Direct production of ethanol from neoagarobiose using recombinant yeast that secretes α-neoagarooligosaccharide hydrolase.

An α-neoagarooligosaccharide hydrolase, AgaNash, was purified from Cellvibrio sp. OA-2007, which utilizes agarose as a substrate. The agaNash gene, which encodes AgaNash, was obtained by comparing the N-terminal amino acid sequence of AgaNash with that deduced from the nucleotide sequence of the full-length OA-2007 genome. The agaNash gene combined with the Saccharomyces cerevisiae signal peptide α-mating factor was transformed into the YPH499 strain of S. cerevisiae to generate YPH499/pTEF-MF-agaNash, and the recombinant yeast was confirmed to produce AgaNash, though it was mainly retained within the recombinant cell. To enhance AgaNash secretion from the cell, the signal peptide was replaced with a combination of the signal peptide and a threonine- and serine-rich tract (T-S region) of the S. diastaticus STA1 gene. The new recombinant yeast, YPH499/pTEF-STA1SP-agaNash, was demonstrated to secrete AgaNash and hydrolyze neoagarobiose with an efficiency of as high as 84%, thereby producing galactose, which is a fermentable sugar for the yeast, and ethanol, at concentrations of up to 1.8 g/L, directly from neoagarobiose.

Direct ethanol production from cellulosic materials by consolidated biological processing using the wood rot fungus Schizophyllum commune.

In the present study, ethanol production from polysaccharides or wood chips was conducted in a single reactor under anaerobic conditions using the white rot fungus Schizophyllum commune NBRC 4928, which produces enzymes that degrade lignin, cellulose and hemicellulose. The ethanol yields produced from glucose and xylose were 80.5%, and 52.5%, respectively. The absolute yields of ethanol per microcrystalline cellulose (MCC), xylan and arabinogalactan were 0.26g/g-MCC, 0.0419g/g-xylan and 0.0508g/g-arabinogalactan, respectively. By comparing the actual ethanol yields from polysaccharides with monosaccharide fermentation, it was shown that the rate of saccharification was slower than that in fermentation. S. commune NBRC 4928 is concluded to be suitable for CBP because it can produce ethanol from various types of sugar. From the autoclaved cedar chip, only little ethanol was produced by S. commune NBRC 4928 alone but ethanol production was enhanced by combined use of ethanol fermenting and lignin degrading fungi.

Draft Genome Sequence of the Nonmarine Agarolytic Bacterium Cellvibrio sp. OA-2007.

Cellvibrio sp. OA-2007 is a Gram-negative, aerobic, and agarolytic bacterium isolated from activated sludge. We present the draft genome sequence of strain OA-2007, composed of 97 contigs, totaling 4,595,379 bp in size, and containing 4,094 open reading frames, with a G+C content of 47.71%.

Purification and characterization of α-neoagarooligosaccharide hydrolase from Cellvibrio sp. OA-2007.

α-Neoagarooligosaccharide (α-NAOS) hydrolase was purified from Cellvibrio sp. OA-2007 by using chromatographic techniques after hydroxyapatite adsorption. The molecular masses of α-NAOS hydrolase estimated using SDS-PAGE and gel filtration chromatography were 40 and 93 kDa, respectively, and the optimal temperature and pH for the enzyme activity were 32ºC and 7.0-7.2. α-NAOS hydrolase lost 43% of its original activity when incubated at 35ºC for 30 min. The enzyme hydrolyzed neoagarobiose, neoagarotetraose, and neoagarohexaose to galactose, agarotriose, and agaropentaose, respectively, and produced 3,6-anhydro-L-galactose concomitantly; however, it did not degrade agarose.

Cloning of agarase gene from non-marine agarolytic bacterium Cellvibrio sp.

Agarase genes of non-marine agarolytic bacterium Cellvibrio sp. were cloned into Escherichia coli and one of the genes obtained using HindIII was sequenced. From nucleotide and putative amino acid sequences (713 aa, molecular mass; 78,771 Da) of the gene, designated as agarase AgaA, the gene was found to have closest homology to the Saccharophagus degradans (formerly, Microbulbifer degradans) 2-40 aga86 gene, belonging to glycoside hydrolase family 86 (GH86). The putative protein appears to be a non-secreted protein because of the absence of a signal sequence. The recombinant protein was purified with anion exchange and gel filtration columns after ammonium sulfate precipitation and the molecular mass (79 kDa) determined by SDS-PAGE and subsequent enzymography agreed with the estimated value, suggesting that the enzyme is monomeric. The optimal pH and temperature for enzymatic hydrolysis of agarose were 6.5 and 42.5 degrees C, and the enzyme was stable under 40 degrees C. LC-MS and NMR analyses revealed production of a neoagarobiose and a neoagarotetraose with a small amount of a neoagarohexaose during hydrolysis of agarose, indicating that the enzyme is a beta-agarase.

Degradation of fats during thermophilic composting of organic waste.

The degradation of fats during thermophilic composting was investigated by adding lard of four different mixing ratios (0, 33.3, 42.9 and 50% on a dry weight basis) to dog food used as a model substrate for organic waste. The lard added at the mixing ratio of 33.3% did not inhibit the decomposition of organic matter in the dog food, with lard itself beginning decomposition after decay of more easily decomposable organic compounds of the dog food, 84 h from the start of composting. The percentage of lard decomposition reached as high as 29.3% by the end of 8 days of composting. By contrast, the decomposition of organic matter in the processed dog food was apparently inhibited when the portion of lard was greater than 33.3%, especially at the earliest stage of composting. It is possible, however, that lard would decompose vigorously once decomposition has begun, even when the ratio of lard is as high as 50%. The percentages of lard decomposition in composting mixtures with 42.9 and 50% lard were 15.7 and 9.50%, respectively, thus the higher the mixing ratio of lard, the lower the percentage of lard decomposition. However, it was found that the maximum decomposition rate of the lard was similar for all of the ratios tested; that is, approximately 5.0 x 10(-3) g carbon h(-1).