#TITLE_ALTERNATIVE#

• Main Author: PRIMA SARI (NIM : 20512047) ; Pembimbing M. Asyari, M.Si, IRA
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/18716
• Institution: Institut Teknologi Bandung

The existence of rubber or gum in crude oil results of extraction of sunflower <br /> <br /> <br /> <br /> <br /> seed, soybeans, and palm oils causing a decrease in the quality oil production. <br /> <br /> <br /> <br /> <br /> This is because such contaminant can form bubles and the oil become murky. It <br /> <br /> <br /> <br /> <br /> is therefore, the removal of gum or deguming is the important key in the process <br /> <br /> <br /> <br /> <br /> of refining crude oil. One of the efforts that are being developed currently is the <br /> <br /> <br /> <br /> <br /> use of enzymes that can eliminate gum, phospholipase group of enzymes. <br /> <br /> <br /> <br /> <br /> Phospholipase is belong to the hydrolases class of enzymes that hydrolyze <br /> <br /> <br /> <br /> <br /> phospholipid in accordance with the position of the hydrolysis by each enzyme <br /> <br /> <br /> <br /> <br /> (PLA1, PLA2, PLB, PLC, PLD). This process is more environmentally friendly <br /> <br /> <br /> <br /> <br /> as it can reduce the use of chemicals. In addition, the quality of the oil produced <br /> <br /> <br /> <br /> <br /> is better than any other process. Beside for degumming, phospholipase is also <br /> <br /> <br /> <br /> <br /> widely used in food industry, such as breads, sauces, mayonaise and as <br /> <br /> <br /> <br /> <br /> emulsifiers, processing and production of milk and cheese. <br /> <br /> <br /> <br /> <br /> Based on the analysis of the gene sequence from the bacterial genomics of <br /> <br /> <br /> <br /> <br /> Halomonas elongata, it was revealed that BK-AB8 strains have a gene sequence <br /> <br /> <br /> <br /> <br /> of phospholipase A1 (PLA1). Phospholipase A1 (EC 3.1.1.32) is a group of <br /> <br /> <br /> <br /> <br /> enzymes that hydrolyze the bond in phospholipid at sn-1 position. The objective <br /> <br /> <br /> <br /> <br /> of this work is to the isolate gene of phospholipase A1 from H. elongata BKAB8 <br /> <br /> <br /> <br /> <br /> strains such that it can be well expressed and producing a new variant of <br /> <br /> <br /> <br /> <br /> the phospholipase A1. The genes isolations was carried out by the following step <br /> <br /> <br /> <br /> <br /> : primers design, DNA chromosome isolation, PCR, and cloning. <br /> <br /> <br /> <br /> <br /> The primer design was determined based on phospholipase A1 gene sequences of <br /> <br /> <br /> <br /> <br /> the bacterial sample. The sequence of 5 '-ATG AAG CCC TCA CGA TTT CTC <br /> <br /> <br /> <br /> <br /> CTC CC-3 ' was selected as a forward primer (fPLA1H) and the sequence of the <br /> <br /> <br /> <br /> <br /> iv <br /> <br /> <br /> <br /> <br /> 5 '-GCG TCA CTC CTC GAA GCC CGA G-3 ' was selected as a reverse primer <br /> <br /> <br /> <br /> <br /> (rPLA1H). Chromosomal DNA isolation that has been successfully used as a <br /> <br /> <br /> <br /> <br /> template for gene amplification of phospholipase A1. The gene amplification <br /> <br /> <br /> <br /> <br /> was carried out by PCR method, in which the temperature for denaturation, <br /> <br /> <br /> <br /> <br /> annealing, and polymerization stages were set at 94oC, 58,8oC, 72oC <br /> <br /> <br /> <br /> <br /> respectively, to 34 times. DNA fragment of ~ 1 kb in length was observed in <br /> <br /> <br /> <br /> <br /> electrophoresis indicating that phospholipase A1 gene has been successfully <br /> <br /> <br /> <br /> <br /> amplified and purified. <br /> <br /> <br /> <br /> <br /> Fragments of phospholipase A1 was then ligated into the cloning vector pGEMT <br /> <br /> <br /> <br /> <br /> Easy and transformed into host cells of E. coli TOP10. The selection was <br /> <br /> <br /> <br /> <br /> carried out based on the resistance of transformants against to ampiciline. <br /> <br /> <br /> <br /> <br /> Selected transformans were confirmed by PCR colonies and restriction analysis. <br /> <br /> <br /> <br /> <br /> Two transformant’s colonies has been confirmed to carry recombinant <br /> <br /> <br /> <br /> <br /> pGT_plA1. The sequencing results to the colony suspected to carry pGT_plA1 <br /> <br /> <br /> <br /> <br /> plasmid confirmed that the transformant was positively carring the intact of <br /> <br /> <br /> <br /> <br /> phospholipase A1 gene. It was known from the presence of start and a stop <br /> <br /> <br /> <br /> <br /> codon in the gene sequence. Nucleotide sequence of phospholipase A1 H. <br /> <br /> <br /> <br /> <br /> elongata BK-AB8 has identity level about 99% with phospholipase A1 from H. <br /> <br /> <br /> <br /> <br /> elongata DSM 2581. Phospholipase A1 H. elongata BK-AB8 encode by 343 <br /> <br /> <br /> <br /> <br /> amino acids residues with an approximate molecular weight of about 39,5 kDa. <br /> <br /> <br /> <br /> <br /> 3D structural model of phospholipase A1 H. elongata BK-AB8 homolog with <br /> <br /> <br /> <br /> <br /> outer membrane phospholipase A1 structure from E.coli (PDB: 1im0) with the <br /> <br /> <br /> <br /> <br /> identity level about 38,02%. The result of our work confirmed that <br /> <br /> <br /> <br /> <br /> phospholipase A1 gene was successfully isolated from H. elongata BK-AB8 <br /> <br /> <br /> <br /> <br /> strains.