Production of a Fungal Punicalagin-Degrading Enzyme by Solid-State Fermentation: Studies of Purification and Characterization.

The present work describes the purification of an enzyme capable of degrading punicalagin. The enzyme was produced by Aspergillus niger GH1 by solid-state fermentation, and the enzyme production was induced by using ellagitannins as the sole carbon source. The purification steps included the concentration by lyophilization, desalting, anionic exchange, and gel filtration chromatography. The enzyme kinetic constants were calculated by using punicalagin, methyl gallate, and sugar beet arabinans. The molecular mass of the protein was estimated by SDS-PAGE. The identified bands were excised and digested using trypsin, and the peptides were submitted to HPLC-MS/MS analysis. The docking analysis was conducted, and a 3D model was created. The purification fold increases 75 times compared with the cell-free extract. The obtained Km values were 0.053 mM, 0.53% and 6.66 mM for punicalagin, sugar beet arabinans and methyl gallate, respectively. The optimal pH and temperature for the reaction were 5 and 40 °C, respectively. The SDS-PAGE and native PAGE analysis revealed the presence of two bands identified as α-l-arabinofuranosidase. Both enzymes were capable of degrading punicalagin and releasing ellagic acid.

Effects of chronic exposure to low levels of IR on Medaka (Oryzias latipes): a proteomic and bioinformatic approach.

PURPOSE: Chronic exposure to ionizing radiation (IR) at low doses (<100 mGy) has been insufficiently studied to understand fully the risk to health. Relatively little knowledge exists regarding how species and healthy tissues respond at the protein level to chronic exposure to low doses of IR, and mass spectrometric-based profiling of protein expression is a powerful tool for studying changes in protein abundance. MATERIALS AND METHODS: SDS gel electrophoresis, LC-MS/MS mass spectrometry-based approaches and bioinformatic data analytics were used to detect proteomic changes following chronic exposure to moderate/low doses of radiation in adults and normally developed Medaka fish (Oryzias latipes). RESULTS: Significant variations in the abundance of proteins involved in thyroid hormone signaling and lipid metabolism were detected, which could be related to the gonadal regression phenotype observed after 21.04 mGy and 204.3 mGy/day exposure. The global proteomic change was towards overexpression of proteins in muscle and skin, while the opposite effect was observed in internal organs. CONCLUSION: The present study provides information on the impacts of biologically relevant low doses of IR, which will be useful in future research for the identification of potential biomarkers of IR exposure and allow for a better assessment of radiation biosafety regulations.

Chronic exposure to low doses of ionizing radiation impacts the processing of glycoprotein N-linked glycans in Medaka (Oryzias latipes).

PURPOSE: Ionizing radiation is found naturally in the environment. Low doses of IR may have beneficial applications, yet there is also potential for detrimental long-term health effects. Impacts following exposure to low levels of IR have been refractory to identification and quantification. Glycoprotein glycosylation is vital to cell-cell communication and organismal function, and sensitive to changes in an organism's macro- and cellular environment. We investigated whether accumulated low doses of IR (LoDIR) affect the N-linked glycoprotein glycans using Medaka fish (Oryzias latipes). MATERIALS AND METHODS: State-of-the-art methods in radiation exposure and glycan analysis were applied to study N-glycan changes after 190 day exposure at three different rates of gamma irradiation (2.25, 21.01, and 204.3 mGy/day) in wild-type adult Medaka. Tissue N-glycans were analyzed following enzymatic release from extracted proteins. RESULTS: N-linked glycan profiles are dominated by complex type N-glycans modified with terminal sialic acid and core fucose. Fucosylation and sialylation of N-linked glycoprotein glycans are affected by LoDIR and a subset of N-glycans are involved in the organismal radio-response. CONCLUSION: This is the first indication that the glycome can be interrogated for biomarkers that report the impact of chronic exposure to environmental stressors, such as low-level IR.

Isolation of a polygalacturonase-inhibiting protein (PGIP) from wheat.

Evidence for the presence of a polygalacturonase-inhibiting protein (PGIP) from a monocotyledonous cereal is presented. A 40.3-kDa PGIP that was closely associated with the cell wall was acetone-extracted and purified from wheat (Triticum aestivum L.) leaves and stems. Wheat PGIP exhibited a highly selective inhibitory activity against endopolygalacturonase (EPG) from various fungi. Of nine EPG tested, wheat PGIP only inhibited EPG from Cochliobolus sativus, a pathogen of the tribe Poaceae. A short N-terminal amino acid sequence of wheat PGIP shows no similarity to any other characterized PGIP.

Characterization of a tomato protein that inhibits a xyloglucan-specific endoglucanase.

A basic, 51 kDa protein was purified from suspension-cultured tomato and shown to inhibit the hydrolytic activity of a xyloglucan-specific endoglucanase (XEG) from the fungus Aspergillus aculeatus. The tomato (Lycopersicon esculentum) protein, termed XEG inhibitor protein (XEGIP), inhibits XEG activity by forming a 1 : 1 protein:protein complex with a Ki approximately 0.5 nm. To our knowledge, XEGIP is the first reported proteinaceous inhibitor of any endo-beta-1,4-glucanase, including the cellulases. The cDNA encoding XEGIP was cloned and sequenced. Database analysis revealed homology with carrot extracellular dermal glycoprotein (EDGP), which has a putative role in plant defense. XEGIP also has sequence similarity to ESTs from a broad range of plant species, suggesting that XEGIP-like genes are widely distributed in the plant kingdom. Although Southern analysis detected only a single XEGIP gene in tomato, at least five other XEGIP-like tomato sequences have been identified. Similar small families of XEGIP-like sequences are present in other plants, including Arabidopsis. XEGIP also has some sequence similarity to two previously characterized proteins, basic globulin 7S protein from soybean and conglutin gamma from lupin. Several amino acids in the XEGIP sequence, notably 8 of the 12 cysteines, are generally conserved in all the XEGIP-like proteins we have encountered, suggesting a fundamental structural similarity. Northern analysis revealed that XEGIP is widely expressed in tomato vegetative tissues and is present in expanding and maturing fruit, but is downregulated during ripening.

Effect of pectic oligomers on physiological responses of chilling injury in discs excised from zucchini (Cucurbita pepo L.).

The effect of pectic oligomers (OG) on ethylene biosynthesis, electrolyte leakage (EL), and CO(2) production was studied in discs excised from zucchini fruit (Cucurbita pepo L.) and stored at 20 or 2.5 degrees C. At 20 degrees C, OG enhanced ethylene biosynthesis and had a transient effect on decreasing EL, but showed little effect on respiratory rate; both the amount and size of the oligomer were important in changing both ethylene synthesis and EL. At 2.5 degrees C, OG increased both ethylene biosynthesis and respiratory rate with a maximum effect at 100 microg of oligomer and peaking at 6 h; shorter oligomers demonstrated an even greater effect on ethylene biosynthesis, but differences were smaller in respiratory rate. EL at 2.5 degrees C was affected most by 1 microg of OG and by monomeric galacturonic acid, with transient increases that peaked at 8 h. We suggest a signaling role for OG in the early steps of cold acclimation or chilling injury.