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GC-based Detection of Aldononitrile Acetate Derivatized Glucosamine and Muramic Acid for Microbial Residue Determination in Soil
Published on: May 19, 2012
Polymerase chain reaction for identification of aldoxime dehydratase in aldoxime- or nitrile-degrading microorganisms
Yasuo Kato1, Satoshi Yoshida, Yasuhisa Asano
1Biotechnology Research Center, Faculty of Engineering, Toyama Prefectural University, 5180 Kurokawa, Kosugi, Toyama 939-0398, Japan.
Abstract:
We developed a molecular screening procedure using Southern hybridization and polymerase chain reaction (PCR) to identify aldoxime dehydratase (Oxd) encoding genes (oxds) among 14 aldoxime- or nitrile-degrading microorganisms. When an oxd gene of Rhodococcus erythropolis N-771 was used as a probe, positive hybridization signals were seen with the chromosomal DNA of eight strains, suggesting that these strains have similar oxd genes to R. erythoropolis N-771. By analyzing the PCR-amplified fragments with degenerate consensus primers, the occurrence of homologous Oxd coexisting with Fe-containing NHase in the active eight strains was demonstrated coinciding with the results of Southern hybridization. Whole length of oxd gene was cloned as an example from one of the positive strains, Pseudomonas sp. K-9, sequenced, and expressed in E. coli. Analysis of the primary structure of the protein (OxdK) encoded by the oxd gene of Pseudomonas sp. K-9 led to identify an Oxd having a new primary structure. Thus, the PCR-based analysis of oxd gene is a useful tool to detect and analyze the "aldoxime-nitrile pathway" in nature, since Oxd is the key enzyme for the pathway.
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