CLONING, CHARACTERIZATION AND EXPRESSION OF GENE ENCODING THERMOSTABLE LIPASE FROM COMPOST THROUGH METAGENOMIC APPROACH

• Main Author: Nurhasanah
• Format: Dissertations
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/32825
• Institution: Institut Teknologi Bandung

Lipase is one of hydrolase enzyme that has many roles within the industries. This is due to lipase has the ability to carry out the reaction in both interface and other solvents. This enzyme has a distinctive character that is widely used in various industrial fields such as food industry, pharmaceuticals, detergents, paper, biodiesel, waste treatment, textile, medical and synthetic of organic compounds. The fact that lipase is broad application and the world’s increasing need on this enzyme, drive the researcher to explore more lipase from natural resourses. One approach that is currently done for exploring the lipase is through culture independent approach called metagenom approach. This study aims to get the diversity of the gene encoding thermostable lipases of compost and know the nature of the lipase produced. In this study, the samples were collected from thermophilic phase of compost (45 ° C - 68 ° C). Total DNA was isolated from compost community directly without cultivation. The gene encoding lipase isolated by PCR using several pairs of primers have been designed by some previous researchers in the laboratory of Biochemistry ITB. PCR products were formed from each primary have been cloned to the cloning vector pJet1.2/blunt and followed by the determination nucleotide sequence. Nucleotide sequence alignment several clones obtained showed that five genes encoded lipases. The results of translation in silico of the fifth nucleotide sequences showed high identity to the genus of Pseudomonas and have the closest identitiy with clones of Pseudomonas stutzeri lipase (AID66451.1) with the level of identity between 96-100%. While the aligment among samples produces the highest identitiy of about 99.6% and the lowest was around 85.2%. The results of phylogenetic analysis also showed that the five samples form different branches, although still has a closeness with P. stutzeri lipase (AID66451.1). It is suggested that the five samples obtained from the composting thermophilic phase are different samples. Differences several amino acid residues and position occur among samples or with P. stutzeri lipase (AID66451.1). The biggest difference was found in two samples are LK3 and LK5. LK3 sample have a shorter size is 280 amino acids and the sample experienced deletions in the 31 amino acid residues of the N terminal region causing LK3 sample do not have signal peptide. While the LK5 sample as well as three other samples have 311 amino acids but they are different in several substitutions of amino acid residues. The different found in the most sustainable region around the catalytic aspartate residue. The differences in several amino acid residues were found among samples leads to generate different a nature and character lipases compare with P. stutzeri lipase (AID66451.1) from halophilic environments. Analysis of amino acid sequence identity among the five samples with some family I.1 lipase of several microorganism showed the sustainable region owned by the five samples. Those sustainable are tetrapeptida area, pentapeptide, catalytic triad and also some residues that has important role in the formation of protein folding. Based on the results of this analysis, it was concluded that the lipase of fifth samples obtained from thermophilic compost included in the group of family I.1 lipase. Structure prediction analysis of the five samples lipase generally does not showed any significant different except LK5 sample which experienced several different of amino acid residues around the catalytic center of aspartate. Superimposed results between LK5 and P. aeruginosa lipase (PDB ID 1EX9) as template showed conformational different that occur in the active center of the enzyme that is believed to have an impact on the activity of the enzyme produced. Three samples were chosen to be cloned into the expression vector pET30a. The results of three samples on the condition of the addition 1 mM IPTG as inducer, for 4 hours of induction time and induction temperature of 37 ° C, 150 rpm showed expression of fusion proteins that has molecular weight 34 kDa for LK2, 31 kDa for LK3 and 28 kDa for LK5. Quantitaton of the protein expression showed that the fusion protein expressed in the sample around 30,89 % for LK2, 44,38 % for LK3 and 21,67 % for LK5. The activity test of this lipase using p-nitrophenyl lauric (C12) as substrate in the conditions of the reaction temperature at 50 ° C and posphat buffer pH 8, showed that specific activity for LK2 = 1,493 U / mg; LK3 = 0,938 U / mg and LK5 = 1,101 U / mg. These results indicate that the soluble protein fraction is an active protein with good activities. In this study, the metagenome approach of thermophilic compost has successful to get lipase gene with high diversity. The five of samples were thermostable lipase gene from Pseudomonas genus founded in domestic compost of Indonesia. The thermostable lipases are expected to have differences in the nature and character of lipases that have been studied previously. Further analysis of the protein produced is still need to get information about the nature and character of lipase of this compost environment.