ISOLATION AND CHARACTERIZATION OF SACYLTRANSFERASES FRAGMENT GENE FROM TROPICAL MARINE MICROALGAE Chlorella sp.

• Main Author: WIYATI PURNANINGTYAS (NIM : 20514050), DAYU
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/21596
• Institution: Institut Teknologi Bandung

Along with the increasing number of population in the world, the need for energy especially fossil energy has been increasing from year to year. The condition leads <br /> <br /> to energy crisis because of decreasing fossil energy. The discoveries of alternative renewable energy that can contribute to energy supply beside of fossil fuels are <br /> <br /> indispensable. Microalgae is one of the marine life that has potential as an alternative source of renewable energy. Microalgae biomass contains lipids that can be processed into biodiesel. However, its unit production costs using the existing technology are relative high. Efforts to increase the production of lipids from microalgae as a raw materials for biodiesel have been done in several ways such as conditioning of microalgae cultivation in a starving medium (reducing sources of nitrogen, carbon, sulfur, zinc, phosphorus, etc.), and by limiting the metabolic production of carbohydrates. The drawbacks of this methode is relatively low microalgae life cycle, low quality metabolism product including the quality of lipids and biomass. Other alternative to increase the production of lipids from microalgae is using genetic engineering. Report and publication related to the use of genetic engineering in the production of lipid are today still very limited. The studies of lipid biosynthesis and molecular genetics at the microalgae are also not widely published yet. Protein S-asiltransferase (PAT) is a protein that plays an important role in the process of lipid biosynthesis. The protein is a chain acyl transfer catalyst to the acceptor molecular (monogliserol or diacylglycerol) in the biosynthesis of triacylglycerol (TAG). So that without these proteins cycle, the lipid biosynthesis will stop until the formation of fatty origin only. Study of the gene encoding PAT majority has been performed on green microalgae, due to the availability of genetic information wich is more complete than the other types of microalgae. A green microalgae are easily found in the territorial Indonesian waters. They have fairly large potential of lipid which is about 21-25% of wet biomass weight. One of the green microalgae biochemical KK collection ITB there is Chlorella sp. The purpose of this study is to identify the species of green microalgae Chlorella sp. as well as to investigate the characteristics of the gene encoding Sasiltransferase of the Chlorella sp. The stages of studies gen S-asiltransferase involve purification of the Chlorella sp.’s culture, species identification of microalgae with 18S rRNA gene, isolation of the fragment gene encoding PAT through the RT-PCR technique (Reverse Transcription Polymerase Chain Reaction) and amplification of the first strand cDNA using primary collection Biochemistry ITB’s group. Cloning of the fragment gene encoding PAT into E.coli TOP10 as a host cell was done using a vector pGEM-T. Determination of the nucleotide sequence and analysis the fragment gene encoding PAT were done using some software DNA Baser, BLAST, MEGA6, Bioedit, Expasy, and the Swiss model. Identification species of microalgae Chlorella sp. using 18S rRNA gene has shown affinity with Chlorella vulgaris species KF574391.1 (nucleotide similarity are 99%). RT-PCR and first strand cDNA amplification using primers ZcoF3 and ZCoChR1a generate cDNA fragments amplicon size of ± 600 pb. The results of nucleotide and amino acid sequences analysis indicate the closeness of the translation with the PAT protein sequences from Chlorella variabilis XP 005749864.1 (amino acid similarity of 51%). While the 3-dimensional structure <br /> <br /> prediction of PAT fragments indicated the same motive functionality as PAT fragment of Chlorella variabilis XP 005749864.1, namely as a transporter protein.