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A multimodular hyperthermophilic β‑glucuronidase (TpGUS) from Thermotoga petrophila RKU-1T, belongs to glycoside hydrolase family 2 (GH2), was cloned and overexpressed in Escherichia coli BL21 CodonPlus (DE3)-RIPL. Expression and production of extracellular TpGUS was enhanced through various specific cultivation and induction strategies. Extracellular TpGUS activity was improved by 3.44 and 7 fold in 4 × ZB medium induced with 0.5 mM IPTG and 100 mM lactose, respectively. The enzyme was purified to homogeneity with a single band of 65.6 kDa on SDS-PAGE, using two subsequent steps of anion exchange and hydrophobic interaction chromatography after heat precipitation (70 °C, 1 h). Optimal activity of TpGUS was observed at 95 °C and pH 6.0; and it displayed prodigious thermal stability over a temperature range of 50–85 °C for 12 h at pH 6.0–7.5. Km, Vmax, Vmax Km−1, kcat, and kcat Km−1 were calculated to be 0.7 mM, 227 mmol mg−1 min−1, 324.3 min−1, 164,492.7 s−1 and 234,989.6 mM−1 s−1, respectively using pNPGU as a substrate. Recombinant TpGUS exhibited favorable properties which make this a promising candidate for various biotechnological and pharmacological applications. |
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