Motoki Kubo

Phylogenetic analysis of long-chain hydrocarbon-degrading bacteria and evaluation of their hydrocarbon-degradation by the 2,6-DCPIP assay

Thirty-six bacteria that degraded long-chain hydrocarbons were isolated from natural environments using long-chain hydrocarbons (waste car engine oil, base oil or the c-alkane fraction of base oil) as the sole carbon and energy source. A phylogenetic tree of the isolates constructed using their 16S rDNA sequences revealed that the isolates were divided into six genera plus one family (Acinetobacter, Rhodococcus, Gordonia, Pseudomonas, Ralstonia, Bacillus and Alcaligenaceae, respectively). Furthermore, most of the isolates (27 of 36) were classified into the genera Acinetobacter, Rhodococcus or Gordonia. The hydrocarbon-degradation similarity in each strain was confirmed by the 2,6-dichlorophenol indophenol (2,6-DCPIP) assay. Isolates belonging to the genus Acinetobacter degraded long-chain normal alkanes (n-alkanes) but did not degrade short-chain n-alkanes or cyclic alkanes (c-alkanes), while isolates belonging to the genera Rhodococcus and Gordonia degraded both long-chain n-alkanes and c-alkanes.

Treatment of Hypersaline-Containing Wastewater with Salt-Tolerant Microorganisms.

Two salt-tolerant bacteria were isolated from soil samples to treat a high-salt-content wastewater from a pickled plum production plant. The strains, which were identified as Staphylococcus sp. and Bacillus cereus, grew well in a medium containing 0 to 15% NaCl. When wastewater in a flask was treated with the strains for 72 h, the COD removal was about 70%, and this was increased to about 90% when they were used in a pilot plant (working volume 1 m3) in a batch culture carried out for 7 d. The Staphylococcus sp. and Bacillus cereus strains occupied almost all of the pilot plant, in which they were present in a ratio of 3:1 (at 23-27 degrees C).

Isolation and characterization of an ether‐type polyurethane‐degrading micro‐organism and analysis of degradation mechanism by Alternaria sp.

Aims:  To degrade ether‐type polyurethane (ether‐PUR), ether‐PUR–degrading micro‐organism was isolated. Moreover, ether‐PUR–degrading mechanisms were analysed using model compounds of ether‐PUR.

Characterization of PCB-degrading bacteria immobilized in polyurethane foam

This study is carried out to investigate (1) conditions for the synthesis of polyurethane foam to be used for immobilizing microorganisms, (2) the viability of microorganisms immobilized simultaneously into the pores of a polyurethane foam when the foam is synthesized, and (3) the difference in the ability to degrade PCBs between the immobilized and suspended microorganisms. The results of this study show that polyurethane foam is suitable for synthesizing 10% NCO-prepolymer, water and surfactant in the ratio of 100:2.6:1.2 (w/w), respectively, and the viability of microorganisms (input microbes) immobilized in the foam is high. The input microbes, designated as strain SY5, are isolated from a municipal sewage treatment plant. In addition, immobilized strain SY5 degrades 5-40% more PCB of a PCB mixture (Aroclor 1242) than the suspended strain SY5.

Soybean Peptide: Novel Plant Growth Promoting Peptide from Soybean

Soybean is one of the most important agricultural products and its global production was more than 200 million tons per year in 2005 (Table 1) (Ministry of Agriculture, Forestry and Fisheries (Japan), 2007; Uchida, 2007). Soybean is used mainly as a vegetable oil (31.6 million tons a year in 2005) and the production ratio is the highest (30%) among vegetable oils. Soybean waste, which remains after extraction of vegetable oil, contains about 50% proteins, which consist of a well-balanced mix of amino acids. Therefore, soybean waste is a valuable biomass for animal feedstuffs. Soybean is used directly as food in Japan and several Asian countries but soybean proteins are used less widely elsewhere in the world.

Expression in Escherichia coli, Refolding, and Purification of the Recombinant Mature Form of Human Matrix Metalloproteinase 7 (MMP-7)

In the latent pro-form of matrix metalloproteinase 7 (MMP-7), the cysteine residue in the pro-peptide binds the active-site zinc ion. Hence, recombinant active MMP-7 was prepared from pro-MMP-7 by modification of this cysteine residue with a mercuric reagent. In this study, mature MMP-7 was expressed in Escherichia coli as inclusion bodies, solubilized, and refolded with 1 M L-arginine. The purified product was indistinguishable from the one prepared from pro-MMP-7 as assessed by hydrolysis of (7-methoxycoumarin-4-yl)acetyl-L-Pro-L-Leu-Gly-L-Leu-[N 3-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl]-L-Ala-L-Arg-NH2.

Culture condition ofPseudomonas aeruginosa F722 for biosurfactant production

Pseudomonas aeruginosa F722 produces a biosurfactant (BS) during its degradation of carbon and hydrocarbon compounds. The culture conditions for upgrading the biosurfactant productivity were investigated. The concentration of the biosurfactant produced byP. aeruginosa F722 was 0.78 g/L in C-medium; however, this increased to 1.66 g/L in BS medium, which was experimentally adjusted to optimal conditions. NaNO2 was found to be most effective for microbial growth, with an O.D600nm of 1.18 for 0.1% NaNO2. Microbial growths, according to the O.D600nm were 2.53, 2.68, 2.89, and 2.87 for glucose, glycerol,n-C10, andn-C22, respectively. Clear zone diameters (cm), indicating biosurfactant activity, were 9.0, 8.8, 5.7, and 8.5 for glucose, glycerol,n-C10, andn-C22, respectively. Microbial growth was not consistent with the biosurfactant activity. The best biosurfactant activity was found with a C/N ratio of 20. Under optimal culture condition, the average surface tension decreased from 70 to 30 mN/m after 5 days. With aeration of 1.0 vvm, the biosurfactant produced increased to 1.94 g/L (up to 20%) compared to that of 1.66 g/L with no aeration. With aeration, the velocities of glucose degradation during both the log and stationary growth phases increased from 0.25 and 0.18 h−1 to 0.33 and 0.29 h−1, respectively, and the time for the culture to arrive at the maximum clear zone diameter became shorter, from 80 down to 60 h with no aeration.

Isolation and characterization of a bluegill-degrading microorganism, and analysis of the root hair-promoting effect of the degraded products.

Bluegill-degrading bacteria were isolated from various environmental sources. Brevibacillus sp. BGM1 degraded bluegill efficiently at 50 °C, and its culture supernatant showed the highest peptide and amino acid concentrations as trichloroacetic acid (TCA) soluble fraction (ASF) (10.7 mg/ml) of all supernatants obtained with bluegill as a substrate. Strain BGM1 secreted a protease(s) into the medium, and the concentration of peptides and amino acids gradually increased. The fertile effect of the degraded bluegill products (DGP) on Brassica rapa was also investigated. The root hair density of B. rapa grown with DGP at a concentration of 30 μg peptides and amino acids/ml was about 1.7 times higher than when grown with the same concentration of undegraded bluegill. DGP was shown to increase root hair numbers and adventitious root formation. The results of this study suggest that a specific peptide(s) for promotion of root hair is produced from the order Perciformes with a protease(s) from BGM1.

Relationship among Phosphorus Circulation Activity, Bacterial Biomass, pH, and Mineral Concentration in Agricultural Soil

Improvement of phosphorus circulation in the soil is necessary to enhance phosphorus availability to plants. Phosphorus circulation activity is an index of soil’s ability to supply soluble phosphorus from organic phosphorus in the soil solution. To understand the relationship among phosphorus circulation activity; bacterial biomass; pH; and Fe, Al, and Ca concentrations (described as mineral concentration in this paper) in agricultural soil, 232 soil samples from various agricultural fields were collected and analyzed. A weak relationship between phosphorus circulation activity and bacterial biomass was observed in all soil samples (R2 = 0.25), and this relationship became significantly stronger at near-neutral pH (6.0–7.3; R2 = 0.67). No relationship between phosphorus circulation activity and bacterial biomass was observed at acidic (pH < 6.0) or alkaline (pH > 7.3) pH. A negative correlation between Fe and Al concentrations and phosphorus circulation activity was observed at acidic pH (R2 = 0.72 and 0.73, respectively), as well as for Ca at alkaline pH (R2 = 0.64). Therefore, bacterial biomass, pH, and mineral concentration should be considered together for activation of phosphorus circulation activity in the soil. A relationship model was proposed based on the effects of bacterial biomass and mineral concentration on phosphorus circulation activity. The suitable conditions of bacterial biomass, pH, and mineral concentration for phosphorus circulation activity could be estimated from the relationship model.

Mechanism of thermostability in thermolysin : analysis of subsite S2 mutant enzymes of thermolysin

An aromatic amino acid at position 115 (tryptophan residue; subsite S2) in thermolysin is known to be essential for proteolytic activity of thermolysin. Mutant enzymes substituted by phenylalanine (W115F) and tyrosine (W115Y) at position 115 were expressed at similar levels as the wild type (WT) enzyme in Bacillus subtilis. The thermostability of the W115Y mutant enzyme was equal to that of the WT. However, that of the W115F mutant enzyme was significantly lower than the WT. Enzymatic kcat/Km values of W115F increased to about twice those of the WT, but W115F also seemed to promote increased autodegradation compared with the WT and W115Y enzymes.