Direct Stimulation by Methanol Addition on the Cultured Medium for Methanol Dehydrogenase Protein Purification from Bradyrhizobium japonicum USDA110

https://doi.org/10.21059/buletinpeternak.v42i3.28155

Novita Kurniawati(1*), Ambar Pertiwiningrum(2), Yuny Erwanto(3), Nanung Agus Fitriyanto(4), Mohammad Zainal Abidin(5)

(1) Animal Products Technology, Faculty of Animal Science, Universitas Gadjah Mada, Yogyakarta, 55281, Indonesia
(2) Animal Products Technology, Faculty of Animal Science, Universitas Gadjah Mada, Yogyakarta, 55281, Indonesia
(3) Animal Products Technology, Faculty of Animal Science, Universitas Gadjah Mada, Yogyakarta, 55281, Indonesia
(4) Animal Products Technology, Faculty of Animal Science, Universitas Gadjah Mada, Yogyakarta, 55281, Indonesia
(5) Animal Products Technology, Faculty of Animal Science, Universitas Gadjah Mada, Yogyakarta, 55281, Indonesia
(*) Corresponding Author

Abstract


Methanol dehydrogenase (MDH) enzyme was purified from Bradyrhizobium japonicum USDA110 cell-free extract. The bacteria were grown in a culture medium with direct 0.5% methanol addition aimed to stimulates the MDH catalytic enzyme activation. Bradyrhizobium japonicum USDA110 MDH enzyme was purified by using 25 mM 2-(N-morpholine) ethanesulfonic acid/MES pH 5.5 buffer and 1 M sodium chloride/NaCl which separated into three columns, the first column was PD-10 for buffer exchange; the second column was HiTrap Sepharose HP to obtain unbonded fraction in the column; and the third column was Mono S 5/50 GL integrated with two pumps HPLC (high-performance liquid chromatography) to obtain pure MDH enzyme for serial changing of 1 M NaCl-25mM MES pH 5.5 with the flow rate at 1 ml/min. The protein concentration and MDH catalytic enzyme activity were observed on each purification process starting from the cell-free extract to pure MDH enzyme. The pure MDH enzyme was obtained by Mono S 5/50 GL-HPLC purification which showed a single band on SDS PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The MDH enzyme purification from Bradyrhizobium japonicum USDA110 showed 90-fold purification, a sub-molecular weight of 63 kDa, specific activity at 2.69 U/mg, and optimum activity at a 35oC temperature and pH 9.

     


Keywords


High-performance liquid chromatography; MDH; Sodium chloride; 2-(N- morpholine) ethanesulfonic acid

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DOI: https://doi.org/10.21059/buletinpeternak.v42i3.28155

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