Karakterisasi alga hijau Dunaliella sp. dan isolat sianobakteria serta deteksi gen DXS penyandi enzim kunci biosintesis karotenoid

KUSUMANINGRUM, Hermin Pancasakti, Promotor Prof. Dr. Ir. Joedoro Soedarsono

2008 | Disertasi |

Biosintesis karotenoid diketahui menggunakan jalur mevalonat (MVA). Alga hijau cenderung menggunakan jalur non-MVA (non-MVA/ D-1-deoksixilulose-5-fosfat (DOXP)/2C-metil-D- Eritritol-4-Fosfat (MEP). Tahap penting biosintesis karotenoid pada jalur non-MVA ditentukan oleh enzim Dxs yang disandi oleh gen D-1-deoksixilulose-5- fosfat Sintase (DXS). Alga hijau Dunaliella sp. dan Isolat Sianobakteria dari BBAP Jepara merupakan pakan alami sumber karotenoid pada hewan akuakultur. Karakterisasi mikrobiologis, ekofisiologis dan molekuler telah dilakukan terhadap kedua jasad. Penelitian yang dilakukan bertujuan untuk mendeteksi gen DXS, guna mengetahui apakah Dunaliella sp. dan Isolat Sianobakteria juga me ngikuti jalur non-MVA untuk biosintesis karotenoidnya. Metode penelitian menggunakan teknik Polymerase Chain Reaction (PCR) menggunakan primer E. coli, tanaman dan Synechocystis. Uji pendukung dilakukan menggunakan senyawa penghambat jalur MVA lovastatin dan hibridisasi. Penelitian telah memperoleh isolat murni Sianobakteria. Karakterisasi morfologis, ekofisiologis dan molekuler menunjukkan Isolat Sianobakteria merupakan alga prokariot dan Dunaliella sp. merupakan alga eukariot. Isolat Sianobakteria berkeserupaan tinggi dengan Cyanobacterium MBIC 1210 dan Synechocystis. Dunaliella sp. Merupakan spesies Dunaliella salina dengan keserupaan 99%. Hasil amplifikasi memperoleh beberapa fragmen DNA berkeserupaan sebesar 9 – 50% dengan gen DXS tanaman, alga, bakteri dan sianobakteria. Keserupaan yang rendah didukung oleh penghambatan lovastatin dan hasil hibridisasi memperlihatkan kedua isolat alga tidak memiliki gen DXS.

Carotenoid is known to be synthesised through the well known mevalonate (MVA) pathway. Recently, a non-mevalonate pathway (non-MVA) was discovered in a green algae. The limiting step for carotenoid biosynthesis in the non-MVA/DOXP/MEP pathway was catalysed by 1-Deoxy-D-xylulose-5-phosphate Synthase enzyme, encoded by the DXS gene. The aims of this study were to characterize two local isolates of a green algal species from Jepara, Dunaliella sp. and a Cyanobacterial isolates and to elucidate the possible carotenoid synthesis pathway employed in the two isolates. Morphological, ecophysiological and molecular characterization have been conducted on two local isolates. Elucidation of the pathway was carried out by analysing the presence of the DXS gene on the two isolates as it has been widely understood that the DXS gene is the key and limiting enzyme in the non-MVA pathway. Detection of the DXS gene was conducted by PCR amplification of the gene of interest using Escherichia coli, plants and Synechocystis gene amplification primers. The mevalonate inhibitor lovastatin and hibridization methods was carried out to reaffirming PCR result. Morphological, ecophysological, and molecular characterization demonstrated that Cyanobacterial isolate was a prokaryotic algae, while Dunaliella sp. was a eukaryotic algae. Cyanobacterial isolate demonstrates striking homology with Cyanobacterium MBIC 1210 and Synechocystis. Dunaliella sp., on the other hand, was a species of Dunaliella salina showing 99% homologies. Detection of the DXS gene in the two isolates showed about 9 – 50% homology with the known the DXS gene. Low homology with the current DNA amplification approach supported with biochemical experiment using lovastatin and hybridization result suggesting that the DXS gene was absent in the two isolates.

Kata Kunci : Karotenoid,Non-MVA,DXS,Sianobakteria,Dunaliella sp