#TITLE_ALTERNATIVE#

• Main Author: ANDRIANI (NIM : 20512028) ; Pembimbing Prof. Dr. Euis Holisotan Hakim, Dr. Rukman Hertadi, LILI
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/18838
• Institution: Institut Teknologi Bandung

Morus is one of the most important genus belongs to the family Moraceae, beside <br /> <br /> <br /> <br /> <br /> Artocarpus and Ficus. Some of these spesies are used as food source for <br /> <br /> <br /> <br /> <br /> silkworms, as fruit, and its stem has been used to building materials. Plants of <br /> <br /> <br /> <br /> <br /> Morus comprise plenty of phenolic constituents, including Diels-Alder type <br /> <br /> <br /> <br /> <br /> adducts, flavonoids, 2-arylbenzofurans, and stilbenes, which showed various <br /> <br /> <br /> <br /> <br /> bioactivities (e.g antifungal, antioxidant, etc). Study on Morus which is developed <br /> <br /> <br /> <br /> <br /> by the tissue culture technique, produced phenolic compounds, mainly Diels- <br /> <br /> <br /> <br /> <br /> Alder type adducts. Diels-Alder type adducts compounds produced by this plant <br /> <br /> <br /> <br /> <br /> are formed by cycloaddition reaction between chalcone as dienophille and <br /> <br /> <br /> <br /> <br /> dehydroprenyl phenol (e.g dehydroprenyl stilbene, dehydroprenyl flavonoids, or <br /> <br /> <br /> <br /> <br /> dehydroprenyl-2-arylbenzofuran) as diene. This reaction is suggested to be <br /> <br /> <br /> <br /> <br /> catalyzed by Diels-Alderase in the biosynthetic pathway. Previous study showed <br /> <br /> <br /> <br /> <br /> that Diels-Alder type adducts were mainly produced in root culture of Morus, so <br /> <br /> <br /> <br /> <br /> hence the present work used the root culture of M. cathayana as the source of <br /> <br /> <br /> <br /> <br /> enzyme, while as substrate use isobavachalcone. First investigation about <br /> <br /> <br /> <br /> <br /> detection of enzymatic activity from Morus have been reported, and also optimum <br /> <br /> <br /> <br /> <br /> condition of Diels-Alderase. To date, no report for the characterization of an <br /> <br /> <br /> <br /> <br /> enzyme, and catalyzing a Diels-Alder has been made. Therefore, in the objective <br /> <br /> <br /> <br /> <br /> of this study it to isolate and characterize Diels-Alderase from Morus plant. The <br /> <br /> <br /> <br /> <br /> steps of enzyme isolation include homogenation, extraction, fractionation and <br /> <br /> <br /> <br /> <br /> purification. Homogenation of enzyme was carried by adding liquid nitrogen, and <br /> <br /> <br /> <br /> <br /> iv <br /> <br /> <br /> <br /> <br /> the culture was ruptured via mechanic process use mortar and pastle. Addition the <br /> <br /> <br /> <br /> <br /> extraction buffer to stabilize extracted protein, and supernatant as crude enzyme <br /> <br /> <br /> <br /> <br /> of Diels-Alderase. The substrate was diluted with phosphate buffer and was added <br /> <br /> <br /> <br /> <br /> crude enzyme, the assay mixture was incubated at 37oC for 16 hour. The extracts <br /> <br /> <br /> <br /> <br /> were analyzed by reverse phase HPLC, with solvent system methanol : water <br /> <br /> <br /> <br /> <br /> (0,1% phosphate acid). The product of the enzymatic reaction was verified by coinjection <br /> <br /> <br /> <br /> <br /> of authentic samples in HPLC analysis, and monitored by UV detectors <br /> <br /> <br /> <br /> <br /> at 370 nm. Chromatogram standard for substrate and product arise at 4 min and 8 <br /> <br /> <br /> <br /> <br /> min respectively. The HPLC profile showed a retention time of kuwanon V (the <br /> <br /> <br /> <br /> <br /> reaction product) at 8 min, which was simillar to that of the standard product. <br /> <br /> <br /> <br /> <br /> Further analyses by LC-MS revealed that the indicated peak appear at m/z value = <br /> <br /> <br /> <br /> <br /> 647 (M+H)+,which is corresponds with the molecular weight of kuwanon V. After <br /> <br /> <br /> <br /> <br /> succesfully detection of enzyme Diels-Alder activity, then fractionation using <br /> <br /> <br /> <br /> <br /> ammonium sulfate precipitation, and fraction 40-60% which gave the highest <br /> <br /> <br /> <br /> <br /> Diels-Alderase activity. SDS PAGE also gave further confirmation, which show <br /> <br /> <br /> <br /> <br /> the major band at this fraction. After that, reproduce samples of Diels-Alderase <br /> <br /> <br /> <br /> <br /> and determination of protein concentration using Bradford method. Partial <br /> <br /> <br /> <br /> <br /> purification of Diels-Alderase was conducted using ion exchange <br /> <br /> <br /> <br /> <br /> chromatography, and the result was evaluated with SDS PAGE. Elution with <br /> <br /> <br /> <br /> <br /> phospate buffer containing NaCl concentrations of 300 mM and 500 mM showed <br /> <br /> <br /> <br /> <br /> major band with molecular weight of 40-60 kDa. The enzyme fractions from the <br /> <br /> <br /> <br /> <br /> NaCl 500 mM elution showed the highest activity, with a specific activity of <br /> <br /> <br /> <br /> <br /> 790,08 Unit/mg of protein, where this value is equivalent to 14 times purity <br /> <br /> <br /> <br /> <br /> compare to ammonium sulfate precipitation. From these results, it can be <br /> <br /> <br /> <br /> <br /> concluded that, Diels-Alderase has been succesfully isolated from the root culture <br /> <br /> <br /> <br /> <br /> of M. cathayana, and the partially purified enzyme approximately has molecular <br /> <br /> <br /> <br /> <br /> weight of 40-60 kDa.