Identification and Structural Analysis of Amino Acid Substitutions that Increase the Stability and Activity of Aspergillus niger Glucose Oxidase

Abstract

Glucose oxidase is one of the most conspicuous commercial enzymes due to its many different applications in diverse industries such as food, chemical, energy and textile. Among these applications, the most remarkable is the manufacture of glucose biosensors and in particular sensor strips used to measure glucose levels in serum. The generation of ameliorated versions of glucose oxidase is therefore a significant biotechnological objective. We have used a strategy that combined random and rational approaches to isolate uncharacterized mutations of Aspergillus niger glucose oxidase with improved properties. As a result, we have identified two changes that increase significantly the enzyme's thermal stability. One (T554M) generates a sulfur-pi interaction and the other (Q90R/Y509E) introduces a new salt bridge near the interphase of the dimeric protein structure. An additional double substitution (Q124R/L569E) has no significant effect on stability but causes a twofold increase of the enzyme's specific activity. Our results disclose structural motifs of the protein which are critical for its stability. The combination of mutations in the Q90R/Y509E/T554M triple mutant yielded a version of A. niger glucose oxidase with higher stability than those previously described.

Homology Modeling and Structural Dynamics of the Glucose Oxidase

“…This region facilitates the path for electron transfer from the flavin oxygen-4 (O4) [12]. In respect to the sulfur atoms in the A. niger GOD crystallographic structure, Marín-Navarro et al [11] elucidated a new sulfur-pi interaction formed by residual mutation of threonine to methionine in position 554 [30]. The hydrogen bond is shown in green dashed line (T554M) that has greater thermally stabilization energy (4.2-12.6 kJ/mol) when compared with that of hydrogen bond-associated (1.3-6.3 kJ/mol).…”

“…Among the other computational protein prediction methods, the most reliable approach is by using homology modeling, in which a target sequence is modeled using known structures of candidate proteins where the similarity between their sequences are judged to be similar [9][10]. This methods also have the important benefits in the sense of gaining insightful information about the structure-function relationship in GOD, as previously reported [11].…”

Enhanced Thermostability of Glucose Oxidase through Computer-Aided Molecular Design

“…Alternatively, available protein structure information of GOD makes it possible to obtain thermostable mutants through rational design. Marín-Navarro et al obtained GOD variants with improved thermostability based on rational design aimed at introducing stabilizing salt bridges [ 12 ]. In the present study, amino acids putatively related to the thermostability of the enzyme were predicted through a rational computer-aided molecular design strategy based on ΔΔG and the analysis of amino acids related to the coenzyme FAD, and two mutants with significantly improved thermostability, S100A and D408W, were obtained through site-directed saturation mutagenesis.…”

“…The specific structures of proteins are closely related to their thermal stability. Many factors affect the specific structure of proteins, such as amino acid composition characteristics, hydrogen bonds, hydrophobic interactions, electrostatic forces, salt bridges and the stability of loops [ 9 , 10 , 11 , 12 ]. Protein structure is the combined effect of these factors in an enzyme molecule, and any changes to these forces may lead to changes in stability.…”

From Protein Features to Sensing Surfaces

“…Covalent bonds such as disulfide bonds are not the only contributors to protein stabilization as the introduction of multiple weak interactions, such as salt bridges on the surface of the proteins, to counteract thermal denaturation has been well established. The modification of the surface of glucose oxidase to carry both a novel sulfur–pi interaction and a salt bridge led to a three-fold increase in thermal stability [ 58 ]. These surface substitutions did not affect the glycosylation pattern of the enzyme which was also reported to enhance the thermal stability through the introduction of structural rigidity [ 59 ].…”