ENRICHMENT OF CIMANGGU HOTSPRING METAGENOME WITH ENDOGLUCANASE GENE
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, suc...
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Format: | Theses |
Language: | Indonesia |
Online Access: | https://digilib.itb.ac.id/gdl/view/34897 |
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Institution: | Institut Teknologi Bandung |
Language: | Indonesia |
Summary: | 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. |
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