Bura Gohain Manorama, Talukdar Shruti, Talukdar Madhumita, Yadav Archana, Gogoi Binod Kumar, Bora Tarun Chandra, Kiran Shashi, Gulati Arvind
Biotechnology Division, CSIR-North East Institute of Science and Technology, Jorhat, India.
Biotechnol Appl Biochem. 2015 Mar-Apr;62(2):226-36. doi: 10.1002/bab.1260. Epub 2014 Nov 23.
In recent years, nitrilases from fungus have received increasing attention, and most of the studies are performed on nitrilases of bacterial origin. Frequently used methods are based on analytical methods such as high-performance liquid chromatography, liquid chromatography-mass spectrometry, and gas chromatography; therefore, an efficient, user friendly, and rapid method has been developed to screen nitrilase enzyme based on the principle of color change of a pH indicator. Phenol red amended with the minimal medium appears light yellow at neutral pH, which changes into pink with the formation of ammonia, indicating nitrilase activity in the reaction medium. A highly potent strain ED-3 identified as Fusarium oxysporum f. sp. lycopercisi (specific activity 17.5 µmol/Min/mg dcw) was isolated using this method. The nitrilase activity of F. oxysporum f. sp. lycopercisi ED-3 strain showed wide substrate specificity toward aliphatic nitriles, aromatic nitriles, and orthosubstituted heterocyclic nitriles. 4-Aminobenzonitrile was found to be a superior substrate among all the nitriles used in this study. This nitrilase was active within pH 5-10 and temperature ranging from 25 to 60 °C with optimal at pH 7.0 and temperature at 50 °C. The nitrilase activity was enhanced to several folds through optimization of culture and biotransformation conditions from 1,121 to 1,941 µmol/Min.
近年来，来自真菌的腈水解酶受到越来越多的关注，而大多数研究是针对细菌来源的腈水解酶进行的。常用方法基于高效液相色谱、液相色谱 - 质谱和气相色谱等分析方法；因此，基于pH指示剂颜色变化原理，开发了一种高效、用户友好且快速的方法来筛选腈水解酶。用基本培养基改良的酚红在中性pH下呈浅黄色，随着氨的形成变为粉红色，表明反应介质中有腈水解酶活性。使用该方法分离出了一种高效菌株ED - 3，鉴定为尖孢镰刀菌番茄专化型（比活性为17.5 μmol/分钟/毫克干重）。尖孢镰刀菌番茄专化型ED - 3菌株的腈水解酶活性对脂肪族腈、芳香族腈和邻位取代杂环腈表现出广泛的底物特异性。在本研究中使用的所有腈类中，4 - 氨基苯甲腈被发现是一种优质底物。这种腈水解酶在pH 5 - 10和温度25至60°C范围内有活性，最适pH为7.0，最适温度为50°C。通过优化培养和生物转化条件，腈水解酶活性从1121 μmol/分钟提高到1941 μmol/分钟，提高了几倍。
