Dataset Information

A Thermostable Bilirubin-Oxidizing Enzyme from Activated Sludge Isolated by a Metagenomic Approach.

ABSTRACT: A gene coding for a multicopper oxidase (BopA) was identified through the screening of a metagenomic library constructed from wastewater treatment activated sludge. The recombinant BopA protein produced in Escherichia coli exhibited oxidation activity toward 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS) in the presence of copper, suggesting that BopA is laccase. A bioinformatic analysis of the bopA gene sequence indicated that it has a phylogenetically bacterial origin, possibly derived from a bacterium within the phylum Deinococcus-Thermus. Purified BopA exhibited maximum activity at pH 7.5 with bilirubin as its substrate and was found to be active over a markedly broad pH range from 6 to 11. It also showed notable thermostability; its activity remained intact even after a heat treatment at 90°C for 60 min. This enzyme is a thermostable-bilirubin oxidase that exhibits markedly higher thermostability than that previously reported for laccases.

SUBMITTER: Kimura N

PROVIDER: S-EPMC5158116 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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A Thermostable Bilirubin-Oxidizing Enzyme from Activated Sludge Isolated by a Metagenomic Approach.

Kimura Nobutada N Kamagata Yoichi Y

Microbes and environments 20161123 4

A gene coding for a multicopper oxidase (BopA) was identified through the screening of a metagenomic library constructed from wastewater treatment activated sludge. The recombinant BopA protein produced in Escherichia coli exhibited oxidation activity toward 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS) in the presence of copper, suggesting that BopA is laccase. A bioinformatic analysis of the bopA gene sequence indicated that it has a phylogenetically bacterial origin, possibly der ...[more]

PMID: 27885197

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Project description:BACKGROUND: Pyrethroid pesticides are broad-spectrum pest control agents in agricultural production. Both agricultural and residential usage is continuing to grow, leading to the development of insecticide resistance in the pest and toxic effects on a number of nontarget organisms. Thus, it is necessary to hunt suitable enzymes including hydrolases for degrading pesticide residues, which is an efficient "green" solution to biodegrade polluting chemicals. Although many pyrethroid esterases have consistently been purified and characterized from various resources including metagenomes and organisms, the thermostable pyrethroid esterases have not been reported up to the present. RESULTS: In this study, we identified a novel pyrethroid-hydrolyzing enzyme Sys410 belonging to familyV esterases/lipases with activity-based functional screening from Turban Basin metagenomic library. Sys410 contained 280 amino acids with a predicted molecular mass (Mr) of 30.8 kDa and was overexpressed in Escherichia coli BL21 (DE3) in soluble form. The optimum pH and temperature of the recombinant Sys410 were 6.5 and 55°C, respectively. The enzyme was stable in the pH range of 4.5-8.5 and at temperatures below 50°C. The activity of Sys410 decreased a little when stored at 4°C for 10 weeks, and the residual activity reached 94.1%. Even after incubation at 25°C for 10 weeks, it kept 68.3% of its activity. The recombinant Sys410 could hydrolyze a wide range of ?-nitrophenyl esters, but its best substrate is ?-nitrophenyl acetate with the highest activity (772.9 U/mg). The enzyme efficiently degraded cyhalothrin, cypermethrin, sumicidin, and deltamethrin under assay conditions of 37°C for 15 min, with exceeding 95% hydrolysis rate. CONCLUSION: This is the first report to construct metagenomic libraries from Turban Basin to obtain the thermostable pyrethroid-hydrolyzing enzyme. The recombinant Sys410 with broad substrate specificities and high activity was the most thermostable one of the pyrethroid-hydrolyzing esterases studied before, which made it an ideal candidate for the detoxification of pyrethroids.

Project description:It is well acknowledged that the activities of activated sludge (AS) are influenced by seasonal temperature variation. However, the underlying mechanisms remain largely unknown. Here, the activities of activated sludge under three simulated temperature variation trends were compared in lab-scale. The TN, HN3-H, and COD removal activities of activated sludge were improved as temperature elevated from 20 °C to 35 °C. While, the TN, HN3-H, COD and total phosphorus removal activities of activated sludge were inhibited as temperature declined from 20 °C to 5 °C. Both the extracellular polymer substances (EPS) composition (e.g., total amount, PS, PN and DNA) and sludge index of activated sludge were altered by simulated seasonal temperature variation. The variation of microbial community structures and the functional potentials of activated sludge were further explored by metagenomics. Proteobacteria, Actinobacteria, Acidobacteria and Bacteroidetes were the dominant phyla for each activated sludge sample under different temperatures. However, the predominant genera of activated sludge were significantly modulated by simulated temperature variation. The functional genes encoding enzymes for nitrogen metabolism in microorganisms were analyzed. The enzyme genes related to ammonification had the highest abundance despite the changing temperature, especially for gene encoding glutamine synthetase. With the temperature raising from 20 °C to 35 °C. The abundance of amoCAB genes encoding ammonia monooxygenase (EC:1.14.99.39) increased by 305.8%. Meanwhile, all the enzyme genes associate with denitrification were reduced. As the temperature declined from 20 °C to 5 °C, the abundance of enzyme genes related to nitrogen metabolism were raised except for carbamate kinase (EC:2.7.2.2), glutamate dehydrogenase (EC:1.4.1.3), glutamine synthetase (EC:6.3.1.2). Metagenomic data indicate that succession of the dominant genera in microbial community structure is, to some extent, beneficial to maintain the functional stability of activated sludge under the temperature variation within a certain temperature range. This study provides novel insights into the effects of seasonal temperature variation on the activities of activated sludge.

Dataset Information

A Thermostable Bilirubin-Oxidizing Enzyme from Activated Sludge Isolated by a Metagenomic Approach.

Publications

A Thermostable Bilirubin-Oxidizing Enzyme from Activated Sludge Isolated by a Metagenomic Approach.

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