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( Hongwei Guo ),( Jinyao Han ),( Jingjing Wu ),( Hongwen Chen ) 한국미생물생명공학회(구 한국산업미생물학회) 2019 Journal of microbiology and biotechnology Vol.29 No.4
The engineered Aspergillus oryzae has a high NADPH demand for xylose utilization and overproduction of target metabolites. Glucose-6-phosphate dehydrogenase (G6PDH, E.C. 1.1.1.49) is one of two key enzymes in the oxidative part of the pentose phosphate pathway, and is also the main enzyme involved in NADPH regeneration. The open reading frame and cDNA of the putative A. oryzae G6PDH (AoG6PDH) were obtained, followed by heterogeneous expression in Escherichia coli and purification as a his6-tagged protein. The purified protein was characterized to be in possession of G6PDH activity with a molecular mass of 118.0 kDa. The enzyme displayed maximal activity at pH 7.5 and the optimal temperature was 50°C. This enzyme also had a half-life of 33.3 min at 40°C. Kinetics assay showed that AoG6PDH was strictly dependent on NADP+ (K<sub>m</sub> = 6.3 μM, k<sub>cat</sub> = 1000.0 s<sup>-1</sup>, k<sub>cat</sub>/K<sub>m</sub> =158.7 s<sup>-1</sup>·μM<sup>-1</sup>) as cofactor. The K<sub>m</sub> and k<sub>cat</sub>/K<sub>m</sub> values of glucose-6-phosphate were 109.7 s<sup>-1</sup>·μM<sup>-1</sup> and 9.1 s<sup>-1</sup>·μM<sup>-1</sup> respectively. Initial velocity and product inhibition analyses indicated the catalytic reaction followed a two-substrate, steady-state, ordered BiBi mechanism, where NADP<sup>+</sup> was the first substrate bound to the enzyme and NADPH was the second product released from the catalytic complex. The established kinetic model could be applied in further regulation of the pentose phosphate pathway and NADPH regeneration of A. oryzae to improve its xylose utilization and yields of valued metabolites.