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Kyong-Cheol Ko Ju Hee LeeJong Hyun Choi Jae Jun Song 《Biochemical and biophysical research communications》2013
Metagenomic resources representing ruminal bacteria were screened for novel exocellulases using a robotic, high-throughput screening system, the novel CelEx-BR12 gene was identified and the predicted CelEx-BR12 protein was characterized. The CelEx-BR12 gene had an open reading frame (ORF) of 1140 base pairs that encoded a 380-amino-acid-protein with a predicted molecular mass of 41.8 kDa. The amino acid sequence was 83% identical to that of a family 5 glycosyl hydrolase from Prevotella ruminicola 23. Codon-optimized CelEx-BR12 was overexpressed in Escherichia coli and purified using Ni–NTA affinity chromatography. The Michaelis–Menten constant (Km value) and maximal reaction velocity (Vmax values) for exocellulase activity were 12.92 μM and 1.55 × 10−4 μmol min−1, respectively, and the enzyme was optimally active at pH 5.0 and 37 °C. Multifunctional activities were observed against fluorogenic and natural glycosides, such as 4-methylumbelliferyl-β-d-cellobioside (0.3 U mg−1), CMC (105.9 U mg−1), birch wood xylan (132.3 U mg−1), oat spelt xylan (67.9 U mg−1), and 2-hydroxyethyl-cellulose (26.3 U mg−1). Based on these findings, we believe that CelEx-BR12 is an efficient multifunctional enzyme as endocellulase/exocellulase/xylanase activities that may prove useful for biotechnological applications. 相似文献
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《Microbes and infection / Institut Pasteur》2022,24(2):104892
Gut microbiota is believed to play a crucial role in obesity. However, the consistent findings among published studies regarding microbiome–obesity interaction are relatively rare, and one of the underlying causes could be the limited sample size of cohort studies. In order to identify gut microbiota changes between normal-weight individuals and obese individuals, fecal samples along with phenotype information from 2262 Chinese individuals were collected and analyzed. Compared with normal-weight individuals, the obese individuals exhibit lower diversity of species and higher diversity of metabolic pathways. In addition, various machine learning models were employed to quantify the relationship between obesity status and Body mass index (BMI) values, of which support vector machine model achieves best performance with 0.716 classification accuracy and 0.485 R2 score. In addition to two well-established obesity-associated species, three species that have potential to be obesity-related biomarkers, including Bacteroides caccae, Odoribacter splanchnicus and Roseburia hominis were identified. Further analyses of functional pathways also reveal some enriched pathways in obese individuals. Collectively, our data demonstrates tight relationship between obesity and gut microbiota in a large-scale Chinese population. These findings may provide potential targets for the prevention and treatment of obesity. 相似文献
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《Systematic and applied microbiology》2019,42(1):67-76
Candidate bacterial phylum BRC1 has been identified in a broad range of mostly organic-rich oxic and anoxic environments through molecular analysis of microbial communities. None of the members of BRC1 have been cultivated and only a few draft genome sequences have been obtained from metagenomes or as a result of single-cell sequencing. We have reconstructed complete genome of BRC1 bacterium, BY40, from metagenome of the microbial community of a deep subsurface thermal aquifer in the Tomsk Region of the Western Siberia, Russia, and used it for metabolic reconstruction and comparison with existing genomic data. Analysis of 3.3 Mb genome of BY40 bacterium revealed numerous glycoside hydrolases that could enable utilization of carbohydrates, including enzymes of chitin-degradation pathway. The bacterium lacks flagellar machinery but the twitching motility is encoded. The reconstructed central metabolism revealed pathways enabling the fermentation of organic substrates, as well as their complete oxidation through aerobic and anaerobic respiration. Phylogenetic analysis using BY40 genome supported the phylum level classification of BRC1 lineage. Based on phylogenetic and genomic analyses, the novel bacterium is proposed to be classified as Candidatus Sumerlaea chitinivorans, within a candidate phylum Sumerlaeota. 相似文献
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Lee DG Jeon JH Jang MK Kim NY Lee JH Lee JH Kim SJ Kim GD Lee SH 《Biotechnology letters》2007,29(3):465-472
A metagenomic library was constructed using total genomic DNA extracted from the mud in the west coast of Korea and was used
together with a fosmid vector, pCC1FOS in order to uncover novel gene sources. One clone from approximately 30,000 recombinant
Escherichia coli clones was identified that showed proteolytic activity. The gene for the proteolytic enzyme was subcloned into pUC19 and
sequenced, and a database search for homologies revealed it to be a zinc-dependent metalloprotease. The cloned gene included
the intact coding gene for a novel metalloproteinase and its own promoter. It comprised an open reading frame of 1,080 base
pairs, which encodes a protein of 39,490 Da consisting of 359 amino acid residues. A His-Glu-X-X-His sequence, which is a
conserved sequence in the active site of zinc-dependent metalloproteases, was found in the deduced amino acid sequence of
the gene, suggesting that the enzyme is a zinc-dependent metalloprotease. The purified enzyme showed optimal activity at 50°C
for 1 h and pH 7.0. The enzyme activity was inhibited by metal-chelating reagents, such as EDTA, EGTA and 1,10-phenanthroline.
The enzyme hydrolyzed azocasein as well as fibrin. Thus, the enzyme could be useful as a therapeutic agent to treat thrombosis.
The sequence reported in this paper has been deposited in the GenBank database (Accession number: EF100137). 相似文献
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Novel bacterial sulfur oxygenase reductases from bioreactors treating gold-bearing concentrates 总被引:1,自引:0,他引:1
Chen ZW Liu YY Wu JF She Q Jiang CY Liu SJ 《Applied microbiology and biotechnology》2007,74(3):688-698
The microbial community and sulfur oxygenase reductases of metagenomic DNA from bioreactors treating gold-bearing concentrates
were studied by 16S rRNA library, real-time polymerase chain reaction (RT-PCR), conventional cultivation, and molecular cloning.
Results indicated that major bacterial species were belonging to the genera Acidithiobacillus, Leptospirillum, Sulfobacillus, and Sphingomonas, accounting for 6.3, 66.7, 18.8, and 8.3%, respectively; the sole archaeal species was Ferroplasma sp. (100%). Quantitative RT-PCR revealed that the 16S rRNA gene copy numbers (per gram of concentrates) of bacteria and archaea
were 4.59 × 109 and 6.68 × 105, respectively. Bacterial strains representing Acidithiobacillus, Leptospirillum, and Sulfobacillus were isolated from the bioreactors. To study sulfur oxidation in the reactors, pairs of new PCR primers were designed for
the detection of sulfur oxygenase reductase (SOR) genes. Three sor-like genes, namely, sor
Fx, sor
SA, and sor
SB were identified from metagenomic DNAs of the bioreactors. The sor
Fx is an inactivated SOR gene and is identical to the pseudo-SOR gene of Ferroplasma acidarmanus. The sor
SA and sor
SB showed no significant identity to any genes in GenBank databases. The sor
SB was cloned and expressed in Escherichia
coli, and SOR activity was determined. Quantitative RT-PCR determination of the gene densities of sor
SA and sor
SB were 1,000 times higher than archaeal 16S rRNA gene copy numbers, indicating that these genes were mostly impossible from
archaea. Furthermore, with primers specific to the sor
SB gene, this gene was PCR-amplified from the newly isolated Acidithiobacillus sp. strain SM-1. So far as we know, this is the first time to determine SOR activity originating from bacteria and to document
SOR gene in bioleaching reactors and Acidithiobacillus species. 相似文献
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Background
Horizontal gene transfer (HGT) has been widely identified in complete prokaryotic genomes. However, the roles of HGT among members of a microbial community and in evolution remain largely unknown. With the emergence of metagenomics, it is nontrivial to investigate such horizontal flow of genetic materials among members in a microbial community from the natural environment. Because of the lack of suitable methods for metagenomics gene transfer detection, microorganisms from a low-complexity community acid mine drainage (AMD) with near-complete genomes were used to detect possible gene transfer events and suggest the biological significance.Results
Using the annotation of coding regions by the current tools, a phylogenetic approach, and an approximately unbiased test, we found that HGTs in AMD organisms are not rare, and we predicted 119 putative transferred genes. Among them, 14 HGT events were determined to be transfer events among the AMD members. Further analysis of the 14 transferred genes revealed that the HGT events affected the functional evolution of archaea or bacteria in AMD, and it probably shaped the community structure, such as the dominance of G-plasma in archaea in AMD through HGT.Conclusions
Our study provides a novel insight into HGT events among microorganisms in natural communities. The interconnectedness between HGT and community evolution is essential to understand microbial community formation and development.Electronic supplementary material
The online version of this article (doi:10.1186/s12864-015-1720-0) contains supplementary material, which is available to authorized users. 相似文献9.
采用传统分离培养筛选微生物新活性物质的方法受到很大制约,自然界99%以上的微生物不能培养,其资源开发受到很大限制。环境微生物宏基因组技术应用避开了微生物分离纯培养问题,极大拓展了微生物资源的利用空间,增加获得新活性物质的机会和途径。本文着重介绍宏基因组的概念、研究策略包括DNA提取、文库构建与筛选等及在微生物活性物质筛选中的应用,并对宏基因组研究中存在的问题进行探讨。 相似文献
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【目的】通过建立宏基因组文库的高通量保存与基于探针洗脱的多次膜杂交筛选方法,从植物共生菌宏基因组文库筛选具有生物催化潜力的新酶基因。【方法】首先根据滴度将初始文库噬菌体包装颗粒感染到EPI300-T1R E.coli,过夜培养后对应保存于96孔板;提取粘粒进行文库的杂交筛选。【结果】描述的洗脱条件可完全去除尼龙膜上与靶DNA结合的探针,并且尼龙膜上的靶DNA至少可用于7次探针杂交,从而明显提高宏基因组文库的筛选效率。【结论】以Enoate reductase(ER)和短链脱氢酶(SDR)的同源基因片段为探针,运用该方法经两轮筛选获得候选单克隆并进行了部分粘粒的测序,发现了新的ER和SDR同源基因,并克隆到相应的全长基因序列用于后续的表达与酶化学研究。 相似文献