Abstract
Our previous study on the binding activity between Cel5H and clay minerals showed highest binding efficiency among other cellulase enzymes cloned. Here, based on previous studies, we hypothesized that the positive amino acids on the surface of Cel5H protein may play an important role in binding to clay surfaces. To examine this, protein sequences of Bacillus licheniformis Cel5H (BlCel5H) and Paenibacillus polymyxa Cel5A (PpCel5A) were analyzed and then selected amino acids were mutated. These mutated proteins were investigated for binding activity and force measurement via atomic force microscopy (AFM). A total of seven amino acids which are only present in BlCel5H but not in PpCel5A were selected for mutational studies and the positive residues which are present in both were omitted. Of the seven selected surface lysine residues, only three mutants K196A(M2), K54A(M3) and K157T(M4) showed 12%, 7% and 8% less clay mineral binding ability, respectively compared with wild-type. The probable reason why other mutants did not show altered binding efficiency might be due to relative location of amino acids on the protein surface. Meanwhile, measurement of adhesion forces on mica sheets showed a well-defined maximum at 69 ± 19 pN for wild-type, 58 ± 19 pN for M2, 53 ± 19 pN for M3, and 49 ± 19 pN for M4 proteins. Hence, our results demonstrated that relative location of surface amino acids of Cel5H protein especially positive charged amino acids are important in the process of clay mineral-protein binding interaction through electrostatic exchange of charges.
Highlights
Several explanations have been provided on the mechanism of protein adsorption on clay mineral surfaces, which states that - electrostatic interactions occur between proteins and surfaces
It is assumed that if the protein is to bind on clay mineral surface, only the hydrolytic domain (GH) might be responsible for interacting with the clay surface rather than the substrate binding region (Math et al 2020)
Many theories had been proposed stating the importance of protein pI in binding to clay surfaces through biochemical assays (Burns and Dick 2002; Math et al 2019)
Summary
Several explanations have been provided on the mechanism of protein adsorption on clay mineral surfaces, which states that - electrostatic interactions occur between proteins and surfaces. The protein becomes more positively charged as the pH decreases below the isoelectric point (pI) and the clay remains negatively charged (Burns and Dick 2002; Math et al 2020). Our understanding of the clay mineralsprotein interactions and their forces on clay surfaces. None of these studies provided visualization and force measurement data. Studies involving AFM analysis might expand the horizons understanding on clay mineral-protein complexes and forces involved during the process (Zhai et al 2019)
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