Applied Biochemistry and Biotechnology (v.171, #4)
Zeaxanthin Production by Novel Marine Isolates from Coastal sand of India and its Antioxidant Properties by Sudharshan Prabhu; Rekha PD; Chiu-Chung Young; Asif Hameed; Shih-Yao Lin; Arun AB (817-831).
Zeaxanthin carotenoids are class of commercially important natural products and diverse biomolecules produced by plants and many microorganisms. Bacteria often produce a cocktail of polar and nonpolar carotenoids limiting their industrial applications. Marine members of the family Flavobacteriaceae are known to produce potential carotenoids such as astaxanthin and zeaxanthin. A few bacterial species have been reported for the predominant production zeaxanthin. Here, we report the molecular identification of the zeaxanthin as a major carotenoid produced by two novel bacteria (YUAB-SO-11 and YUAB-SO-45) isolated from sandy beaches of South West Coast of India and the effect of carbon sources on the production of zeaxanthin. The strains were identified based on the 16S rRNA gene sequencing as a member of genus Muricauda. The closest relatives of YUAB-SO-11 and YUAB-SO-45 were Muricauda aquimarina (JCM 11811T) (98.9 %) and Muricauda olearia (JCM 15563T) (99.2 %), respectively, indicating that both of these strains might represent a novel species. The highest level of zeaxanthin production was achieved (YUAB-SO-11, 1.20 ± 0.11 mg g−1) and (YUAB-SO-45, 1.02 ± 0.13 mg g−1) when cultivated in marine broth supplemented with 2 % NaCl (pH 7) and incubated at 30 °C. Addition of 0.1 M glutamic acid, an intermediate of citric acid cycle, enhanced the zeaxanthin production as 18 and 14 % by the strains YUAB-SO-11 and YUAB-SO-45 respectively. The zeaxanthin showed in vitro nitric oxide scavenging, inhibition of lipid peroxidation, and 2,2-diphenyl-1-picryl hydrazyl scavenging activities higher than the commercial zeaxanthin. The results of this study suggest that two novel strains YUAB-SO-11 and YUAB-SO-45 belonging to genus Muricauda produce zeaxanthin as a predominant carotenoid, and higher production of zeaxanthin was achieved on glutamic acid supplementation. The pigment showed good in vitro antioxidant activity, which can be exploited further for commercial applications.
Keywords: Muricauda olearia ; Zeaxanthin; Flavobacterium; Antioxidant activity
Dynamic Changes in Xylanases and β-1,4-Endoglucanases Secreted by Aspergillus niger An-76 in Response to Hydrolysates of Lignocellulose Polysaccharide by Sheng Xing; Guoli Li; Xulu Sun; Su Ma; Guanjun Chen; Lushan Wang; Peiji Gao (832-846).
Aspergillus niger is an effective secretor of glycoside hydrolases that facilitate the saprophytic lifestyle of the fungus by degrading plant cell wall polysaccharides. In the present study, a series of dynamic zymography assays were applied to quantify the secreted glycoside hydrolases of A. niger cultured in media containing different carbon sources. Differences in the diversity and concentrations of polysaccharide hydrolysates dynamically regulated the secretion of glycoside hydrolases. The secretion of β-1,4-endoglucanase isozymes was observed to lag at least 24 h behind, rather than coincide with, the secretion of xylanase isozymes. Low concentrations of xylose could induce many endoxylanases (such as Xyn1/XynA, Xyn2, and Xyn3/XynB). High concentrations of xylose could sustain the induction of Xyn2 and Xyn3/XynB but repress Xyn1/XynA (GH10 endoxylanase), which has a broad substrate specificity, and also triggers the low-level secretion of Egl3/EglA, which also has a broad substrate specificity. Mixed polysaccharide hydrolysates sustained the induction of Egl1, whereas the other β-1,4-endoglucanases were sustainably induced by the specific polysaccharide hydrolysates released during the hydrolysis process (such as Egl2 and Egl4). These results indicate that the secretion of glycoside hydrolases may be specifically regulated by the production of polysaccharide hydrolysates released during the process of biomass degradation.
Keywords: Aspergillus niger ; Glycoside hydrolase; Carbon source; Dynamic zymography assay; Hydrolysates of lignocellulose
RNAi for Insect Control: Current Perspective and Future Challenges by Rajan Katoch; Amit Sethi; Neelam Thakur; Larry L. Murdock (847-873).
The research on the RNA interference (RNAi) for the control of insect pests has made significant growth in recent years. The availability of the genomic sequences of insects has further widened the horizons for the testing of this technology to various insect groups. Different modes of application of double-stranded RNA (dsRNA) have been tested; however, the practicability of delivery of dsRNA in insects still remains the biggest challenge. Till date, the oral delivery of dsRNA in insects is one of the efficient approaches for the practical application of this technique. The uptake of dsRNA from the insect gut is mediated either by SID-1/SID-2 transmembrane proteins or by endocytosis; however, the systemic RNAi machinery still remains to be revealed in insect species. The RNAi-mediated gene knockdown has shown striking results in different insect groups, pointing it to be the upcoming technique for insect control. However, before the successful application of this technique for insect control, some potential issues need to be resolved. This review presents the account of prospects and challenges for the use of this technology for insect control.
Keywords: RNAi; Dicer; Gene knockdown; Insect control; dsRNA
Exploring the Structural Insights on Human Laforin Mutation K87A in Lafora Disease—A Molecular Dynamics Study by P. S. Srikumar; K. Rohini (874-882).
Lafora disease (LD) is an autosomal recessive, progressive form of myoclonus epilepsy which affects worldwide. LD occurs mainly in countries like southern Europe, northern Africa, South India, and in the Middle East. LD occurs with its onset mainly in teenagers and leads to decline and death within 2 to 10 years. The genes EPM2A and EPM2B are commonly involved in 90 % of LD cases. EPM2A codes for protein laforin which contains an amino terminal carbohydrate binding module (CBM) belonging to the CBM20 family and a carboxy terminal dual specificity phosphatase domain. Mutations in laforin are found to abolish glycogen binding and have been reported in wet lab methods. In order to investigate on structural insights on laforin mutation K81A, we performed molecular dynamics (MD) simulation studies for native and mutant protein. MD simulation results showed loss of stability due to mutation K87A which confirmed the structural reason for conformational changes observed in laforin. The conformational change of mutant laforin was confirmed by analysis using root mean square deviation, root mean square fluctuation, solvent accessibility surface area, radius of gyration, hydrogen bond, and principle component analysis. Our results identified that the flexibility of K87A mutated laforin structure, with replacement of acidic amino acid to aliphatic amino acid in functional CBM domain, have more impact in abolishing glycogen binding that favors LD.
Keywords: Laforin; Lafora Disease; Mutant; Conformation; Molecular Dynamics Simulations; Principal Component Analysis
Prevalence of Genetically Modified Rice, Maize, and Soy in Saudi Food Products by Rafaat M. Elsanhoty; A. I. Al-Turki; Mohamed Fawzy Ramadan (883-899).
Qualitative and quantitative DNA-based methods were applied to detect genetically modified foods in samples from markets in the Kingdom of Saudi Arabia. Two hundred samples were collected from Al-Qassim, Riyadh, and Mahdina in 2009 and 2010. GMOScreen 35S and NOS test kits for the detection of genetically modified organism varieties in samples were used. The positive results obtained from GMOScreen 35S and NOS were identified using specific primer pairs. The results indicated that all rice samples gave negative results for the presence of 35S and NOS terminator. About 26 % of samples containing soybean were positive for 35S and NOS terminator and 44 % of samples containing maize were positive for the presence of 35S and/or NOS terminator. The results showed that 20.4 % of samples was positive for maize line Bt176, 8.8 % was positive for maize line Bt11, 8.8 % was positive for maize line T25, 5.9 % was positive for maize line MON 810, and 5.9 % was positive for StarLink maize. Twelve samples were shown to contain <3 % of genetically modified (GM) soy and 6 samples >10 % of GM soy. Four samples containing GM maize were shown to contain >5 % of GM maize MON 810. Four samples containing GM maize were shown to contain >1 % of StarLink maize. Establishing strong regulations and certified laboratories to monitor GM foods or crops in Saudi market is recommended.
Keywords: Genetically modified foods; GMO; DNA extraction; Real-time PCR; Saudi market
Site-Directed Mutagenesis Improves the Thermostability and Catalytic Efficiency of Aspergillus niger N25 Phytase Mutated by I44E and T252R by Yan Liao; Chun-mei Li; Hui Chen; Qi Wu; Zhi Shan; Xue-yi Han (900-915).
Aspergillus niger phytase (PhyA) has been used as a feed supplement to improve the bioavailability of phytate phosphorus to swine and poultry. However, it is unable to maintain its stability due to high temperature during the feed pelleting process. In this study, we performed site-directed mutagenesis in the Aspergillus niger N25 phyA m gene at residue 44I and 252 T, and they were replaced by glutamic acid and arginine. Single-site mutants I44E-PhyA and T252R-PhyA, as well as double-site mutant I44E/T252R-PhyA, were constructed to improve the thermostability of PhyA through hydrogen bondings and ionic interactions. The three mutant enzymes all showed more than 20 % improvement in thermostability compared to the wild-type enzyme after being heated at 80 °C for 10 min. Their melting temperatures (T m) were increased by 1, 1, and 1.2 °C, respectively. The k m values of I44E-PhyA, T252R-PhyA, and I44E/T252R-PhyA for sodium phytate were 78, 44, and 79 % lower (P <0.05) than that of the wild-type enzyme. Overall catalytic efficiency (k cat/k m) of I44E-PhyA, T252R-PhyA, and I44E/T252R-PhyA was improved by 310, 155, and 84 % (P <0.05) than that of the wild type, respectively. The catalytic efficiency did not seem to be negatively affected by the improvement in thermostability.
Keywords: Phytase; Site-directed mutagenesis; Thermostability; Catalytic properties; Three-dimensional structure
Production of a Functional Human Acid Maltase in Tobacco Seeds: Biochemical Analysis, Uptake by Human GSDII Cells, and In Vivo Studies in GAA Knockout Mice by Frank Martiniuk; Serena Reggi; Kam-Meng Tchou-Wong; William N. Rom; Matteo Busconi; Corrado Fogher (916-926).
Genetic deficiency of acid alpha glucosidase (GAA) results in glycogen storage disease type II (GSDII) or Pompe’s disease. To investigate whether we could generate a functional recombinant human GAA enzyme (tobrhGAA) in tobacco seeds for future enzyme replacement therapy, we subcloned the human GAA cDNA into the plant expression plasmid-pBI101 under the control of the soybean β-conglycinin seed-specific promoter and biochemically analyzed the tobrhGAA. Tobacco seeds contain the metabolic machinery that is more compatible with mammalian glycosylation−phosphorylation and processing. We found the tobrhGAA to be enzymatically active was readily taken up by GSDII fibroblasts and in white blood cells from whole blood to reverse the defect. The tobrhGAA corrected the enzyme defect in tissues at 7 days after a single dose following intraperitoneal (IP) administration in GAA knockout (GAA−/−) mice. Additionally, we could purify the tobrhGAA since it bound tightly to the matrix of Sephadex G100 and can be eluted by competition with maltose. These data demonstrate indirectly that the tobrhGAA is fully functional, predominantly proteolytically cleaved and contains the minimal phosphorylation and mannose-6-phosphate residues essential for biological activity.
Keywords: Recombinant human acid maltase; Transgenic tobacco plants; Pompe’s disease; Enzyme replacement
Emerging Approaches in Fermentative Production of Statins by Sudheer Kumar Singh; Ashok Pandey (927-938).
Microbial metabolites have many important applications in pharmaceutical and health-care industry. The products of microbial origin are usually produced by submerged fermentation. The solid-state fermentation represents an alternative mode of fermentation, which is increasingly being employed as an alternative to submerged fermentation for metabolite production. The prospect of producing high-value product using low-value raw material offers a substantial premium to switch to these technologies. The cost of statins being one major factor, solid-state fermentation with agro-industrial residues as carbon, nitrogen and support matrix, promises to substantially lower the cost of production. Hence, newer approaches are required to exploit the agro-industrial residues for statin production. The development of these technologies offers an opportunity to exploit low-cost substrates without substantial investment in newer production methodologies. The emerging evidence of beneficial effect of statins in applications other than lipid lowering such as in Alzheimer disease, HIV, age-related dementia, and cancer chemotherapy makes it very important to develop methods for economic production of statins.
Keywords: Statins; Agro-industrial residues; Submerged fermentation; Solid-state fermentation
Metabolic Behavior and Enzymatic Aspects of Denitrifying EBPR Sludge in a Continuous-Flow Anaerobic–Anoxic System by Ilias Zafiriadis; Spyridon Ntougias; Anastasios G. Kapagiannidis; Alexander Aivasidis (939-953).
The metabolic aspects of enhanced biological phosphorus removal (EBPR) were investigated for the first time in a continuous-flow anaerobic–anoxic plant fed with acetate, propionate, or substrates which are involved in the tricarboxylic acid and/or glyoxylate cycle, i.e., fumarate, malate, or oxaloacetate, as the sole carbon source. Although the polyphosphate-accumulating organisms (PAOs) population remained stable with any carbon source examined, no typical EBPR metabolism was observed during fumarate, malate, or oxaloacetate utilization. Specific enzymatic activities related to EBPR were determined in activated sludge homogenates and directly correlated with the nutrient metabolic rates. The experimental results indicated the direct involvement of alkaline phosphatase, pyrophosphatase, and exopolyphosphatase in the denitrifying EBPR process. Metabolic aspects of glyoxylate cycle enzymes are discussed with regard to the biomass anaerobic and anoxic activity. Process performance was highly influenced by the kind of substrate utilized, indicating that specific metabolic pathways should be followed to favor efficient EBPR.
Keywords: Denitrification; Enhanced biological phosphorus removal (EBPR); Glyoxylate cycle enzymes; Phosphatase activity; Polyphosphate-accumulating organisms (PAOs)
Expression of Low Endotoxin 3-O-Sulfotransferase in Bacillus subtilis and Bacillus megaterium by Wenya Wang; Jacob A. Englaender; Peng Xu; Krunal K. Mehta; Jiraporn Suwan; Jonathan S. Dordick; Fuming Zhang; Qipeng Yuan; Robert J. Linhardt; Mattheos Koffas (954-962).
A key enzyme for the biosynthesis and bioengineering of heparin, 3-O-sulfotransferase-1 (3-OST-1), was expressed and purified in Gram-positive Bacillus subtilis and Bacillus megaterium. Western blotting, protein sequence analysis, and enzyme activity measurement confirmed the expression. The enzymatic activity of 3-OST-1 expressed in Bacillus species were found to be similar to those found when expressed in Escherichia coli. The endotoxin level in 3-OST-1 from B. subtilis and B. megaterium were 104–105-fold lower than that of the E. coli-expressed 3-OST-1, which makes the Bacillus expression system of particular interest for the generation of pharmaceutical grade raw heparin from nonanimal sources.
Keywords: 3-O-Sulfotransferase; Bioengineered heparin; Endotoxin free; Bacillus subtilis ; Bacillus megaterium ; Nonanimal sources
Preferential Lectin Binding of Cancer Cells upon Sialic Acid Treatment Under Nutrient Deprivation by Haitham A. Badr; Abdelaleim I. ElSayed; Hafiz Ahmed; Miriam V. Dwek; Chen-zhong Li; Leyla B. Djansugurova (963-974).
The terminal monosaccharide of glycoconjugates on a eukaryotic cell surface is typically a sialic acid (Neu5Ac). Increased sialylation usually indicates progression and poor prognosis of most carcinomas. Here, we utilize two human mammary epithelial cell lines, HB4A (breast normal cells) and T47D (breast cancer cells), as a model system to demonstrate differential surface glycans when treated with sialic acid under nutrient deprivation. Under a starved condition, sialic acid treatment of both cells resulted in increased activities of α2→3/6 sialyltransferases as demonstrated by solid phase assay using lectin binding. However, a very strong Maackia amurensis agglutinin I (MAL-I) staining on the membrane of sialic acid-treated T47D cells was observed, indicating an increase of Neu5Acα2→3Gal on the cell surface. To our knowledge, this is a first report showing the utility of lectins, particularly MAL-I, as a means to discriminate between normal and cancer cells after sialic acid treatment under nutrient deprivation. This method is sensitive and allows selective detection of glycan sialylation on a cancer cell surface.
Keywords: Sialic acid; Metabolism; Glycosylation; Lectin; Diagnosis; Cancer cell
The Desaturase OPIN17 from Phytophthora infestans Converts Arachidonic Acid to Eicosapentaenoic Acid in CHO Cells by Yong Fu; Xinzhong Fan; Xinzheng Li; Hui Wang; Hongju Chen (975-988).
We demonstrate the ability to increase the amount of eicosapentaenoic acid (EPA, 20:5n-3) in mammalian cells using OPIN17 desaturase gene. This gene was codon optimized based on genomic sequence of Δ17 from Phytophthora infestans and introduced into Chinese hamster ovary cells using liposome-mediated transfection protocol. Reverse transcription polymerase chain reaction was utilized to evaluate co-expression of AcGFP1 and OPIN17. Our results indicate that the OPIN17 gene can be expressed in mammalian cells. Heterologous expression of this gene was evaluated by assessing the fatty acid content of OPIN17-transfected cells. A total cellular lipid analysis of transfected cells which were fed with arachidonic acid (AA, 20:4n-6) as a substrate resulted in an 86.5–246 % (p < 0.05) increase in the amount of EPA in transfected cells compared with that in control cells. The ratio of AA to EPA was reduced from approximately 4.07:1 in control cells to 2.2:1 in transfected cells (p < 0.05), which indicates an EPA percent conversion of 30.94 %. Our study demonstrates that the codon-optimized OPIN17 gene can be functionally expressed in mammalian cells, converting AA into EPA and elevating the level of ω-3 polyunsaturated fatty acids efficiently. These results provide an additional support for the use of this gene in generating transgenic livestock.
Keywords: Arachidonic acid; CHO cells; Eicosapentaenoic acid; OPIN17 desaturase; Phytophthora infestans
A Cold-Adapted and Organic Solvent-Tolerant Lipase from a Psychrotrophic Bacterium Pseudomonas sp. Strain YY31: Identification, Cloning, and Characterization by Yoko Yamashiro; Akihiro Sakatoku; Daisuke Tanaka; Shogo Nakamura (989-1000).
A novel cold-adapted lipase (designated as LipYY31) was obtained from a psychrotrophic Pseudomonas sp. YY31. The strain YY31 was gram-negative, rod shaped, motile by means of one polar flagellum, and exhibited chemotaxis toward oil droplets under a microscope. The strain displayed remarkable degradation of edible oil and fat even at 5 °C. The LipYY31 DNA fragment contains an open reading frame of 1,410 bp which encoded a protein of 470 amino acids with an estimated molecular mass of 49,584 Da. LipYY31 showed high sequence similarity to those of subfamily Ι.3 lipase and had a conserved GXSXG motif around the catalytic Ser residue. Its optimal temperature was 25–30 °C, and it retained 20–40 % of its activity at 0–5 °C. The optimal pH value was 8.0. The activity was strongly inhibited by Cd2+, Zn2+, EDTA and was highly dependent on Ca2+. Tricaprin and p-nitrophenyl caprate were the most favorable substrates among the triglycerides and p-nitrophenyl esters, respectively. LipYY31 also had high activity towards natural substrates including edible vegetable oils and animal fat. Furthermore, LipYY31 was very active and stable in the presence of several detergents and organic solvents. In particular, the lipase exhibited high stability against organic solvents such as methanol, ethanol, and isopropanol.
Keywords: Pseudomonas sp.; Cold-adapted lipase; Organic solvent-tolerant lipase
Towards Industrially Feasible Treatment of Potato Starch Processing Waste by Mixed Cultures by Bingnan Liu; Jinzhu Song; Ying Li; Jia Niu; Zhenyu Wang; Qian Yang (1001-1010).
The present study aimed at reducing the pollution of the waste generated by the potato starch industry to the environment and transform the potato pulp and wastewater into single-cell protein (SCP) to be used as animal feed. The chemical oxygen demand of the wastewater was reduced from 26,700 to 9,100 mg/L by batch fermentation with mixed cultures in an aerated 10-L fermenter. The SCP products, with a crude protein content of 46.09 % (higher than soybean meal), were found palatable and safe for mice. During the treatment process, the microbial community was analyzed using the terminal restriction fragment length polymorphism for bacterial 16S rRNA genes. The results of the analysis suggested that Curacaobacter/Pseudoalteromonas and Paenibacillus/Bacillus were the main microorganisms in treating potato starch processing wastes. The 150-m3-scale fermentation demonstrated a potential for treatment in industrial applications. Fermentation of potato pulp and wastewater without adding an extra nitrogen source was a novel approach in treating the potato starch processing waste.
Keywords: Batch processing; Single-cell protein; Scale-up; Waste treatment; Microbial community
Proteomic Analysis of Sarcoplasmic Peptides of Two Related Fish Species for Food Authentication by Sudhir Kumar Barik; Sudeshna Banerjee; Soma Bhattacharjee; Sujoy Kumar Das Gupta; Sasmita Mohanty; Bimal Prasanna Mohanty (1011-1021).
Detection of species-specific sarcoplasmic peptides can be used as proteomic markers for fish food authentication and identification of species of origin in processed products. In the present study, proteomics technology was employed for differential characterization of sarcoplasmic peptides of two closely related fish species, Sperata seenghala and Sperata aor. Species-specific peptides were searched in white muscle extracts of the two species for identification of unique peptides that might aid in differentiation of the species, under two-dimensional gel electrophoresis platform. A total of 19 proteins were identified by combined matrix-assisted laser desorption ionization time-of-flight mass spectrometry and liquid chromatography-tandem mass spectrometry, of which nine and two proteins were found to be unique to S. seenghala and S. aor, respectively. One of the proteins, triosephosphate isomerase (TPI) was found to have three isoforms, out of which two were specific to S. aor, and one was specific to S. seenghala. All the three isoforms of TPI were present in the mixed samples of raw protein extracts of S. seenghala and S. aor, an observation that can be exploited to differentiate between the species and detection of deceptive practices of fraudulent substitution of commercially valuable fish species with inferior ones and differential characterization between closely related fish species.
Keywords: Food authentication; Fish species identification; 2-D GE; PMF; MALDI-TOF MS; LC-MS/MS
A New Isolate from Fusarium proliferatum (AUF-2) for Efficient Nitrilase Production by Farnaz Yusuf; Asha Chaubey; Urmila Jamwal; Rajinder Parshad (1022-1031).
A fungal isolate from Fusarium proliferatum strain AUF-2 has been found to have a high nitrilase activity (≥1,000 U/l culture). The present work describes optimization of growth conditions and production medium to achieve maximum nitrilase production. The most effective carbon and nitrogen sources for nitrilase production were glucose and sodium nitrate, respectively. ε-Caprolactam was found to be the best inducer for maximum nitrilase production with 80 g/l wet cell biomass and 26 U/g nitrilase activity. An overall nitrilase activity of ≥2,000 U/l culture was obtained in this study, which is one of the best activities reported so far in any Fusarium strain. Chemo-profiling has shown that the strain is versatile in its ability to hydrolyze both aliphatic as well as aromatic nitriles. Efforts are being made to use the strain for biotransformation of pharmaceutical substrates.
Keywords: Fungi; Nitrilase; Fusarium proliferatum ; Nitrile Hydrolysis; Optimization
Monitoring of PQQ-Dependent Glucose Dehydrogenase Substrate Specificity for Its Potential Use in Biocatalysis and Bioanalysis by Miroslav Streďanský; Rastislav Monošík; Vladimír Mastihuba; Ernest Šturdík (1032-1041).
Substrate specificity of 2,7,9-tricarboxypyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase was investigated in biosensor arrangement for understanding the suitability and the limitations of its use in bioanalysis and bioproduction of chemicals. The study demonstrated a very broad substrate specificity of biosensor utilising soluble form of PQQ-dependent glucose dehydrogenase. Nineteen saccharides out of 31 were oxidised by the sensor. Investigation confirmed strong importance of hydroxyl configuration in the positions 2 and 5 of oxidised saccharides. The broad specificity suggests that the PQQ-dependent glucose dehydrogenase could be utilised for analysis of other sugars than glucose in food samples for various production processes and for biofuel cells. In addition, the results showed that the substrate specificity of enzymes can be effectively and generally studied by biosensor arrangement for research purposes. This layout utilising immobilised enzyme allowed performing comprehensive study using a small amount of enzymes and thus saving the costs and time.
Keywords: PQQ glucose dehydrogenase; Biosensor; Specificity; Saccharides; Bioanalysis
Cell-Free Supernatants Obtained from Fermentation of Cheese Whey Hydrolyzates and Phenylpyruvic Acid by Lactobacillus plantarum as a Source of Antimicrobial Compounds, Bacteriocins, and Natural Aromas by Noelia Rodríguez-Pazo; Laura Vázquez-Araújo; Noelia Pérez-Rodríguez; Sandra Cortés-Diéguez; José Manuel Domínguez (1042-1060).
Cheese whey hydrolyzates supplemented with phenylpyruvic acid (PPA) and commercial nutrients can be efficiently metabolized by Lactobacillus plantarum CECT-221 to biosynthesize some compounds with attractive applications in the food market. The main metabolites of cell-free extracts were antimicrobial compounds such as phenyllactic acid (PLA) and lactic acid (LA). The production of PLA by L. plantarum CECT-221 was evaluated in the Man–Rogosa–Sharpe broth supplemented with two biosynthetic precursors: phenylalanine or PPA. Using 30.5 mM PPA, the microorganism increased sevenfold the concentration of PLA producing 16.4 mM PLA in 46 h. A concentration of 40 mM PPA was a threshold to avoid substrate inhibition. The biosynthesis of whey hydrolyzates as a carbon source was enhanced by fed-batch fermentation of PPA; the average productivity of PLA increased up to 45.4 ± 3.02 mM after 120 h with a product yield of 0.244 mM mM−1; meanwhile, LA reached 26.1 ± 1.3 g L−1 with a product yield of 0.72 g g−1. Cell-free fed-batch extracts charged in wells showed bacteriocin activity with halos of 7.49 ± 1.44 mm in plates inoculated with Carnobacterium piscicola and antimicrobial activity against Staphylococcus aureus (11.54 ± 1.14 mm), Pseudomonas aeruginosa (10.17 ± 2.46 mm), Listeria monocytogenes (7.75 ± 1.31 mm), and Salmonella enterica (3.60 ± 1.52 mm). Additionally, the analysis of the volatile composition of the headspace of this cell-free extract revealed that L. plantarum is a potential producer for natural aromas, such as acetophenone, with high price in the market. This is the first report of PLA production from cheese whey and PPA. The extracts showed bacteriocin activity and potential to be applied as an antimicrobial in the elaboration of safer foods.
Keywords: Cheese whey; Phenylpyruvic acid; Phenyllactic acid; Lactobacillus plantarum
Apoptosis of Human Gastric Carcinoma SGC-7901 Induced by Deoxycholic Acid via the Mitochondrial-Dependent Pathway by Wei Song; Hai-Bo Yang; Pu Chen; Shu-Min Wang; Li-Pei Zhao; Wen-Hao Xu; Hai-Fang Fan; Xu Gu; Lan-Ying Chen (1061-1071).
The study aimed to evaluate the effects of deoxycholic acid (DCA) on human gastric carcinoma cell lines and to explore its mechanisms. In the present study, effects of DCA on SGC-7901 cell growth, cell cycle, and apoptosis were investigated by MTT assay, inverted microscopy, fluorescence microscopy, PI single- and FITC/PI double-staining flow cytometry, and western blotting. The study have revealed that DCA significantly inhibited the growth of SGC-7901 cells in a dose- and time-dependent manner and arrested cell cycle at G0/G1 phase. SGC-7901 cells showed typical apoptotic morphological changes after treated with DCA for 48 h. The intensity of typical apoptosis pattern- “ladders” formed by DNA in fragments of multiples of 200 base pairs was also observed. Apoptosis of SGC-7901 cells induced by DCA were associated with collapse of the mitochondrial membrane potential. DCA treatment could also increase the ratio of Bax to Bcl-2 in SGC-7901 cells. Meanwhile, the expression of p53, cyclinD1, and c-Myc were changed after DCA treatment. These results suggest that DCA induces apoptosis of gastric carcinoma cells through an intrinsic mitochondrial-dependent pathway, and the increase in the Bax/Bcl-2 ratio and collapse of the mitochondrial membrane potential may play important roles in DCA-induced apoptosis of gastric carcinoma cells.
Keywords: Deoxycholic acid; Antiproliferation; Apoptosis; Mitochondrial pathway
Effect of Enhanced UV-B Radiation and Low-Energy N+ Ion Beam Radiation on the Response of Photosynthesis, Antioxidant Enzymes, and Lipid Peroxidation in Rice (Oryza sativa) Seedlings by Linyu Li; Qunce Huang; Shuyin Zhang; Shuaipeng Zhao (1072-1083).
To understand the effect of enhanced UV-B radiation and low-energy N+ ion beam radiation on the response of photosynthesis, antioxidant enzymes, and lipid peroxidation in rice seedlings, Oryza sativa was exposed to three different doses of low-energy N+ ion beam and enhanced UV-B alone and in combination. Enhanced UV-B caused a marked decline in some photosynthetic parameters (net photosynthetic rate, transpiration rate, and stomatal conductance) and photosynthetic pigments, whereas it induced an increase in hydrogen peroxide (H2O2) accumulation, the rate of superoxide radical production, and the content of malondialdehyde (MDA). Enhanced UV-B also induced an increase in the activity of antioxidant enzymes (superoxide dismutase [SOD], peroxidase (POD), and catalase [CAT]) and some nonenzymatic antioxidants such as proline. Under the combined treatment of enhanced UV-B and low-energy N+ ion beam at the dose of 3.0 × 1017 N+ cm−2, the activity of antioxidant compounds (SOD, POD, CAT, proline, and glutathione), photosynthetic pigments, and some photosynthetic parameters (net photosynthetic rate, transpiration rate, and stomatal conductance) increased significantly; however, the MDA content, H2O2 accumulation, and rate of superoxide radical production showed a remarkable decrease compared with the enhanced UV-B treatment alone. These results implied that the appropriate dose of low-energy N+ ion beam treatment may alleviate the damage caused by the enhanced UV-B radiation on rice.
Keywords: UV-B radiation; N+ ion beam; Superoxide dismutase (SOD); Peroxidase (POD); Catalase (CAT); Proline; Photosynthetic pigment