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Subramanian Mohan Raj,Chelladurai Rathnasingh,Woo-Chel Jung,Edwardraja Selvakumar,박성훈 한국생물공학회 2010 Biotechnology and Bioprocess Engineering Vol.15 No.1
3-Hydroxypropionic acid (3-HP), a versatile and valuable platform chemical, has diverse industrial applications; but its biological production from glycerol is often limited by the capability of the enzyme aldehyde dehydrogenase (ALDH) to convert an intermediary compound, 3-hydroxypropionaldehyde (3-HPA), to 3-HP. In this study, we report a new ALDH, PuuC, from Klebsiella pneumoniae DSM 2026, that efficiently converts 3-HPA to 3-HP. The identified gene puuC was cloned, expressed in Escherichia coli, purified, and characterized for its properties. The recombinant enzyme with a molecular weight of 53.8 kDa exhibited broad substrate specificity for various aliphatic aldehydes, especially C2-C5 aldehydes. NAD+ was the preferred coenzyme for the oxidation of most aliphatic and aromatic aldehydes tested. The optimum pH and temperature for PuuC activity were pH 8.0 and 45oC. The Km values for 3-HPA and NAD+ were 0.48 and 0.09 mM, respectively. The activity of PuuC was enhanced in the presence of reducing agents such as 2-mercaptoethanol or dithiothreitol, while several metal ions, particularly Hg2+, Ag+, and Cu2+ inhibited its activity. The predicted structure of PuuC indicated the presence of K191 and E194 in close proximity to the glycine motif, suggesting that PuuC belongs to class 2 ALDHs.
Rathnasingh, Chelladurai,Raj, Subramanian Mohan,Jo, Ji-Eun,Park, Sunghoon Wiley Subscription Services, Inc., A Wiley Company 2009 Biotechnology and bioengineering Vol.104 No.4
<P>3-Hydroxypropionic acid (3-HP) is a commercially valuable chemical with the potential to be a key building block for deriving many industrially important chemicals. However, its biological production has not been well documented. Our previous study demonstrated the feasibility of producing 3-HP from glycerol using the recombinant Escherichia coli SH254 expressing glycerol dehydratase (DhaB) and aldehyde dehydrogenase (AldH), and reported that an “imbalance between the two enzymes” and the “instability of the first enzyme DhaB” were the major factors limiting 3-HP production. In this study, the efficiency of the recombinant strain(s) was improved by expressing DhaB and AldH in two compatible isopropyl-thio-β-galactoside (IPTG) inducible plasmids along with glycerol dehydratase reactivase (GDR). The expression levels of the two proteins were measured. It was found that the changes in protein expression were associated with their enzymatic activity and balance. While cloning an alternate aldehyde dehydrogenase (ALDH), α-ketoglutaric semialdehyde dehydrogenase (KGSADH), instead of AldH, the recombinant E. coli SH-BGK1 showed the highest level of 3-HP production (2.8 g/L) under shake-flask conditions. When an aerobic fed-batch process was carried out under bioreactor conditions at pH 7.0, the recombinant SH-BGK1 produced 38.7 g 3-HP/L with an average yield of 35%. This article reports the highest level of 3-HP production from glycerol thus far. Biotechnol. Bioeng. 2009; 104: 729–739 © 2009 Wiley Periodicals, Inc.</P>
Lee, Philah,Raj, Subramanian Mohan,Zhou, Shengfang,Ashok, Somasundar,Edwardraja, Selvakumar,Park, Sunghoon 한국생물공학회 2014 Biotechnology and Bioprocess Engineering Vol.19 No.1
This study examined the role and physiological relevance of 3-hydroxyisobutyrate dehydrogenase-I (3HIBDH-I) of Pseudomonas denitrificans ATCC 13867 in the degradation of 3-hydroxypropionic acid (3-HP) during 3-HP production. The gene encoding 3HIBDH-I of P. denitrificans ATCC 13867 was cloned and expressed in Escherichia coli BL21 (DE3). The recombinant 3HIBDH-I was then purified on a Ni-NTA-HP column and characterized for its choice of substrates, cofactors, metals, reductants, and the optimal temperature and pH. The recombinant 3HIBDH-I showed a high catalytic constant ($k_{cat}/K_m$) of $604.1{\pm}71.1$ mM/S on (S)-3-hydroxyisobutyrate, but no detectable activity on (R)-3-hydroxyisobutyrate. 3HIBDH-I preferred $NAD^+$ over $NADP^+$ as a cofactor for its catalytic activity. The $k_{cat}/K_m$ determined for 3-HP was $15.40{\pm}1.43$ mM/S in the presence of $NAD^+$ at $37^{\circ}C$ and pH 9.0. In addition to (S)-3-hydroxyisobutyrate and 3-HP, 3HIBDH-I utilized L-serine, methyl-D,L-serine, and methyl-(S)-(+)-3-hydroxy-2-methylpropionate; on the other hand, the kcat/Km values determined for these substrates were less than 5.0mM/S. Ethylenediaminetetraacetic acid, 2-mercaptoethanol, dithiothreitol and $Mn^{2+}$ increased the activity of 3HIBDHI significantly, whereas the presence of $Fe^{2+}$, $Hg^{2+}$ and $Ag^+$ in the reaction mixture at 1.0 mM completely inhibited its activity. This study revealed the characteristics of 3HIBDH-I and its significance in 3-HP degradation.