Characterization of a D-mannitol oxidase from Paenibacillus sp. and its application in the preparation of D-mannose / 生物工程学报

D-mannose has many functional activities and is widely used in food, medicine, agriculture and other industries. D-mannitol oxidase that can efficiently convert D-mannitol into D-mannose has potential application in the enzymatic preparation of D-mannose. A D-mannitol oxidase (PsOX) was found from Paenibacillus sp. HGF5. The similarity between PsOX and the D-mannitol oxidase (AldO) from Streptomyces coelicolor was 50.94%. The molecular weight of PsOX was about 47.4 kDa. A recombinant expression plasmid pET-28a-PsOX was constructed and expressed in Escherichia coli BL21(DE3). The Km and kcat/Km values of PsOX for D-mannitol were 5.6 mmol/L and 0.68 L/(s·mmol). Further characterization of PsOX showed its optimal pH and temperature were 7.0 and 35 ℃, respectively, while its enzyme activity could be stably remained below 60 ℃. The molar conversion rate of 400 mmol/L D-mannitol by PsOX was 95.2%. The whole cells of PsOX and AldO were used to catalyze 73 g/L D-mannitol respectively. The reaction catalyzed by PsOX completed in 9 h and 70 g/L D-mannose was produced. PsOX showed a higher catalytic efficiency compared to that of AldO. PsOX may facilitate the enzymatic preparation of D-mannose as a novel D-mannose oxidase.

Haemagglutination and siderophore production as the urovirulence markers of uropathogenic Escherichia coli.

A total of 160 strains of Escherichia coli isolated from urine of patients with clinically diagnosed urinary tract infection were included in the study and 50 faecal isolates of E. coli were studied. They were studied for virulence factors, namely mannose-resistant and mannose-sensitive haemagglutination (MRHA, MSHA) and siderophore production.Among 160 urinary isolates of E. coli , 40 (25%) showed MRHA, siderophore production was seen in 156 (97.5%). In 50 faecal isolates, two (4%) were MRHA, four (8%) MSHA and siderophore production in two (4%). The results suggest that MRHA and siderophore production positive strains can be considered as UPEC.

Isolation of temperature-sensitive yeast GPI-anchoring mutants

We are using a genetic approach to explore the synthesis and function of glycosylphosphatidylinositol (GPI). We have developed a novel strategy to isolate Saccharomyces cerevisiae mutants blocked in GPI anchoring by screening colonies of mutagenized yeast cells for those that fail to incorporate [3H]inositol into protein. Among our isolates are strains blocked in mannosylation of the GPI-anchor precursor, and strains defective in the synthesis of N-acetylglucosaminylphosphatidylinositol (GlcNAc-PI). We have characterized one mutant, gpil, further. This strain is defective in GlcNAC-PI synthesis and is temperature-sensitive for growth. Completion of the first step in GPI assembly is therefore required for the growth of the unicellular eukaryote S. cerevisiae. We have isolated plasmids that complement the gpil mutation from S. cerevisiae genomic DNA- and fission yeast cDNA libraries

Metabolism of GPIs in mammalian cells

In Trypanosoma brucei, glycosylphosphatidylinositol (GPI) anchors of proteins and free GPIs with identical structures have been characterized. This identity provides strong presumptive evidence that the free GPIs are in fact precursors of the GPI anchors on proteins. In mammalian tissues, however, rather consistent differences in the structures of free GPIs and GPI anchors are observed. The terminal GPIs produced by the mammalian biosynthetic pathway differ from GPI anchors in being almost exclusively fatty acid acylated on the inositol residue, having a greater number of phosphoethanolamine residues, and perhaps in containing a greater percentage of diacylglycerol components. While in principle these differences could be reconciled by remodeling reactions before or after attachment of GPI anchors, it is possible that some of the mammalian free GPIs play cellular roles other than as anchor precursors. We have approached this question by studying the lifetimes of the last three GPIs on the biosynthetic pathway, denoted H6, H7 and H8, in K562 cells and in K562 mutant designated class K that is devoid of GPI-anchored proteins. Pulse-chase metabolic labeling with [3H]-mannose indicated that H6 was a precursor of H7 and H8 and that the H8 lifetime was more than one hour in the parental cells and even longer in the mutant. Preliminary data indicated that the majority of each of the three GPIs was localized in the plasma membrane fraction rather than the endoplasmic reticulum. These observations argue that mammalian GPIs are not utilized exclusively as GPI anchor precursors

El enlace O-glicosídico en glicoproteínas de plasma seminal humano / The linkage O-glycosidic in glycoproteins of human seminal plasma

La composición y estructura de los extremos terminales no reductores portadores de los determinantes antigénicos en las cadenas de oligosacáridos de las glicoproteínas del plasma seminal humano, han permitido deducir que estos componentes son fragmentos producidos por la degradación proteolítica endógena de las mucinas. Dado que las cadenas de hidratos de carbono unidas O-glicosídicamente a la serina y la treonina de la cadena peptídica dan lugar a una reacción de beta-eliminación en condiciones alcalinas suaves, hicimos uso del tratamiento alcalino para demostrar la presencia de enlaces lábiles al álcali en las muestras de plasma seminal humano y algunas fracciones glicoproteicas aisladas a partir del mismo. El análisis de los hidratos de carbono que se liberan durante ell tratamiento han permitido obtener información sobre la composición y estructura de este tipo de unidades de oligosacarídos. Hemos identificado al monosacárido involucrado en el enlace O-glicosídico a serina y treonina y hemos podido caracterizar, a través de la reacción de beta-eliminación en condiciones reductoras y no reductoras, las unidades de monosacáridos del extremo terminal reductor