Gonzalez-Franco Ana C, Deobald Lee A, Spivak Aaron, Crawford Don L
Department of Microbiology, Molecular Biology and Biochemistry, University of Idaho, Moscow 83844-3052, USA.
Can J Microbiol. 2003 Nov;49(11):683-98. doi: 10.1139/w03-089.
The objective of this study was to determine if antifungal actinomycetes isolated from rhizosphere and non-rhizosphere soils exhibit different chitinase-like production and (or) induction patterns. Selected isolates from both habitats were compared. Chitinase-like levels and isoform characteristic patterns were evaluated over time in culture fluids of isolates grown on media containing different combinations of colloidal chitin and fungal cell wall (FCW) preparation. Supernatants were also subjected to native and non-native polyacrylamide gel electrophoresis (PAGE), using glycol chitin amended gels. For non-native PAGE, protein samples were denatured by two different approaches. Multiple active bands, ranging from 20 to 53 kDa and present in varying amounts, were detected in gels for most strains. Different substrate preferences were observed among strains, and different chitinase-like enzymes were produced, depending upon the substrate combinations used. The presence of FCW in the medium induced specific chitinase-like enzymes not observed otherwise. Enzymatic activities and profiles of the isolates, however, were strain and substrate specific rather than habitat specific. However, a sagebrush rhizosphere soil had a larger actinomycete community with higher chitinolytic activities than the nearby bulk soil. The use of PAGE to compare chitinase-like proteins induced in media with and without FCW was useful for identifying chitinase-like enzymes potentially involved in antifungal activity.
本研究的目的是确定从根际和非根际土壤中分离出的抗真菌放线菌是否表现出不同的几丁质酶样产生和（或）诱导模式。对来自这两种生境的选定分离株进行了比较。在含有不同组合的胶体几丁质和真菌细胞壁（FCW）制剂的培养基上生长的分离株的培养液中，随时间评估几丁质酶样水平和同工型特征模式。还使用乙二醇几丁质改良的凝胶对上清液进行了非变性和变性聚丙烯酰胺凝胶电泳（PAGE）。对于变性PAGE，蛋白质样品通过两种不同的方法进行变性。在大多数菌株的凝胶中检测到多个活性条带，大小在20至53 kDa之间，含量各不相同。菌株之间观察到不同的底物偏好，并且根据所使用的底物组合产生不同的几丁质酶样酶。培养基中FCW的存在诱导了其他情况下未观察到的特定几丁质酶样酶。然而，分离株的酶活性和图谱是菌株和底物特异性的，而不是生境特异性的。然而，与附近的块状土壤相比，鼠尾草属植物根际土壤具有更大的放线菌群落和更高的几丁质分解活性。使用PAGE比较在有和没有FCW的培养基中诱导的几丁质酶样蛋白，有助于鉴定可能参与抗真菌活性的几丁质酶样酶。
