Limits...
Characterization of the Kluyveromyces marxianus strain DMB1 YGL157w gene product as a broad specificity NADPH-dependent aldehyde reductase.

Akita H, Watanabe M, Suzuki T, Nakashima N, Hoshino T - AMB Express (2015)

Bottom Line: In the present study, the YGL157w gene product (KmGRE2) was purified to homogeneity from overexpressing Escherichia coli cells and found to be a monomer.The optimal pH for methylglyoxal reduction was 5.5.Taken together, these results indicate that KmGRE2 is potentially useful as an inhibit decomposer in E. coli cells.

View Article: PubMed Central - PubMed

Affiliation: Biomass Refinery Research Center, National Institute of Advanced Industrial Sciences and Technology (AIST), 3-11-32 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-0046 Japan.

ABSTRACT
The open reading frame YGL157w in the genome of the yeast Kluyveromyces marxianus strain DMB1 encodes a putative uncharacterized oxidoreductase. However, this protein shows 46% identity with the Saccharomyces cerevisiae S288c NADPH-dependent methylglyoxal reductase, which exhibits broad substrate specificity for aldehydes. In the present study, the YGL157w gene product (KmGRE2) was purified to homogeneity from overexpressing Escherichia coli cells and found to be a monomer. The enzyme was strictly specific for NADPH and was active with a wide variety of substrates, including aliphatic (branched-chain and linear) and aromatic aldehydes. The optimal pH for methylglyoxal reduction was 5.5. With methylglyoxal as a substrate, the optimal temperature for enzyme activity at pH 5.5 was 45°C. The enzyme retained more than 70% of its activity after incubation for 30 min at temperatures below 35°C or at pHs between 5.5 and 9.0. In addition, the KmGRE2-overexpressing E. coli showed improved growth when cultivated in cedar hydrolysate, as compared to cells not expressing the enzyme. Taken together, these results indicate that KmGRE2 is potentially useful as an inhibit decomposer in E. coli cells.

No MeSH data available.


Related in: MedlinePlus

Effects of pH and temperature on KmGRE2 activity and stability. The markers of buffer were indicated following: circles, acetate; squares, citrate; diamonds, phosphate, isosceles triangles, borate-NaOH and right triangles, bicarbonate-NaOH. (A) Effect of pH on KmGRE2 activity. (B) Effect of temperature on KmGRE2 activity. (C) KmGRE2 activity after incubation for 30 min at various temperatures in the 20 mM Tris–HCl buffer (pH 7.2). (D) KmGRE2 activity after incubation for 30 min at 35°C in buffer solutions of various pHs. Error bars indicate SE (n = 3).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4385108&req=5

Fig3: Effects of pH and temperature on KmGRE2 activity and stability. The markers of buffer were indicated following: circles, acetate; squares, citrate; diamonds, phosphate, isosceles triangles, borate-NaOH and right triangles, bicarbonate-NaOH. (A) Effect of pH on KmGRE2 activity. (B) Effect of temperature on KmGRE2 activity. (C) KmGRE2 activity after incubation for 30 min at various temperatures in the 20 mM Tris–HCl buffer (pH 7.2). (D) KmGRE2 activity after incubation for 30 min at 35°C in buffer solutions of various pHs. Error bars indicate SE (n = 3).

Mentions: The effect of pH on the reduction of methylglyoxal was determined by assessing the enzyme activity at several pHs. At a temperature of 25°C, the optimum pH was about 5.5 (Figure 3A). When the temperature dependence at pH 5.5 was examined, maximum activity was observed at around 45°C (Figure 3B). Moreover, when KmGRE2 was incubated for 30 min at various temperatures in 20 mM Tris–HCl buffer (pH 7.2), KmGRE2 retained more than 80% of its activity at temperatures below 35°C (Figure 3C). On the other hand, there was a complete loss of activity when the enzyme was incubated at temperatures above 45°C. When the effect of pH on the stability of the enzyme was evaluated based on the activity remaining after incubation at 35°C for 30 min, we found that KmGRE2 retained more than 70% of its activity at pHs between 5.5 and 9.0 (Figure 3D). Somewhat disappointingly, however, KmGRE2 showed nearly the same temperature and pH stability as S. cerevisiae GRE2 (Park et al. 2010).Figure 3


Characterization of the Kluyveromyces marxianus strain DMB1 YGL157w gene product as a broad specificity NADPH-dependent aldehyde reductase.

Akita H, Watanabe M, Suzuki T, Nakashima N, Hoshino T - AMB Express (2015)

Effects of pH and temperature on KmGRE2 activity and stability. The markers of buffer were indicated following: circles, acetate; squares, citrate; diamonds, phosphate, isosceles triangles, borate-NaOH and right triangles, bicarbonate-NaOH. (A) Effect of pH on KmGRE2 activity. (B) Effect of temperature on KmGRE2 activity. (C) KmGRE2 activity after incubation for 30 min at various temperatures in the 20 mM Tris–HCl buffer (pH 7.2). (D) KmGRE2 activity after incubation for 30 min at 35°C in buffer solutions of various pHs. Error bars indicate SE (n = 3).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4385108&req=5

Fig3: Effects of pH and temperature on KmGRE2 activity and stability. The markers of buffer were indicated following: circles, acetate; squares, citrate; diamonds, phosphate, isosceles triangles, borate-NaOH and right triangles, bicarbonate-NaOH. (A) Effect of pH on KmGRE2 activity. (B) Effect of temperature on KmGRE2 activity. (C) KmGRE2 activity after incubation for 30 min at various temperatures in the 20 mM Tris–HCl buffer (pH 7.2). (D) KmGRE2 activity after incubation for 30 min at 35°C in buffer solutions of various pHs. Error bars indicate SE (n = 3).
Mentions: The effect of pH on the reduction of methylglyoxal was determined by assessing the enzyme activity at several pHs. At a temperature of 25°C, the optimum pH was about 5.5 (Figure 3A). When the temperature dependence at pH 5.5 was examined, maximum activity was observed at around 45°C (Figure 3B). Moreover, when KmGRE2 was incubated for 30 min at various temperatures in 20 mM Tris–HCl buffer (pH 7.2), KmGRE2 retained more than 80% of its activity at temperatures below 35°C (Figure 3C). On the other hand, there was a complete loss of activity when the enzyme was incubated at temperatures above 45°C. When the effect of pH on the stability of the enzyme was evaluated based on the activity remaining after incubation at 35°C for 30 min, we found that KmGRE2 retained more than 70% of its activity at pHs between 5.5 and 9.0 (Figure 3D). Somewhat disappointingly, however, KmGRE2 showed nearly the same temperature and pH stability as S. cerevisiae GRE2 (Park et al. 2010).Figure 3

Bottom Line: In the present study, the YGL157w gene product (KmGRE2) was purified to homogeneity from overexpressing Escherichia coli cells and found to be a monomer.The optimal pH for methylglyoxal reduction was 5.5.Taken together, these results indicate that KmGRE2 is potentially useful as an inhibit decomposer in E. coli cells.

View Article: PubMed Central - PubMed

Affiliation: Biomass Refinery Research Center, National Institute of Advanced Industrial Sciences and Technology (AIST), 3-11-32 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-0046 Japan.

ABSTRACT
The open reading frame YGL157w in the genome of the yeast Kluyveromyces marxianus strain DMB1 encodes a putative uncharacterized oxidoreductase. However, this protein shows 46% identity with the Saccharomyces cerevisiae S288c NADPH-dependent methylglyoxal reductase, which exhibits broad substrate specificity for aldehydes. In the present study, the YGL157w gene product (KmGRE2) was purified to homogeneity from overexpressing Escherichia coli cells and found to be a monomer. The enzyme was strictly specific for NADPH and was active with a wide variety of substrates, including aliphatic (branched-chain and linear) and aromatic aldehydes. The optimal pH for methylglyoxal reduction was 5.5. With methylglyoxal as a substrate, the optimal temperature for enzyme activity at pH 5.5 was 45°C. The enzyme retained more than 70% of its activity after incubation for 30 min at temperatures below 35°C or at pHs between 5.5 and 9.0. In addition, the KmGRE2-overexpressing E. coli showed improved growth when cultivated in cedar hydrolysate, as compared to cells not expressing the enzyme. Taken together, these results indicate that KmGRE2 is potentially useful as an inhibit decomposer in E. coli cells.

No MeSH data available.


Related in: MedlinePlus