NDLI: Rational Redesign of Glucose Oxidase for Improved Catalytic Function and Stability

Content Provider	PubMed Central
Author	Holland, J. Todd Harper, Jason C. Dolan, Patricia L. Manginell, Monica M. Arango, Dulce C. Rawlings, Julia A. Apblett, Christopher A. Brozik, Susan M.
Editor	Luis, Menéndez-arias
Copyright Year	2012
Abstract	Glucose oxidase (GOx) is an enzymatic workhorse used in the food and wine industries to combat microbial contamination, to produce wines with lowered alcohol content, as the recognition element in amperometric glucose sensors, and as an anodic catalyst in biofuel cells. It is naturally produced by several species of fungi, and genetic variants are known to differ considerably in both stability and activity. Two of the more widely studied glucose oxidases come from the species Aspergillus niger (A. niger) and Penicillium amagasakiense (P. amag.), which have both had their respective genes isolated and sequenced. GOx from A. niger is known to be more stable than GOx from P. amag., while GOx from P. amag. has a six-fold superior substrate affinity (K M) and nearly four-fold greater catalytic rate (k cat). Here we sought to combine genetic elements from these two varieties to produce an enzyme displaying both superior catalytic capacity and stability. A comparison of the genes from the two organisms revealed 17 residues that differ between their active sites and cofactor binding regions. Fifteen of these residues in a parental A. niger GOx were altered to either mirror the corresponding residues in P. amag. GOx, or mutated into all possible amino acids via saturation mutagenesis. Ultimately, four mutants were identified with significantly improved catalytic activity. A single point mutation from threonine to serine at amino acid 132 (mutant T132S, numbering includes leader peptide) led to a three-fold improvement in k cat at the expense of a 3% loss of substrate affinity (increase in apparent K M for glucose) resulting in a specify constant (k cat/K M) of 23.8 (mM−1 · s−1) compared to 8.39 for the parental (A. niger) GOx and 170 for the P. amag. GOx. Three other mutant enzymes were also identified that had improvements in overall catalysis: V42Y, and the double mutants T132S/T56V and T132S/V42Y, with specificity constants of 31.5, 32.2, and 31.8 mM−1 · s−1, respectively. The thermal stability of these mutants was also measured and showed moderate improvement over the parental strain.
Related Links	http://dx.doi.org/10.1371/journal.pone.0037924
Starting Page	37924
File Format	PDF
ISSN	19326203
e-ISSN	19326203
Journal	PLoS ONE
Issue Number	6
Volume Number	7
Language	English
Publisher	Public Library of Science
Publisher Date	2012-06-01
Access Restriction	Open
Rights Holder	Public Library of Science
Subject Keyword	Biochemistry, Genetics and Molecular Biology(all) Agricultural and Biological Sciences(all) Medicine(all) Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Multidisciplinary

Sl.	Authority	Responsibilities	Communication Details
1	Ministry of Education (GoI), Department of Higher Education	Sanctioning Authority	https://www.education.gov.in/ict-initiatives
2	Indian Institute of Technology Kharagpur	Host Institute of the Project: The host institute of the project is responsible for providing infrastructure support and hosting the project	https://www.iitkgp.ac.in
3	National Digital Library of India Office, Indian Institute of Technology Kharagpur	The administrative and infrastructural headquarters of the project	Dr. B. Sutradhar bsutra@ndl.gov.in
4	Project PI / Joint PI	Principal Investigator and Joint Principal Investigators of the project	Dr. B. Sutradhar bsutra@ndl.gov.in Prof. Saswat Chakrabarti will be added soon
5	Website/Portal (Helpdesk)	Queries regarding NDLI and its services	support@ndl.gov.in
6	Contents and Copyright Issues	Queries related to content curation and copyright issues	content@ndl.gov.in
7	National Digital Library of India Club (NDLI Club)	Queries related to NDLI Club formation, support, user awareness program, seminar/symposium, collaboration, social media, promotion, and outreach	clubsupport@ndl.gov.in
8	Digital Preservation Centre (DPC)	Assistance with digitizing and archiving copyright-free printed books	dpc@ndl.gov.in
9	IDR Setup or Support	Queries related to establishment and support of Institutional Digital Repository (IDR) and IDR workshops	idr@ndl.gov.in

Structural Basis for Binding of Fluorinated Glucose and Galactose to Trametes multicolor Pyranose 2-Oxidase Variants with Improved Galactose Conversion

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

Effects of High Pressure Homogenization on the Activity, Stability, Kinetics and Three-Dimensional Conformation of a Glucose Oxidase Produced by Aspergillus niger

Improved Assessment of Orbital Stability of Rhythmic Motion with Noise

Hydrologic-Process-Based Soil Texture Classifications for Improved Visualization of Landscape Function

Rapid Catalytic Template Searching as an Enzyme Function Prediction Procedure

Partitioning of Minimotifs Based on Function with Improved Prediction Accuracy

Urotensin II Inhibits Skeletal Muscle Glucose Transport Signaling Pathways via the NADPH Oxidase Pathway

Oscillations in Rational Economies

Rational Redesign of Glucose Oxidase for Improved Catalytic Function and Stability

Similar Documents

Structural Basis for Binding of Fluorinated Glucose and Galactose to Trametes multicolor Pyranose 2-Oxidase Variants with Improved Galactose Conversion

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

Effects of High Pressure Homogenization on the Activity, Stability, Kinetics and Three-Dimensional Conformation of a Glucose Oxidase Produced by Aspergillus niger

Improved Assessment of Orbital Stability of Rhythmic Motion with Noise

Hydrologic-Process-Based Soil Texture Classifications for Improved Visualization of Landscape Function

Rapid Catalytic Template Searching as an Enzyme Function Prediction Procedure

Partitioning of Minimotifs Based on Function with Improved Prediction Accuracy

Urotensin II Inhibits Skeletal Muscle Glucose Transport Signaling Pathways via the NADPH Oxidase Pathway

Oscillations in Rational Economies

Rational Redesign of Glucose Oxidase for Improved Catalytic Function and Stability