CONSTRUCTION AND EXPRESSION OF PARTIAL GENE bglp_15 ENCODING Β-GLUCOSIDASE FROM DEEP SEA METAGENOME OF KAWIO ARCHIPELAGO NORTH SULAWESI
<p align="justify">Marine ecosystem is a potential source of biodiversity that can be explored to support industrial development, because of its unique chemical substances. One of these unique substances is the thermo stable enzyme such as β-glucosidase. This class of enzyme...
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| Format: | Final Project |
| Language: | Indonesia |
| Online Access: | https://digilib.itb.ac.id/gdl/view/31339 |
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| Institution: | Institut Teknologi Bandung |
| Language: | Indonesia |
| Summary: | <p align="justify">Marine ecosystem is a potential source of biodiversity that can be explored to support industrial development, because of its unique chemical substances. One of these unique substances is the thermo stable enzyme such as β-glucosidase. This class of enzyme has been widely applied in bioethanol production. Isolation and characterization of βglucosidase gene from deep sea metagenome of Kawio Archipelago, North Sulawesi had been performed and named bglp_15. However, this gene has an open reading frame (ORF) in the downstream of ribosom binding site (RBS) that produces peptide containing 9 amino acids, so it is impossible to produce an active enzyme. According to SDS PAGE and measurement of enzyme activity, both protein band with molecular mass approximately 25 kDa and enzyme activity can be detected. Another ORF in the middle of the gene had been found and suspected to produce the 25 kDa protein by Shine Dalgarno-independent translation. So, it is needed to confirm the ORF, whether it is functional or not by deleting the nucleotide sequence between RBS and deserved ORF. The new partial gene produced after the deletion is named bglp_15p. PCR is used to isolate the deserved ORF and also used to add new restriction site, so it can be inserted to the expression vector, pET-32b plasmid, and transformed to Escherichia coli BL21 (DE3). Transformed E. coli harvested then SDS PAGE and enzyme activity assay conducted. The assay performed in the optimum temperature and pH, 60oC and 9,4 respectively. The result shows that bglp_15p produce functional enzyme with molecular mass approximately 25 kDa. bglp_15 activity is also significantly higher than bglp_15 according to t-test with 5% significance. <p align="justify"> <br />
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