OPTIMIZING CULTURE CONDITION FOR EXPRESSION OF CYP124 IN ESCHERICHIA COLI USING RESPONSE SURFACE METHODOLOGY APPROACH
Metabolic engineering in bacteria can produce heterologous secondary metabolites. Therefore, Mycobacterium tuberculosis can hydroxylate farnesyl pyrophosphate (FPP) at C?produce 12OH-FPP. Hydroxylated FPP is precursor for amorphadiene synthase (ADS) producing dihydroartemisinic acid so biosynthe...
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| Format: | Theses |
| Language: | Indonesia |
| Online Access: | https://digilib.itb.ac.id/gdl/view/85202 |
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| Institution: | Institut Teknologi Bandung |
| Language: | Indonesia |
| Summary: | Metabolic engineering in bacteria can produce heterologous secondary
metabolites. Therefore, Mycobacterium tuberculosis can hydroxylate farnesyl
pyrophosphate (FPP) at C?produce 12OH-FPP. Hydroxylated FPP is precursor
for amorphadiene synthase (ADS) producing dihydroartemisinic acid so
biosynthetic steps on artemisinin production can be reduced. Optimization of
CYP124 expression was carried out using Response Surface Methodology by
varying FeCl3, 5-ALA, and IPTG concentration in medium. The parameters
measured from this treatment were R/Z, CYP124 mass from SDS-PAGE results,
and CYP124 characterization. SDS-PAGE purified CYP124 showed a single band
around 45 kDa. Factor affecting CYP mass is interaction IPTG and 5-ALA. 5-ALA
also affecting R/Z value. Validation for optimal design result was match with the
predicted CYP mass and R/Z value. So, optimal design for CYP124 expression is
addition 0.10 mM FeCl3, 1 mM 5-ALA, and 0.2427 mM IPTG. To ensure that
produce CYP124 enzyme is functional, an activity assay can be carried out.
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