A 1602 bp fragment was cloned from a soil bacterium Ochrobactrum sp. 531. It contained an open reading frame (ORF) of 1092 bp which was identified as a multicopper oxidase (MCO) with potential laccase activity. After ...A 1602 bp fragment was cloned from a soil bacterium Ochrobactrum sp. 531. It contained an open reading frame (ORF) of 1092 bp which was identified as a multicopper oxidase (MCO) with potential laccase activity. After inserting the cloned gene into the expression vector pET23a, it was expressed in E. coli BL21(DE3)pLysS, and its product was purified to homogeneity through chromatography. The Ochrobactrum sp. 531 MCO, consisting of 533 amino acids with a molecular mass of 57.8 kDa, was quite stable in neutral pH and showed laccase-like activity oxidizing 2,6-dimethoxyphenol (DMP), 2,2’-azino-bis(3-ethylbe- nzthiazolinesulfonic acid) (ABTS), and syringaldazine (SGZ). The enzyme showed optimum activity towards DMP, ABTS, and SGZ at the pH 8.0, 3.6, and 7.5 respectively. Kinetic studies gave this enzyme Km, kcat and kcat//Km values of: 0.09 mM, 7.94 s–1, and 88.22 s–1?mM–1 for DMP;0.072 mM, 2.95 s–1, and 40.97 s–1.mM–1 for ABTS;and 0.015 mM, 2.4 s–1, and 160 s–1.mM–1 for SGZ. Our results demonstrate that Ochrobactrum sp. 531 MCO is a bacterial laccase which oxidized phenolic substrates DMP and SGZ effectively under alkaline conditions. These unusual properties make the enzyme an interesting biocatalyst in applications for which classical laccases are unsuitable.展开更多
A bacterial strain BAP5 with a relatively high degradation ability of benzo[a]pyrene （BaP） was isolated from marine sediments of Xiamen Western Sea, China and identified as Ochrobactrum sp. according to 16S rRNA gen...A bacterial strain BAP5 with a relatively high degradation ability of benzo[a]pyrene （BaP） was isolated from marine sediments of Xiamen Western Sea, China and identified as Ochrobactrum sp. according to 16S rRNA gene sequence as well as Biolog microbial identification system. Strain BAP5 could grow in mineral salt medium with 50 mg/L of BaP and degrade about 20% BaP after 30 d of incubation. Ochrobactrum sp. BAP5 was able to utilize other polycyclic aromatic hydrocarbons （PAHs） （such as phenanthrene, pyrene and fluoranthene） as the sole carbon source and energy source, suggesting its potential application in PAHs bioremediation. The profile of total soluble protein from Ochrobactrum sp. BAP5 was also investigated. Some over- and special-expressed proteins of strain BAP5 when incubated with the presence of BaP were detected by two-dimensional polyacrylamide gel electrophoresis, and found to be related with PAHs metabolism, DNA translation, and energy production based on peptide fingerprint analysis through matrix-assisted laser desorption/ionization-time of flight mass spectrometry.展开更多
麦草畏是理想的抗除草剂转基因工程的靶标除草剂;发掘新的麦草畏高效降解菌株和基因具有非常重要的理论和应用价值.从南京土壤样品中分离到一株麦草畏高效降解菌株,命名为3-3.根据生理生化特征和16S r DNA序列相似性分析,将其初步鉴定...麦草畏是理想的抗除草剂转基因工程的靶标除草剂;发掘新的麦草畏高效降解菌株和基因具有非常重要的理论和应用价值.从南京土壤样品中分离到一株麦草畏高效降解菌株,命名为3-3.根据生理生化特征和16S r DNA序列相似性分析,将其初步鉴定为苍白杆菌属(Ochrobactrum sp.).菌株3-3在48 h内完全降解100 mg/L的麦草畏.该菌株降解麦草畏的最适温度为30℃,最适p H为7.0.代谢产物高效液相和质谱鉴定结果表明该菌株降解麦草畏的起始步骤是脱甲基,形成没有除草活性的3,6-二氯水杨酸(DCSA).菌株粗酶液只在NADH存在时才有麦草畏脱甲基酶活性.PCR扩增和该菌基因组生物信息学分析表明该菌株没有已报道的麦草畏脱甲基酶基因DMO、Mtv及Dmt或其同源序列.总之,本研究首次分离筛选到苍白杆菌属的麦草畏降解菌,且该菌可能存在一个新的氧化酶类麦草畏脱甲基酶基因.展开更多
A bacterial strain,designated as LS,was isolated from a contaminated soil and was found to be capable of utilizing quinclorac,bensulfuronmethyl,and a mixture of the two as carbon and energy sources for growth. Strain ...A bacterial strain,designated as LS,was isolated from a contaminated soil and was found to be capable of utilizing quinclorac,bensulfuronmethyl,and a mixture of the two as carbon and energy sources for growth. Strain LS was identified as Ochrobactrum sp. based on its physiological-biochemical properties,16S rDNA sequences,and phylogenetic analysis. The extent of degradation of quinclorac and bensulfuronmethyl at initial concentrations of 1.5 and 0.1 g L-1 was 90% and 67%,respectively,as measured by high performance liquid chromatography(HPLC) . When a herbicide mixture of 0.34 g L-1 quinclorac and 0.02 g L-1 bensulfuronmethyl was applied as carbon sources,quinclorac and bensulfuronmethyl were degraded at 95.7% and 67.5%,respectively. It appears that quinclorac is utilized more easily in a mixture than in a single state. The optimal temperature for growth of strain LS was 37 ℃. Strain LS grew well at pH 6 to 9 and had the highest degradation level for quinclorac and bensulfuronmethyl at an initial pH of 7 and 8,respectively. Addition of 0.25 g L-1 yeast extract could promote the growth and extent of degradation of quinclorac and bensulfuronmethyl by strain LS. Strain LS also showed the capability to degrade other aromatic compounds such as catechol,propisochlor,4-chloro-2-methylphenoxyacetic acid sodium(MCPA-Na) and imazethapy. The isolate LS shows a huge potential to be used in bioremediation for treating complex herbicide residues.展开更多
文摘A 1602 bp fragment was cloned from a soil bacterium Ochrobactrum sp. 531. It contained an open reading frame (ORF) of 1092 bp which was identified as a multicopper oxidase (MCO) with potential laccase activity. After inserting the cloned gene into the expression vector pET23a, it was expressed in E. coli BL21(DE3)pLysS, and its product was purified to homogeneity through chromatography. The Ochrobactrum sp. 531 MCO, consisting of 533 amino acids with a molecular mass of 57.8 kDa, was quite stable in neutral pH and showed laccase-like activity oxidizing 2,6-dimethoxyphenol (DMP), 2,2’-azino-bis(3-ethylbe- nzthiazolinesulfonic acid) (ABTS), and syringaldazine (SGZ). The enzyme showed optimum activity towards DMP, ABTS, and SGZ at the pH 8.0, 3.6, and 7.5 respectively. Kinetic studies gave this enzyme Km, kcat and kcat//Km values of: 0.09 mM, 7.94 s–1, and 88.22 s–1?mM–1 for DMP;0.072 mM, 2.95 s–1, and 40.97 s–1.mM–1 for ABTS;and 0.015 mM, 2.4 s–1, and 160 s–1.mM–1 for SGZ. Our results demonstrate that Ochrobactrum sp. 531 MCO is a bacterial laccase which oxidized phenolic substrates DMP and SGZ effectively under alkaline conditions. These unusual properties make the enzyme an interesting biocatalyst in applications for which classical laccases are unsuitable.
基金supported by the National Natural Sci-ence Foundation of China (No 40206015, 30970106)the Fork Ying Tong Education Foundation (No 94002) the Science and Technology Project of Guangdong Province,China (No 2006A36502001, 2007A032600003)
文摘A bacterial strain BAP5 with a relatively high degradation ability of benzo[a]pyrene （BaP） was isolated from marine sediments of Xiamen Western Sea, China and identified as Ochrobactrum sp. according to 16S rRNA gene sequence as well as Biolog microbial identification system. Strain BAP5 could grow in mineral salt medium with 50 mg/L of BaP and degrade about 20% BaP after 30 d of incubation. Ochrobactrum sp. BAP5 was able to utilize other polycyclic aromatic hydrocarbons （PAHs） （such as phenanthrene, pyrene and fluoranthene） as the sole carbon source and energy source, suggesting its potential application in PAHs bioremediation. The profile of total soluble protein from Ochrobactrum sp. BAP5 was also investigated. Some over- and special-expressed proteins of strain BAP5 when incubated with the presence of BaP were detected by two-dimensional polyacrylamide gel electrophoresis, and found to be related with PAHs metabolism, DNA translation, and energy production based on peptide fingerprint analysis through matrix-assisted laser desorption/ionization-time of flight mass spectrometry.
基金the National Natural Science Foundation of China (Nos.40501037 and 30570053)the National Key Technologies Research and Development Program of China during the 11th Five-Year Plan Period(No.2006BAJ08B01).
文摘A bacterial strain,designated as LS,was isolated from a contaminated soil and was found to be capable of utilizing quinclorac,bensulfuronmethyl,and a mixture of the two as carbon and energy sources for growth. Strain LS was identified as Ochrobactrum sp. based on its physiological-biochemical properties,16S rDNA sequences,and phylogenetic analysis. The extent of degradation of quinclorac and bensulfuronmethyl at initial concentrations of 1.5 and 0.1 g L-1 was 90% and 67%,respectively,as measured by high performance liquid chromatography(HPLC) . When a herbicide mixture of 0.34 g L-1 quinclorac and 0.02 g L-1 bensulfuronmethyl was applied as carbon sources,quinclorac and bensulfuronmethyl were degraded at 95.7% and 67.5%,respectively. It appears that quinclorac is utilized more easily in a mixture than in a single state. The optimal temperature for growth of strain LS was 37 ℃. Strain LS grew well at pH 6 to 9 and had the highest degradation level for quinclorac and bensulfuronmethyl at an initial pH of 7 and 8,respectively. Addition of 0.25 g L-1 yeast extract could promote the growth and extent of degradation of quinclorac and bensulfuronmethyl by strain LS. Strain LS also showed the capability to degrade other aromatic compounds such as catechol,propisochlor,4-chloro-2-methylphenoxyacetic acid sodium(MCPA-Na) and imazethapy. The isolate LS shows a huge potential to be used in bioremediation for treating complex herbicide residues.
