Kenzo Tonomura

Lack of homology between two haloacetate dehalogenase genes encoded on a plasmid from Moraxella sp. strain B

Two genes encoding haloacetate dehalogenases, H-1 and H-2, are closely linked on a plasmid from Moraxella sp. strain B. H-1 predominantly acts on fluoroacetate, but H-2 does not. To elucidate the molecular relationship between the two enzymes, we compared their structural genes. Two restriction fragments of the plasmid DNA were subcloned on M13 phages and their nucleotide sequences were determined. The sequence of each fragment contained an open reading frame that was identified as the structural gene for each of the two dehalogenases on the basis of the following criteria; N-terminal amino acid sequence, amino acid composition, and molecular mass. The genes for H-1 and H-2, designated dehH1 and dehH2, respectively, had different sizes (885 bp and 675 bp) and G+C contents (58.3% and 53.4%). Sequence analysis revealed no homology between the two genes. We concluded that the dehalogenases H-1 and H-2 have no enzyme-evolutionary relationship. The deduced amino acid sequence of the dehH1 gene showed significant similarity to those of three hydrolases of Pseudomonas putida and a haloalkane dehalogenase of Xanthobacter autotrophicus. The dehH2 coding region was sandwiched between two repeated sequences about 1.8 kb long, which might play a part in the frequent spontaneous deletion of dehH2 from the plasmid.

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.

Degradation of Polylactide by Commercial Proteases

Fifty-six commercially available proteases were tested for polylactide-degrading activity. Little or no activity was found in acid and neutral proteases, while some alkaline proteases formed appreciable amounts of lactic acid from polylactide. These polylactide-degrading proteases were derived from Bacillus species and had catalytic activity even under neutral, as well as alkaline, conditions. Savinase (Novo Nordisk) degraded polylactide the fastest among the enzymes tested and its specific activity corresponded to about one-half of proteinase K. Polylactide-degrading activity was not always present in the enzymes that affected keratin, while polylactide-degrading proteases commonly hydrolyzed keratin. A significant correlation was observed between degrading activities of polylactide and keratin in alkaline proteases.

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.

Cloning, Sequencing, and Characterization of the Intracellular Invertase Gene from Zymomonas mobilis

The structural gene for the intracellular invertase E1 of Zymomonas mobilis strain Z6C was cloned in a 2.25-kb DNA fragment on pUSH11, and expressed in Escherichia coli HB101. The enzyme produced by the E. coli carrying pUSH11 was purified about 1,122 fold to homogenicity with a yield of 4%. The molecular weight and substrate specificity of the enzyme were identical with those of the intracellular invertase E1 from Z. mobilis. The nucleotides of the cloned DNA were sequenced; they included an open reading frame of 1,536 bp, coding for a protein with a molecular weight of 58,728. The N-terminal amino acid sequence predicted was identical with the sequence of the first 20 N-terminal amino acid residues of the protein obtained by Edman degradation. Comparison of the predicted amino acid sequence of E1 protein with those of the four other known beta-D-fructofuranosidases from Escherichia coli, Bacillus subtilis, and Saccharomyces cerevisiae indicated a stronger homology in the N-terminal portion than in the C-terminal portion.

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 to homogeneity from the culture filtrate of Paecilomyces lilacinus D218 by column chromatography on CM-Toyopearl 650M and hydroxylapatite. The molecular weight of the enzyme was estimated to be 48,000 by SDS-PAGE. Maximal activity was observed near pH 7.0 and 45°C. The Km and Vmax values for PHB were 0.13 (mg/ml) and 3750 (U/mg protein), respectively. The enzyme hydrolyzed PHB and p-nitrophenyl fatty acids but not polycaprolactone and triglycerides.

Expression of the Escherichia coli α-galactosidase and lactose permease genes in Zymomonas mobilis and its raffinose fermentation

Abstract A 0.33-kilobase (kb) DNA fragment encoding the promoter was isolated from Zymomonas chromosomal DNA as an in-frame fusion producing functional β-galactosidase. The DNA sequence of the fragment was analyzed, and a transcriptional initiation site was found. To confer the fermentation of raffinose on Z. mobilis , two recombinant plasmids were constructed: pZER193 that contains the α-galactosidase gene of E. coli inserted immediately downstream of the isolated promoter, and pZY1 that contains the lactose permease gene of E. coli . They were introduced into the strain Z6C of Zymomonas , capable of fermenting the fructose moiety of raffinose but not the melibiose moiety. Cells of the strain carrying both plasmids could ferment melibiose to ethanol, indicating that expression of α-galactosidase and lactose permease in Z6C strain expanded the substrate spectrum of Z. mobilis for ethanol production.

Enzyme System Involved in the Decomposition of Phenyl Mercuric Acetate by Mercury-resistant Pseudomonas

The enzymatic decomposition of phenyl mercuric acetate (PMA) to metallic mercury by a mercury-resistant Pseudomonas has been studied. The formation of the system involved in the decomposition was found to be inducible. Glucose dehydrogenase (D-glucose: NAD oxidoreductase), arabinose dehydrogenase (L-arabinose: NADP oxidoreductase), cytochrome c and the “decomposing enzyme” which catalyzes the splitting of the C–Hg linkage, were separated from cell free extract by gel filtration on a column of Sephadex G–150. The cytochrome fraction was further separated into two types, c-I and c-II, by chromatography on a column of CM-Sephadex. Each of these cytochromes showed absorption peaks at 416, 519 and 547 mμ in the reduced form, but they were different in the molecular weight; cytochrome c-I was estimated to be about 26,000, and cytochrome c-II about 14,000. The reconstruction of these enzymes demonstrated that a reduced NAD(P) generating system, glucose dehydrogenase or arabinose dehydrogenase, cytochrome c-I and...