#TITLE_ALTERNATIVE#

• Main Author: WAYA MERAY (NIM : 20511034)Pembimbing : Prof. Akhmaloka, Ph.D ;Dr. Santi Nurbaiti, NISHIA
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/19061
• Institution: Institut Teknologi Bandung

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 /> thermostable enzyme keep increase continuously, exploration of thermophilic <br /> <br /> <br /> <br /> <br /> microorganisms is continuously carried out. From previous study, 1.9 kb gene <br /> <br /> <br /> <br /> <br /> fragment has been isolated from Domas crater, West Java with metagenom <br /> <br /> <br /> <br /> <br /> approach. The gene fragment was succesfully cloned into pJET1.2/blunt vector. <br /> <br /> <br /> <br /> <br /> Based on bioinformatics analysis, the gene fragment carrying the intact Open <br /> <br /> <br /> <br /> <br /> Reading Frame (ORF) of class II aldolase protein. In this research, cloning and <br /> <br /> <br /> <br /> <br /> expression of a gene encoding class II aldolase from 1.9 kb fragment gene was <br /> <br /> <br /> <br /> <br /> perform. The results of this research expected can be increase a database of <br /> <br /> <br /> <br /> <br /> diversity of hyperthermophilics microorganisms and hyperthermostble enzyme <br /> <br /> <br /> <br /> <br /> from Indonesian. <br /> <br /> <br /> <br /> <br /> Bioinformatics analysis showed that the 1.9 kb gene fragment contain three ORF, <br /> <br /> <br /> <br /> <br /> among other Fe-S oxidoreductase, class II aldolase and citrate synthase. By using <br /> <br /> <br /> <br /> <br /> Basic Local Alignment Search Tool (BLAST) from NCBI, homology of those <br /> <br /> <br /> <br /> <br /> protein known have similiarity with the same protein of Acidilobus <br /> <br /> <br /> <br /> <br /> saccharovorans with straight percent of homology is 78 %, 61 % and 91 % for <br /> <br /> <br /> <br /> <br /> Fe-S oxidoreductase, class II aldolase and citrate synthase. All three protein then <br /> <br /> <br /> <br /> <br /> known has responsible in metabolic processes in A. saccharovorans mainly in <br /> <br /> <br /> <br /> <br /> glycolysis. However, only ORF of class II aldolase was the intact ORF with size <br /> <br /> <br /> <br /> <br /> about 639 bp. For the expression purposes, the intact gene encoding class II <br /> <br /> <br /> <br /> <br /> aldolase (aldII) has been amplified and ligated into the cloning vector pGEM-T <br /> <br /> <br /> <br /> <br /> Easy Vector generating a recombinant vector called pGEM-aldII. The aldII gene <br /> <br /> <br /> <br /> <br /> then isolated and was subcloned into expression vector pET-30a(+). <br /> <br /> <br /> <br /> <br /> Deduction of nucleotide sequence of aldII, produced the amino acids sequences of <br /> <br /> <br /> <br /> <br /> class II aldolase protein (AldII) with 196 of amino acids. Determination of <br /> <br /> <br /> <br /> <br /> biochemical characteristic with in silico approach of AldII by analyzing the <br /> <br /> <br /> <br /> <br /> sequence is by using Protparam from Expasy. The result of analysis sugest that <br /> <br /> <br /> <br /> <br /> molecular weight of AldII is ~21.2 kDa with molecular formulas <br /> <br /> <br /> <br /> <br /> C940H1539N261O281S8. Total negatively charged residues (Asp + Glu) is 22 residues, <br /> <br /> <br /> <br /> <br /> while total positively charged residues (Arg + Lys) is 18 residues. Theoritical pI <br /> <br /> <br /> <br /> <br /> value of this protein is 5.86. This protein are classified as a stable protein. <br /> <br /> <br /> <br /> <br /> Prediction of 3D structural modeling of AldII was perform by QUARK with ab <br /> <br /> <br /> <br /> <br /> v <br /> <br /> <br /> <br /> <br /> initio approach. This protein was predicted has a topological structure with 6 beta <br /> <br /> <br /> <br /> <br /> sheet and 6 alpha helix. <br /> <br /> <br /> <br /> <br /> Recombinant vector of pET-aldII which expressed in host cell E. coli BL21 <br /> <br /> <br /> <br /> <br /> (DE3) with different concentration of isopropylthiogalactoside (IPTG) (0.5 mM <br /> <br /> <br /> <br /> <br /> and 1 mM) produced recombinant class II aldolase protein (AldII) with molecular <br /> <br /> <br /> <br /> <br /> weight ~26 kDa. Concentration of AldII crude protein was determined by using <br /> <br /> <br /> <br /> <br /> Bradford method and give a concentration value 11 mg/&#956;L and 7.38 mg/&#956;L for <br /> <br /> <br /> <br /> <br /> 0.5 mM and 1 mM concentration of IPTG. Determination of expression level <br /> <br /> <br /> <br /> <br /> using ImageJ showed that the AldII protein expressed amount 39.78 % and 31.87 <br /> <br /> <br /> <br /> <br /> % for 0.5 mM and 1 mM concentration of IPTG. AldII protein proved to be a <br /> <br /> <br /> <br /> <br /> thermostable protein after heating for 30 minutes at 60 oC and has been <br /> <br /> <br /> <br /> <br /> succesfully purified using Ni-NTA, with a purity level 88.44% before heating and <br /> <br /> <br /> <br /> <br /> increased to 97.6 % after heating.