ALINE PUSPITA KUSUMADJAJA, 090610357 D (2012) PENAPISAN, KARAKTERISASI FITASE DAN ANALISIS HOMOLOGI GEN PENYANDI FITASE DARI BAKTERI TERMOFILIK KAWAH IJEN BANYUWANGI. Disertasi thesis, UNIVERSITAS AIRLANGGA.

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Abstract

Phytase is an enzyme that hydrolyse phosphodiester bond in phytic acid (myo-inositol hexakis phosphate), producing inorganic phosphate and phosphate esters of lower myo-inositol. Utilization of phytase in animal feed industry and food industry can solve the problem of nutrition and the environment arising from incompletely digested phytic acid/ salt by livestock and humans. Phytase that resistant to high temperatures, the thermophilic phytase, is highly advantageous to be used in animal feed industry and food industry. The research conducted aimed to obtain isolates of thermophilic microorganisms of Kawah Ijen Banyuwangi which has the highest phytase activity, determine the species of selected thermophilic isolates based on analysis of genes encoding 16S rRNA, determine the character of crude extract of phytase from selected thermophilic isolates, as well as PCR amplification and determine the sequence of genes encoding phytase of the selected thermophilic isolates. In the conducted research, 33 thermophilic isolates that have phytase activity had been obtained from Kawah Ijen Banyuwangi. Isolate AP-17 is a phytase-producing isolate with the highest activity, ie 0.0285 U/mL. The analysis results of genes encoding 16S rRNA showed isolates AP-17 has 99% homology with Bacillus amyloliquefaciens FZB42. Characterization of crude phytase extract from isolates AP-17 showed that the enzyme has an optimum temperature of 75 °C with the activity of 0.1413 U/mL and the optimum pH of 6 with the activity of 0.0875 U/mL. Heating at 75 °C for 1 and 3 hours led to the remaining enzyme activity by 89% and 60%, respectively. Heating for 20 minutes at 85 °C and 95 °C led to the remaining enzyme activity by 58% and 43%, consecutively. Meanwhile, heating for 80 minutes at 85 °C led to the remaining enzyme activity by 39%, while heating at a temperature of 95 °C for 80 minutes cause the enzyme only had activity by 25%. The enzyme has good stability at pH range 5-8 with a residual activity of more than 90%. From the results of SDS PAGE and electroelution, the relative molecular mass (Mr) of enzyme was estimated at 43.9 kDa. Addition of Ca2+ ion can increase the enzyme activity up to 118%, while Cd2+ and Mn2+ ions decrease the activity of enzyme to 19% and 25%, respectively. In the PCR process to amplify the gene encoding phytase from isolates of AP-17, using a pair of specific primers designed based on homology with genes encoding phytase from Bacillus group, amplicon/fragment size of approximately 1200 bp DNA was obtained. Homology assay showed that this DNA fragment had 59% homology with the gene encoding phytase from B. amyloliquefaciens FZB42.

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