Polymerase chain reaction for identification of aldoxime dehydratase in aldoxime- or nitrile-degrading microorganisms
Polymerase chain reaction for identification of aldoxime dehydratase in aldoxime- or nitrile-degrading microorganisms
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DOI：
10.1016/j.femsle.2005.04.011
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发表时间：
2005-05-15
影响因子：
2.1
通讯作者：
Asano, Y
中科院分区：
文献类型：
--
作者：
Kato, Y;Yoshida, S;Asano, Y
我们开发了一种利用Southern杂交和聚合酶链式反应(PCR)的分子筛选方法，从14株乙醛或丁腈降解菌中鉴定乙醛肟脱水酶(Oxd)编码基因(Oxds)。以红球红球菌N-771的oxd基因为探针，8株菌株的染色体DNA出现阳性杂交信号，表明这些菌株具有与红球红球菌N-771相似的oxd基因。用简并的共同性引物对扩增片段进行分析，结果表明8株活性菌株中存在同源Oxd与含铁NHase共存的现象，这与Southern杂交结果一致。从其中一株阳性菌株假单胞菌中克隆了oxd基因的全长序列。K-9基因测序，并在大肠杆菌中表达。假单胞菌oxD基因编码蛋白OxdK的一级结构分析K-9鉴定出一个具有新一级结构的Oxd。因此，基于PCR的oxd基因分析是检测和分析自然界中的“醛-腈途径”的有用工具，因为Oxd是该途径的关键酶。(C)2005年欧洲微生物学会联合会。爱思唯尔出版，版权所有。
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. (c) 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.