Identification and functional analysis of genes required for desulfurization of alkyl dibenzothiophenes of Mycobacterium sp. G3.

Mycobacterium sp. G3 was reported as a dibenzothiophene (DBT)-degrading microorganism and the first strain to have the ability to degrade high-molecular-weight alkyl DBTs, such as 4,6-dibutyl DBT and 4,6-dipentyl DBT, by the C-S bond cleavage pathway. Three genes (mdsA, mdsB, and mdsC) for desulfurization, which form a cluster, were cloned from Mycobacterium sp. G3. The expression of each gene in Escherichia coli JM109 showed that MdsC oxidized DBT to DBT sulfone, MdsA transformed DBT sulfone into 2-(2'-hydroxyphenyl)benzene sulfinate (HPBS), and MdsB desulfinated HPBS into 2-hydroxybiphenyl (HBP), indicating that the gene products of mdsABC are functional in the recombinant. MdsC oxidized 4,6-dimethyl DBT, 4,6-diethyl DBT, 4,6-dipropyl DBT and 4,6-dibutyl DBT to each sulfone form, suggesting that MdsC covers a broad specificity for alkyl DBTs.

De-repression and comparison of oil-water separation activity of the dibenzothiophene desulfurizing bacterium, Mycobacterium sp. G3.

Expression of the desulfurization genes (dsz) in Mycobacterium sp. G3 is repressed by sulfate, which is the product of biodesulfurization. An expression clone, pSMTABC, was constructed by placing the dsz genes downstream of the hsp60 promoter and the constructed plasmid was electroporated into G3. The recombinant strain G3-1 desulfurized dibenzothiophene in the presence of 0.5 mM: sulfate while the Dsz phenotype was completely repressed in the wild-type strain. However, there was no significant increase in the amount of desulfurization enzymes in G3-1. In addition, G3 had superior separation of diesel oil-water separation activity compared to E. coli, which is superior to desulfurizing rhodococci.

Analyses of microbial desulfurization reaction of alkylated dibenzothiophenes dissolved in oil phase.

The kinetics of the oil/water two-phase reaction system was analyzed, and the reaction was carried out with the desulfurization of alkylated dibenzothiophenes (Cx-DBTs) using the desulfurizing microorganism Mycobacterium sp. G3. In the water-phase reaction system, the desulfurization activities were constant with respect to species of Cx-DBTs as substrates. However, the desulfurization activities in the oil/water two-phase reaction system against DBT, 4,6-dimethyl DBT, 4,6-diethyl DBT, 4,6-dipropyl DBT, and 4,6-dibutyl DBT were 49.0, 45.9, 11.5, 1.35, and 0.00 micromol g DCW(-1) h(-1), respectively. The kinetic parameters for the degradation of DBT, 4,6-dimethyl DBT, and 4,6-diethyl DBT were also obtained (V(max) values 90.0, 68.7, and 22.7 micromol g DCW(-1) h(-1) and K(m) values 0.21, 0.70, and 3.03 mM, respectively). The reason for the decrease in activity against Cx-DBTs of high molecular weight was a decrease in the V(max) value and an increase in the K(m) value, the latter being a particularly serious problem. Furthermore, the hydrophobicity of the substrate was evaluated as the capacity factor measured by high-performance liquid chromatography (HPLC). The correlation between substrate hydrophobicity and desulfurization activity indicated that the desulfurization reaction in the oil/water two-phase reaction system is greatly influenced by the hydrophobicity of the substrates. In addition, the influence of the solvent on desulfurization activity was examined, and it was found that not only the hydrophobicity of substrates, but also that of solvents, affected the desulfurization reaction.

Cloning and sequencing of a poly(DL-lactic acid) depolymerase gene from Paenibacillus amylolyticus strain TB-13 and its functional expression in Escherichia coli.

The gene encoding a poly(DL-lactic acid) (PLA) depolymerase from Paenibacillus amylolyticus strain TB-13 was cloned and overexpressed in Escherichia coli. The purified recombinant PLA depolymerase, PlaA, exhibited degradation activities toward various biodegradable polyesters, such as poly(butylene succinate), poly(butylene succinate-co-adipate), poly(ethylene succinate), and poly(epsilon-caprolactone), as well as PLA. The monomeric lactic acid was detected as the degradation product of PLA. The substrate specificity toward triglycerides and p-nitrophenyl esters indicated that PlaA is a type of lipase. The gene encoded 201 amino acid residues, including the conserved pentapeptide Ala-His-Ser-Met-Gly, present in the lipases of mesophilic Bacillus species. The identity of the amino acid sequence of PlaA with Bacillus lipases was no more than 45 to 50%, and some of its properties were different from those of these lipases.

A simple screening procedure for heterotrophic nitrifying bacteria with oxygen-tolerant denitrification activity.

Various naturally occurring strains of heterotrophic nitrifying bacteria were isolated by enrichment culture using acetamide as the C and N source, and 21 strains were identified as heterotrophic nitrifiers. Using a new simple procedure, these 21 strains were also investigated for the ability to carry out denitrifcation in the presence of oxygen. Several of the nitrifying strains were found to exhibit a distinct activity that allows for denitrifcation via nitrite (NO2-) in the presence of oxygen, indicating that they have an oxygen-tolerant denitrifcation system. A wide variety of bacteria possessing both nitrification and denitrifcation capabilities in the presence of oxygen were isolated and partially characterized by using the simple screening combinatorial procedure described in this paper.

Succession of bacterial community structure along the Changjiang River determined by denaturing gradient gel electrophoresis and clone library analysis.

Bacterial community structure along the Changjiang River (which is more than 2,500 km long) was studied by using denaturing gradient gel electrophoresis (DGGE) and clone library analysis of PCR-amplified 16S ribosomal DNA (rDNA) with universal bacterial primer sets. DGGE profiles and principal-component analysis (PCA) demonstrated that the bacterial community gradually changed from upstream to downstream in both 1998 and 1999. Bacterial diversity, as determined by the Shannon index (H'), gradually decreased from upstream to downstream. The PCA plots revealed that the differences in the bacterial communities among riverine stations were not appreciable compared with the differences in two adjacent lakes, Lake Dongting and Lake Poyang. The relative stability of the bacterial communities at the riverine stations was probably due to the buffering action of the large amount of water flowing down the river. Clone library analysis of 16S rDNA revealed that the dominant bacterial groups changed from beta-proteobacteria and the Cytophaga-Flexibacter-Bacteroides group upstream to high-G+C-content gram-positive bacteria downstream and also that the bacterial community structure differed among the stations in the river and the lakes. The results obtained in this study should provide a reference for future changes caused by construction of the Three Gorges Dam.

Coimmobilization of gluconolactonase with glucose oxidase for improvement in kinetic property of enzymatically induced volume collapse in ionic gels.

The object of this paper is to provide an enzymatic means to attain faster swelling or shrinking kinetics of polyelectrolyte gels that undergo volume phase transition as an immobilized enzyme reaction sets in. For this, we studied the coimmobilization of gluconolactonase (GL) with glucose oxidase (GOD). A gel used was in the shape of a small cylinder (several hundred micrometers in diameter) and composed of a lightly cross-linked copolymer of N-isopropylacrylamide and acrylic acid. GL was isolated from Aspergillus niger and purified about 100-fold. It was found that in a substrate solution containing glucose, the gel with the coimmobilized GL and GOD shrinks very rapidly. The shrinking rate was identical to that of the enzyme-free gel that undergoes a shrinking transition in response to a sudden pH change of the outer medium from 7 to 5. This indicates the rate-limiting step in the shrinking process to be diffusion of the networks, but not the enzyme reaction. In the gel with singly immobilized GOD, a very slow shrinking was observed because the process is governed by the enzyme reaction. These results were discussed in full in connection with an enzymatically induced decrease in pH within and in the vicinity of the gel phase. As a result, it has become apparent that the faster shrinking kinetics in the coimmobilized enzyme system is attained by the GL-catalyzed hydrolysis of D-glucono-delta-lactone resulting from the oxidation of glucose with GOD.

Analyses of substrate specificity of the desulfurizing bacterium Mycobacterium sp. G3.

The substrate specificity of Mycobacterium sp. G3 with desulfurization activity against dibenzothiophene (DBT) was investigated. Desulfurization reactions were carried out using a concentrated cell suspension of G3. The conversion from 4,6-dipropyl DBT, one of the sulfur-containing compounds that is difficult to desulfurize in diesel oil, to 2-hydroxy-3,3'-dipropylbiphenyl as an end -product of the desulfurization reaction was found in the water reaction system and in the oil/water two-phase reaction system. 4,6-Dibutyl DBT and 4,6-dipentyl DBT were metabolized to the hydroxybiphenyl form via the sulfone form in the water reaction system. These results indicate that G3 has high membrane permeability and superior substrate specificity for high molecular weight alkyl DBTs, which are represented by 4,6-dipentyl DBT as C10-DBT. Furthermore, G3 could desulfurize diesel oil, and the sulfur concentration was decreased from 116 mg l(-1) to 48 mg l(-1) within 24 h.

Cloning and sequence analysis of poly(tetramethylene succinate) depolymerase from Acidovorax delafieldii strain BS-3.

A gene encoding poly(tetramethylene succinate), PBS, depolymerase, pbsA, has been cloned from Acidovorax delafieldii strain BS-3 chromosomal DNA. The clone expressed in Escherichia coli showed the ability to degrade both PBS and poly[(tetramethylene succinate)-co-adipate] that are kinds of biodegradable plastics. PBS depolymerase was considered to be a kind of lipase, since it also degrades olive oil. It had no apparent hydrophobic-amino-acid-rich region which exists in other known plastic-degrading enzymes. From the result of amino acid homology search, PbsA was found to have some similarities with lipases of Streptomyces sp. and Mollaxella sp. In the motif surrounding the active site Ser residue (Gly-X1-Ser-X2-Gly), PbsA was revealed to have a Trp residue in the X1 position instead of His which is most likely found in other bacterial lipases.

Analysis of dibenzothiophene metabolic pathway in Mycobacterium strain G3.

The dibenzothiophene (DBT) metabolic pathway in Mycobacterium strain G3, which is classified as a desulfurizing microorganism with the 4S pathway, was analyzed. 2-Hydroxybiphenyl (HBP), which is an end metabolite in the DBT desulfurization reaction, and 2-methoxybiphenyl (MBP) were found in the reaction mixture, and the methoxylation pathway from HBP to MBP was clarified. Although the substrate in the methoxylation reaction was HBP, there was no relationship between expression of the methoxylation activity and that of the desulfurization activity. Then, 4,6-dimethyl DBT, 4,6-diethyl DBT and benzo[b]naphtho[2,1-d]thiophene were metabolized to their methoxy forms via the desulfurization pathway. We established the methoxylation pathway in Mycobacterium G3.

Difference in degrading p-nitrophenol between indigenous bacteria in a reactor.

p-Nitrophenol (PNP) -degrading bacteria were isolated from a reactor using a mineral salt medium containing a low and high PNP concentration. We isolated two bacterial species, Pseudomonas sp. YTK17 and Rhodococcus opacus YTK32, that utilize PNP as their sole source of carbon and energy. These strains exhibited differences in PNP degradation activity in relation to PNP concentration. Strain YTK17 showed a high level of degradation following pre-exposure to a low PNP concentration, whereas strain YTK32 required a relatively high PNP concentration for degradation to occur. These results indicated that phylogenetically and physiologically different types of PNP-degrading bacteria coexisted in a reactor.

Functions and potential applications of glycolipid biosurfactants--from energy-saving materials to gene delivery carriers.

Biosurfactants (BS) produced by various microorganisms show unique properties (e.g., mild production conditions, lower toxicity, higher biodegradability and environmental compatibility) compared to their chemical counterparts. The numerous advantages of BS have prompted applications not only in the food, cosmetic, and pharmaceutical industries but in environmental protection and energy-saving technology as well. Glycolipid BS are the most promising, due to high productivity from renewable resources and versatile biochemical properties. Mannosylerythritol lipids (MEL), which are glycolipid BS produced by a yeast Candida antarctrica, exhibit not only excellent interfacial properties but also remarkable differentiation-inducing activities against human leukemia cells. MEL also show a potential anti-agglomeration effect on ice particles in ice slurry used for cold thermal storage. Recently, the cationic liposome bearing MEL has been demonstrated to increase dramatically the efficiency of gene transfection into mammalian cells. These features of BS should broaden its applications in new advanced technologies. The current status of research and development on glycolipid BS, especially their function and potential applications, is discussed.