Karakterisasi Molekuler Gen Penyandi Kitinase Bakteri Asal Limbah Udang dan Kepiting sebagai Anti Saprolegnia
Ibnu Dwi Buwono, Hanif Sri Wahyuni dan Roffi Grandiosa 2013

Abstract
Mortality in the eggs of freshwater fish can reach 80% due to fungal pathogen infection (Saprolegnia sp.) resulted in low fry production and fish eggs. Saprolegnia growth inhibition using the biologically effective chitinolytic bacteria applied, therefore chitinase enzyme produced is able to lysis fungal cell walls that contain chitin as the main composition. Isolation and characterization of genes encoding chitinase of bacterial origin shrimp and crabs waste is needed to verify the functional domain coding sequences containing sequences encoding the enzyme catalytic site and the binding of chitin as an anti Saprolegnia. This research uses experimental exploratory method to obtain sequences of genes encoding chitinase bacterial origin of shrimp and crabs waste. The encoding gene sequencing results were analyzed by bioinformatics with blastn for nucleotide sequence alignment and blastx for amino acid sequences. Functional domains of chitinase and prediction of protein three-dimensional structures of enzymes were analyzed using the program SIB (Swiss Institute of Bioinformatic) Expasy (http://www.expasy.org/vg/index.protein). Isolate pure bacterial origin shrimp and crabs waste has chitinolytic index was relatively high (2.12 mm and 1.99 mm) and inhibition zone of crabs waste bacterial (11.59 mm) was higher than the original shrimps waste (6.005 mm) on the growth of mycelium and Saprolegnia hyphae. Genes encoding chitinase bacterial origin of shrimp and crabs waste obtained with the process starting from genomic DNA extraction from pure bacterial culture results are then used as a template for chitinase DNA synthesis by PCR (Polymerase Chain Reaction). Amplification results of the chitinase gene with primers Chie-F (5'-CTAGACAACTTTTTGTATAGGAGTGTTGATATG-3 ') and Chie-R (5'-CGATTGATGAGGGCTAATTATAGTTTTACTTTG-3') of 1200 bp. Analysis of amino acid residues of chitinase using blastx program (forward) showed that bacterial chitinase genes from shrimp waste has a high similarity (96%) with Bacillus thuringiensis chitinase sequences (no. accession YP003665928.1) and crabs waste bacterial chitinase sequences 99% identical with Bacillus cereus (no. accession WP000932552.1). Detection of functional domains B. thuringiensis and B. cereus with SIB Expasy obtained catalytic site, active site binding of chitin, the signal peptide, N-glycosylation, two cysteine residues and transmembrane helix as a molecular characteristic enzyme. Differences in three-dimensional molecular structure of chitinase B. thuringiensis and B. cereus associated with differences in the amount of each composition amino acids.