Modeling the clay minerals–enzyme binding by fusion fluorescent proteins and under atomic force microscope

• Published in: Microscopy research and technique 2019-06, Vol.82 (6), p.884-891
• Main Authors: Math, Renukaradhya K., Reddy, Srinivasa, Dae Yun, Han, Kambiranda, Devaiah, Ghebreiyessus, Yemane
• Format: Article
• Language: English
• Subjects: Atomic force microscopes; Atomic force microscopy; Binding; Cellulase; Clay; Clay minerals; Confocal microscopy; Enzymes; Fluorescence; fusion fluorescent protein; Image processing; Kaolinite; Microscopes; Microscopy; Minerals; Molecular conformation; Montmorillonite; protein binding; Protein structure; Proteins
• ISSN: 1059-910X; 1097-0029
• DOI: 10.1002/jemt.23233

In the present study, binding of cellulase protein to different clay minerals were tested using fluorescent–protein complex and microscopic techniques. Cellulase gene (Cel5H) was cloned into three fluorescent vectors and expressed as fusion enzymes. Binding of Cel5H–mineral particles was confirmed by confocal microscopy, and enzyme assay. Among the Cel5H–fusion enzymes, green–fusion enzyme showed higher intensity compared with other red and yellow fusion–proteins. Intensity of fusion–proteins was dependent on the pH of the medium. Confocal microscopy revealed binding of the all three fusion proteins with different clay minerals. However, montmorillonite displayed higher binding capacity than kaolinite clay. Likewise, atomic force microscopy (AFM) image profile analysis showed proteins appeared globular molecules in free‐state on mica surface with an average cross sectional diameter of 110 ± 2 nm and rough surface of montmorillonite made protein appear flattened due to structural alteration. Even surface of kaolinite also exerted some strain on protein molecular conformation after binding to surface. Our results provide further evidence for 3D visualization of enzyme–soil complex and encourage furthering study of the force involved interactions. Therefore, our results indicate that binding of proteins to clay minerals was external and provides a molecular method to observe the interaction of clay minerals–enzyme complex. Cellulase gene (Cel5H) was cloned into three fluorescent vectors and expressed as fusion enzymes. Binding of Cel5H–mineral particles was confirmed by confocal microscopy, and enzyme assay. Among the Cel5H–fusion enzymes, green‐fusion enzyme showed higher intensity compared with other red‐ and yellow‐fusion–proteins. Intensity of fusion–proteins was dependent on the pH of the medium. Confocal microscopy revealed binding of the all three fusion proteins with different clay minerals. However, montmorillonite displayed higher binding capacity than kaolinite clay. Likewise, atomic force microscopy (AFM) image profile analysis showed proteins appeared globular molecules in free‐state on mica surface with an average cross sectional diameter of 110 ± 2 nm and rough surface of montmorillonite made protein appear flattened due to structural alteration. Our results provide further evidence for 3D visualization of enzyme–soil complex and encourage furthering study of the force involved interactions.