Yuji Oda

A Phylogenetic Analysis of Saccharomyces Species by the Sequence of 18S–28S rRNA Spacer Regions

Sequences of two internally transcribed spacer regions between 18S and 28S rRNA genes were determined to assess the phylogenetic relationship in the strains belonging to the genus Saccharomyces. The sequences of S. bayanus and S. pastorianus were quite similar, but not identical. Two phylogenetic trees constructed by the neighbor‐joining method showed that all the species examined were distinguished from one another. The Saccharomyces sensu stricto species: S. cerevisiae, S. bayanus, S. paradoxus and S. pastorianus, were closely related and far from the Saccharomyces sensu lato species including S. barnetti, S. castellii, S. dairensis, S. exiguus, S. servazzii, S. spencerorum and S. unisporus, and an outlying species, S. kluyveri.

Microbial degradation of poly(3-hydroxybutyrate) and polycaprolactone by filamentous fungi

Five strains were isolated from soil as fungi able to degrade both poly(3-hydroxybutyrate) (PHB) and polycaprolactone (PCL), and one of the strains, D218, identified as Paecilomyces lilacinus, was selected. In 10 d, D218 degraded PHB almost completely and 10% of the PCL, each at 0.1% in the culture media. Strain D218 excreted PHB and PCL depolymerases in media containing either PHB, PCL or PHB plus PCL. Both depolymerase activities were reduced when the medium was supplemented with either soluble starch, lactose or glucose. The optimum conditions for PHB depolymerase were pH 6.5 to 7.5 at 50°C, while those for PCL depolymerase were pH 3.5 to 4.5 at 30°C. In the reaction mixtures used for the enzyme assays, the formation of 3-hydroxybutyrate from PHB and e-caprolactone from PCL was confirmed by high performance liquid chromatography.

Recycling of bakery wastes using an amylolytic lactic acid bacterium

Production of lactic acid from discarded bread by using an amylolytic lactic acid bacterium, Lactobacillus amylovorus, was investigated to recycle bakery wastes. Addition of 2.0% yeast extract in the medium containing 3.58% bread crust caused maximum acid production. The stimulation of lactic acid production by less expensive materials such as corn steep liquor, defatted soybean powder, rice bran and wheat bran at additional levels of 2.0% was also observed, but limited. Acid production in the medium supplemented with 2.0% corn steep liquor was enhanced by the further addition of 2.0% defatted soybean powder and reached the levels comparable to medium containing 1.4% yeast extract. In the medium supplemented with 2.0% corn steep liquor and 2.0% defatted soybean powder, 47.2% of total sugars was converted to dl-lactic acid in 72 h under static incubation. When the baking test was carried out, the bread made with the addition of the culture filtrate was significantly (P < 0.05) preferred over those with and without addition of commercial fermented seasoning with respect to taste and overall acceptability.

Purification and properties of extracellular β-mannanases produced by Enterococcus casseliflavus FL2121 isolated from decayed Konjac

Abstract Two β-mannanases (M-I and M-II) were purified from the culture filtrate of Enterococcus casseliflavus FL2121 by ammonium sulfate precipitation, column chromatography of DEAE-Toyopearl 650M and Phenyl-Toyopearl 650M and preparative polyacrylamide gel electrophoresis to homogenities. The molecular weights of M-I and M-II were estimated to be 142,000 and 137,000 by SDS-polyacrylamide gel electrophoresis and 127,000 and 113,000 by gel filtration, respectively. M-I and M-II exhibited an optimum pH at 6.0 and an optimum temperature at 50°C. The enzymes were activated slightly by CoCl 2 and MnCl 2 , and inhibited strongly by AgNO 3 , HgCl 2 , EDTA, and N -bromosuccinimide. The K m values of M-I and M-II for konjac β-1,4-glucomannan were 0.14 and 0.30 (mg/ml), and the maximum velocities were 1,110 and 1,700 (U/mg protein), respectively. Both enzymes were endo-type β-mannanases hydrolyzing mannosides larger than β-1,4- d -mannotetraose.

Purification and Properties of Poly(3-Hydroxybutyrate) Depolymerase from the Fungus Paecilomyces lilacinus D218

Abstract. Poly(3-hydroxybutyrate) depolymerase was purified tonnhomogeneity from the culture filtrate of Paecilomyces lilacinus D218nnby column chromatography on CM-Toyopearl 650M and hydroxylapatite. Thennmolecular weight of the enzyme was estimated to be 48,000 by SDS-PAGE.nnMaximal activity was observed near pH 7.0 and 45°C. ThennKm and Vmax values for PHB were 0.13nn(mg/ml) and 3750 (U/mg protein), respectively. The enzyme hydrolyzed PHB andnnp-nitrophenyl fatty acids but not polycaprolactone and triglycerides.n

Construction of a sucrose-fermenting bakers' yeast incapable of hydrolysing fructooligosaccharides.

Fructooligosaccharides stimulate the growth of intestinal bifidobacteria which are related to the favorable health and nutrition of humans and other animals. Since the efficient amount of fructooligosaccharide for an adult human is relatively large (about 5 g per day), its addition to daily foods like bakery goods might be beneficial. However, commercial Bakers yeast hydrolyses fructooligosaccharides by the action of invertase encoded in SUC genes and ferments the resulting monosaccharides. According to the findings that strains carrying the MAL-constitutive gene and lacking the SUC gene fermented sucrose and not fructooligosaccharide, we constructed a sucrose-fermenting strain, YOY920, incapable of hydrolysing fructooligosaccharide, by cross-breeding a baking strain and a laboratory strain. In a molasses medium, the cell yield of YOY920 was comparable to that of a baking strain FSC6001, and much higher than that of the non-sucrose-fermenting strains. Although fructooligosaccharide inhibited the dough leavening ability of YOY920, white bread containing fructooligosaccharide could be produced in the defined dough formula using the new strain.

Molecular genetic properties of the yeast Torulaspora pretoriensis: characterization of chromosomal DNA and genetic transformation by Saccharomyces cerevisiae-based plasmids.

Chromosomal DNA banding patterns were obtained for three strains of Torulaspora pretoriensis by contour-clamped homogenous-electric-field gel electrophoresis. Chromosomes were resolved into six or seven bands in the range of 800 to 2000 kb, and a polymorphism of these lengths was observed. By Southern-blot analysis, the three strains were shown to lack the DNA sequences homologous to the URA3, LEU2, TRP1, and HO genes of Saccharomyces cerevisiae. A uracil auxotrophic mutant derived from T. pretoriensis was transformed with three plasmids (YEp24, YRpHI, and YCp50) carrying the URA3 gene of S. cerevisiae by the lithium acetate method.

Isolation and characterization of MELt gene from Torulaspora delbrueckii IFO 1255.

Torulaspora delbrueckii IFO 1255 is a melibiose‐fermenting strain in Torulaspora species. From the genome of strain IFO 1255, we obtained a 770u2009bp fragment by PCR with oligonucleotides synthesized based on the MEL genes of Saccharomyces cerevisiae and its related species. The region encompassing the 770u2009bp fragment was cloned by inverse PCR and sequenced. The nucleotide sequence revealed an open reading frame of 1422u2009bp encoding a 474 amino acid protein with a molecular weight of 52u2009360. The similarity of the presumed mature protein to Saccharomyces species and Zygosaccharomyces cidri α‐galactosidases was 69·7–73·2% and 58·4%, respectively. The phylogenetic relationship between these species is discussed. The sequence is deposited in the DDBJ/EMBL/GenBank database under Accession No. AB027130. Copyright

Characterization of a Mutant from Lactobacillus amylovorus JCM 1126T with Improved Utilization of Sucrose

A strain YF43, which can grow on sucrose as rapidly as glucose, was isolated by mutation from Lactobacillus amylovorus JCM 1126, the type strain defective in sucrose utilization. Exogenous sucrose stimulated the production of invertase by strains YF43 and JCM 1126 simultaneously. In a medium containing fructooligosaccharide as the sole carbon source, the cells of strain YF43 showed high invertase activity in spite of poor growth. The two invertases produced in the cells grown on sucrose and fructooligosaccharide were an identical β-fructofuranosidase, as judged from properties of partially purified enzymes. These observations indicated that strain YF43 is a mutant improved for permeation of sucrose and not derepressed for the synthesis of invertase.

Liberation of sucrose-hydrolyzing enzymes from cells by the zliS gene product that mediates protein secretion in Zymomonas mobilis

Abstract A 1.7 kb DNA fragment containing both the zliE and zliS genes complements the sucrose-deficiency of the mutant, Z6C-S2, derived from Zymomonas mobilis ATCC 29191, and the mutant containing both genes produces sucrose-hydrolyzing enzymes extracellularly. The mutant containing zliE accumulates the enzymes within the cells, while that containing zliS does not produce the enzymes (Kondo, T. et al. , Biosci. Biotech. Biochem., 58, 526–530, 1994). In the mutant cells containing zliE , most of the sucrose-hydrolyzing enzymes were bound on the cell surface, and these enzymes were liberated when incubated with either washing, suspension or extract of the cells containing zliS . These results suggest that a product of the zliS gene affects the cell membrane to stimulate the liberation of cell-associated proteins.