Characterization of a new endo-type polyM-specific alginate lyase from Pseudomonas sp.

An alginate lyase gene, algA, encoding a new poly ß-D-mannuronate (polyM)-specific alginate lyase AlgA, was cloned from Pseudomonas sp. E03. The recombinant AlgA with (His)6-tag, consisting of 364 amino acids (40.4 kDa),was purified using Ni-NTA Sepharose. The purified lyase had maximal activity (222 EU/mg) at pH 8 and 30 °C and also maintained activity between pH 7-9 and below 45 °C. It exclusively and endolytically depolymerized polyM by ß-elimination into oligosaccharides with degrees of polymerization (DP) of 2-5. Due to its high substrate specificity, AlgA could be a valuable tool for production of polyM oligosaccharides with low DP and for determining the fine structure of alginate.

Characterization of a new alginate lyase from newly isolated Flavobacterium sp. S20.

Alginate lyase is a promising biocatalyst because of its application in saccharification of alginate for the production of biochemicals and renewable biofuels. This study described the isolation of a new alginate metabolizing bacterium, Flavobacterium sp. S20, from sludge samples and the characterization of its alginate lyase Alg2A. The alginate lyase gene, alg2A, was obtained by constructing and screening the genomic library of the strain S20 and overexpressed in Escherichia coli. Substrate specificity assays indicated Alg2A preferred poly-α-L-guluronate as a substrate over poly-ß-D-mannuronate. In the saccharification process of a high content (10 %, w/v) of sodium alginate, the recombinant alginate lyase Alg2A yielded 152 of mM the reducing sugars after 69 h of reaction, and the amounts of oligosaccharides with a different degree of polymerization (DP) generated by Alg2A gradually accumulated without significant variation in the distribution of oligosaccharide compositions. These results indicated that Alg2A possessed high enzymatic capability for saccharifying the alginate, which could be used in saccharifying the alginate biomass prior to the main fermentation process for biofuels. In addition, Alg2A had a different endolytic reaction mode from both the two commercial alginate lyases and other alginate lyases from polysaccharide lyase family 7 owing to high yields of penta-, hex-, and hepta-saccharides in the hydrolysis products of Alg2A. Thus, Alg2A could be a good tool for the large-scale preparation of alginate oligosaccharides with high DP.

Improvement of mannitol production by Lactobacillus brevis mutant 3-A5 based on dual-stage pH control and fed-batch fermentations.

Lactobacillus brevis 3-A5 was isolated and expected to produce mannitol efficiently by regulating pH in batch and fed-batch fermentations. In 48 h batch fermentations with free and constant pH, the optimal pH for cell growth and mannitol production in the first 24 h of incubation was 5.5, whereas that for mannitol production in the second 24 h of incubation was 4.5. To achieve high cell density and mannitol yield simultaneously, a dual-stage pH control strategy was proposed based on the kinetic analysis of mannitol production. The pH value was controlled at 5.5 for the first 12 h of fermentation and subsequently shifted to 4.5 until the fermentation was completed. Under dual-stage pH control fermentation, a 103 g/L yield of mannitol with a volumetric production rate of 3.7 g/L/h was achieved after 28 h. The dual-stage pH control fed-batch fermentation strategy was further developed to improve mannitol yield, wherein the yield increased by 109 % to 215 g/L after 98 h of fermentation. This value is the highest yield of mannitol ever reported using L. brevis.

Peptide-Based Therapeutic HPV Cancer Vaccine Synthesized via Bacterial Outer Membrane Vesicles.

Background: Peptide-based vaccines have broad application prospects because of their safety, simple preparation, and effectiveness, especially in the development of personalized cancer vaccines, which have shown great advantages. However, the current peptide-based vaccines often require artificial synthesis and intricate delivery technology, which increases the cost and complexity of preparation. Methods: Here, we developed a simple technique for combining a peptide and a delivery system using the natural secretion system of bacteria. Specifically, we biosynthesized an antigenic peptide in bacteria, which was then extracellularly released through the bacterial secretory vesicles, thus simultaneously achieving the biosynthesis and delivery of the peptide. Results: The system utilizes the natural properties of bacterial vesicles to promote antigen uptake and dendritic cell (DC) maturation. Therefore, tumor-specific CD4+ Th1 and CD8+ cytotoxic T lymphocyte (CTL) responses were induced in TC-1 tumor-bearing mice, thereby efficiently suppressing tumor growth. Conclusion: This research promotes innovation and extends the application of peptide-based vaccine biosynthesis technology. Importantly, it provides a new method for personalized cancer immunotherapy that uses screened peptides as antigens in the future.

Meroterpenoids with a large conjugated system from Ganoderma lucidum and their inhibitory activities against renal fibrosis.

Baoslingzhines A-E (1-5), five new meroterpenoids were isolated from the fruiting bodies of Ganoderma lucidum. The structures including their absolute configurations were characterized by using spectroscopic and computational methods. Compound 1 is a novel trinormeroterpenoid featuring the presence of an unusual dihydronaphthalene representing an unprecedented meroterpenoid skeleton. Compounds 2-4 are mononormeroterpenoids characteristic of a large conjugated system. Among them, racemic 3 and 4 were separated by HPLC on chiral phase. Biological evaluation toward kidney fibrosis found that compounds 2 and (+)-3 could inhibit the expression of fibronectin and collagen I dose dependently in TGF-ß1-induced rat kidney proximal tubular cells (NRK-52e). Additionally, (+)-3 could also down regulate ɑ-SMA in a concentration dependent manner. Further investigation showed that 2 could inhibit Smad2 phosphorylation.

Draft genomic sequence of Armillaria gallica 012m: insights into its symbiotic relationship with Gastrodia elata.

Armillaria species (Basidiomycota, Physalacriaceae) are well known as plant pathogens related to serious root rot disease on various trees in forests and plantations. Interestingly, some Armillaria species are essential symbionts of the rare Chinese medicinal herb Gastrodia elata, a rootless and leafless orchid used for over 2000 years. In this work, an 87.3-M draft genome of Armillaria gallica 012m strain, which was symbiotic with G. elata, was assembled. The genome includes approximately 23.6% repetitive sequences and encodes 26,261 predicted genes. In comparison with other four genomes of Armillaria, the following gene families related to pathogenicity/saprophytic phase, including cytochrome P450 monooxygenases, carbohydrate-active enzyme AA3, and hydrophobins, were significantly contracted in A. gallica 012m. These characteristics may be beneficial for G. elata to get less injuries. The genome-guided analysis of differential expression between rhizomorph (RH) and vegetative mycelium (VM) showed that a total of 2549 genes were differentially expressed, including 632 downregulated genes and 1917 upregulated genes. In the RH, most differentially expressed genes (DEGs) related to pathogenicity were significantly upregulated. To further elucidate gene function, Gene Ontology enrichment analysis showed that the upregulated DEGs significantly grouped into monooxygenase activity, hydrolase activity, glucosidase activity, extracellular region, fungal cell wall, response to xenobiotic stimulus, response to toxic substance, etc. These phenomena indicate that RH had better infection ability than VM. The infection ability of RH may be beneficial for G. elata to obtain nutrition, because the rhizomorph constantly infected the nutritional stems of G. elata and formed the hyphae that can be digested by G. elata. These results clarified the characteristics of A. gallica 012m and the reason why the strain 012m can establish a symbiotic relationship with G. elata in some extent from the perspective of genomics.