Purification, characterization and gene identification of a membrane-bound glucose dehydrogenase from 2-keto-d-gluconic acid industrial producing strain Pseudomonas plecoglossicida JUIM01

Abstract

The membrane-bound glucose dehydrogenase (mGDH) is a rate-limiting enzyme for the industrial production of 2-keto-d-gluconic acid (2KGA) from glucose. In this study, mGDH was firstly purified from a 2KGA industrial producing strain Pseudomonas plecoglossicida JUIM01. The purified mGDH exhibited a specific activity of 16.85 U/mg and was identified as monomeric membrane-bound PQQ-dependent dehydrogenase with a molecular mass of ~87 kDa. The K_m and V_max value of d-glucose were 0.042 mM and 14.620 μM/min, and the optimal pH and temperature were of 6.0 and 35 °C with favorable acid resistance and poor heat tolerance. Ca²⁺/Mg²⁺ showed a significantly positive effect on mGDH activity with 20% increase, whereas EDTA/EGTA had a negative influence, and Ca²⁺ was essential for enzyme activity. Furthermore, a 2412 bp-length gcd was amplified by genome walking technique and heterologously expressed in Escherichia coli. Bioinformatics analysis and heterologous expression further confirmed it as a mGDH encoding gene. mGDH contained binding sites of Ca²⁺, cofactor PQQ and polypeptide binding sites concluded from alignment results of mGDHs from different genera. This study would lay the foundation for improving 2KGA productivity through further strain modification.

Keywords: 2-Keto-d-gluconic acid (2KGA); Gene identification; Glucose dehydrogenase; Pseudomonas plecoglossicida; Purification.