Isolation and characterization of a novel metagenomic enzyme capable of degrading bacterial phytotoxin toxoflavin.

Toxoflavin, a 7-azapteridine phytotoxin produced by the bacterial pathogens such as Burkholderia glumae and Burkholderia gladioli, has been known as one of the key virulence factors in crop diseases. Because the toxoflavin had an antibacterial activity, a metagenomic E. coli clone capable of growing well in the presence of toxoflavin (30 µg/ml) was isolated and the first metagenome-derived toxoflavin-degrading enzyme, TxeA of 140 amino acid residues, was identified from the positive E. coli clone. The conserved amino acids for metal-binding and extradiol dioxygenase activity, Glu-12, His-8 and Glu-130, were revealed by the sequence analysis of TxeA. The optimum conditions for toxoflavin degradation were evaluated with the TxeA purified in E. coli. Toxoflavin was totally degraded at an initial toxoflavin concentration of 100 µg/ml and at pH 5.0 in the presence of Mn2+, dithiothreitol and oxygen. The final degradation products of toxoflavin and methyltoxoflavin were fully identified by MS and NMR as triazines. Therefore, we suggested that the new metagenomic enzyme, TxeA, provided the clue to applying the new metagenomic enzyme to resistance development of crop plants to toxoflavin-mediated disease as well as to biocatalysis for Baeyer-Villiger type oxidation.

Bioethanol Production from Hydrodictyon reticulatum by Fed-Batch Fermentation Using Saccharomyces cerevisiae KCTC7017.

The aim of this study was to develop a potential process for bioethanol production from Hydrodictyon reticulatum (HR), a filamentous freshwater alga, using Saccharomyces cerevisiae (KCTC7017). From the sugar solutions prepared by the four different hydrolysis methods, bioethanol production ranged from 11.0 g/100 g dried material (acid hydrolysis) to 22.3 g/ 100 g dried material (enzymatic hydrolysis, EH). Bioethanol was fermented from a highly concentrated sugar solution obtained by a decompression-mediated (vacuum) enrichment method (VE). As the results, ethanol was more efficiently produced from HR when sugar solutions were concentrated by VE following EH (EH/VE). Using multiple feeding of the sugar solution prepared by EH/VE from HR, ethanol reached up to a concentration of 54.3 g/l, corresponding to 24.9 g/100 g dried material, which attained the economic level of product concentration (approximately 5%). The results indicate that by using HR, it is feasible to establish a bioethanol production process, which is effective for using microalgae as the raw material for ethanol production.

Acid hydrolysis of Curcuma longa residue for ethanol and lactic acid fermentation.

This research examines the acid hydrolysis of Curcuma longa waste, to obtain the hydrolysate containing lactic acid and ethanol fermentative sugars. A central composite design for describing regression equations of variables was used. The selected optimum condition was 4.91% sulphuric acid, 122.68°C and 50 min using the desirability function under the following conditions: the maximum reducing sugar (RS) yield is within the limited range of the 5-hydroxymethylfurfural (HMF) and furfural concentrations. Under the condition, the obtained solution contained 144 g RS/L, 0.79 g furfural/L and 2.59 g HMF/L and was directly fermented without a detoxification step. The maximum product concentration, average productivity, RS conversion and product yield were 115.36 g/L, 2.88 g/L/h, 89.43% and 64% for L-lactic acid; 113.92 g/L, 2.59 g/L/h, 88.31% and 63.29% for D-lactic acid; and 55.03 g/L, 1.38 g/L/h, 42.66 and 30.57%, respectively, for ethanol using a 7-L jar fermenter.

Production of L- and D-lactic acid from waste Curcuma longa biomass through simultaneous saccharification and cofermentation.

Simultaneous saccharification and cofermentation (SSCF) of Curcuma longa waste biomass obtained after turmeric extraction to L- and D-lactic acid by Lactobacillus coryniformis and Lactobacillus paracasei, respectively, was investigated. This is a rich, starchy, agro-industrial waste with potential for use in industrial applications. After optimizing the fermentation of the biomass by adjusting nitrogen sources, enzyme compositions, nitrogen concentrations, and raw material concentrations, the SSCF process was conducted in a 7-l jar fermentor at 140 g dried material/L. The maximum lactic acid concentration, average productivity, reducing sugar conversion and lactic acid yield were 97.13 g/L, 2.7 g/L/h, 95.99% and 69.38 g/100 g dried material for L-lactic acid production, respectively and 91.61 g/L, 2.08 g/L/h, 90.53% and 65.43 g/100 g dried material for D-lactic acid production, respectively. The simple and efficient process described in this study could be utilized by C. longa residue-based lactic acid industries without requiring the alteration of plant equipment.

D-lactic acid production from dry biomass of Hydrodictyon reticulatum by simultaneous saccharification and co-fermentation using Lactobacillus coryniformis subsp. torquens.

D-lactic acid production from dry biomass of the microalga, Hydrodictyon reticulatum, was carried out in a 5-l jar fermentor (initial pH 6, 34 °C using CaCO(3) as a neutralizing agent) through simultaneous saccharification and co-fermentation using the Lactobacillus coryniformis subsp. torquens. After 36 h, 36.6 g lactic acid/l was produced from 80 g H. reticulatum/l in the medium containing 3 g yeast extract/l and 3 g peptone/l in the absence of mineral salts. The maximum productivity, average productivity and yield were 2.38 g/l h, 1.02 g/l h and 45.8 %, respectively. The optical purity of D-Lactic acid ranged from 95.8-99.6 %. H. reticulatum is thus a promising biomass material for the production of D-Lactic acid.

Production of l-lactic acid from a green microalga, Hydrodictyon reticulum, by Lactobacillus paracasei LA104 isolated from the traditional Korean food, makgeolli.

The freshwater microalga, Hydrodictyon reticulum, that contained 47.5% reducing sugars including 35% glucose was used as substrate for the production of l-lactic acid (LA) by LA-producing bacteria. Lactobacillus paracasei LA104 was selected for fermentation in a 5-l fermentor since it was able to grow at pH 3, 60g LA/l, 200g glucose/l, 125g NaCl/l, and 45°C and produced over 97.3% optically pure l-lactic acid with glucose as a substrate. Simultaneous saccharification and cofermentation from H. reticulum to l-LA using LA104 was investigated in a jar fermentor. The yield reached 46g/100g H. reticulum dry material, with a final concentration of 37.11g/l and a productivity of 1.03g/l/h. This is the first report of the production of l-LA from a microalga, and H. reticulum could be a potential feedstock for large-scale production of l-LA by LA104.

Nematicidal and antifungal activities of annonaceous acetogenins from Annona squamosa against various plant pathogens.

The methanol extract of Annona squamosa seeds was highly active against two phytoparasitic nematodes, Bursaphelenchus xylophilus and Meloidogyne incognita. It efficiently suppressed plant diseases, caused by Phytophthora infestans and Puccinia recondita. Ten annonaceous acetogenins (AAs) were isolated, and their chemical structures were identified by mass and nuclear magnetic resonance spectral data. Out of 10 substances, eight displayed strong in vitro nematicidal activity against B. xylophilus with LD(50) values ranging 0.006 to 0.048 µg/mL. Squamocin-G showed potent nematicidal activity against M. incognita. Squamocin, squamocin-G, and squamostatin-A also displayed potent in vitro and in vivo antifungal activities against P. infestans causing tomato late blight. In addition, squamostatin-A effectively controlled the development of wheat leaf rust caused by P. recondita. Our findings suggested that A. squamosa seeds and its bioactive AAs can be an alternative resource of a promising botanical nematicide and fungicide to control various plant diseases.