ENRICHMENT OF CIMANGGU HOTSPRING METAGENOME WITH ENDOGLUCANASE GENE

• Main Author: Rahman Sadjuri, Arif
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/34897
• Institution: Institut Teknologi Bandung

The large consumption of fossil fuel has caused energy and environmental crises. The high demand of fossil fuel is not followed by high supply of the fuel. High fossil fuel combustion also causes an increase in CO2 concentration in the atmosphere which leads to the greenhouse effect. Biofuel, such as bioethanol, is one alternative solution to overcome this problem. Bioethanol combustion is cleaner and more efficient than fossil fuel. The raw material, glucose, to produce 1st generation bioethanol (starch-based bioethanol) is competing with food demand. Lignocellulose from biomass can be an alternative source of glucose for production of 2nd generation bioethanol (lignocellulose-based bioethanol). Lignocellulose, which is the main component of plant cell wall, is abundant and renewable. However, current technologies of lignocellulose processing are very expensive. The high cost is mainly due to difficulties in the degradation of lignocellulose which has a compact structure. The degradation of lignocellulose needs high temperature and low pH condition in order to proceed more efficiently. Thermostable cellulase enzymes (cellobiohydrolase, endoglucanase, ?– glucosidase) from thermophilic microbes can be used for better hydrolysis of cellulose. The specific habitats of thermophilic microbes make them difficult to culture. Isolation of metagenomic DNA is a method to obtain genes coding for thermostable cellulose enzymes without the need for prior culturing or identification of the microbes. Partial endoglucanase genes from metagenome of Cimanggu hot spring, Ciwidey, West Java had been isolated by another researcher. The next stage was to try to isolate the full length endoglucanase gene from the same place. Metagenomic DNA was isolated from the sediment of the hot spring by a large scale method. The metagenomic DNA sample was separated from inhibitors by electrophoresis followed by extraction from gel using Gene Aid Kit. The presence of partial endoglucanase gene in the metagenomic DNA was confirmed by amplification using partial endoglucanase gene primers. The metagenomic DNA was then partially digested and ligated with adapters ORM28 and ORM29 at both ends of the DNA fragments. Adapter-ligated metagenomic DNAs were then amplified using ORM28 primer. Enrichment process was conducted by hybridization of adapter-ligated metagenomic DNAs to partial endoglucanase gene fragments. The eluted hybridized DNA was amplified by ORM28 primer. The success of enrichment was confirmed by amplification with specific primers of partial endoglucanase gene. The enriched DNA fragments were then cloned in pJET1.2 vector. The presence of DNA inserts in the recombinant clones was confirmed by PCR screening and restriction analysis. The longest DNA inserts was sequenced and analyzed by several bioinformatic programs. Analysis results showed that the inserted DNA was homologous with part of polyamine transport ATP-binding protein (PotG) and part of inner membrane permease polyamine transport protein (PotH). This result did not match the expectation and goal of this research.