Molecular cloning and characterization of a thermostable esterase/lipase produced by a novel Anoxybacillus flavithermus strain.
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A thermophilic strain producing an extracellular esterase/lipase was isolated from a hot spring in Tasnad, Romania, and was identified phenotypically and by 16S rDNA sequencing as Anoxybacillus flavithermus (GenBank ID: JQ267733). The gene encoding the putative carboxyl esterase (GenBank ID: JX494348) was cloned by direct PCR amplification from genomic DNA. The protein, consisting of 246 amino acids and having a predicted molecular weight of 28.03 kDa, is encoded by an ORF of 741 bps. Expression was achieved in Escherichia coli and a recombinant protein with esterolytic activity and estimated molecular weight of 25 kDa was recovered and purified from the periplasmic fraction by IMAC. The purified enzyme, most active at 60-65 degrees C and in the near-neutral range (pH 6.5-8), displayed a half-life at 60 degrees C of about 5 h. Est/Lip displayed a relative tolerance to methanol, DMSO, acetonitrile, and low detergent concentrations (SDS, Triton) increased its thermostability. Highest activity was attained with p-nitrophenyl butyrate, but the enzyme was also able to hydrolyze long chain fatty acid esters, as well as triolein. The primary sequence and predicted tridimensional structure of the enzyme are very similar to those of other Anoxybacillus and Geobacillus carboxyl esterases in a distinct, recently described lipase family. Est/Lip was highly enantioselective, with preference for the (S)-enantiomer of substrates. (+info)
Taxonomic classification of Anoxybacillus isolates from geothermal regions in Turkey by 16S rRNA gene sequences and ARDRA, ITS-PCR, Rep-PCR analyses.
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A total of 115 endospore-forming bacilli were taken for 16S rRNA gene sequence analyses and clustered among 7 genera. In this paper, the most abundant thermophiles belonging to genus Anoxybacillus with its 53 isolates are presented. The Anoxybacillus species, some of which were producing biotechnologically valuable enzymes, mostly displayed amylolytic and glucosidic activities and the ability of carbohydrate degradation made them superior in number among the other bacilli in these extreme habitats. In comparative sequence analyses, similarities ranged from 91.1% to 99.9% between the isolates and the type strains. Isolates were clustered into eight phylogenetic lineages within the type strains of A. kamchatkensis, A. flavithermus, A. kamchatchensis subsp. asaccharedens, and A. salavatliensis. In addition, C161ab and A321 were proposed as novel species which displayed < 97.0% similarities to their closest relatives. Moreover, their individual AluI, HaeIII, and TaqI ARDRA restriction patterns, ITS-, (GTG)5-, and BOX-PCR fingerprintings generated 27, 28, 31, 35, 40, and 41 clusters, respectively. The twelve type strains and 35 of the isolates showed unique distinctive patterns from all the others at least in two of these analyses. These phenotypic and genomic characters allowed us to differentiate their genotypic diversity from the reference strains. (+info)