Properties of a chimeric glucose dehydrogenase improved by site directed mutagenesis.
Glucose dehydrogenase, a membrane bound enzyme oxidizing glucose to gluconic acid in the periplasmic space of Gram-negative bacteria plays a key role in mineral phosphate solubilization and is also an industrially important enzyme, being used as a glucose biosensor. A chimeric glucose dehydrogenase (ES chimera) encoding the N-terminal transmembrane domain from Escherichia coli and the C-terminal periplasmic domain from Serratia marcescens was constructed and the expression was studied on MacConkey glucose medium. The phosphate solubilizing ability of the chimeric GDH was also evaluated, substantiating the role of GDH in mineral phosphate solubilization (MPS). Four mutants of ES chimeric GDH were generated by site directed mutagenesis and the enzyme properties studied. Though the substrate affinity was unaltered for E742K and Y771M, the affinity of H775A and EYH/KMA to glucose and galactose decreased marginally and the affinity to maltose increased. Though Y771M showed a decreased GDH activity there was an increase in the heat tolerance. All the mutants showed an increase in the EDTA tolerance. The triple mutant EYH/KMA showed improved heat and EDTA tolerance and also an increase in affinity to maltose over the ES chimeric GDH.
Department of Plant Sciences, University of Hyderabad, P.O. Central University, Hyderabad 500046, Andhra Pradesh, India.
Chaturvedula
C
Tripura
Appa Rao
AR
Podile
D019943:Amino Acid Substitution; D004492:Edetic Acid / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005816:Genetic Complementation Test; D042843:Glucose 1-Dehydrogenase / Q000235:genetics* / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010710:Phosphates / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D012706:Serratia marcescens / Q000201:enzymology / Q000235:genetics; D013379:Substrate Specificity; D013696:Temperature
D010710:Phosphates; D011993:Recombinant Fusion Proteins; D004492:Edetic Acid; D042843:Glucose 1-Dehydrogenase