Cloning and Characterization of Partial Nucleotide Sequence of Levanase Gene from a Thermotolerant Bacillus licheniformis

Azhar, Minda and Syukur, Sumaryati and Natalia, Dessy and Jamsari, Jamsari (2012) Cloning and Characterization of Partial Nucleotide Sequence of Levanase Gene from a Thermotolerant Bacillus licheniformis. [Image]

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Abstract

Gene that encodes thermostable inulin degrading enzyme are potential enzyme to convert inulin into fructose and FOS prebiotic. Cloning and characterization of partial nucleotide sequence of levanase gene from a thermotolerant Bacillus licheniformis have been undertaken. The fragments of levanase gene were isolated from B. licheniformis genome by using PCR method. Degenerate PCR primers were designed from conserved sequences of domain A, D and E in inulin degrading enzyme. E.coli TOP10F’ and pGEM-T Easy were used as host strain and cloning vector, respectively. Fragment of levanase gene have been obtained 398 bp and 539 bp. In the nucleotide sequence of DNA fragment 539 bp contained sequence of DNA fragments 398 bp.The nucleotide sequence 539 bp had high similarity degree 97% with levanase Bacillus subtilis BSn5 (Genbank, YP004204503.1). The DNA fragment 539 bp had spesific hits GH32 ß-fructosidase with three active site residues and five substrate binding residues. In the protein fragment (179 amino acid residues) was found conserved domain A, B, B1, C, D and E of GH32 family. Tertier structure of the protein fragment showed ß-propeller fold that have 50% homology with exoinulinase from Aspergillus awamori.

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