ALBERT

Description: Histamine is an immune mediator that is mainly secreted when an immediate, rapid response is needed in the body, and an excessive secretion of histamine or lack of enzymes that degrade histamine can result in various side effects. Histamine binding protein (HBP) is secreted by a mite species to prevent the host’s histamine-induced immune responses by binding the histamine molecule in the blood. Cloning was performed to express HBP on the yeast surface (MBTL-GWL-1), and immunofluorescence (IF) and western blot was performed to confirm the expression of the recombinant protein. The histamine inhibitory ability of GWL-1 cells was tested according to the cell concentration. The highest inhibitory ability of 1.30 × 107 CFU/ml of GWL-1 cells was of about 60 %. The GWL-1 cell concentration and the degree of histamine inhibition were confirmed to be dose-dependent, and dead cell debris was shown to have a histamine inhibitory effect, although not as much as that of whole cells. Phagocytosis assays were performed to determine whether histamine affected the RAW 264.7 cell’s phagocytosis, and to indirectly confirm the GWL-1 cell’s histamine inhibition. By confirming that, we found that GWL-1 captures histamine. Therefore, it can be expected to become a competitive material in the anti-allergy market.

Description: Red-osier dogwood (ROD) extract contains a lot of polyphenols that have the potential for modulation of gut microbiota. However, little information is available about its prebiotic properties. This study investigated the impact of ROD polyphenol extract on the ileal microbiota with dietary supplementation of ROD polyphenol extract in a pig model. The data indicated that supplementation of ROD polyphenol extract significantly increased class Bacilli, order Lactobacillales and family lactobacillaceae. Within family lactobacillaceae, Lactobacillus was the main responder by increasing from 5.92% to 35.09%. Further analysis showed that ROD polyphenol extract improved two species Lactobacillus delbrueckii and Lactobacillus mucus. The results of this study suggested that ROD polyphenol extract has the potential to play prebiotic role and confer health benefit through modifying gut microbiota.

Description: Wild-type cultivations are of invaluable relevance for industrial biotechnology when it comes to the agricultural or food sector. Here, genetic engineering is hardly applicable due to legal barriers and consumer’s demand for GMO-free products. An important pillar for wild-type cultivations displays the genus Bacillus. One of the challenges for Bacillus cultivations is the global ComX-dependent quorum sensing system. Here, molecular process control can serve as a tool to optimize the production process without genetic engineering. To realize this approach, quantitative knowledge of the mechanism is essential, which, however, is often available only to a limited extent. The presented work provides a case study based on the production of cyclic lipopeptide surfactin, whose expression is in dependence of ComX, using natural producer B. subtilis DSM 10 T. First, a surfactin reference process with 40 g/L of glucose was performed as batch fermentation in a pilot scale bioreactor system to gain novel insights into kinetic behavior of ComX in relation to surfactin production. Interestingly, the specific surfactin productivity did not increase linearly with ComX activity. The data were then used to derive a mathematic model for the time course of ComX in dependence of existing biomass, biomass growth as well as a putative ComX-specific protease. The newly adapted model was validated and transferred to other batch fermentations, employing 20 and 60 g/L glucose. The applied approach can serve as a model system for molecular process control strategies, which can thus be extended to other quorum sensing dependent wild-type cultivations.

Description: This study sets out to compare the antibacterial and antibiofilm profiles of Ci/Ca EOs alone and in combination together against infectious bacterial strains. MIC assay was carried out to survey the effectiveness of prepared EOs by two-fold serial dilution method and MTT evaluation. Synergic antibacterial properties of EOs against target strains were studied by using checkerboard titration method. Biofilm growth and development were evaluated using CV and XTT reduction assays. Antibacterial activity was observed for EOs against both bacterial strains with stronger activity for CiEO against both bacteria. The synergistic antibacterial effect was observed only against B. subtilis. Based on the FIC index, combinations could not inhibit the growth of E. coli. The pure EOs and their combination inhibited cell attachment for both studied bacteria with stronger effect on E. coli. CV and XTT reduction assays results showed that Ci EO and its combination with CaEO had the highest antibiofilm activity at lowest MIC value 0.08% and 0.04/0.02% against biofilm formed by E. coli and B. subtilis respectively, indicating a high antibiofilm potential. Computational docking analyses also postulated that the active constituents of evaluated EOs have the potential to interact with different bacterial targets, suggested binding mode of action of EOs metabolites. By and large, synergistic anti-biofilm properties of EOs may provide further options for developing novel formula to inhibit a variety of infectious clinical and industrial strains without (or less) toxicity effects on human body. Graphical Abstract

Description: Microorganisms play an essential role in sulfide removal. Alkaline absorption solution facilitates the sulfide’s dissolution and oxidative degradation, so haloalkaliphile is a prospective source for environmental-friendly and cost-effective biodesulfurization. In this research, 484 sulfide oxidation genes were identified from the metagenomes of the soda-saline lakes and a haloalkaliphilic heterotrophic bacterium Halomonas salifodinae IM328 (=CGMCC 22183) was isolated from the same habitat as the host for expression of a representative sequence. The genetic manipulation was successfully achieved through the conjugation transformation method, and sulfide: quinone oxidoreductase gene (sqr) was expressed via pBBR1MCS derivative plasmid. Furthermore, a whole-cell catalyst system was developed by using the engineered strain that exhibited a higher rate of sulfide oxidation under the optimal alkaline pH of 9.0. The whole-cell catalyst could be recycled six times to maintain the sulfide oxidation rates from 41.451 to 80.216 µmol·min−1·g−1 dry cell mass. To summarize, a whole-cell catalyst system based on the engineered haloalkaliphilic bacterium is potentiated to be applied in the sulfide treatment at a reduced cost.

Description: The Chinese mitten crab (Eriocheir sinensis) is a popular aquaculture product in East Asia, especially in China. In the last decade, rice–crab co-culture has rapidly expanded in China. Under this model, crabs are raised in rice fields instead of in traditional aquaculture ponds. In this study, we cultured two varieties of Chinese mitten crabs (Changjiang and Liaohe) in an alkaline region in northwest China and used Illumina MiSeq sequencing to compare the intestinal bacterial alpha diversity and community structure between traditional and co-culture aquaculture models, between two crab varieties, and between female and male crabs. Significant variations in intestinal bacterial communities were found between crab varieties and between female and male crabs but not between aquaculture models. These results show that rice–crab co-culture operations did not obviously impact the crab intestinal bacterial community compared with traditional pond aquaculture. Firmicutes was the most abundant bacterial phylum in the crab intestines (78%, relative abundance). Three dominant operational taxonomic units (OTUs) represented 73.2% of Firmicutes sequences and 56.8% of all sequences. A dominant OTU assigned as Firmicutes that was negatively correlated with crab body length, width, and weight was found in the source water for the experimental area. The results of this study suggest that the aquaculture of Chinese mitten crabs in alkaline regions requires more study to improve cultivation techniques.

Description: Uncultured microbes are an important resource for the discovery of novel enzymes. In this study, an amylase gene (amy2587) that codes a protein with 587 amino acids (Amy2587) was obtained from the metagenomic library of macroalgae-associated bacteria. Recombinant Amy2587 was expressed in Escherichia coli BL21 (DE3) and was found to simultaneously possess α-amylase, agarase, carrageenase, cellulase, and alginate lyase activities. Moreover, recombinant Amy2587 showed high thermostability and alkali resistance which are important characteristics for industrial application. To investigate the multifunctional mechanism of Amy2587, three motifs (functional domains) in the Amy2587 sequence were deleted to generate three truncated Amy2587 variants. The results showed that, even though these functional domains affected the multiple substrates degrading activity of Amy2587, they did not wholly explain its multifunctional characteristics. To apply the multifunctional activity of Amy2587, three seaweed substrates (Grateloupia filicina, Chondrus ocellatus, and Scagassum) were digested using Amy2587. After 2 h, 6 h, and 24 h of digestion, 121.2 ± 4 µg/ml, 134.8 ± 6 µg/ml, and 70.3 ± 3.5 µg/ml of reducing sugars were released, respectively. These results show that Amy2587 directly and effectively degraded three kinds of raw seaweeds. This finding provides a theoretical basis for one-step enzymatic digestion of raw seaweeds to obtain seaweed oligosaccharides.

Description: Robust statistical tools such as the Skellam model and Bayesian networks can capture the count properties of transcriptome sequencing data and clusters of genes among treatments, thereby improving our knowledge of gene functions and networks. In this study, we successfully implemented a model to analyze a transcriptome dataset of Cucumis sativus and Botrytis cinerea before and after their interaction. First, 4200 differentially expressed genes (DEGs) from C. sativus were clustered into 17 distinct groups, and 670 DEGs from B. cinerea were clustered into 12 groups. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were applied on these DEGs to assess the interactions between C. sativus and B. cinerea. In C. sativus, more DEGs were divided into terms in the molecular function and biological process domains than into cellular components, and 277 DEGs were allocated to 19 KEGG pathways. In B. cinerea, more DEGs were divided into terms in the biological process and cellular component domains than into molecular functions, and 150 DEGs were allocated to 26 KEGG pathways. In this study, we constructed networks of genes that interact with each other to screen hub genes based on a directed graphical model known as Bayesian networks. Through a detailed GO analysis, we excavated hub genes which were biologically meaningful. These results verify that availability of Skellam model and Bayesian networks in clustering gene expression data and sorting out hub genes. These models are instrumental in increasing our knowledge of gene functions and networks in plant–pathogen interaction.

Description: Ajuga integrifolia Buch. Ham. ex D.Don, a member of Lamiaceae family is pharmaceutically an active perennial herb widely spread in China, Afghanistan and Pakistan Himalayan region. The application of biotic elicitors is a promising approach to cover limitations of in vitro cell technology and challenges faced by pharmaceuticals industry for bulk up production. The current study involved the induction of agitated micro-shoot cultures with the aim to investigate the growth-promoting as well as phytochemicals enhancement role of yeast extract (YE) and pectin (PE). The results showed that both elicitors induced a considerable physiological response. Biomass accumulation was observed maximum (DW: 18.3 g/L) against PE (10 mg/L) compared to YE and control. Eleven secondary phytocompounds were quantified using high-performance liquid chromatography. PE (50 mg/L) was found to be effective in elicitation of rosmarinic acid (680.20 µg/g), chlorogenic acid (294.12 µg/g), apigenin (579.61 µg/g) and quercetin (596.89 µg/g). However, maximum caffeic acid (359.52 µg/g) and luteolin (546.12 µg/g accumulation was noted in PE (1 mg/L) treatment. Harpagide, aucubin, harpagoside and 8-O-acetyl-harpagoside production was suppressed by both elicitors except for YE (100 mg/L). Catalpol accumulation in micro-shoot cultures was also downregulated except in response to YE (50 and 100 mg/L). Antioxidant activity and anti-inflammatory activity remained higher under PE (50 mg/L) and YE (100 mg/L) respectively. Therefore, results suggested that Ajuga integrifolia micro-shoot cultures treated with yeast extract and pectin might be an efficient bio-factory to produce commercially potent specific secondary metabolites.

Description: The glycosidases within GH5-23 cleave the glycosidic bond of β-glucosylated or rutinosylated flavonoids. Moreover, by virtue of their transglycosylation activity, glycoconjugates with glucosyl and rutinosyl moieties are accessible. Here we report the biochemical characterization and biotechnological assessment of two heterologously expressed members of GH5-23—McGlc from Mucor circinelloides and PcGlc from Penicillium chrysogenum. Both enzymes exhibited the highest hydrolytic activities with quercetin-3-β-O-glucopyranoside, whereas lower specificity constants were determined with the rutinosides narcissin, rutin and hesperidin. High stabilities against thermal, ethanol and dimethyl sulfoxide-induced inactivation, a very limited secondary hydrolysis of the formed transglycosylation products, and no detectable product inhibition were additional features appropriate for biotechnological applications. The enzymes were compared in their efficiencies to hydrolyze rutin and to synthesize 2-phenylethyl rutinoside under homogeneous and heterogeneous reaction conditions using high rutin concentrations of 100 and 300 mM. Highest transglycosylation efficiencies were achieved with fully dissolved rutin in reaction mixtures containing 25% dimethyl sulfoxide. Molecular docking and multiple sequence alignments suggest that the hydrophobic environment of aromatic residues within the + 1 subsite of GH5-23 glycosidases is very important for the binding of flavonoid glucosides and rutinosides.