Literature summary for 1.1.3.4 extracted from

Marin-Navarro, J.; Roupain, N.; Talens-Perales, D.; Polaina, J.
Identification and structural analysis of amino acid substitutions that increase the stability and activity of Aspergillus niger glucose oxidase (2015), PLoS ONE, 10, e0144289 .

Application

Application	Comment	Organism
diagnostics	the enzyme is used for the manufacture of glucose biosensors and in particular sensor strips used to measure glucose levels in serum	Aspergillus niger
industry	in the textile industry, enzyme GOX is used for bio-bleaching and in oral care products as antimicrobial agent	Aspergillus niger

Cloned(Commentary)

Cloned (Comment)	Organism
recombinant expression of wild-type and mutant enzymes in Saccharomyces cerevisiae strain BY4741 (MATa his3 leu2 met15 ura3) from plasmid pSSP-GOX, the GOX coding sequence is fused to the STA1 signal peptide from Saccharomyces cerevisiae var. diastaticus and is under the control of the galactose-inducible GAL10/CYC1 promoter	Aspergillus niger

Protein Variants

Protein Variants	Comment	Organism
H172K	site-directed mutagenesis, mutant H172K shows increased thermosensitivity compared to the wild-type enzyme	Aspergillus niger
H172K/H220D	site-directed mutagenesis, mutant H172K/H220D does not show significant differences in thermal stability but about 70% increased initial activity compared to the wild-type enzyme	Aspergillus niger
H220D	site-directed mutagenesis, mutant H220D shows increased thermosensitivity and reduced activity compared to the wild-type enzyme	Aspergillus niger
H447K	site-directed mutagenesis, introduction of two symmetrical, intermolecular salt bridges at the dimer interface, between K447 and D70	Aspergillus niger
H447K	site-directed mutagenesis, the shows similar initial activity but higher thermal sensitivity compared to the wild-type enzyme	Aspergillus niger
L500D	site-directed mutagenesis, inactive mutant	Aspergillus niger
L569E	site-directed mutagenesis, the mutant shows about 50% increased initial activity compared to the wild-type enzyme	Aspergillus niger
L569E	site-directed mutagenesis, the thermal stability of the mutant is similar to the wild-type enzyme, but the initial activity is increased compared to the wild-type enzyme	Aspergillus niger
additional information	usage of a strategy that combined random and rational approaches to isolate uncharacterized mutations of Aspergillus niger glucose oxidase with improved properties. GOX library construction in Saccharomyces cerevisiae and random mutagenesis and screening for mutants with improved thermal stability	Aspergillus niger
Q124R/L569E	site-directed mutagenesis, the mutation has no significant effect on stability but causes a twofold increase of the enzyme's specific activity	Aspergillus niger
Q345K	site-directed mutagenesis, introduction of the mutation to create a salt bridge with D177	Aspergillus niger
Q345K	site-directed mutagenesis, the mutant shows highly reduced thermal stability and about 50% increased initial activity compared to the wild-type enzyme	Aspergillus niger
Q469K	site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme	Aspergillus niger
Q469K/L500D	site-directed mutagenesis, the mutant shows strongly reduced activity compared to the wild-type enzyme	Aspergillus niger
Q469K/L500D	site-directed mutagenesis, the thermal stability of the mutant is similar to the wild-type enzyme, but the initial activity is reduced compared to the wild-type enzyme	Aspergillus niger
Q90R	site-directed mutagenesis, the mutant shows increased sensitivity to thermal denaturation, with R1 and R2 values 60% and 80% lower than wild-type enzyme respectively	Aspergillus niger
Q90R/Y509E	site-directed mutagenesis, the mutation does not cause a significant change in the thermal stability of the enzyme, but causes increased enzyme activity compared to the wild-type enzyme	Aspergillus niger
Q90R/Y509E	site-directed mutagenesis, the mutation introduces a new salt bridge near the interphase of the dimeric protein structure, the mutation does not cause a significant change in the thermal stability of the enzyme, but causes increased enzyme activity compared to the wild-type enzyme	Aspergillus niger
Q90R/Y509E/T554M	the triple mutant is a glucose oxidase with high stability	Aspergillus niger
T30S/I94V	site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme	Aspergillus niger
T30S/I94V	site-directed mutagenesis, a thermoresistant mutant	Aspergillus niger
T554M	random mutagenesis, the mutation generates a sulfur-pi interaction, the mutant shows 60% reduced activity and 40% increased thermal stability compared to the wild-type enzyme	Aspergillus niger
Y509E	site-directed mutagenesis, the mutation does not cause a significant change in the thermal stability of the enzyme, but causes increased enzyme activity compared to the wild-type enzyme	Aspergillus niger

Molecular Weight [Da]

Molecular Weight [Da]	Molecular Weight Maximum [Da]	Comment	Organism
160000	-	-	Aspergillus niger

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
beta-D-glucose + O2	Aspergillus niger	-	D-glucono-1,5-lactone + H2O2	-	?
beta-D-glucose + O2	Aspergillus niger CECT 2775	-	D-glucono-1,5-lactone + H2O2	-	?

Organism

Organism	UniProt	Comment	Textmining
Aspergillus niger	A0A068CB13	sequence with exchanges at V167T and K282E	-
Aspergillus niger CECT 2775	A0A068CB13	sequence with exchanges at V167T and K282E	-

Posttranslational Modification

Posttranslational Modification	Comment	Organism
glycoprotein	-	Aspergillus niger

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
beta-D-glucose + O2	-	Aspergillus niger	D-glucono-1,5-lactone + H2O2	-	?
beta-D-glucose + O2	cofactor FAD is transiently reduced along the reaction mechanism	Aspergillus niger	D-glucono-1,5-lactone + H2O2	-	?
beta-D-glucose + O2	-	Aspergillus niger CECT 2775	D-glucono-1,5-lactone + H2O2	-	?
beta-D-glucose + O2	cofactor FAD is transiently reduced along the reaction mechanism	Aspergillus niger CECT 2775	D-glucono-1,5-lactone + H2O2	-	?

Subunits

Subunits	Comment	Organism
homodimer	each monomer contains two domains, one consists of a five-stranded beta-sheet sandwiched between a three-stranded beta-sheet and three alpha-helices, and the other is composed by a large six-stranded antiparallel beta-sheet supported by six alpha-helices. The two units of the dimer are connected through hydrophobic and hydrophilic contacts, the latter including salt bridges and hydrogen bonds	Aspergillus niger

Synonyms

Synonyms	Comment	Organism
GOX	-	Aspergillus niger

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
37	-	assay at	Aspergillus niger

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
6	-	assay at	Aspergillus niger

Cofactor

Cofactor	Comment	Organism	Structure
FAD	dependent on, the FAD cofactor is not covalently but tightly bound to the enzyme domain, that consists of a five-stranded beta-sheet sandwiched between a three-stranded beta-sheet and three alpha-helices	Aspergillus niger

General Information

General Information	Comment	Organism
additional information	identification and analysis of structural motifs of the protein which are critical for its stability	Aspergillus niger
physiological function	hydrogen peroxide generated by GOX action has anti-microbial effect	Aspergillus niger