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ABSTRACT: The construction of a complex genomic library is one of the comprehensive ways to study a complex bacterial community and to access the variety of metabolic pathways present in the rich soil environment. In this report, we developed a new protocol whereby we are able to retrieve nearly complete microbe genomic fragments from soil samples, which are employed to generate a metagenomic library for visualizing the basic scaffolding of the soil microbial community. The use of direct cell lysis within soil-embedded agarose plugs, along with a double-size selection, enabled us to successfully isolate pure and high-molecular weight DNA (0.1-1 Mb) without the need for any further purification. A metagenomic library containing 1.2 Gbp of DNA in total was constructed. Furthermore, analysis of the microbial community structure using 16S rDNA partial sequences found the dominant phylotypes to consist of alpha-Proteobacteria and Actinobacteria, which are similar to those seen in forest and agricultural soils, and numerous uncultured microbes from a wide variety of bacterial taxa as well. In conclusion, this study presents a novel protocol for generating a metagenomic library that carries much larger and diverse DNA fragments from soil bacteria that will be applied for the reconstruction of soil microbial genomes and the discovery of novel habitat-specific pathways.
Journal of Microbiological Methods 03/2008; 72(2):197-205. · 2.09 Impact Factor
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ABSTRACT: An N-acyl-d-amino acid amidohydrolase (N-D-AAase) was identified in cell extracts of a strain, Iso1, isolated from an environment containing N-acetyl-d-methionine. The bacterium was classified as Variovorax paradoxus by phylogenetic analysis. The gene was cloned and sequenced. The gene consisted of a 1467-bp ORF encoding a polypeptide of 488 amino acids. The V. paradoxusN-D-AAase showed significant amino acid similarity to the N-acyl-d-amino acid amidohydrolases of the two eubacteria Alcaligenes xylosoxydans A-6 (44-56% identity), Alcaligenes facelis DA1 (54% identity) and the hyperthermophilic archaeon Pyrococcus abyssi (42% identity). After over-expression of the N-D-AAase protein in Escherichia coli, the enzyme was purified by multistep chromatography. The native molecular mass was 52.8 kDa, which agreed with the predicted molecular mass of 52 798 Da and the enzyme appeared to be a monomer protein by gel-filtration chromatography. A homogenous protein with a specific activity of 516 U.mg-1 was finally obtained. After peptide sequencing by LC/MS/MS, the results were in agreement with the deduced amino acid sequence of the N-D-AAase. The pI of the enzyme was 5.12 and it had an optimal pH and temperature of 7.5 and 50 degrees C, respectively. After 30 min heat treatment at 45 degrees C, between pH 6 and pH 8, 80% activity remained. The N-D-AAase had higher hydrolysing activity against N-acetyl-d-amino acid derivates containing d-methionine, d-leucine and d-alanine and against N-chloroacetyl-d-phenylalanine. Importantly, the enzyme does not act on the N-acetyl-l-amino acid derivatives. The enzyme was inhibited by chelating agents and certain metal ions, but was activated by 1 mm of Co2+ and Mg2+. Thus, the N-D-AAase from V. paradoxus can be considered a chiral specific and metal-dependent enzyme.
European Journal of Biochemistry 11/2002; 269(19):4868-78. · 3.58 Impact Factor
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ABSTRACT: A gene encoding N-acylamino acid racemase (NAAAR) from Amycolatopsis azurea CCRC13413 was cloned. An analysis of its sequence revealed an open reading frame encoding 368 amino acid residues. The deduced amino acid sequence of NAAAR showed an 89% identity with that of Amycolatopsis sp. TS-1–60. The N-acylamino acid racemase gene (aaar) was subcloned into expression vector pET-17b, and was transformed into E. coli BL21 (DE3). A 41-kDa protein band was present on the SDS-PAGE gel from the intracellular soluble part of E. coli. The recombinant NAAAR produced in E. coli was purified by Toyopearl DEAE-650M and Sephacryl S-200 h chromatography. The molecular weight of NAAAR, determined by gel-filtration chromatography, was 320 kDa, indicating that NAAAR was composed of eight identical subunits. NAAAR had maximal activity at 40°C and pH 7.4. The enzyme activity was enhanced notably by Co2+ and Mn2+. Substrate specificity revealed that N-acetyl-D-methionine, N-acetyl- L-methionine, N-acetyl-D-tyrosine, N-acetyl-L-valine, and N-chloroacetyl -D-phenylalanine were the effective substrates. For the substrate N- acetyl-D-methionine, Km was 27.8 mM with a Vmax of 0.07 μmol−1 min−1 and a kcat/Km of 311.4 M−1 s−1. Our data revealed that this N-acylamino acid racemase has a broad substrate range. In addition, we procured the distribution of sequence-conserved regions from known NAAARs and putative NAAARs in this study.
Enzyme and Microbial Technology.
