EXPLORATION OF LIPOLYTIC THERMOSTBALE ENZYME THROUGH GENOMIC APPROACH IN BACTERIA FROM ISOLATE LOCAL

• Main Author: Nur Afifah, Deviyanthi
• Format: Dissertations
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/88002
• Institution: Institut Teknologi Bandung

The genome of every organism contains all the biological information required for cellular construction, sustaining life and passing genetic traits to subsequent generations. Next Generation Sequencing (NGS) technology has facilitated high-throughput data collection, enabling rapid determination of genomes, epigenomes, transcriptomes, and proteomes. Genomic analyses have successfully revealed complete gene sequences in various microorganisms. The complete nucleotide sequence of the genome from the AL17 isolate has been determined. The AL17 genome measures 3,064,463 base pairs in length, consisting of 2,833 genes, 2,775 coding sequences (CDS), 6 rRNA genes, 48 tRNA genes, and 17 pseudogenes, with a GC content of 72%. Bioinformatics analyses based on nucleotide sequences have established that the AL17 isolate is closely related to Pseudoxanthomonas taiwanensis. A detailed examination of the genes in the AL17 genome reveals several genes with the potential to encode commercial enzymes, including hydratases, transferases, dehydrogenases, exopeptidases and hydrolases. Further investigation of the hydrolase genes showed that the AL17 genome lacks truly lipase-encoding genes but contains genes encoding lipolytic enzymes, such as GDSL esterase/lipase (ITBGDSL_1) and GDSL lysophospholipase (ITBLPL_1). ITBGDSL_1 and ITBLPL_1 were successfully cloned using PCR amplification from the total genomic DNA of the AL17 isolate. Both genes were expressed in Escherichia coli BL21 (DE3) using the pET30a(+) vector. Crude extracts of the expressed proteins exhibited lipolytic activity on para-nitrophenyl acetate (pNP-C2) substrates. The ITBGDSL_1 gene and its encoded protein sequence exhibit low homology to previously cloned or characterized GDSL proteins, with the highest similarity observed with GDSL protein from Bacillus sp. K91 at 30 %. However, a higher similarity was observed with a putative GDSL esterase/lipase gene from Pseudoxanthomonas taiwanensis (not previously cloned). Heterologous expression of ITBGDSL_1 in Escherichia coli BL21 (DE3) resulted in a protein with an approximate molecular weight of 46 kDa, confirmed through SDS- Polyacrylamide Gel Electrophoresis (PAGE) analysis. The crude enzyme extract displayed hydrolytic activity toward the para-nitrophenyl acetate (pNP-C2) substrate, with a 6.6-fold increase following purification using Ni-NTA Immobilized Metal Affinity Chromatography (IMAC). Further characterization revealed that ITBGDSL_1 exhibited optimal activity on the pNP-C2 substrate at 55 °C and pH 8, findings confirmed by molecular docking analysis. ITBGDSL_1 demonstrated thermal stability for up to 60 hours at 55 °C, preferred polar solvents and exhibited activity modulated by metal ions. Monovalent metal ions inhibited ITBGDSL_1 activity, while divalent metal ions enhanced it. These ions' inhibition and enhancement effects were confirmed through molecular docking analysis. ITBGDSL_1 activity was also inhibited by chemical compounds such as EDTA, PMSF, and ?-mercaptoethanol. Similarly, the ITBLPL_1 gene and its encoded protein sequence exhibit low homology to previously cloned and characterized lysophospholipases, with the highest similarity observed with a lysophospholipase from Streptomyces sp. NA684 at less than 30%. However, a higher similarity was noted with a putative lysophospholipase gene from the Lisobacteraceae bacterium. Heterologous expression of ITBLPL_1 in Escherichia coli BL21 (DE3) yielded a protein with an approximate molecular weight of 27 kDa, confirmed by SDS-PAGE analysis. Crude enzyme extracts exhibited hydrolytic activity on the para-nitrophenyl acetate (pNP-C2) substrate, with 39.2 fold increase following purification using Ni-NTA IMAC. Characterization revealed that ITBLPL_1 displayed optimal activity on the pNP-C2 substrate at 55 °C and pH 7, as confirmed by molecular docking analysis. ITBLPL_1 demonstrated thermal stability for up to 12 hours at 55 °C. The enzyme showed reduced activity in polar and non-polar solvents and its activity was influenced by metal ions, with both monovalent and divalent ions inhibiting its activity. The inhibitory effects of divalent ions were confirmed via molecular docking analysis. ITBLPL_1 activity was also inhibited by chemical compounds such as EDTA, PMSF, and ?-mercaptoethanol. The findings suggest that ITBGDSL_1 and ITBLPL_1 are novel enzymes and hold significant potential as biocatalysts.