Abstract
Culture-independent approaches to analyze metagenome are practical choices for rapid exploring useful genes. The mg-MSDH gene, acquired from the hot spring metagenomic, was retrieved full lengths of functional gene using semi-nest touch-down PCR. Two pairs of degenerate primers were used to separate seven conserve partial sequences by semi-nest touch-down PCR. One of them showed similarity with aldehyde dehydrogenase was used as a target fragment for isolating full-length sequence. The full-length mg-MSDH sequence contained a 1,473 bp coding sequence encoding a 490-amino-acid polypeptide and assigned an accession number JQ715422 in Genbank. The upstream sequences TAGGAG of the start codon (GTG), suggested that was a ribosome binding site. The coding sequence of mg-MSDH was ligated to pET-303 vector and the reconstructive plasmid was successfully overexpressed in E. coli. The purified recombinant mg-MSDH enzyme showed propionaldehyde oxidative activity of 3.0 U mg−1 at 37 °C.
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This research was financially supported by National Natural Science Foundation of China (21006018), Science and Technology Department of Zhejiang Province (2009C31086), Technology Research and Development Program for Institute of Hangzhou (20090331N03) and Young Nature Fund of Hangzhou Normal University (2010QN19).
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Chen, R., Li, C., Pei, X. et al. Isolation an Aldehyde Dehydrogenase Gene from Metagenomics Based on Semi-nest Touch-Down PCR. Indian J Microbiol 54, 74–79 (2014). https://doi.org/10.1007/s12088-013-0405-0
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DOI: https://doi.org/10.1007/s12088-013-0405-0