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Isolation of an organic solvent-tolerant lipolytic enzyme from uncultivated microorganism.
Roh, Changhyun,Schmid, Rolf D Humana Press 2013 Applied biochemistry and biotechnology Vol.171 No.7
<P>Although the use of lipases as biocatalysts has frequently been proposed, it is yet scarcely being implemented in industrial processes. This is mainly due to the difficulties associated with the discovery and engineering of new enzymes and the lack of versatile screening methods. In this study, we screened the available strategy from a metagenomic pool for the organic solvent-tolerant lipase with enhanced performance for industrial processes. A novel lipase was identified through functional screening from a metagenomic library of activated sludge in an Escherichia coli system. The gene encoding the lipase from the metagenomic pool, metalip1, was sequenced and cloned by PCR. Metalip1 encoding a polypeptide of 316 amino acids had typical residues essential for lipase such as pentapeptide (GXSXGG) and catalytic triad sequences (Ser160, Asp260, and His291). The deduced amino acid sequence of metalip1 showed high similarity to a putative lipase from Pseudomonas sp. CL-61 (80 %, ABC25547). Metalip1 was expressed in E. coli BL21 (DE3) with a his-tag and purified using a Ni-NTA chelating column and characterized. This enzyme showed high expression level and solubility in the heterologous E. coli host. This enzyme was active over broad organic solvents. Among organic solvents examined, dimethyl formamide was the best organic solvent for metalip1. We showed that function-based strategy is an effective method for fishing out an organic solvent-tolerant lipase from the metagenomic library. Also, it revealed high catalytic turnover rates, which make them a very interesting candidate for industrial application.</P>
Screening of Crude Plant Extracts with Anti-Obesity Activity
Roh, Changhyun,Jung, Uhee Molecular Diversity Preservation International (MD 2012 INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES Vol.13 No.2
<P>Obesity is a global health problem. It is also known to be a risk factor for the development of metabolic disorders, type 2 diabetes, systemic hypertension, cardiovascular disease, dyslipidemia, and atherosclerosis. In this study, we screened crude extracts from 400 plants to test their anti-obesity activity using porcine pancreatic lipase assay (PPL; triacylglycerol lipase, EC 3.1.1.3) <I>in vitro</I> activity. Among the 400 plants species examined, 44 extracts from plants, showed high anti-lipase activity using 2,4-dinitrophenylbutyrate as a substrate in porcine pancreatic lipase assay. Furthermore, 44 plant extracts were investigated for their inhibition of lipid accumulation in 3T3-L1 cells. Among these 44 extracts examined, crude extracts from 4 natural plant species were active. <I>Salicis Radicis Cortex</I> had the highest fat inhibitory activity, whereas <I>Rubi Fructus</I>, <I>Corni Fructus</I>, and <I>Geranium nepalense</I> exhibited fat inhibitory capacity higher than 30% at 100 μg/mL in 3T3-L1 adipocytes, suggesting anti-obesity activity. These results suggest that four potent plant extracts might be of therapeutic interest with respect to the treatment of obesity.</P>
Nanoparticles induced by ultraviolet rays support electrons for enzymatic reaction
Roh, Changhyun,Yun, Jae Young,Kang, Chankyu,Hong, Jong Wook John Wiley Sons, Ltd. 2012 Journal of chemical technology and biotechnology Vol.87 No.7
<P><B>Abstract</B></P><P><B>BACKGROUND:</B> The NAD(P)H cofactor, which is the reduced form of nicotinamide adenine dinucleotide phosphate, plays the important role of electron donor in enzymatic metabolism. The primary drawback of metabolism reactions involving enzymes is the dependence on this highly expensive cofactor.</P><P><B>RESULTS:</B> Electron formation was effectively triggered by inducing Qdots with (UV) irradiation at 365 nm. The analytical potential of the Qdots‐based electron‐transfer system was demonstrated by examining two different types of cofactors, i.e. chemical (NADPH) and nanoparticle (Qdots). The Qdots‐based method gave promising results in comparison with the NADPH‐dependent system. A Qdots‐based electron supply to a CYP2B4 and 7‐pentoxyresorufin incubation mixture produced a resorufin metabolite. The kinetic rates of the cytochrome P450 reactions induced by photoactivated Qdots were determined</P><P><B>CONCLUSIONS:</B> The influence of electron formation from ultraviolet (UV)‐induced quantum dots (Qdots) on the cytochrome P450 (CYP2B4) reaction was elucidated. The process comprised UV‐induced electron formation by photoactivated Qdots and subsequent enzyme reactions mediated by the resultant electron supply. Copyright © 2012 Society of Chemical Industry</P>