武尾 正弘

Rhodococcus (opacus) erythropolis HL PM-1 grows on 2,4,6-trinitrophenol or 2,4-dinitrophenol (2,4-DNP) as a sole nitrogen source. The NADPH-dependent F(420) reductase (NDFR; encoded by npdG) and the hydride transferase II (HTII; encoded by npdI) of the strain were previously shown to convert both nitrophenols to their respective hydride Meisenheimer complexes. In the present study, npdG and npdI were amplified from six 2,4-DNP degrading Rhodococcus spp. The genes showed sequence similarities of 86 to 99% to the respective npd genes of strain HL PM-1. Heterologous expression of the npdG and npdI genes showed that they were involved in 2,4-DNP degradation. Sequence analyses of both the NDFRs and the HTIIs revealed conserved domains which may be involved in binding of NADPH or F(420). Phylogenetic analyses of the NDFRs showed that they represent a new group in the family of F(420)-dependent NADPH reductases. Phylogenetic analyses of the HTIIs revealed that they form an additional group in the family of F(420)-dependent glucose-6-phosphate dehydrogenases and F(420)-dependent N(5),N(10)-methylenetetrahydromethanopterin reductases. Thus, the NDFRs and the HTIIs may each represent a novel group of F(420)-dependent enzymes involved in catabolism.

A 4-nitrophenol (4-NP)-degrading bacterium was isolated from activated sludge and identified as a Rhodococcus sp. This bacterium, designated as strain PN1, could utilize 4-NP as a sole carbon, nitrogen and energy source. Degradation tests of 4-NP using cell suspensions of strain PN1 revealed that the degradation was induced by 4-NP and that 4-nitrocatechol (4-NC) was one of the metabolites. A gene library was constructed from the total DNA of strain PN1 and introduced into Rhodococcus rhodochrous ATCC 12674. Two recombinant strains showed 4-NP hydroxylase activity, and a 9.1-kb DNA fragment encoding the activity was isolated from one of the strains. In addition, a 2.4-kb smaller fragment expressing the activity was subcloned from the 9.1-kb fragment and sequenced. The sequence analysis showed that the fragment encodes a two-component 4-NP hydroxylase, the predicted amino acid sequence of which exhibits significant similarity to those of phenol hydroxylases and 4-hydroxyphenylacetate 3-hydroxylases belonging to the two-component flavin diffusible monooxygenase (TC-FDM) family proposed by Galán et al. (J. Bacteriol., 182, 627-636, 2000).

The metabolism of 2,6-naphthalenedisulfonate (26NDS) by Pigmentiphaga sp. NDS-2 was analyzed by isolating a mutant (NDS2-002) which slowly grew on a minimal medium containing 26NDS as the sole source of carbon. Liquid chromatography/mass spectrometry (LC/MS) analysis of metabolic intermediates in this strain revealed that 5-sulfosalicylate (5SSA) is accumulated during the degradation of 26NDS. To analyze the lower metabolic pathway, a mutant strain NDS2-008, which could not grow either on 26NDS, 5SSA or gentisate, but could on succinate as the carbon source, was isolated. When the resting cells of NDS2-008 were incubated with 5SSA or gentisate, a substance deduced to be maleylpyruvate (Zhou et al., J. Bacteriol., 183, 700-708, 2001) was commonly detected upon HPLC analysis. These results suggest that Pigmentiphaga sp. NDS-2 degrades 26NDS via 5SSA, gentisate and maleylpyruvate as intermediates.

回収牛毛を可溶化して,膜などの構造材料として利用出来る分子量の大きい可溶性タンパク質を得るために,亜硫酸ナトリウムで前処理し,パパインまたはトリプシンを用いる酵素還元加水分解法(SP法)およびアルカリ加水分解法により可溶化を行う場合の可溶化条件と可溶化タンパク質の理化学的性質を調べた.また,可溶化タンパク質の酸沈澱における挙動についても検討を加えた.SP法(パパイン)では可溶化率約44%,最終収量は22.2%で可溶化タンパク質を得た.トリプシン使用の場合には,それぞれ39%および8.4%であった.また,アルカリ加水分解法では93%および49%であった.SP法およびアルカリ加水分解法により得られた可溶化タンパク質の酸沈澱における溶解度曲線は異なったパターンを示した.両可溶化タンパク質とも共通して酸不溶性画分(沈澱物)より酸可溶性画分(上澄み液溶解物)の方が多く,また,SP法由来の酸不溶性画分はアルカリ加水分解法由来のそれに比べ,多く存在した.SDS-PAGE, SEC-HPLC分析により分子量組成を調べたところ,それぞれ2～3の画分がみられ,分子量として15KD～25KDのものが多い.アミノ酸組成はSP法による可溶化タンノパク質中にはランチオニンが多く存在し,アルカリ可溶化物では比較的少なかった.SP法とアルカリ可溶化物と比べると,システイン酸,セリンおよびプロリン等が多く,アルカリ可溶化物はセリン,トレオニンおよびランチオニンが少ないことが認められた.

Populations of phenol-degrading bacteria in soil and their trichloroethylene (TCE)-degrading capabilities were evaluated to find effective and reasonable in situ bioremediation methods. In eight soil samples, it is observed that bacteria able to grow on inorganic medium including 125 mg·l-1 of phenol accounted for 6 to 20 % of total aerobic heterotrophic bacteria. Further, TCE-degrading capabilities of fourteen phenol-degrading strains including six isolates from the soil samples described above were compared. Under phenol-induced conditions, the majority of the bacteria could reduce 1mg·l-1 of TCE to an undetectable limit, while the rests could not completely remove TCE or at all. However, all the strains with only one exception showed TCE-degrading activity to a certain extent (ultimate TCE removal was 20 to 100%), and their specific degradation rates for TCE corresponded to those for phenol. These results suggest the possibility of TCE removal by in situ biostimulation in the presence of effective native phenol-degrading bacteria.