RATNA MELINDA, 081042006 (2012) KARAKTERISASI SIFAT BIOKIMIA DAN KAJIAN PENGARUH MUTASI TERHADAP STRUKTUR -L-ARABINOFURANOSIDASE (AbfA) VARIAN SECARA IN SILICO. Thesis thesis, UNIVERSITAS AIRLANGGA.

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Abstract

Geobacillus thermoleovorans IT-08 a-L-arabinofuranosidase (AbfA) is an enzyme with optimum activity around neutral pH (6-8). Its variant (variant A9)exhibit an increased activity at pH 9. In this study, a nucleotide alignment to the wildtype (DQ387046.1) was done to determine the modification in abfa variant A9 gene. The enzyme expressed in E. coli BL21 (DE3)/pBM5abf were characterized for its biochemical properties. Protein tertiary structure model which built by aligning the sequence to Geobacillus stearothermophilus T-6 -L-arabinofuranosidase protein crystal structure (PDB accession number: 1PZ3), was subjected to determine the structure alteration. The relation between change in biochemical property and structure alteration was also investigated. Substitution at three bases in abfa variant A9 gene (A137G; T615A; A853G) resulted in amino acids replacement (Gln46Arg; Asp205Glu; Lys285Glu). Expression of abfa variant gene was detected using SDS PAGE, shown a 61 KDa band corresponds to AbfA variant A9. The partially purified enzyme displayed optimum activity at pH 7 and 70oC. AbfA variant A9 was stable for 24 hours at pH 6-9 (in 4oC) and lost almost 70% of its activity on 16 hours incubation at 70oC. Superimpose of Abfa variant with the wildtype tertiary structure model shown RMSD value 0.05%. In silico analysis of non covalent interaction revealed that non covalent interactions within the protein were reduced due to amino acid replacement. Substitution of Arg46 to Gln46 (ΔΔG: +0,22) decrease 3 Van der Waals interaction, Asp205Glu substitution (ΔΔG: +2.32) resulted in decrease of 3 Hydrogen bonds and 1 Van der Waals bond and generation of 1 electrostatic interaction, substitution of Lys285 by Glu (ΔΔG: -0.09) decreased 1 Van der Waals interaction but reduced the repulsive effect arises from positive side chains around residue 285. The decrease in AbfA variant thermostability was related to the reduction of some non covalent interactions especially Hydrogen bonds and Van der Waals interaction, because of changes in structure.

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