Heteroexpression and functional characterization of glucose 6-phosphate dehydrogenase from industrial Aspergillus oryzae

Journal of Microbiology and Biotechnology
Hongwei GuoHongwen Chen

Abstract

The engineered Aspergillus oryzae has a high NADPH demand for the xylose utilization and overproduction of target metabolites. Glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) is one of two key enzymes in the oxidative part of the pentose phosphate pathway, which is the main enzyme responding for the NADPH regeneration. The open reading frame and cDNA of putative A. oryzae G6PDH (AoG6PDH) were obtained, which was followed by heterogeneous expression in Escherichia coli and was purified as a his6-tagged protein presently. The purified protein was characterized to be in possession of G6PDH activity with a molecular mass of 118.0 kDa. The enzyme displayed the maximal activity at pH 7.5 and the optimal temperature was 50 °C. This enzyme had half-life time of 33.3 min at 40 °C. Kinetics assay showed that AoG6PDH was strictly dependent on NADP+ (Km = 6.3 μM, kcat = 1000.0 s-1, kcat/Km =158.7 s-1·μM-1) as cofactor. The Km and kcat/Km value of glucose-6-phosphate were 109.7 s-1·μM-1 and 9.1 -1·μM-1 respectively. Initial velocity and product inhibition analyses indicated the catalytic reaction followed a two-substrate steady-state ordered BiBi mechanism, where NADP+ was the first substrate bound to the enzyme and NADPH was the sec...Continue Reading

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