BIOTRANSFORMATION OF PRENYLATED PHENOLIC COMPOUND USING DIELS-ALDERASE FROM ROOTS CULTURE OF Morus shalun

• Main Author: KURNIAWAN (NIM : 20515024), RAHMAT
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/30148
• Institution: Institut Teknologi Bandung

<p align="justify">Morus is one of the important genus of the Moraceae family that produces secondary metabolites of various phenolic compound such as flavonoids, stilbenoid, 2-arylbenzofuran, and Diels-Alder adduct. The phenolic compounds of the Morus are unique by the presence of a prenyl group, this group leads to the structural diversity of prenylphenol compounds. Prenylphenol compounds generally have higher bioactivity compared to their non-prenylated precursor. Prenyl group may undergo oxidation reaction to generate dehydroprenyl phenol, which is an important precursor in the formation of Diels-Alder adduct. Diels-Alder adduct of the Morus plant are typically formed from a dienes of dehydroprenyl phenol with a dienophiles of &#945;,&#946;-unsaturated carbonyl of a chalcone. This group of compounds has bioactivity as an antioxidant, antibacterial, anti-inflammatory, and anticancer. <br /> Plant tissue culture technique was used for propagation of Morus root culture sample. This biotechnology method is friendly to environment and can be an alternative for the development of samples from plants. Plant tissue culture has various advantages such as can produce plant culture that is free from contamination from microorganisms. Studies on Morus plants developed in tissue culture showed that Diels-Alder adduct were main metabolites and had cytotoxic activity against murine leukemia P388 cells. In this study an investigation of the Diels-Alderase enzyme that involved in the biosynthesis of the Diels-Alder adduct by the cycloadditon [4 + 2] reaction. Root culture of M. shalun were used as the source of the enzyme. Diels-Alderase enzyme was obtained by homogenizing the root culture of M. shalun by using mortar in the presence of liquid nitrogen. Crude enzyme was extracted by phosphate buffer pH 6.5 followed by sonication and centrifugation at 5000 rpm for 30 min in 4 o C. Ammonium sulfate precipitation was carried out for enzyme fractionation, followed by dialysis by using 20 mM phosphate buffer pH 6.5. Enzyme purification was performed by ion exchange chromatography method using DEAE anion resin. This purification covers preparation step and rsin equilibration, protein-resin binding and elution NaCl solution with various concentration. Protein content was determined by using Bradford method and protein molecule mass was amalyzed by SDS PAGE. The assesment of the activity of Diels-Alderase was performed by incubating the enzyme fraction with 1 mM morachalcone A as a substrate at 37 o C for 2 hours and the product kuwanon J performs detection with HPLC. Morachalcone A is a secondary metabolite of the chalcone group isolated from the Morus plant. This compound has a dienophile from &#945;,&#946;-unsaturated carbonyl of a chalcone structure and a diene from the prenyl group that is oxidized to be dehydroprenylphenol. <br /> Morachalcone A and dehydroprenyl-moracalkon A are expected to undergo an intermolecular cycloaddition reaction resulting in a kuwanon J, a Diels-Alder adduct that has cytotoxic activity against murine leukemia P388 cells (IC50 5.9 &#956;g/mL) and as anti-HIF 1 (hypocia inducible factor ). Isolation of Diels-Alderase enzyme from root culture Morus shalun was first reported from this plant tissue. The Diels-Alder enzyme is only reported from microorganism that catalyze the intramolecules cycloadition [4+2] reaction, while the isolated Diels-Alderase enzyme catalyzes the intermolecular cycloadition [4+2] reaction between a dehydroprenyl-moracalkon A as a diene with &#945;,&#946;-carbonyl unsaturated groups from moracalkon A as dienophile produces the kuwanon J compound. The purified Diels-Alderase enzyme has a molecular mass around 20 kDa and 40 kDa, with a specific activity of 6139,05 Units/mg and an enzyme purity improvement of 32 times compared to the ammonium sulfate fractions. Analysis of the results of enzymatic reactions with LCMS showed peaks in chromatograms having a retention time equal to the standard kuwanon J with molecular mass m/z 679.42 [M+H]+ confirming the activity of Diels-Alderase enzyme from root culture of M. shalun.<p align="justify">