Produksi dan Karakterisasi Enzim Arabinosa Isomerase dari Gen Bakteri Geobacillus stearothermophilus Lokal

Abstract

Enzim arabinosa isomerase (AI) dapat mengkatalisis secara revesible reaksi isomerisasi D-galaktosa menjadi D-tagatosa. Tagatosa telah digunakan sebagai pemanis rendah kalori (1,5 kkal/g) yang memiliki tingkat kemanisan 92% dibandingkan sukrosa. Tagatosa memberikan berbagai manfaat kesehatan diantaranya seperti menurunkan berat badan, prebiotik, anti-histolisis serta mereduksi sejumlah gejala yang berhubungan dengan diabetes tipe 2, hiperglikemia, obesitas, anemia dan hemophilia. Peran tagatosa sebagai antidiabetes akan bermanfaat sebagai gula alternatif di Indonesia, mengingat Indonesia menempati peringkat ke-4 dengan jumlah penderita diabetes terbesar di dunia.

Arabinose isomerase (AI) is an enzyme that catalyzes isomerization of galactose to tagatose. Besides being used as a low-calorie sweeteners, tagatose has been developed as a functional food because it provides many health benefits such as promoting of weight-loss, anti-halitosis, prebiotic, treating of obesity and reducing in symptoms associated with type 2 diabetes, hyperglycemia, anemia, and hemophilia. Thermostable AIs are potential for tagatose production. AI enzymes encoded by araA gene. The araA gene Geobacillus stearothermophilus originated from Tanjung Api, Poso, Indonesia has been successfully cloned and exspressed at previously study in E. coli BL21 (DE3) pLysS. However expression level of AI still low by SDS-PAGE analysis. The E. coli BL21 was incubated in 37°C at 150 rpm. This research was conducted to optimize the araA gene expression. Result from this research showed that the medium tofu liquid waste consisting yeast extract 0.5% (TLW+YE) increased enzyme productivity. Optimation production was obtained by 16 hours induction. The purification was carried out with three steps of freeze-thaw at -70°C, heat treatment (60°C, 30 minutes) and DEAE ion exchange chromatography (elution buffer 0-1000 mM NaCl). The purified enzyme exhibited optimum activity at 60°C and pH 7. The AI activity in the presence of CaCl2 and MnCl2 was increased to 152% and 563% respectively. Heat stability of enzymes in the presence of CaCl2 and MnCl2 was increased. Half-life (t 1/2) AI in the presence 1 mM of CaCl2 and MnCl2 was increased becomes 301 and 990 minutes respectively.