#TITLE_ALTERNATIVE#

• Main Author: Egy Rahman Firdaus (NIM : 10512005), Moh.
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/29023
• Institution: Institut Teknologi Bandung

<palign="justify">GDSL esterases/lipases are new subclass of lipolytic enzymes that are unique. The catalytic serine of GDSL is located near the N terminus of polypeptide, whereas the catalytic serine of other lipolytic enzymes is rarely found at the N terminal region. Moreover, GDSL exhibit various activities due to their broaden types of substrates. This study aims to subclone a gene encoding GDSL lipase family from Bacillus aquimaris MKSC 6.2 with and without the signal sequences. The existence of the signal sequences will be used to study secretion and stability of lipolytic enzymes in the near future. The genes encoding GDSL with and without the signal sequences were amplified by the polymerase chain reaction (PCR) method using two primers containing the nucleotide sequences recognized by endonucleases NdeI and XhoI, respectively. The PCR fragment was then cloned to pGEM-T, resulting the recombinant plasmid pGEM-T-gdsl, which was confirmed by the colony PCR method, restriction analysis, and nucleotide sequencing. The next step is to ligate the gdsl fragment that had been digested by NdeI and XhoI with the expression plasmid pET30a, producing the recombinant plasmid pET30a-gdsl. The presence of gdsl in the recombinant plasmid pET30a-gdsl was confirmed by colony PCR. In silico analysis on GDSL from B. aquimaris MKSC 6.2 showed that the protein shared 90% homology to lipase from Bacillus sp and have 78% identity to GDSL lipase family from Bacillus vietnamensis. Therefore, GDSL from B. aquimaris MKSC 6.2 hopefully has unique substrates with novel biochemical and biophysical properties.<palign="justify">