Patterns of oligosaccharide production by polygalacturonases

The products of hydrolytic action of 18 enzyme preparations at pH 3·5 and 5·5 on pectate were analyzed by gel-filtration chromatography early in the course of reaction (8–15% hydrolysis), and at a time 10 times that required for 10% hydrolysis. The degree of hydrolysis at the latter time varied from 25 to 74%. Three patterns of oligosaccharide production could be distinguished: endo-hydrolysis, exo-hydrolysis, and that due to S-polygalacturonase. The initial products of endo-hydrolysis were mixed oligosaccharides 5–30 units long; monomer and dimer appeared early but represented less than 2% of the products until late in the reaction. exo-Polygalacturonase (not entirely free of endo-) showed predominant production of the monomer and was clearly evident when mixed with four parts of endo-polygalacturonase. The time course of reducing group production by highly purified S-polygalacturonase could be reproduced by the above mixture of exo- and endo-polygalacturonases, but the pattern of products and the pH relations could not. The initial products of S-polygalacturonase were monomer, dimer and pentamer with lesser amounts of trimer and tetramer. After the hydolysis of the polymer and large oligomers, the pentamer was attacked by S-polygalacturonase, in the same way that the accumulated hexamer, etc. were finally hydrolysed by the endo-polygalacturonase.     

Tissue distribution of a plasmid DNA encoding Hsp65 gene is dependent on the dose administered through intramuscular delivery

In order to assess a new strategy of DNA vaccine for a more complete understanding of its action in immune response, it is important to determine the in vivo biodistribution fate and antigen expression. In previous studies, our group focused on the prophylactic and therapeutic use of a plasmid DNA encoding the Mycobacterium leprae 65-kDa heat shock protein (Hsp65) and achieved an efficient immune response induction as well as protection against virulent M. tuberculosis challenge. In the present study, we examined in vivo tissue distribution of naked DNA-Hsp65 vaccine, the Hsp65 message, genome integration and methylation status of plasmid DNA. The DNA-Hsp65 was detectable in several tissue types, indicating that DNA-Hsp65 disseminates widely throughout the body. The biodistribution was dose-dependent. In contrast, RT-PCR detected the Hsp65 message for at least 15 days in muscle or liver tissue from immunized mice. We also analyzed the methylation status and integration of the injected plasmid DNA into the host cellular genome. The bacterial methylation pattern persisted for at least 6 months, indicating that the plasmid DNA-Hsp65 does not replicate in mammalian tissue, and Southern blot analysis showed that plasmid DNA was not integrated. These results have important implications for the use of DNA-Hsp65 vaccine in a clinical setting and open new perspectives for DNA vaccines and new considerations about the inoculation site and delivery system.     

Novel bacterial ferulic acid esterase from <Emphasis Type="Italic">Cellvibrio japonicus</Emphasis> and its application in ferulic acid release and xylan hydrolysis

Recent genome sequencing of Cellvibrio japonicas revealed the presence of two highly homologous ferulic acid esterases (FAEs), encoded by fee1A and fee1B. In this work, the putative FAE, Fee1B, was successfully cloned and expressed in an E. coli system and the purified enzyme was characterized as a type-D FAE with a pH and temperature optima of 6.5 and 35−40°C, respectively. Additionally, the two tandem N-terminal carbohydrate binding modules of the multi-domain enzyme were shown to be crucial for optimum enzyme activity. The potential of the enzyme in biomass processing was demonstrated with its high synergy with a xylanase in the release of reducing sugar from arabinoxylan and its ability to liberate ferulic acid from various complex xylan substrates.