Production of synthetically created phospholipase A(2) variants with industrial impact.
Phospholipases A(2) (PLA(2)) play an important role for the production of lysophospholipids. Presently they are mainly obtained from porcine or bovine pancreas but these mammalian sources are not accepted in several fields of application. To make accessible a non-mammalian PLA(2) to industrial application, synthetic genes encoding PLA(2) from honey bee (Apis mellifera) with modified N-termini were constructed and expressed in Escherichia coli. While expression of the gene with an N-terminal leader sequence to direct the protein into the periplasm failed, four variants with slightly modified N-termini (I1A-PLA(2), I1V-PLA(2), His(6)-tagged PLA(2) and PLA(2) still containing the start methionine) were successfully expressed. In all cases, the PLA(2) variants were produced as inclusion bodies. Their protein content amounted to 26-35% of total cell protein. The optimized renaturation procedure and subsequent purification by cation-exchange chromatography yielded pure active enzymes in yields of 4-11 mg L(-1). The recombinant PLA(2) variants showed activities, far-UV CD and fluorescence spectra similar to the glycosylated PLA(2) isolated from the venom glands of honey bee (bv-PLA(2)). The thermodynamic stabilities of the recombinant enzymes calculated from the transition curves of guanidine hydrochloride induced unfolding were also nearly identical to the stability of bv-PLA(2). For the variant I1A-PLA(2) high-cell density fermentation in 10 L-scale using mineral salt medium was shown to increase the volumetric enzyme yield considerably.
Markert
Yvonne
Y
Institute of Biochemistry and Biotechnology, Martin-Luther University Halle-Wittenberg, Kurt-Mothes Strasse 3, 06120 Halle, Germany.
Mansfeld
Johanna
J
Schierhorn
Angelika
A
Rücknagel
Karl Peter
KP
Ulbrich-Hofmann
Renate
R
D000818:Animals; D001516:Bees / Q000201:enzymology* / Q000235:genetics*; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D007218:Industrial Microbiology / Q000379:methods; D010741:Phospholipases A / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
D011994:Recombinant Proteins; D010741:Phospholipases A