Metal ion effects on Polyphenol Oxidase Covalently immobilized on a Bio-Composite

Biosensors can be developed using different immobilization methods. Interest in immobilization methods have increased because biosensors have been important for science. Polyphenol oxidase (PPO) was used generally in biosensor applications. For this purpose, Polyphenol oxidase from banana was purified and covalently immobilized on chitosan-gelatin bio-composite. The properties of immobilized enzyme were investigated and compared to free enzyme. Various parameters were studied such as pH, temperature and storage stability on immobilized and free enzyme. Kinetic parameters were also evaluated by different substrates on immobilized and free enzyme. Catechol was determined the best substrate for immobilized enzyme with optimum condition. In vitro effects of metal ions were studied on immobilized enzyme. Concentration range of metal ions is 1.0-10.0 x10-6 mol/L. The activity of immobilized PPO was increased by Fe+2 and Ag+1 ion. Co+3 and Cu+1 had very strong inhibitory effects with IC50 values of 19.69x10-3 mol/L and 23.49 x10-3 mol/L, respectively. Inhibition constants (Ki) and inhibition types of metal ions were determined with immobilized enzyme. Zn+2 and Cr+3 ions were showed competitive inhibition and Pb+2 ions were determined non-competitive inhibition with immobilized enzyme. Mixed type inhibition was obtained with Co+3 ion using catechol as substrate with 3.33x10-5 mol/L Ki value on immobilized PPO. Immobilized PPO can be evaluated for biosensor for the purpose of measurements of metal ions.

BIOCHEMICAL CHARACTERIZATION OF RECOMBINANT PHYTASE ENZYME (phyK) FROM Klebsiella sp. ASR1 ENCAPSULATED WITH ALGINATE

Karakterisasi Biokimia Enzim Fitase Rekombinan (phyK) dari Klebsiella sp. ASR1 Yang Dienkapsulasi Dengan AlginatEnzim fitase melepas molekul fosfor pada atom C dari benzena Inositol fitat. Tetapi fitase memiliki kelemahan tidak mampu bertahan terhadap kondisi ekstrim dalam lambung nonruminansia. Solusi dalam penelitian ini yaitu fitase dienkapsulasi menggunakan alginat. Penelitian ini bertujuan mengkarakterisasi fitase setelah dienkapsulasi menggunakan alginate. Hasil penelitian ini yaitu fitase yang dienkapsulasi memiliki aktivitas tertinggi pada pH 6,0, sedangkan fitase tanpa enkapsulasi pada pH 5,0. Suhu optimum untuk aktivitas tertinggi fitase yang dienkapsulasi yaitu 70ºC, sedangkan fitase tanpa enkapsulasi 37ºC. Untuk perlakuan penambahan ion logam, aktivitas tertinggi fitase yang dienkapsulasi terjadi dengan penambahan 0,1 mM Fe2+ dan 1,0 mM Ca2+, sedangkan fitase tanpa enkapsulasi dengan penambahan 0,1 mM Fe2+. Berdasarkan hasil penelitian ini, fitase yang dienkapsulasi memiliki keunggulan lebih banyak dibandingkan dengan fitase tanpa enkapsulasi, karena mampu bertahan pada pH dan suhu tinggi, dan beberapa efek ion logam.Kata Kunci: alginat, asam fitat, enkapsulasi, fitase, fitase rekombinanABSTRACTPhytase enzymes release phosphorus molecules on the C atom from benzene inositol phytate. But phytase has the disadvantage of being unable to withstand extreme conditions in the non-ruminant stomach. The solution in this research was phytase encapsulated using alginate. This study aims to characterize phytase after being encapsulated using alginate. The results of this study were the encapsulated phytase had the highest activity at pH 6.0, while the unencapsulated phytase at pH 5.0. The optimum temperature for the highest activity of the encapsulated phytase was 70ºC, while the unencapsulated phytase 37ºC. For treatment of metal ion addition, the highest activity of the encapsulated phytase occurred with the addition of 0.1 mM Fe2+ and 1.0 mM Ca2+, while the unencapsulated phytase with the addition of 0.1 mM Fe2+. Based on the results of this study, the encapsulated phytase had more advantages compared to the unencapsulated phytase, as the former withstand high pH and temperature, and some metal ion effects.