STUDY OF DIELS-ADERASE WHICH RESPONSIBLE IN BIOSYNTHESIS OF SECONDARY METABOLITES IN ROOT CULTURE OF MORUS CATHAYANA
Morus plants, or locally known as “murbei”, have been widely cultivated for feeding silkworm in silk industry in many countries. Phytochemistry study showed this plants produce phenolic compounds as secondary metabolite, mainly stilbenes, 2- arylbenzofurans, and flavonoids. Research on Morus which...
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| Format: | Theses |
| Language: | Indonesia |
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| Online Access: | https://digilib.itb.ac.id/gdl/view/34926 |
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| Institution: | Institut Teknologi Bandung |
| Language: | Indonesia |
| Summary: | Morus plants, or locally known as “murbei”, have been widely cultivated for feeding silkworm in silk industry in many countries. Phytochemistry study showed this plants produce phenolic compounds as secondary metabolite, mainly stilbenes, 2- arylbenzofurans, and flavonoids. Research on Morus which plant samples developed by using tissue culture technique showed this plants are also rich of adducts Diels- Alder, cycloaddition product between dehydroprenylphenol as diene source and ?,?- unsaturated carbonyl from chalcone as dienophile. Currently there is no information regarding enzyme responsible in biosynthesis of adduct Diels-Alder namely Diels- Alderase. Initial study of Diels-Alderase was carried in this research, by analyzing Diels-Alderase activity in crude enzyme of root culture of M. cathayana. This research covers isolation of morachalcone A, a prenylchalcone which has ability as precursor to produce adduct Diels-Alder, in this case is kuwanon J. Morachalcone A was isolated by several chromatography methods. Thin layer chromatography is used to analyze isolated morachalcone A comparing with standard compound. ESI-MS analysis confirmed this morachalcone A by determination of peaks in m/z 341,77 and
204,93. Crude enzyme of Diels-Alderase was isolated from root culture of M. cathayana. Homogenation and extraction of enzyme was carried in cool temperature to maintain protein stability. Extraction buffer contains ingredients which needed to
stabilize extracted protein, such as NaCl to maintain ionic strength of protein solution, glycerol to help storing in cold temperature, dithiothreitol to prevent enzyme’s oxidation, and phenylmethylsulphonyl floride (PMSF) as protease inhibitor. Enzyme assay of Diels-Alderase was carried by incubating crude extract with morachalcone A for 24 hours in 30°C, and the reaction product was extracted by ethyl acetate. Kuwanon J as reaction product was detected by HPLC using reversed phase column. Eluen system was methanol-water in gradient composition. After successfully detection of enzyme Diels-Alder activity, fractionation of crude enzyme using ammonium sulfate precipitation resulted fraction 40-60% which gave the highest Diels-Alderase activity. Since currently there is no standard data of Diels- Alderase, SDS PAGE analysis cannot be used to predict molecular size of this enzyme. There are two optimum value of pH and temperature to produce adduct Diels-Alder. This indicates there are two enzymes which have similar catalytic function. Unfortunately, this data cannot describe whether one of this catalytic function refer to oxydase activity. In order to confirm the number of enzyme responsible in this reaction, oxydase activity assay should be done to similar crude enzyme, which continued by separation and purification of both enzyme. The result of this research can be used as preliminary data to study biosynthesis of adduct Diels- Alder from Morus plants.
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