[Properties of a triphenylmethane dyes decolorization enzyme (TpmD) from Aeromonas hydrophila strain DN322].

A novel bacterial enzyme for decolorization of triphenylmethane dyes from Aeromonas hydrophila strain DN322 was purified and named TpmD. The basic properties of this enzyme including molecular weight, isoelectric point Km as well as the optimum temperature and pH were determined and the enzyme was identified as an NADH/NADPH-dependent oxygenase in previous research. Based on previous results, the effect of different inhibitor including Vc, metyrapone, rotenone, antimycin A and NaN3 as well as the effect of FAD and FMN on the activity of TpmD were measured. The results indicated that the activity of the decolorization enzyme was inhibited by Vc and metyrapone in a concentration-dependent manner, but wasn't inhibited by rotenone, antimycin A and NaN3. The activity of the decolorization enzyme was not enhanced by addition of FAD or FMN. The solution of the enzyme protein displayed only a single peak at 408nm in the Soret region, a characteristic peak of porphyrin, but did not show the characteristic peak of the cytochrome P450 proteins at 450nm in sodium dithionite (DTN)-reduced enzyme solution after treatment with carbon monoxide. The amino acid sequence of N-terminal of TpmD provided further evidence that the enzyme is an oxygenase. All these results suggest that decolorization enzyme TpmD is a new hemo-containing oxygenase. The decolorization enzyme would be a good material for further research of the enzymological mechanism of triphenylmethane dyes decolorization by bacteria.

[Properties of a triphenylmethane dyes decolorization enzyme TpmD from Aeromonas hydrophila strain DN322].

A novel bacterial decolorization enzyme for triphenylmethane dyes from Aeromonas hydrophila strain DN322 was purified, named TpmD. The purified enzyme catalyzes the decolorization of several triphenylmethane dyes, i.e., crystal violet, basic fuchsin, brilliant green and malachite green. The enzyme was identified by the clear transparent band development of zymogram stained with crystal violet, basic fuchsin, brilliant green and malachite green after polyacrylamide gel electrophoresis (PAGE) respectively. The decolorization enzyme was enzymologically characterized. The results showed that the molecular weight of TpmD is 29.4kDa and its isoelectric point (pI) is 5.6. The maximal activity of TpmD for above four triphenylmethane dyes was observed at 50 degrees C - 55 degrees C and pH 7.4 - 8.0. The temperature for losing half of the activity (t1/2) within 4h is 62 degrees C. The activities of decolorization enzyme are relatively stable at pH range of 5.5 - 9.0. The K(m) and V(max) of TpmD for decolorizing crystal violet, basic fuchsin, brilliant green and malachite green are 24.3, 40.6, 54.2, 68.5 micromol/L respectively, V(max) are 19.6, 74.1, 82.8, 115.6 micromol x L(-1) x s(-1) respectively. Both NADH/NADPH and molecular oxygen are necessary for the enzyme to decolorize triphenylmethane dyes, indicate the enzyme is an NADH/NADPH-dependent oxygenase.

[Anaerobic humus respiration by Shewanella cinica D14T].

Experimental results suggested Shewanella cinica D14T is capable of humus respiration utilizing various organic acids and some important environmental pollutants (e.g., toluene. etc) as electron donors and AQS or AQDS as a sole terminal electron acceptor under anaerobic condition. The dissimilatory reduction of 1mmol/L AQDS can couple to the production of enough ATP to support cell growth about 60 generations; The oxidization of electron donors was coupled to the reduction of humus, as reduced humus increased corresponding with increasing of electron donor; The typical inhibitors such as Cu2+ which inhibited Fe-S center, Stigmatellin which was methyl-naphthoquinone model, Dicumarol which inhibited oxidized methyl-naphthoquinone transform to reduced one, Metyrapone which was specific inhibitor for P450 enzyme blocked the humus respiration seriously. These were powerful evidences for humus-respiration by D14.

[Isolation and characterization of two aniline-degrading strains and compare of functional genes].

Two aniline-degrading bacterial strains, AN30 and DN425, were isolated from activated sludge of textile-printing wastewater treatment plant and identified as Pseudomonas sp. and Shewanella sp. , respectively. Under shaking condition, 250 mg/L aniline was removed 96.1% and 13.8% within 72h by strains AN30 and DN425, respectively. Under static condition, their degrading rates were 39.6% and 8.6% , respectively. Under static condition, the decolorizing rate of strain DN425 reached 96% for azo dye Great Red GR in 4h, exhibiting a remarkable color removal capability. However, strain AN30 was not capable of decolorizing Great Red GR. With two sets of specific primers for tdnQ gene and fre gene, the two genes were detected by PCR amplification. The results indicated the two strains possess both tdnQ gene and fre gene.

[Cloning and analysis of genes encoding 2-naphthoate monooxygenase and NADH:flavin oxidoreductase].

In Burkholderia sp. JT1500, a key step of 2-naphthoate biodegradation pathway is carried out by 2-naphthoate monooxygenase (Nmo) in which 2-naphthoate is oxidized to 1-hydroxy-2-naphthoate. A gene cluster of 4.8kb from Burkholderia sp. JT1500 was cloned and sequenced, four open reading frames named orfB, orfC, orfD and orfA were identified in this region. Sequence alignment showed that orfA had a high homology of nucleotide acid composition to monooxygenase genes from both Japonicum USDA 110 and Ralstonia eutropha HF 39, orfB had some homology to the component of flavin reductase genes from Bordetlla pertussis Tohama I, Ralstonia solanacearum GMI1000 and Bordetella bronchiseptica RB50. Enzyme activity analysis showed that the cell extracts of recombinant E. coli S(A) (only harboring orfA) showed very low oxygenase oxidation activity as detected by NADH decreasing, while the cell extracts of recombinant S(B) (only harboring orfB) did not show any oxidation activity at all. But when the cell extracts of S(B) and S(A) were mixed, which showed very strong oxidation activity when flavin (FMN or FAD) provided; the recombinant S(B + A) cells harboring both orfB and orfA genes also showed strong oxidation activity when flavin provided; weak flavin deoxidization activity could be detected from the cell extracts of E. coli S(B) under anaerobic conditions. Based on above message, a conclusion was drawn that Nmo is consisted of two components: a flavin oxidoreductase (NmoB) and a monooxygenase (NmoA). First NmoB uses NADH to reduce flavin and supplies reduced flavin to NmoA to catalyze O2 oxidizing 2-NAT. NmoB is NmoA' s coupling protein.

[Bacterial Fe(III) reduction].

Bacterial Fe(III) reduction is an important pathway of bioenergy metabolism in the process of life evolution. Many kinds of archaebacteria and eubacteria are capable of reducing Fe(III) to conserve energy. Anaerobic Fe(III) respiration pathway involves many membranous proteins and regulating factors, especially the muti-haem c-type cytochromes are very important in the course of electron transportation. In addition, bacterial Fe(III) reduction play important roles in the biological geochemistry circulation and environmental remediation, therefore has vital environmental significance.

[Microbial community composition and phylogenetic analysis of deodor biofilter under different pH conditions].

Molecular biological methods were applied to analyze the genetic diversity of microbe community in biofilter. V3 variable fragments of genes coding for 16S rRNA were amplified by Polymerase Chain Reaction. Then PCR-DGGE combined technique were used to analyze the microbial population composition and phylogeny of deodorant biofilter. The results indicate that there are prominent differences in microbial diversity and abundance between acid and neutral conditions. Owing to the selective pressure of extreme acidity, there is less diversity of microbe compared with that in neutral environment. It is demonstrated the obvious distinction of spacial diversity in different biofilter bed as well. PCR and T-vector cloning sequencing results shows that the sulfur-oxidizing bacteria are the predominant population. The research offers the valid scientific basis for better treatment of odorant gas and the theoretical foundation for application of bio-deodorization.

Shewanella decolorationis sp. nov., a dye-decolorizing bacterium isolated from activated sludge of a waste-water treatment plant.

A highly efficient dye-decolorizing bacterium, strain S12T, was isolated from activated sludge of a textile-printing waste-water treatment plant in Guangzhou, China. The cells were Gram-negative and motile by means of a single polar flagellum. The strain was capable of anaerobic growth either by fermentation of glucose or by anaerobic respiration and utilized a variety of electron acceptors, including nitrate, iron oxide and thiosulfate. The physiological properties, tested by using the Biolog GN2 system, were similar to those of the genus of Shewanella. Analysis of the nearly complete 16S rRNA gene sequence of strain S12T showed the highest similarity (98 and 97 %, respectively) to Shewanella baltica and Shewanella putrefaciens. However, the level of gyrB similarity between strain S12T and S. putrefaciens was 87 %. DNA from strain S12T showed 41.8 and 41.9 % DNA relatedness, respectively, to the DNA of S. baltica DSM 9439T and S. putrefaciens DSM 6067T. The DNA G+C content of strain S12T was 49.3 mol%. The predominant menaquinone was MK-7 and the predominant ubiquinones were Q-7 and Q-8. The dominant fatty acids were 15 : 0, 16 : 0, iso-15 : 0 and 16 : 1omega7c, similar to the profiles of other Shewanella species. On the basis of its physiological and molecular properties, strain S12T appears to represent a novel species of the genus Shewanella, for which the name Shewanella decolorationis sp. nov. is proposed. The type strain is S12T (=CCTCC M 203093T=IAM 15094T).

[Study on the ammonia-oxidizing bacteria from activated sludge samples by the molecular analysis].

The molecular analysis methods of PCR amplification, random cloning and sequencing were used to investigate the ammonia-oxidizing bacterial community composition and the activity of ammonia-monooxygenase (AMO) from the activated sludge samples of an industrial wastewater treatment plant receiving sewage with high ammonia concentration. It is the first time to use PCR-DGGE combined technique to analysis the difference of dominant bacterial community compositions of the activated sludge samples in China. The result showed that the ammonia-oxidizing bacteria (AOB) detected from the activated sludge samples all belong to Nitrosomonas sp. The activity of AMO, the stability of bacteria community composition and the treatment efficiency of the wastewater treatment system were improved evidently, after the activated sludge system was operated for a certain extant. It is suggested that the molecular techniques will contribute to our understanding of the diversity and function of AOB and will benefit to improve the industrial wastewater treatment system.

[Cloning and expression of a 2-naphthoate monooxygenase gene (nmo) in Burkholderia sp. JT1500].

A 4.8kb DNA fragment from one blue colony of the pLARF1 gene library of Burkholderia sp. JT1500 was subcloned to pUC18, designated as pEK123. The sequence of the inserted 4.8kb DNA' of pEK123 was analyzed and submitted to EMBL nucleotide database, the accession # is AJ566333. The transformants of pEK123 could also become blue in LB agar and sequence analysis showed that three open reading frames and a putative promoter sequence were located in this inserted fragment. Then the 4.4kb insert fragment of pEK123 was double digested with Xba I / Kpn I and EcoR I / Xba I respectively to construct plamsids pXK3 and pEX12. The pXK3 contained only one 1158bp open reading frame (ORF) and pEX12 with other two ORFs. Unlike pEK123, the colonies of pEX12 did not show any blue color even incubated for 72h in LB agar, but the transformants of pXK3 did oxidize indole into indigo. The deduced 43kD protein of 1158bp ORF showed 64% homology of amino acid composition to Ralstonia eutropha HF39 hydroxylase (bec). Results of substrate transformation analysis showed that the transformants of pEK123 was able to catalyze the oxidation of 2-naphthoate but not other key intermediates in 2-naphthoate metabolic pathway. These results confirmed that the product of 1158bp ORF is 2-naphthoate monooxygenase. Though the oxygenase activity of pEK123 is much higher than that of pXK3, SDS-PAGE analysis found no difference between the amount of the band of monooxygenase produced by pXK3 or pEK123, but one more band was found produced by pEK123. According to the difference of substrate analysis between pXK3 and pEK123, it is supposed that the products of two open reading frames up stream of nmo gene had strong influence on the activity of the monooxygenase. Benzoate was oxidized by free-cell extracts of the transformants of pEK123 in the transformation experiment with different aromatic substrates. As the DNA sequence and amino acid sequence of 2-naphthoate monoxygenase (nmo) did no show any homology with the DNA sequence and amino acid sequence of benzoate oxygenases reported, the pathway of benzoate oxidation conducted by nmo is on the investigation.