Effect of His6-tag Position on the Expression and Properties of Phenylacetone Monooxygenase from Thermobifida fusca.
Biochemistry (Mosc)
; 85(5): 575-582, 2020 May.
Article
in En
| MEDLINE
| ID: mdl-32571187
ABSTRACT
Phenylacetone monooxygenase (EC 1.14.13.92, PAMÐ) catalyzes oxidation of ketones with molecular oxygen and NADPH with the formation of esters. PAMÐ 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 C, 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 C 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 PAMÐ 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.
Full text:
1
Database:
MEDLINE
Main subject:
Oligopeptides
/
Acetone
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Recombinant Proteins
/
Histidine
/
Mixed Function Oxygenases
Language:
En
Year:
2020
Type:
Article