| Indole is a typical nitrogen heterocyclic organic pollutant existing in coking wastewater and livestock wastewater,and microbial degradation of indole is known to be highly efficient and environmentally friendly.Up to present,researches of indole microbial aerobic degradation have mainly concentrated on genus Pseudomonas and Alcaligenes,with limited microbial resources.Furthermore,few attempts have been made to explore the functional genes of indole degradation.Herein,the process of microbial degradation of indole was studied by degradation characteristics,functional gene analyses and application of indole oxygenase.A strain of IDO3 growing with indole as the sole carbon and nitrogen source was isolated from the activated sludge.Based on 16 S rRNA gene sequence analysis,strain IDO3 was identified as Burkholderia sp.,a novel genus for indole degradation.The removal rate of 100 mg/L indole in culture medium was more than 99% within 14 h.In the range of 20~35 oC,pH 4~11 and rotary speed 0~250 r/min,strain IDO3 had superior degradation capacity.Several metal ions,pyridine,quinoline and additional nutrients inhibited indole degradation process.According to high performance liquid chromatography and liquid chromatography-time of flight-mass spectrometry analyses,indole metabolic intermediates were identified as anthranilate and isatin.The results showed that indole degradation pathway was proposed to be indole-indoxyl-anthranilate-isatin.2~6 kb DNA fragments of strain IDO3 were obtained by random digestion and used to constructed a clone library consisted of 6000 clones.One positive clone was screened by indigo method.Sequencing results revealed that the length of the insert fragment was 3035 bp which contained three open reading frames(ORF).Sequence analysis showed that ORF1 and ORF2 had similarity with the reported indole oxygenase and reductase,named indA and indB.IndA contained FAD binding fingerprint sequences,and its phylogenetic tree belonged to a separate branch,which indicated that IndA was a novel oxygenase.The transcriptional levels of indA and indB in indole mineral salt media were up-regulated 71.9-fold and 14.4-fold according to the results of fluorescence quantitative reverse transcription PCR,which proved that indA and indB were involved in the indole degradation process.Gene indA knockout strain IDO3-ΔindA was obtained by homologous recombination knockout strategy.However,indole degradation ability of strain IDO3-ΔindA showed almost no difference compared with the wild type strain IDO3,indicating that there could exist other indole oxidation pathways in strain IDO3.Gene indAB from strain IDO3 were amplified together to construct recombinant Escherichia coli IND_AB.Protein IndAB was successfully expressed based on the results of SDS-PAGE.The recombinant strain IND_AB was able to produce blue pigments in the LB medium.The blue product should be indigo according to the analyses of high performance liquid chromatography and liquid chromatography-time of flight-mass spectrometry.The optimal conditions for indigo production were: 37 oC,150 r/min,and LB medium with the addition of 50 mg/L indole or 0.1 g/L tryptophan.Under the conditions,strain IND_AB produced 57.7 mg/L and 64.4 mg/L indigo,which increased by 152.0% and 181.2% compared with the initial yield.The indigo synthesis curve indicated that indigo production acceleration period was 6~15 h after inoculation,and indigo yield reached the peak at 18 h. |
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