Effect of His6-tag position on the expression and properties of phenylacetone monooxygenase from Thermobifida fusca

Parshin, P.D.; Pometun, A.A.; Martysuk, U.A.; Kleymenov, S.Y.; Atroshenko, D.L.; Pometun, E.V.; Savin, S.S.; Tishkov, V.I.

Авторы: Parshin P.D., Pometun A.A., Martysuk U.A., Kleymenov S.Yu, Atroshenko D.L., Pometun E.V., Savin S.S., Tishkov V.I.
Журнал: Biochemistry (Moscow)
Том: 85
Номер: 5
Год издания: 2020
Издательство: Pleiades Publishing, Ltd
Местоположение издательства: Road Town, United Kingdom
Первая страница: 575
Последняя страница: 582
DOI: 10.1134/S0006297920050065
Аннотация: Phenylacetone monooxygenase (EC 1.14.13.92, РАМО) catalyzes oxidation of ketones with molecular oxygen and NADPH with the formation of esters. РАМО is a promising enzyme for biotechnological processes. In this work, we generated genetic constructs coding for PAMO from Thermobifida fusca, containing N- or C-terminal His6-tags (PAMO N and PAMO С, respectively), as well as PAMO L with the His6-tag attached to the enzyme C_terminus via a 19_a.a. spacer. All PAMO variants were expressed as catalytically active proteins in Escherichia coli BL21(DE3) cells; however, the expression level of PAMO N was 3 to 5 times higher than for the other two enzymes. The catalytic constants (kcat) of PAMO С and PAMO L were similar to that published for PAMO L produced in a different expression system; the catalytic constant for PAMO N was slightly lower (by 15%). The values of Michaelis constants with NADPH for all РАМО variants were in agreement within the published data for PAMO L (within the experimental error); however, the KM for benzylacetone was several times higher. Thermal inactivation studies and differential scanning calorimetry demonstrated that the thermal stability of PAMO N was 3 to 4 times higher compared to that of the enzymes with the C-terminal His6-tag.
Добавил в систему: Тишков Владимир Иванович

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Effect of His6-tag position on the expression and properties of phenylacetone monooxygenase from Thermobifida fuscaстатья

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