Purification and characterization of quinoprotein glucose dehydrogenase from Acinetobacter calcoaceticus L.M.D. 79.41

Abstract

Quinoprotein glucose dehydrogenase (EC 1.1.99.17) from Acinetobacter calcoaceticus L.M.D. 79.41 was purified to homogeneity. It is a basic protein with an isoelectric point of 9.5 and an Mr of 94,000. Denaturation yields two molecules of PQQ/molecule and a protein with an Mr of 48000, indicating that the enzyme consists of two subunits, which are probably identical because even numbers of aromatic amino acids were found. The oxidized enzyme form has an absorption maximum at 350 nm, and the reduced form, obtained after the addition of glucose, at 338 nm. Since double-reciprocal plots of initial reaction rates with various concentrations of glucose or electron acceptor show parallel lines, and substrate inhibition is observed for glucose as well as for electron acceptor at high concentrations, a ping-pong kinetic behaviour with the two reactants exists. From the plots, Km values for glucose and Wurster's Blue of 22 mM and 0.78 mM respectively, and a Vmax. of 7.730 mumol of glucose oxidized/min per mg of protein were derived. The enzyme shows a broad substrate specificity for aldose sugars. Cationic electron acceptors are active in the assay, anionic acceptors are not. A pH optimum of 9.0 was found with Wurster's Blue and 6.0 with 2,6-dichlorophenol-indophenol. Two types of quinoprotein glucose dehydrogenases seem to exist: type I enzymes are acidic proteins from which PQQ can be removed by dialysis against EDTA-containing buffers (examples are found in Escherichia coli, Klebsiella aerogenes and Pseudomonas sp.); type II enzymes are basic proteins from which PQQ is not removed by dialysis against EDTA-containing buffers (examples are found in A. calcoaceticus and Gluconobacter oxydans).

Isolation and phenotypic characterization of methanol oxidation mutants of the restricted facultative methylotroph Methylophaga marina

“…The purified mutant protein of 4206 is capable of binding PQQ and upon association the absorption maxima of PQQ at 327 and 475 nm are shifted to 365 and 520 nm respectively. The shape of the resulting spectrum and the magnitude of the wavelength shifts is comparable to those observed when apoquinoprotein glucose dehydrogenase is reconstituted with PQQ (Dokter et al, 1986). This indicates a strong interaction between PQQ and the protein.…”

Glucose dehydrogenase of a rhizobacterial strain of Enterobacter asburiae involved in mineral phosphate solubilization shares properties and sequence homology with other members of enterobacteriaceae

“…The enzyme lost more than 55% activity when incubated with 2 mM EDTA. Based on the sensitivity to EDTA and as described by Dokter et al, 3 glucose dehydrogenase of E. asburiae may be grouped under Type I A full-length glucose dehydrogenase of E. asburiae was cloned and expressed as a His-tag fusion facilitating the purifi cation using the Ni-NTA column. Since the enzyme fi had a wide substrate range in vitro, genetic manipulation of gcd to further improve the temperature or EDTA tolerance d without compromising on the enzyme activity holds great promise for a variety of applications.…”

Mutants of Escherichia coli producing pyrroloquinoline quinone

“…To check that potassium ferrocyanide, accumulated during gluconic acid production in the system described above, did not interfere with gluconic acid determination, the membrane preparation was replaced by a sample of crude extract from Acinetobacter calcoaceticus and glucose was replaced by arabinose. A. calcoaceticus extracts contain a high level of GDH (Dokter et al, 1986) which can oxidize several aldoses including arabinose (Goosen et al, 1987). Accumulation of potassium ferrocyanide to a concentration of 1.5 m M did not alter the blank controls of the gluconate assay system, nor the determination of known amounts of added gluconate.…”

“…1 .99.17) from Gram-negative heterotrophic bacteria. The latter enzyme has been studied mainly in Acinetobacter calcoaceticus, which in fact contains two distinct GDHs : a soluble periplasmic one composed of two identical subunits of 48-54 kDa (Dokter et al, 1986;Ggiger & Gorisch, 1986) and a membrane-bound monomeric GDH of 83 kDa (Matsushita et al, , 1988Cleton-Jansen et al, 1988).…”