BIOTRANSFORMATION OF PRENYLATED PHENOLIC COMPOUND USING DIELS-ALDERASE FROM ROOTS CULTURE OF MORUS SHALUN

• Main Author: Kurniawan, Rahmat
• Format: Dissertations
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/46806
• Institution: Institut Teknologi Bandung

Morus plant, known as murbei in West Java, is a genus in the Moraceae family plant. Morus produced some chemical constituents including phenolic compounds e. g. flavonoids, stilbenes, 2-arylbenzofurans and Diels-Alder adducts compounds, which have important bioactivities. The Diels-Alder adduct compounds have some potential bioactivities, such as antioxidant, antifungal, antibacterial and cytotoxic. The presence of isoprenyl group is characteristic of phenolic compounds from Morus, which causes diversity for prenylated-phenolic compounds that had been reported from this genus. The dienes, from isoprenyl- derived group, with dienophile from ?,? unsaturated carbonyl group underwent a Diels-Alder reaction to form the methyl cyclohexene ring system, this system ring is a characteristic of the Diels-Alder adduct compound, that was catalyzed by Diels-Alderase in the plant tissue cell. Morus Shalun developed by micropropagation method has been reported producing Diels-Alder adduct as the mayor constituent from the root cultures. The root culture of M. Shalun was used as a source of the Diels-Alderase enzyme, which is an important utility in the biosynthetic pathway of the Diels-Alder adduct. Diels-Alderase was applied as biocatalyst for the biotransformation of several prenylated phenolic compounds to produce potential bioactive Diels-Alder adducts. The investigation included the micropropagation of M. shalun, Diels-Alderase purification and biotransformation. Enzyme extraction was conducted using phosphate buffer, fractionation using ammonium sulphate precipitation and purification was done using AKTA explorer with Hitrap desalting, CIM DEAE-anion and CIM QA column. The enzyme characterization included the optimization of pH, temperature and time of incubation and the determination of Diels-Alderase molecular weight using SDS-PAGE method. The enzyme activity and biotransformation product were analyzed using HPLC and LCMS analytical method. Substrates, prenylated phenolic compounds, and biotransformation products were used to study cytotoxic activity against three human cancer cell lines. This research presences first new evidence about novel natural Diels-Alderase isolated from plant tissue culture, especially from root culture of M. shalun, and confirmed the Diels-Alder activity on biosynthesis pathway of some Diels-Alder adduct compounds. (formula) Diels-Alderase enzyme from root culture of M. shalun catalyzed endo intermolecular [4+2]-cycloaddition between various prenylated-phenolic compound as source of diene and ?,? unsaturated carbonyl from morachalcone A as dienophile. The purified Diels-Alderase base on SDS-PAGE profile has single band protein about 60 kDa with specific activity value 2553.25 Units/mg and purification fold more than 64 times compared with lysis extracts. The Diels- Alderase optimal condition followed the condition of root culture growth condition at 25-30 °C, pH 7.0 and incubation optimal time at 12 hours. The biotransformation products of Diels-Alderase, were kuwanon J, chalcomoracin and kuwanol E, shown more strong activities against HeLa (cervix cancer), MCF- 7 (breast cancer) and A549 (lung cancer) compared with all the substrates. The cytotoxicity was increased due the Diels-Alder adduct compounds have double all the active functional groups, the hydroxy groups (-OH) and the carbonyl groups (- C=O), compared with the prenylated-phenolic compounds. The products still have substrates characteristic, one isoprenyl group, and add one more methyl cyclohexene system ring. Kuwanol E has potential activity against breast and lung cancer cells with IC50 values of 5.78 ± 2.41 ?M and IC50 9.86 ± 1.24 ?M respectively and chalcomoracin has potential activity against breast cancer cells IC5 7.31 ± 1.16 ?M. Diels-Alderase is non-metalloprotein has potentially develop as future biocatalyst to support ordinary synthesis method for produce some bioactive Diels-Alder adduct compound.