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Exploration of thermophilic microorganism and its thermostable enzyme could <br /> <br /> <br /> <br /> <br /> bursting the development of biotechnological industries. Since the need of <br /> <br /> <br /> <br /> <br /> thermos...
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| Format: | Theses |
| Language: | Indonesia |
| Online Access: | https://digilib.itb.ac.id/gdl/view/19061 |
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| Institution: | Institut Teknologi Bandung |
| Language: | Indonesia |
| Summary: | Exploration of thermophilic microorganism and its thermostable enzyme could <br />
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bursting the development of biotechnological industries. Since the need of <br />
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thermostable enzyme keep increase continuously, exploration of thermophilic <br />
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microorganisms is continuously carried out. From previous study, 1.9 kb gene <br />
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fragment has been isolated from Domas crater, West Java with metagenom <br />
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approach. The gene fragment was succesfully cloned into pJET1.2/blunt vector. <br />
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Based on bioinformatics analysis, the gene fragment carrying the intact Open <br />
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Reading Frame (ORF) of class II aldolase protein. In this research, cloning and <br />
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expression of a gene encoding class II aldolase from 1.9 kb fragment gene was <br />
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perform. The results of this research expected can be increase a database of <br />
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diversity of hyperthermophilics microorganisms and hyperthermostble enzyme <br />
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from Indonesian. <br />
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Bioinformatics analysis showed that the 1.9 kb gene fragment contain three ORF, <br />
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among other Fe-S oxidoreductase, class II aldolase and citrate synthase. By using <br />
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Basic Local Alignment Search Tool (BLAST) from NCBI, homology of those <br />
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protein known have similiarity with the same protein of Acidilobus <br />
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saccharovorans with straight percent of homology is 78 %, 61 % and 91 % for <br />
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Fe-S oxidoreductase, class II aldolase and citrate synthase. All three protein then <br />
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known has responsible in metabolic processes in A. saccharovorans mainly in <br />
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glycolysis. However, only ORF of class II aldolase was the intact ORF with size <br />
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about 639 bp. For the expression purposes, the intact gene encoding class II <br />
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aldolase (aldII) has been amplified and ligated into the cloning vector pGEM-T <br />
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Easy Vector generating a recombinant vector called pGEM-aldII. The aldII gene <br />
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then isolated and was subcloned into expression vector pET-30a(+). <br />
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Deduction of nucleotide sequence of aldII, produced the amino acids sequences of <br />
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class II aldolase protein (AldII) with 196 of amino acids. Determination of <br />
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biochemical characteristic with in silico approach of AldII by analyzing the <br />
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sequence is by using Protparam from Expasy. The result of analysis sugest that <br />
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molecular weight of AldII is ~21.2 kDa with molecular formulas <br />
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C940H1539N261O281S8. Total negatively charged residues (Asp + Glu) is 22 residues, <br />
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while total positively charged residues (Arg + Lys) is 18 residues. Theoritical pI <br />
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value of this protein is 5.86. This protein are classified as a stable protein. <br />
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Prediction of 3D structural modeling of AldII was perform by QUARK with ab <br />
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v <br />
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initio approach. This protein was predicted has a topological structure with 6 beta <br />
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sheet and 6 alpha helix. <br />
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Recombinant vector of pET-aldII which expressed in host cell E. coli BL21 <br />
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(DE3) with different concentration of isopropylthiogalactoside (IPTG) (0.5 mM <br />
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and 1 mM) produced recombinant class II aldolase protein (AldII) with molecular <br />
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weight ~26 kDa. Concentration of AldII crude protein was determined by using <br />
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Bradford method and give a concentration value 11 mg/μL and 7.38 mg/μL for <br />
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0.5 mM and 1 mM concentration of IPTG. Determination of expression level <br />
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using ImageJ showed that the AldII protein expressed amount 39.78 % and 31.87 <br />
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% for 0.5 mM and 1 mM concentration of IPTG. AldII protein proved to be a <br />
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thermostable protein after heating for 30 minutes at 60 oC and has been <br />
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succesfully purified using Ni-NTA, with a purity level 88.44% before heating and <br />
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increased to 97.6 % after heating. |
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