• Journal of Microbiology and Biotechnology
• /
• v.13 no.5
• /
• pp.725-730
• /
• 2003

Three recombinant Saccharomyces cerevisiae strains showing different levels of xylose reductase activity were constructed to investigate the effects of xylose reductase activity and glucose feed rate on xylitol production. Conversion of xylose to xylitol is catalyzed by xylose reductase of Pichia stipitis with cofactor NAD(P)H. A two-substrate fermentation strategy has been employed where glucose is used as an energy source for NADPH regeneration and xylose as substrate for xylitol production. All recombinant S. cerevisiae strains Yielded similar specific xylitol productivity, indicating that xylitol production in the recombinant S. cerevisiae was more profoundly affected by the glucose supply and concomitant It generation of cofactor than the xylose reductase activity itself. It was confirmed in a continuous culture that the elevation of the glucose feeding level in the xylose-conversion period enhanced the xylitol productivity in the recombinant S. cerevisiae.

• Journal of Microbiology and Biotechnology
• /
• v.26 no.10
• /
• pp.1708-1716
• /
• 2016

Glucose dehydrogenase (GDH) is an oxidoreductase enzyme and is used as a biocatalyst to regenerate NAD(P)H in reductase-mediated chiral synthesis reactions. In this study, the glucose 1-dehydrogenase B gene (gdhB) was cloned from Bacillus thuringiensis subsp. kurstaki, and wild-type (GDH-BT^WT) and His-tagged (GDH-BT^N-His, GDH-BT^C-His) enzymes were produced in Escherichia coli BL21 (DE3). All enzymes were produced in the soluble forms from E. coli. GDH-BT^WT and GDH-BT^N-His showed high specific enzymatic activities of 6.6 U/mg and 5.5 U/mg, respectively, whereas GDH-BT^C-His showed a very low specific enzymatic activity of 0.020 U/mg. These results suggest that the intact C-terminal carboxyl group is important for GDH-BT activity. GDH-BT^WT was stable up to 65℃, whereas GDH-BT^N-His and GDH-BT^C-His were stable up to 45℃. Gel permeation chromatography showed that GDH-BT^WT is a dimer, whereas GDH-BT^N-His and GDH-BT^C-His are monomeric. These results suggest that the intact N- and C-termini are required for GDH-BT to maintain thermostability and to form its dimer structure. The homology model of the GDH-BT^WT single subunit was constructed based on the crystal structure of Bacillus megaterium GDH (PDB ID 3AY6), showing that GDH-BT^WT has a Rossmann fold structure with its N- and C-termini located on the subunit surface, which suggests that His-tagging affected the native dimer structure. GDH-BT^WT and GDH-BT^N-His regenerated NADPH in a yeast reductase-mediated chiral synthesis reaction, suggesting that these enzymes can be used as catalysts in fine-chemical and pharmaceutical industries.

• Journal of Microbiology and Biotechnology
• /
• v.27 no.5
• /
• pp.956-964
• /
• 2017

Compactin and pravastatin are competitive cholesterol biosynthesis inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase and belong to the statin drugs; however, the latter shows superior pharmacokinetic characteristics. Previously, we reported that the bacterial P450, CYP105D7, from Streptomyces avermitilis can catalyze the hydroxylation of 1-deoxypentalenic acid, diclofenac, and naringenin. Here, we demonstrate that CYP105D7 could also catalyze compactin hydroxylation in vitro. In the presence of both bacterial and cyanobacterial redox partner systems with an NADPH regeneration system, the reaction produced two hydroxylated products, including pravastatin (hydroxylated at the C6 position). The steady-state kinetic parameters were measured using the redox partners of putidaredoxin and its reductase. The $k_m$ and $k_{cat}$ values for compactin were $39.1{\pm}8.8{\mu}M$ and $1.12{\pm}0.09min^{-1}$, respectively. The $k_{cat}/K_m$ value for compactin ($0.029min^{-1}{\cdot}{\mu}M^{-1}$) was lower than that for diclofenac ($0.114min^{-1}{\cdot}{\mu}M^{-1}$). Spectroscopic analysis showed that CYP105D7 binds to compactin with a $K_d$ value of $17.5{\pm}3.6{\mu}M$. Molecular docking analysis was performed to build a possible binding model of compactin. Comparisons of different substrates with CYP105D7 were conclusively illustrated for the first time.

• Applied Biological Chemistry
• /
• v.42 no.3
• /
• pp.246-251
• /
• 1999

This experiment was carried out to elucidate and investigate the changes in the content of antioxidants and the activities of antioxidant enzymes in the leaves of soybean subjected to water stresses. The results obtained were as follows; Leaves of soybeans subjected to water stresses have showed the differences in the activities of the antioxidant enzymes. In eunhakong, the activity of APOX was increased within a few days, but that of GR was decreased, whereas the activities of APOX and GR were gradually decreased in eunhakong. The activity of MDHAR of the leaves of eunhakong subjected to drought stress was gradually increased within 4days, whereas that of flooding was increased within 2days. We are supposed that the activities of APOX and MDHAR are coupled to maintain ascorbate concentration. In eunhakong, the relative activity of DHAR subjected to flooding was higher than that of drought. These results imply that DHAR is the only enzyme participating in the regeneration of ascorbate when the activity of MDHAR was limited by the deficiency of NADPH. The contents of ascorbate and reduced glutathione subjected to drought stress decreased continually, whereas those subjected to flooding stress recovered after five days of treatment.