APPLIED AND ENVIRONMENTAL MICROBIOLOGY
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2021
·Vol. 87
·期 5
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IF 4.792
Shanshan Chen,Xianyue Jing,Yongliang Yan,Shaofu Huang,Xing Liu,Piao Chen,Shungui Zhou
Diazotrophs can produce bioavailable nitrogen from inert N2 gas by bioelectrochemical nitrogen fixation (e-BNF), which is emerging as an energy-saving and highly selective strategy for agriculture and industry. However, current e-BNF technology is impeded by requirements for NH4+ assimilation inhibitors to facilitate intracellular ammonia secretion and precious metal catalysts to generate H2 as the energy-carrying intermediate. Here, we initially demonstrate inhibitor- and catalystless extracellular NH4+ production by the diazotroph Pseudomonas stutzeri A1501 using an electrode as the sole electron donor. Multiple lines of evidence revealed that P. stutzeri produced 2.32 ± 0.25 mg/liter extracellular NH4+ at a poised potential of −0.3 V (versus standard hydrogen electrode [SHE]) without the addition of inhibitors or expensive catalysts. The electron uptake mechanism was attributed to the endogenous electron shuttle phenazine-1-carboxylic acid, which was excreted by P. stutzeri and mediated electron transfer from electrodes into cells to directly drive N2 fixation. The faradaic efficiency was 20% ± 3%, which was 2 to 4 times that of previous e-BNF attempts using the H2-mediated pathway. This study reports a diazotroph capable of producing secretable NH4+ via extracellular electron uptake, which has important implications for optimizing the performance of e-BNF systems and exploring the novel nitrogen-fixing mode of syntrophic microbial communities in the natural environment.IMPORTANCE Ammonia greatly affects global ecology, agriculture, and the food industry. Diazotrophs with an enhanced capacity of extracellular NH4+ excretion have been proven to be more beneficial to the growth of microalgae and plants, whereas most previously reported diazotrophs produce intracellular organic nitrogen in the absence of chemical suppression and genetic manipulation. Here, we demonstrate that Pseudomonas stutzeri A1501 is capable of extracellular NH4+ production without chemical suppression or genetic manipulation when the extracellular electrode is used as the sole electron donor. We also reveal the electron uptake pathway from the extracellular electron-donating partner to P. stutzeri A1501 via redox electron shuttle phenazines. Since both P. stutzeri A1501 and potential electron-donating partners (such as electroactive microbes and natural semiconductor minerals) are abundant in diverse soils and sediments, P. stutzeri A1501 has broader implications on the improvement of nitrogen fertilization in the natural environment.
研究领域:
微生物学
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环境科学
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农业科学
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PubMed
高影响因子
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
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2021
·Vol. 87
·期 6
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IF 4.792
Minggen Cheng,Dongmei Pei,Xiangrong He,Yongchuang Liu,Pingping Zhu,Xin Yan
The bacterial hydrolytic dehalogenation of 4-chlorobenzoate (4CBA) is a coenzyme A (CoA)-activation-type catabolic pathway that is usually a common part of the microbial mineralization of chlorinated aromatic compounds. Previous studies have shown that the transport and dehalogenation genes for 4CBA are typically clustered as an fcbBAT1T2T3C operon and inducibly expressed in response to 4CBA. However, the associated molecular mechanism remains unknown. In this study, a gene (fcbR) adjacent to the fcb operon was predicted to encode a TetR-type transcriptional regulator in Comamonas sediminis strain CD-2. The fcbR knockout strain exhibited constitutive expression of the fcb cluster. In the host Escherichia coli, the expression of the Pfcb-fused green fluorescent protein (gfp) reporter was repressed by the introduction of the fcbR gene, and genetic studies combining various catabolic genes suggest that the ligand for FcbR may be an intermediate metabolite. Purified FcbR could bind to the Pfcb DNA probe in vitro, and the metabolite 4-chlorobenzyl-CoA (4CBA-CoA) prevented FcbR binding to the Pfcb DNA probe. Isothermal titration calorimetry (ITC) measurements showed that 4CBA-CoA could bind to FcbR at a 1:1 molar ratio. DNase I footprinting showed that FcbR protected a 42-bp DNA motif (5′-GGAAATCAATAGGTCCATAGAAAATCTATTGACTAATCGAAT-3′) that consists of two sequence repeats containing four pseudopalindromic sequences (5′-TCNATNGA-3′). This binding motif overlaps with the −35 box of Pfcb and was proposed to prevent the binding of RNA polymerase. This study characterizes a transcriptional repressor of the fcb operon, together with its ligand, thus identifying halogenated benzoyl-CoA as belonging to the class of ligands of transcriptional regulators.IMPORTANCE The bacterial hydrolytic dehalogenation of 4CBA is a special CoA-activation-type catabolic pathway that plays an important role in the biodegradation of polychlorinated biphenyls and some herbicides. With genetic and biochemical approaches, the present study identified the transcriptional repressor and its cognate effector of a 4CBA hydrolytic dehalogenation operon. This work extends halogenated benzoyl-CoA as a new member of CoA-derived effector compounds that mediate allosteric regulation of transcriptional regulators.
研究领域:
微生物学
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分子生物学
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环境科学
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DOI
PubMed
高影响因子
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
·
2019
·
IF 4.077
Rongshui Wang,Jihong Yi,Jinmeng Shang,Wenjun Yu,Zhifeng Li,Haiyan Huang,Huijun Xie,Shuning Wang
Agrobacterium tumefaciens S33 degrades nicotine via a novel hybrid of the pyridine and the pyrrolidine pathways. The hybrid pathway consists of at least six steps involved in oxidoreductive reactions before the N-heterocycle can be broken down. Collectively, the six steps allow electron transfer from nicotine and its intermediates to the final acceptor O2 via the electron transport chain (ETC). 6-Hydroxypseudooxynicotine oxidase, renamed 6-hydroxypseudooxynicotine dehydrogenase in this study, has been characterized as catalyzing the fourth step using the artificial electron acceptor 2,6-dichlorophenolindophenol. Here, we used biochemical, genetic, and liquid chromatography-mass spectrometry (LC-MS) analyses to determine that 6-hydroxypseudooxynicotine dehydrogenase utilizes the electron transfer flavoprotein (EtfAB) as the physiological electron acceptor to catalyze the dehydrogenation of pseudooxynicotine, an analogue of the true substrate 6-hydroxypseudooxynicotine, in vivo, into 3-succinoyl-semialdehyde-pyridine. NAD(P)+, O2, and ferredoxin could not function as electron acceptors. The oxygen atom in the aldehyde group of the product 3-succinoyl-semialdehyde-pyridine was verified to be derived from H2O. Disruption of the etfAB genes in the nicotine-degrading gene cluster decreased the growth rate of A. tumefaciens S33 on nicotine but not on 6-hydroxy-3-succinoylpyridine, an intermediate downstream of the hybrid pathway, indicating the requirement of EtfAB for efficient nicotine degradation. The electrons were found to be further transferred from the reduced EtfAB to coenzyme Q by the catalysis of electron transfer flavoprotein:ubiquinone oxidoreductase. These results aid in an in-depth understanding of the electron transfer process and energy metabolism involved in the nicotine oxidation and provide novel insights into nicotine catabolism in bacteria.IMPORTANCE Nicotine has been studied as a model for toxic N-heterocyclic aromatic compounds. Microorganisms can catabolize nicotine via various pathways and conserve energy from its oxidation. Although several oxidoreductases have been characterized to participate in nicotine degradation, the electron transfer involved in these processes is poorly understood. In this study, we found that 6-hydroxypseudooxynicotine dehydrogenase, a key enzyme in the hybrid pyridine and pyrrolidine pathway for nicotine degradation in Agrobacterium tumefaciens S33, utilizes EtfAB as a physiological electron acceptor. Catalyzed by the membrane-associated electron transfer flavoprotein:ubiquinone oxidoreductase, the electrons are transferred from the reduced EtfAB to coenzyme Q, which then could enter into the classic ETC. Thus, the route for electron transport from the substrate to O2 could be constructed, by which ATP can be further sythesized via chemiosmosis to support the baterial growth. These findings provide new knowledge regarding the catabolism of N-heterocyclic aromatic compounds in microorganisms.
研究领域:
微生物学
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生物化学
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分子生物学
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环境科学
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DOI
PubMed
高影响因子
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
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2018
·Vol. 84
·期 9
·
IF 3.633
Peng Zhang,Jin-Song Guo,Peng Yan,You-Peng Chen,Wei Wang,You-Zhi Dai,Fang Fang,Gui-Xue Wang,Yu Shen
Pollutant degradation is present mainly in the surface layer of biofilms, and the surface layer is the most vulnerable to impairment by toxic pollutants. In this work, the effects of nanosized TiO2 (n-TiO2) on the average thicknesses of Bacillus subtilis biofilm and on bacterial attachment on different surfaces were investigated. The binding mechanism of n-TiO2 to the cell surface was also probed. The results revealed that n-TiO2 caused biofilm dispersal and the thicknesses decreased by 2.0 to 2.6 μm after several hours of exposure. The attachment abilities of bacteria with extracellular polymeric substances (EPS) on hydrophilic surfaces were significantly reduced by 31% and 81% under 10 and 100 mg/liter of n-TiO2, respectively, whereas those of bacteria without EPS were significantly reduced by 43% and 87%, respectively. The attachment abilities of bacteria with and without EPS on hydrophobic surfaces were significantly reduced by 50% and 56%, respectively, under 100 mg/liter of n-TiO2. The results demonstrated that biofilm dispersal can be attributed to the changes in the cell surface structure and the reduction of microbial attachment ability.IMPORTANCE Nanoparticles can penetrate into the outer layer of biofilm in a relatively short period and can bind onto EPS and bacterial surfaces. The current work probed the effects of nanosized TiO2 (n-TiO2) on biofilm thickness, bacterial migration, and surface properties of the cell in the early stage using the surface plasmon resonance waveguide mode. The results demonstrated that n-TiO2 decreased the adhesive ability of both cell and EPS and induced bacterial migration and biofilm detachment in several hours. The decreased adhesive ability of microbes and EPS worked against microbial aggregation, reducing the effluent quality in the biological wastewater treatment process.
研究领域:
环境科学
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微生物学
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DOI
PubMed
高影响因子
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
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2022
·Vol. 88
·期 6
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IF 5.005
Zihao Liu,Kun Wang,Xuemei Nan,Meng Cai,Liang Yang,Benhai Xiong,Yiguang Zhao
3-Nitrooxypropanol (3-NOP) is effective at reducing ruminal methane emissions in ruminants. But it also causes a drastic increase in hydrogen accumulation, resulting in feed energy waste. Fumarate is a key precursor for propionate formation and plays an important role in rumen hydrogen metabolism. Therefore, this study examined the effects of 3-NOP combined with fumarate on volatile fatty acids, methanogenesis, and microbial community structures in dairy cows in vitro. The in vitro culture experiment was performed using a 2-by-2 factorial design, two 3-NOP levels (0 or 2 mg/g dry matter [DM]) and two fumarate levels (0 or 100 mg/g DM), including 3 runs with 4 treatments, 4 replicates, and 4 blanks containing only the inoculum. Rumen fluid was collected from three lactating Holstein cows with permanent ruminal fistulas. The combination of 3-NOP and fumarate reduced methane emissions by 11.48% without affecting dry matter degradability. The propionate concentration increased and the acetate/propionate ratio decreased significantly. In terms of bacteria, the combination of 3-NOP and fumarate reduced the abundances of Ruminococcus and Lachnospiraceae_NK3A20_group and increased the abundances of Prevotella and Succiniclasticum. For archaea, the combination of 3-NOP and fumarate significantly increased the abundances of Methanobrevibacter_sp._AbM4, while the abundance of operational taxonomic unit 581 (OTU581) (belonging to an uncultured_rumen_methanogen_g__Methanobrevibacter strain) was significantly decreased. These results indicated that the combination of 3-NOP and fumarate could alleviate the accumulation of hydrogen and enhance the inhibition of methanogenesis compared with 3-NOP only in dairy cows.IMPORTANCE The global problem of climate change and the greenhouse effect has become increasingly severe, and the abatement of greenhouse gases has received great attention from the international community. Methane produced by ruminants during digestion not only aggravates the greenhouse effect but also causes a waste of feed energy. As a methane inhibitor, 3-nitrooxypropanol can effectively reduce methane emissions from ruminants. However, when it inhibits methane emissions, the emission of hydrogen increases sharply, resulting in the waste of feed resources. Fumarate is a propionic acid precursor that can promote the metabolism of hydrogen to propionic acid in animals. Therefore, we studied the effects of the combined addition of 3-nitrooxypropanol and fumarate on methanogenesis, rumen fermentation, and rumen flora. It is of great significance to inhibit methane emission from ruminants and slow down the greenhouse effect.
研究领域:
动物科学
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微生物学
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环境科学
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营养学
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DOI
PubMed
高影响因子
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
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2022
·Vol. 88
·期 6
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IF 5.005
Yuan Gong,Jia-Qi Liu,Ming-Jie Xu,Chun-Mei Zhang,Jun Gao,Cheng-Guo Li,Ke Xing,Sheng Qin
Volatile organic compounds (VOCs) produced by microorganisms are considered promising environmental-safety fumigants for controlling postharvest diseases. Ceratocystis fimbriata, the pathogen of black spot disease, seriously affects the quality and yield of sweet potato in the field and postharvest. This study tested the effects of VOCs produced by Streptomyces setonii WY228 on the control of C. fimbriata in vitro and in vivo. The VOCs exhibited strong antifungal activity and significantly inhibited the growth of C. fimbriata. During the 20-day storage, VOC fumigation significantly controlled the occurrence of the pathogen, increased the content of antioxidants and defense-related enzymes and flavonoids, and boosted the starch content so as to maintain the quality of the sweet potatoes. Headspace analysis showed that the volatiles 2-ethyl-5-methylpyrazine and dimethyl disulfide significantly inhibited the mycelial growth and spore germination of C. fimbriata in a dose-dependent manner. Fumigation with 100 μL/L 2-ethyl-5-methylpyrazine completely controlled the pathogen in vivo after 10 days of storage. Transcriptome analysis showed that volatiles mainly downregulated the ribosomal synthesis genes and activated the proteasome system of the pathogen in response to VOC stress, while the genes related to spore development, cell membrane synthesis, mitochondrial function, and hydrolase and toxin synthesis were also downregulated, indicating that WY228-produced VOCs have diverse modes of action for pathogen control. Our study demonstrates that fumigation of sweet potato tuberous roots with S. setonii WY228 or use of formulations based on the VOCs is a promising new strategy to control sweet potato and other food and fruit pathogens during storage and shipment.IMPORTANCE Black spot disease caused by Ceratocystis fimbriata has caused huge economic losses to worldwide sweet potato production. At present, the control of C. fimbriata mainly depends on toxic fungicides, and there is a lack of effective alternative strategies. The research on biological control of sweet potato black spot disease is also very limited. An efficient biocontrol technique against pathogens using microbial volatile organic compounds could be an alternative method to control this disease. Our study revealed the significant biological control effect of volatile organic compounds of Streptomyces setonii WY228 on black spot disease of postharvest sweet potato and the complex antifungal mechanism against C. fimbriata. Our data demonstrated that Streptomyces setonii WY228 and its volatile 2-ethyl-5-methylpyrazine could be a candidate strain and compound for the creation of fumigants and showed the important potential of biotechnology applications in the field of food and agriculture.
研究领域:
微生物学
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植物病理学
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食品科学
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农业生物技术
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DOI
PubMed
高影响因子
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
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2017
·Vol. 83
·期 10
·
IF 3.807
Lei Yu,Ming-yue Cao,Peng-tao Wang,Shi Wang,Ying-rong Yue,Wen-duo Yuan,Wei-chuan Qiao,Fei Wang,Xin Song
Biohydrogen production from the pulp and paper effluent containing rich lignocellulosic material could be achieved by the fermentation process. Xylose, an important hemicellulose hydrolysis product, is used less efficiently as a substrate for biohydrogen production. Moreover, azo dyes are usually added to fabricate anticounterfeiting paper, which further increases the complexity of wastewater. This study reports that xylose could serve as the sole carbon source for a pure culture of Klebsiella oxytoca GS-4-08 to achieve simultaneous decolorization and biohydrogen production. With 2 g liter−1 of xylose as the substrate, a maximum xylose utilization rate (URxyl) and a hydrogen molar yield (HMY) of 93.99% and 0.259 mol of H2 mol of xylose−1, respectively, were obtained. Biohydrogen kinetics and electron equivalent (e− equiv) balance calculations indicated that methyl red (MR) penetrates and intracellularly inhibits both the pentose phosphate pathway and pyruvate fermentation pathway, while methyl orange (MO) acted independently of the glycolysis and biohydrogen pathway. The data demonstrate that biohydrogen pathways in the presence of azo dyes with sulfonate and carboxyl groups were different, but the azo dyes could be completely reduced during the biohydrogen production period in the presence of MO or MR. The feasibility of hydrogen production from industrial pulp and paper effluent by the strain if the xylose is sufficient was also proved and was not affected by toxic substances which usually exist in such wastewater, except for chlorophenol. This study offers a promising energy-recycling strategy for treating pulp and paper wastewaters, especially for those containing azo dyes.IMPORTANCE The pulp and paper industry is a major industry in many developing countries, and the global market of pulp and paper wastewater treatment is expected to increase by 60% between 2012 and 2020. Such wastewater contains large amounts of refractory contaminants, such as lignin, whose reclamation is considered economically crucial and environmentally friendly. Furthermore, azo dyes are usually added in order to fabricate anticounterfeiting paper, which further increases the complexity of the pulp and paper wastewater. This work may offer a better understanding of biohydrogen production from xylose in the presence of azo dyes and provide a promising energy-recycling method for treating pulp and paper wastewater, especially for those containing azo dyes.
研究领域:
环境科学
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微生物学
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产品关联
DOI
PubMed
高影响因子
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
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2017
·Vol. 83
·期 15
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IF 3.807
Erquan Chen,Qi Chen,Shaoming Chen,Bing Xu,Jianhua Ju,Huan Wang
Lantibiotics are antimicrobial peptides belonging to the family of ribosomally synthesized and posttranslationally modified peptides (RiPPs) and feature thioether linkages in their structures. In this study, we identified the biosynthetic gene cluster of a cinnamycin analog, named mathermycin, from Marinactinospora thermotolerans SCSIO 00652 and reconstituted its biosynthesis in Streptomyces lividans and Escherichia coli. Key posttranslational modification enzymes of mathermycin were characterized. Mathermycin exhibited antimicrobial activity and therefore represents an example of cinnamycin-like lantibiotics from Marinactinospora species.IMPORTANCE The discovery of new antimicrobial compounds that can be used as potential drugs is in urgent need due to increasing bacterial resistance to current antibiotics. Lantibiotics are important antimicrobial compounds that have found applications in both the clinic setting and food industry. We report here the discovery of a new lantibiotic, mathermycin, from a marine-derived Marinactinospora thermotolerans strain and elucidation of its biosynthesis. We also demonstrate that mathermycin possesses antimicrobial activity toward a Bacillus strain.
研究领域:
微生物学
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药物化学
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分子生物学
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产品关联
DOI
PubMed
高影响因子
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
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2017
·
IF 3.807
Wenjun Yu,Rongshui Wang,Haiyan Huang,Huijun Xie,Shuning Wang
Agrobacterium tumefaciens S33 can grow with nicotine as the sole source of carbon, nitrogen, and energy via a novel hybrid of the pyridine pathway and the pyrrolidine pathway. Characterization of the enzymes involved in the hybrid pathway is important for understanding its biochemical mechanism. Here, we report that the molybdenum-containing nicotine dehydrogenase (NdhAB), which catalyzes the initial step of nicotine degradation, is located in the periplasm of strain S33, while the 6-hydroxynicotine oxidase and 6-hydroxypseudooxynicoine oxidase are in the cytoplasm. This is consistent with the fact that NdhA has a Tat signal peptide. Interestingly, an open reading frame (ORF) adjacent to the ndhAB gene was verified to encode a copper-containing electron carrier, pseudoazurin (Paz), which has a signal peptide typical of bacterial Paz proteins. Both were transported into the periplasm after being produced in the cytoplasm. We purified NdhAB from the periplasmic fraction of strain S33 and found that with Paz as the physiological electron acceptor, NdhAB catalyzed the hydroxylation of nicotine at a specific rate of 110.52 ± 8.09 μmol · min−1 · mg of protein−1, where the oxygen atom in the hydroxyl group of the product 6-hydroxynicotine was derived from H2O. The apparent Km values for nicotine and Paz were 1.64 ± 0.07 μM and 3.61 ± 0.23 μM, respectively. NAD(P)+, O2, and ferredoxin could not serve as electron acceptors. Disruption of the paz gene disabled the strain for nicotine degradation, indicating that Paz is required for nicotine catabolism in the strain. These findings help our understanding of electron transfer during nicotine degradation in bacteria.IMPORTANCE Nicotine is a toxic and addictive N-heterocyclic aromatic alkaloid produced in tobacco. Its catabolism in organisms and degradation in tobacco wastes have become major concerns for human health and the environment. Bacteria usually decompose nicotine using the classical strategy of hydroxylating the pyridine ring with the help of activated oxygen by nicotine dehydrogenase, which binds one molybdopterin, two [2Fe2S] clusters, and usually one flavin adenine dinucleotide (FAD) as well. However, the physiological electron acceptor for the reaction is still unknown. In this study, we found that the two-component nicotine dehydrogenase from Agrobacterium tumefaciens S33, naturally lacking an FAD-binding domain, is located in the periplasmic space and uses a copper-containing electron carrier, pseudoazurin, as its physiological electron acceptor. We report here the role of pseudoazurin in a reaction catalyzed by a molybdopterin-containing hydroxylase occurring in the periplasmic space. These results provide new biochemical knowledge on microbial degradation of N-heterocyclic aromatic compounds.
研究领域:
微生物学
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生物化学
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环境科学
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分子生物学
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DOI
PubMed
高影响因子
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
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2014
·Vol. 80
·期 8
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IF 3.952
Kundi Zhang,Xiaohua Chen,Wolfgang H. Schwarz,Fuli Li
Two cellulolytic thermophilic bacterial strains, CS-3-2 and CS-4-4, were isolated from decayed cornstalk by the addition of growth-supporting factors to the medium. According to 16S rRNA gene-sequencing results, these strains belonged to the genus Clostridium and showed 98.87% and 98.86% identity with Clostridium stercorarium subsp. leptospartum ATCC 35414T and Clostridium cellulosi AS 1.1777T, respectively. The endoglucanase and exoglucanase activities of strain CS-4-4 were approximately 3 to 5 times those of strain CS-3-2, whereas the β-glucosidase activity of strain CS-3-2 was 18 times higher than that of strain CS-4-4. The xylanase activity of strain CS-3-2 was 9 times that of strain CS-4-4, whereas the β-xylosidase activity of strain CS-4-4 was 27 times that of strain CS-3-2. The enzyme activities in spent cultures following cocultivation of the two strains with cornstalk as the substrate were much greater than those in pure cultures or an artificial mixture of samples, indicating synergism of glycoside hydrolase secretomes between the two strains. Quantitative measurement of the two strains in the cocultivation system indicated that strain CS-3-2 grew robustly during the initial stages, whereas strain CS-4-4 dominated the system in the late-exponential phase. Liquid chromatography-tandem mass spectrometry analysis of protein bands appearing in the native zymograms showed that ORF3880 and ORF3883 from strain CS-4-4 played key roles in the lignocellulose degradation process. Both these open reading frames (ORFs) exhibited endoglucanase and xylanase activities, but ORF3880 showed tighter adhesion to insoluble substrates at 4, 25, and 60°C owing to its five carbohydrate-binding modules (CBMs).
研究领域:
微生物学
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生物化学
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DOI
PubMed
高影响因子
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
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2015
·Vol. 81
·期 6
·
IF 3.668
Zhoukun Li,Jiale Wu,Biying Zhang,Fei Wang,Xianfeng Ye,Yan Huang,Qiang Huang,Zhongli Cui
A novel α-amylase, AmyM, was purified from the culture supernatant of Corallococcus sp. strain EGB. AmyM is a maltohexaose-forming exoamylase with an apparent molecular mass of 43 kDa. Based on the results of matrix-assisted laser desorption ionization–time of flight mass spectrometry and peptide mass fingerprinting of AmyM and by comparison to the genome sequence of Corallococcus coralloides DSM 2259, the AmyM gene was identified and cloned into Escherichia coli. amyM encodes a secretory amylase with a predicted signal peptide of 23 amino acid residues, which showed no significant identity with known and functionally verified amylases. amyM was expressed in E. coli BL21(DE3) cells with a hexahistidine tag. The signal peptide efficiently induced the secretion of mature AmyM in E. coli. Recombinant AmyM (rAmyM) was purified by Ni-nitrilotriacetic acid (NTA) affinity chromatography, with a specific activity of up to 14,000 U/mg. rAmyM was optimally active at 50°C in Tris-HCl buffer (50 mM; pH 7.0) and stable at temperatures of <50°C. rAmyM was stable over a wide range of pH values (from pH 5.0 to 10.0) and highly tolerant to high concentrations of salts, detergents, and various organic solvents. Its activity toward starch was independent of calcium ions. The Km and V max of recombinant AmyM for soluble starch were 6.61 mg ml−1 and 44,301.5 μmol min−1 mg−1, respectively. End product analysis showed that maltohexaose accounted for 59.4% of the maltooligosaccharides produced. These characteristics indicate that AmyM has great potential in industrial applications.
研究领域:
生物化学
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酶学
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微生物学
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DOI
PubMed
高影响因子
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
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2018
·Vol. 84
·期 13
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IF 3.633
Ya-Hui Shao,Li-Zhong Guo,Yu-Qing Zhang,Hao Yu,Bai-Suo Zhao,Hai-Qiang Pang,Wei-Dong Lu
Although some bacteria, including Chromohalobacter salexigens DSM 3043, can use glycine betaine (GB) as a sole source of carbon and energy, little information is available about the genes and their encoded proteins involved in the initial step of the GB degradation pathway. In the present study, the results of conserved domain analysis, construction of in-frame deletion mutants, and an in vivo functional complementation assay suggested that the open reading frames Csal_1004 and Csal_1005, designated bmoA and bmoB, respectively, may act as the terminal oxygenase and the ferredoxin reductase genes in a novel Rieske-type oxygenase system to convert GB to dimethylglycine in C. salexigens DSM 3043. To further verify their function, BmoA and BmoB were heterologously overexpressed in Escherichia coli, and 13C nuclear magnetic resonance analysis revealed that dimethylglycine was accumulated in E. coli BL21(DE3) expressing BmoAB or BmoA. In addition, His-tagged BmoA and BmoB were individually purified to electrophoretic homogeneity and estimated to be a homotrimer and a monomer, respectively. In vitro biochemical analysis indicated that BmoB is an NADH-dependent flavin reductase with one noncovalently bound flavin adenine dinucleotide (FAD) as its prosthetic group. In the presence of BmoB, NADH, and flavin, BmoA could aerobically degrade GB to dimethylglycine with the concomitant production of formaldehyde. BmoA exhibited strict substrate specificity for GB, and its demethylation activity was stimulated by Fe2+. Phylogenetic analysis showed that BmoA belongs to group V of the Rieske nonheme iron oxygenase (RO) family, and all the members in this group were able to use quaternary ammonium compounds as substrates.IMPORTANCE GB is widely distributed in nature. In addition to being accumulated intracellularly as a compatible solute to deal with osmotic stress, it can be utilized by many bacteria as a source of carbon and energy. However, very limited knowledge is presently available about the molecular and biochemical mechanisms for the initial step of the aerobic GB degradation pathway in bacteria. Here, we report the molecular and biochemical characterization of a novel two-component Rieske-type monooxygenase system, GB monooxygenase (BMO), which is responsible for oxidative demethylation of GB to dimethylglycine in C. salexigens DSM 3043. The results gained in this study extend our knowledge on the catalytic reaction of microbial GB degradation to dimethylglycine.
研究领域:
微生物学
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生物化学
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产品关联
DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）