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Xianzhong Chen Huiying Fang Bin Zhuge Zhengxiang Wang Algasan Govender Jian Zhuge 《Process Biochemistry》2013,48(10):1469-1475
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Vashni Gramany Algasan Govender Krishna Bisetty Suren Singh Kugenthiren Permaul 《Journal of biomolecular structure & dynamics》2016,34(8):1681-1692
The aim of this study was to clone, express, and characterize a β-xylosidase (Tlxyn1) from the thermophilic fungus Thermomyces lanuginosus SSBP in Pichia pastoris GS115 as well as analyze optimal activity and stability using computational and experimental methods. The enzyme was constitutively expressed using the GAP promoter and secreted into the medium due to the alpha-mating factor secretion signal present on the expression vector pBGPI. The 1276 bp gene consists of an open reading frame that does not contain introns. A 12% SDS–PAGE gel revealed a major protein band at an estimated molecular mass of 50 kDa which corresponded to zymogram analysis. The three-dimensional structure of β-xylosidase was predicted, and molecular dynamics simulations at different ranges of temperature and pH were performed in order to predict optimal activity and folding energy. The results suggested a strong conformational temperature and pH dependence. The recombinant enzyme exhibited optimal activity at pH 7 and 50°C and retained 80% activity at 50°C, pH 7 for about 45 min. This is the first report of the cloning, functional expression, and simulations study of a β-xylosidase from Thermomyces species in a fungal host. 相似文献
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Govender Algasan Shaik Rehana Pillay Balakrishna 《World journal of microbiology & biotechnology》2011,27(5):1217-1224
1,2-dichloroethane (DCA) is a toxic synthetic haloalkane produced annually in excess of 20 billion tons. Five bacterial isolates
capable of complete mineralization of DCA have recently been isolated from wastewater treatment facilities in South Africa.
Pulsed field gel electrophoresis (PFGE) and random amplification of polymorphic DNA (RAPD) analysis were employed in this
study to identify phylogenetic differences between these closely-related bacteria. Analysis of the 16S rDNA sequences of the
selected isolates revealed similarities to previously characterised isolates of Ancylobacter aquaticus. It has been previously
shown that all isolates follow the same catabolic pathway and possess an identical hydrolytic dehalogenase (DhlA) involved
in the initial carbonchlorine bond cleavage. Analysis of homology matrices deduced from RAPD and restriction profiles, constructed
using the GelCompar software package, revealed that although some of the isolates possessed identical profiles using one primer
or restriction endonuclease, differences were observed when a different primer was used. Furthermore, the results obtained
indicate that the previously characterised isolate A. aquaticus AD25 is significantly different from the isolates used in
this study. PFGE was also able to show that isolates of A. aquaticus do not possess the 200 kb plasmid containing the hydrolytic
dehalogenase gene previously identified in the DCA-degrading bacterium Xanthobacter autotrophicus GJ10. This study has been
able to demonstrate that RAPD and PFGE analysis are suitable molecular tools for the differentiation of closely-related A.
aquaticus isolates and may be routinely used in the differentiation of environmentally important bacteria. 相似文献
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Algasan Govender Rehana Shaik Nathlee Samantha Abbai Balakrishna Pillay 《World journal of microbiology & biotechnology》2011,27(10):2407-2414
In recent years large quantities of mixtures of chlorinated hydrocarbons have accumulated in the environment due to the widespread
use and production of these compounds. Microbes have been found to demonstrate a widespread and diverse potential to adapt
to the dechlorination of such compounds. Therefore the aim of this study was to investigate the presence and diversity of
reductive and hydrolytic dehalogenase genes in a site contaminated with a mixture of chlorinated hydrocarbons. Primers targeting
reductive and hydrolytic bacterial dehalogenase genes were designed. In addition, DGGE analysis was performed in order to
determine the presence of any known dehalogenase-producing organisms. Total DNA isolated from borehole water samples was used
as the template for the amplification reactions. All PCR products obtained with the reductive and hydrolytic gene primers,
as well as the dominant bands present on the DGGE gel were cloned and sequenced. Sequencing of the individual amplicons revealed
significant identities to the tceA gene of Dehalococcoides ethenogenes 195, the vcrA gene of Dehalococcoides sp. VS as well as the dhlA and dhlB genes of Xanthobacter autotrophicus GJ10. DGGE analysis indicated a high level of commonality with the different sampling times and depths. However, sequence
analysis revealed that 66% of the cloned fragments showed significant (95–99%) identity with uncultured microorganisms. Phylogenetic
analysis of the sequences revealed that the DGGE clones clustered into two groups when compared to known bacteria having hydrocarbon
degradative capabilities. This indicated that the sequences of the clones were diverse when compared to known microorganisms.
This diversity represents a largely untapped genetic pool that can be exploited for the discovery of novel biocatalysts that
can be employed in bioremediation. In addition, the presence of both hydrolytic and reductive dehalogenases provided strong
evidence that bacteria capable of dehalogenation of chlorinated hydrocarbons may be present in sites contaminated with these
compounds. 相似文献
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Pyrosequence Analysis of Unamplified and Whole Genome Amplified DNA from Hydrocarbon-Contaminated Groundwater 总被引:2,自引:0,他引:2
Pyrosequence data was used to analyze the composition and metabolic potential of a metagenome from a hydrocarbon-contaminated
site. Unamplified and whole genome amplified (WGA) sequence data was compared from this source. According to MG-RAST, an additional
2,742,252 bp of DNA was obtained with the WGA, indicating that WGA has the ability to generate a large amount of DNA from
a small amount of starting sample. However, it was observed that WGA introduced a bias with respect to the distribution of
the amplified DNA and the types of microbial populations that were accessed from the metagenome. The dominant order in the
WGA metagenome was Flavobacteriales, whereas the unamplified metagenome was dominated by Actinomycetales as determined by
RDPII and CARMA databases. According to the SEED database, the subsystems shown to be present for the individual metagenomes
were associated with the metabolic potential that was expected to be present in the contaminated groundwater, such as the
metabolism of aromatic compounds. A higher percentage (4.4) of genes associated with the metabolism of aromatic compounds
was identified in the unamplified metagenome when compared to the WGA metagenome (0.66%). This could be attributed to the
increased number of hydrocarbon degrading bacteria that had been accessed from this metagenome (Mycobacteria, Nocardia, Brevibacteria,
Clavibacter, Rubrobacter, and Rhodoccocus). Therefore, it was possible to relate the taxonomic groups accessed to the contamination
profile of the metagenome. By collating the sequencing data obtained pre- and post-amplification, this study provided insight
regarding the survival strategies of microbial communities inhabiting contaminated environments. 相似文献
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