Journal of Biotechnology 131 (2007) 197–204 Properties of a chimeric glucose dehydrogenase improved by site directed mutagenesis Chaturvedula Tripura, Appa Rao Podile ∗ Department of Plant Sciences, University of Hyderabad, P.O. Central University, Hyderabad 500046, Andhra Pradesh, India Received 18 February 2007; received in revised form 31 May 2007; accepted 20 June 2007 Abstract 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. © 2007 Elsevier B.V. All rights reserved. Keywords: Glucose dehydrogenase; Escherichia coli; S. marcescens GPS-5; Mineral phosphate solubilization; Site-directed mutagenesis 1. Introduction Glucose dehydrogenase (GDH) of Gram-negative bacteria is a member of the largest group of quinoproteins, and a principal enzyme in the direct oxidation pathway catalyzing the oxida- tion of glucose to gluconic acid (pK a ∼ 3.4) (Duine et al., 1979; Anthony, 1988; Duine, 1991). Membrane GDHs (m-GDHs) from various bacteria are about 88 kDa monomeric proteins that have an N-terminal hydrophobic domain consisting of five transmembrane segments that ensure a strong anchorage of the protein to the membrane, and a large conserved pyrrolo quinoline quinone (PQQ)-binding C-terminal domain that has the catalytic function (Yamada et al., 1993). GDH plays a key regulatory and bioenergetic role in Gram-negative bacteria, in addition to providing carbon for intracellular metabolism. In soils deficient in soluble forms of ∗ Corresponding author. Tel.: +91 40 23134503; fax: +91 40 23010120. E-mail addresses: arpsl@uohyd.ernet.in, podilerao@yahoo.com (A.R. Podile). phosphate, gluconic acid secreted by some Gram-negative bacte- ria brings about the dissolution of mineral phosphate complexes (Goldstein, 1995), thereby playing a significant role in mineral phosphate solubilization (MPS). Due to the oxygen insensitivity and high catalytic activity, GDH is also preferred as a glucose biosensor over glucose oxidase and NAD-glucose dehydroge- nases for in vivo blood glucose monitoring (Matsushita et al., 2002). GDH requires PQQ as a cofactor and also metal ions such as Ca 2+ (or Mg 2+ in vitro) for its activity (Duine et al., 1979). PQQ dependent GDH is present in a wide variety of bacterial species. Acinetobacter calcoaceticus (Hauge, 1966), Gluconobacter suboxydans (Ameyama et al., 1981), Klebsiella aerogenes (Neijssel et al., 1983), and Pseudomonas aerugi- nosa (Midgley and Dawes, 1973), produce the cofactor PQQ themselves while, Escherichia coli (Hommes et al., 1984) and Acinetobacter lwoffi (van Schie et al., 1984), require external supply of PQQ for GDH activity. Based on the EDTA tolerance PQQGDHs are subdivided into Type I-EDTA sensitive (PQQ can be easily removed by dialysis against EDTA containing buffers e.g. GDH of E. coli, P. aeruginosa, P. fluorescens and 0168-1656/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2007.06.015