3, 2B y P5 los cuales fueron aislados de la region andina de Bolivia. El aislado bacteriano FT3 mostro una actividad beta-glucosidasa de 0,35 [UI/mL]. Se tomaron como variables dentro de la optimizacion del medio de cultivo las fuentes de nitrogeno y de carbono, y el pH. Asi tambien se probaron dos sistemas de cultivo: celulas libres y encapsuladas. Empleando extracto de levadura como fuente de nitrogeno se obtuvo una actividad de 0,52 [UI/mL]. En la optimizacion del pH del medio de cultivo se obtuvo una actividad de 0,81 [UI/mL] a pH 5. Como fuente de carbono se eligieron los hidrolizados de paja de trigo y paja de quinoa lleg¨¢ndose a obtener actividades de 1,27 y 1,34 [UI/mL] respectivamente. Se establecio que la localizacion celular de la enzima beta-glucosidasa es extracelular y presenta estabilidad hasta una temperatura de 80 ºC y un pH de 7.

The beta-glucosidases possess hydrolytic and transferase activity or transglucosidase. They have various applications; such as biosynthesis of oligosaccharides, production of ethanol using agricultural residues and wine industry. However for industrial application, stability to high temperatures is needed. Therefore a great interesting in the thermophile microorganism study exist. The purpose of this research is to optimize the culture medium of thermophilic anaerobic bacteria to increase the production of beta-glucosidase. This enzyme is produced by three isolate bacterial FT3, 2B and P5 which were isolated from the Andean region of Bolivia. FT3 isolate showed beta-glucosidase activity of 0.35 [IU/mL]. In regards to the optimization of culture medium variables such as nitrogen source, carbon source and pH were taken into account and also the combination with free and encapsulated bacterial cells. Yeast extract was the selected source of nitrogen obtaining an activity of 0.52 [IU/ mL]. The optimal pH was 5 obtaining an activity of 0.81 [IU/mL]. The selected carbon source was the hydrolyzed wheat straw and quinoa straw obtaining activities of 1.27 and 1.34 [IU/mL], respectively. The cellular localization of beta-glucosidase enzyme is extracellular and provides stability to temperature of 80 ºC and stability at pH 7.

Role of cell surface oligosaccharides of mouse mammary tumor cell lines in cancer metastasis.

Malignant transformation is associated with changes in the glycosylation of cell surface proteins and lipids. In tumor cells, alterations in cellular glycosylation may play a key role in their metastatic behaviour. In the present study, we have assessed the relationship between cell surface oligosaccharides and the metastasis ability of mouse mammary tumor cell lines 67NR and 4TO7. The cell surface oligosaccharides have been analyzed using specific binding assays with some plant lectins and the metastasis ability has been studied using transwell migration and invasion assays. In addition, we investigated the role of terminal sialic acids in the metastatic potential (cell adhesion on fibronectin, cell migration and invasion) in the 4TO7 cells on treatment with neuraminidase. The cell lines used in study have different metastasis abilities in vivo - the 67NR form primary tumors, but no tumor cells are detectable in any distant tissues, while cells of the 4TO7 line are able to spread to lung. In vitro metastasis experiments have revealed higher ability of adhesion, cell migration and invasion in the 4TO7 cells than the 67NR cells. Specific lectins binding assays show that the 4TO7 cells expressed more high-mannose type, multi-antennary complex-type N-glycans, beta-1,6-GlcNAc-branching, alpha-2,6-linked sialic acids, N-acetylgalactosamine and galactosyl(beta-1,3)-N-acetylgalactosamine. Removal of sialic acids on treatment with neuraminidase decreases adhesion, but increases the migration and has shown no significant change in the invasion ability of the 4TO7 cells. The study suggests that the sialic acids are not crucial for the cell migration and invasion in the 4TO7 cells. The findings provide the new insights in understanding the role of cell surface oligosaccharides in cancer metastasis.

Isolation and identification of alpha-amylase producing Bacillus sp. from dhal industry waste.

A bacterial strain was isolated from dhal industry red gram waste and identified as Bacillus. A thermostable extracellular amylase was partially purified from the strain. Optimum temperature and pH for the enzyme were found to be 60 degrees C and 6.5, respectively. The maximum amylase production was achieved with maltose as carbon source. Among the nitrogen sources, peptone and yeast extract produced maximum amylase.

Differential binding properties of Gal/GalNAc specific lectins available for characterization of glycoreceptors.

Differentiating the binding properties of applied lectins should facilitate the selection of lectins for characterization of glycoreceptors on the cell surface. Based on the binding specificities studied by inhibition assays of lectin-glycan interactions, over twenty Gal and/or GalNAc specific lectins have been divided into eight groups according to their specificity for structural units (lectin determinants), which are the disaccharide as all or part of the determinants and of GalNAc alpha 1-->Ser (Thr) of the peptide chain. A scheme of codes for lectin determinants is illustrated as follows: (1) F (GalNAc alpha 1-->3GalNAc), Forssman specific disaccharide--Dolichos biflorus (DBL), Helix pomatia (HPL) and Wistaria floribunda (WFL) lectins. (2) A (GalNAc alpha 1-->3 Gal), blood group A specific disaccharide--Codium fragile subspecies tomentosoides (CFT), Soy bean (SBL), Vicia villosa-A4 (VVL-A4), and Wistaria floribunda (WFL) lectins. (3) Tn (GalNAc alpha 1-->Ser (Thr) of the protein core)--Vicia villosa B4 (VVL-B4), Salvia sclarea (SSL), Maclura pomifera (MPL), Bauhinia purpurea alba (BPL) and Artocarpus integrifolia (Jacalin, AIL). (4) T (Gal beta 1-->3GalNAc), the mucin type sugar sequences on the human erythrocyte membrane(T alpha), T antigen or the disaccharides at the terminal nonreducing end of gangliosides (T beta)--Peanut (PNA), Bauhinia purpurea alba (BPL), Maclura pomifera (MPL), Sophora japonica (SJL), Artocarpus lakoocha (Artocarpin) lectins and Abrus precatorius agglutinin (APA).(5) I and II (Gal beta 1-->3(4)GlcNAc)--the disaccharide residue at the nonreducing end of the carbohydrate chains derived from either N- or O-glycosidic linkage--Ricinus communis agglutinin (RCA1), Datura stramonium (TAL, Thorn apple), Erythrina cristagalli (ECL, Coral tree), and Geodia cydonium (GCL). (6) B (Gal alpha 1-->3Gal), human blood group B specific disaccharide--Griffonia(Banderiaea) simplicifolia B4 (GSI-B4). (7) E (Gal alpha 1-->4Gal), receptors for pathogenic E. coli agglutinin, Shiga toxin and Mistletoe toxic lectin-I (ML-I) and abrin-a.

Sequencing of heparan sulfate proteoglycans: identification of variable and constant oligosaccharide regions in eight heparan sulfate proteoglycans of different origins

The sequence of the disacharide units of eight heparan sulfate proteoglycans of different origins is described. All heparan sulfates contain 5 variable regions made of oligosaccharide blocks of disaccharides, namely GlcUA(1-4) GlcNAc, GlcUA(1-4)GlcNS, IdoUA (104)GlcNS) and monosaccharides (GlcNS, and GlcNS,65) at the non-reducing terminal. The N-acetylated region of the heparan sulfates is linked to the serine of the protein core through a trisaccharide of Xyl-Gal-Gal. Heparan sulfates differ from one another in terms of the number of disaccharides that compose each block

Strategies for the structure determination of parasite glycoconjugates using fast atom bombardment mass spectrometry

Fast atom bombardment mass spectrometry (FAB MS) is a sensitive and powerful technique for the analysis of thermally labile and involatile materials. In this paper we describe the application of the method to the characterization of oligosaccharides isolated from the cell surface glycoconjugates of parasitic protozoa. The spectra typically contain both molecular ions, which define the monosaccharide composition, and fragment ions which are related to the structure of the intact molecules. The use of additional techniques such as chemical derivatization and colisional activation enhances fragmentation and simplifies interpretation of the data, enabling the determination of residue sequence, the positions of branch points, and the location of noncarbohydrate substituents. We have applied these techniques to the characterization of phosphoinositol oligosaccharides from members of the Trypanosoma family, including Leptomonas smueli, Endotrypanum schaudinni and Leishmania adleri. Although it is not usually possible to determine the complete structure of oligosaccharide by mass spectrometric methods alone, the information gained greatly simplifies the interpretation of the results from other techiniques such as nuclear magnetic resonance and methylation analysis.

Characterization of phosphoinositol oligosaccharides from parasitic protozoa by fast atom bombardment and collisional activation mass spectrometry

Fast atom bombardment mass spectrometry (FAB MS) enables the rapid, accurate and sensitive determination of the molecular masses of glycosylphosphatidylinositol(GPI)-derived oligosaccharides, from which the composition in terms of monosaccharide residues and non-carbohydrate substituents can be determined. Interpretation of fragment ions in collisional activation mass spectra further enables the determination of residue sequence, the positions of branch points, and the location of non-carbohydrate substituents. We have applied these techniques to the characterization of phosphoinositol oligosaccharides from Leptomonas samueli, Endotrypanum schaudinni and Leishmania adleri. The mass spectral data permit the postulation of candidate structures for the oligosaccharides, which provide a set of constraints that can assist the interpretation of results from other techniques such as NMR

Structures of four oligosaccharides derived from the glycoinositolphospholipid of Leishmania adleri

Glycoinositolphospholipids (GIPLs) were extracted from the trypanosomatid Leishmania adleri by hot phenol extraction and the carbohydrate moieties isolated after base cleavage. Purification of the crude oligosaccharides by high performance anion exchange (HPAE) chromatography yielded four fractions whose structures were determined by a combination of methylation analysis, fast atom bombardment (FAB) mass spectrometry and two-dimensional nuclear magnetic resonance (NMR) spectroscopy

The use of NMR spectroscopy in the structure determination of a Leptomonas samueli glycosylphosphosphingolipid-derived oligosaccharide

Nuclear magnetic resonance (NMR) spectroscopy provides an extremely powerful technique to determine the structure of oligosaccharides, particularly when used in conjuction with other physical techniques such as methylation analysis and fast atom bombardment mass spectroscopy (FAB-MS). This brief review describes the application of NMR to the determination of the structure of an oligosaccharide isolated from the glycophosphosphingolipid (GPS) from the monogenetic trypanosomatid Leptomonas samueli. Where ambiguities arise in the NMR interpretation, the use of other data will be discussed

Free and protein-linked glycoinositolphospholipids in Trypanosoma cruzi

Two glycoinositol phospholipids (GIPL A and GIPL B) have been purified from epimastigotes of Trypanosoma cruzi at the logarithmic phase of growth (2 days). The GIPLs differ mainly in the lipid moiety and are similar to the lipopeptidophosphoglycan (LPPG) previously isolated from epimastigotes at the stationary phase (4-5 days). [3H]-palmitic acid was incorporated into 1-O-hexadecyl-2-O-palmitoylglycerol in GIPL A and into a sphinganine ceramide with palmitic acid and lignoceric acid as the fatty acids in GIPL B. The lipids could be released by incubation with phosphatidylinositol-specific phospholipase C (PI-PLC) or glycosylphosphatidylinositol phospholipase D (GPI-PLD) from rat serum. The oligosaccharides share the common core structure of the glycosylphosphatidilinositol (GPI) membrane anchors. Microheterogeneity was demonstrated, as well as substitution by galactose, which is mainly in the furanose configuration as was previously described for the LPPG. However, methylation analysis indicated that 20 percent of the galactose is present as terminal pyranose units. In infective trypomastigotes, [3H]-palmitic acid was incorporated into the anchor of the Tc-85 glycoprotein. The lipid cleaved by phospholipase C digestion was identified as 1-O-hexadecylglycerol and the main oligosaccharide has the structure of the conserved core of all GPI anchors. [3H]-palmitic acid-labelled Tc-85 released into the culture medium as membrane vesicles showed 80 percent resistance to the action of PI-PLC. However, after mild alkaline hydrolysis, part of the radioactivity was released by the enzyme

Influence of primary and secondary structures of oligosaccharide chain on the aggregative and geometrical properties of gangliosides.

The aggregative and geometrical properties of gangliosides have been compared and discussed. It is shown that, due to the steric packing features of gangliosides, significative considerations can be made on the primary and secondary structures of their head group, starting from their cooperative behaviour, supporting NMR direct observations.

Developmentally-regulated cell surface N-linked oligosaccharides participate in intercellular cohesion in Dictyostelium discoideum.

The ability of purified plasma membrane glycoconjugates to inhibit the EDTA-resistant agglutination between aggregation-stage cells of Dictyostelium discoideum has suggested that receptor binding of these glycoconjugates provides a basis for cell-cell cohesion during aggregation. This has been tested by analysis of a series of mutants with different defects in the assembly of N-linked oligosaccharides. Mutant HL241 lacks outer branch components of N-linked oligosaccharides and fails to aggregate or express EDTA-resistant cohesion. HL244 makes unsulphated but otherwise normal N-linked oligosaccharides, generates multiple tips on aggregated cell mounds in some clones, and shows abnormally strong EDTA-resistant cohesion. Two mutants that are temperature-sensitive for complete processing of N-linked oligosaccharides are also temperature-sensitive for expression of both aggregation ability and EDTA-resistant cohesion. A revertant that recovered essentially normal N-linked oligosaccharide processing at the restrictive temperature has also recovered its ability to aggregate and to agglutinate in EDTA.

Heterogeneity in buffalo lutropin.

Lutropin (LH-1) from water buffaloes has been shown to exhibit microheterogeneity in the N-terminal amino-acid sequence of its alpha-subunit. The beta-subunit did not exhibit such microheterogeneity. Another protocol of purification yielded a preparation of buffalo LH (bu LH-2) different from the buffalo LH-1 in certain physico-chemical properties like ease of dissociation into subunits, sugar composition, isoelectric point, and elution profile on S-200. Data appear to indicate the presence of more than one form of buffalo lutropin.