title: 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.
authors.affiliation: Instituto de Agroquímica y Tecnología de Alimentos, CSIC, Paterna, Valencia, Spain.
authors.forename: Julia
authors.initials: J
authors.lastname: Marín-Navarro
authors.affiliation: Instituto de Agroquímica y Tecnología de Alimentos, CSIC, Paterna, Valencia, Spain.
authors.forename: Nicole
authors.initials: N
authors.lastname: Roupain
authors.affiliation: Instituto de Agroquímica y Tecnología de Alimentos, CSIC, Paterna, Valencia, Spain.
authors.forename: David
authors.initials: D
authors.lastname: Talens-Perales
authors.affiliation: Instituto de Agroquímica y Tecnología de Alimentos, CSIC, Paterna, Valencia, Spain.
authors.forename: Julio
authors.initials: J
authors.lastname: Polaina
mesh_terms: D019943:Amino Acid Substitution; D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics; D004795:Enzyme Stability / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005949:Glucose Oxidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*
chemical_list: D005656:Fungal Proteins; D005949:Glucose Oxidase