Production of synthetically created phospholipase A2 variants with industrial impact

• Published in: Biotechnology and bioengineering Vol. 98; no. 1; pp. 48 - 59
• Main Authors: Markert, Yvonne, Mansfeld, Johanna, Schierhorn, Angelika, Rücknagel, Karl Peter, Ulbrich-Hofmann, Renate
• Format: Journal Article
• Language: English
• Published: Hoboken Wiley Subscription Services, Inc., A Wiley Company 01.09.2007; Wiley
• Subjects: Apis mellifera; bee venom; Biological and medical sciences; Biotechnology; Escherichia coli; Fundamental and applied biological sciences. Psychology; high cell density fermentation inclusion bodies; Phospholipase A2; renaturation; Phospholipase A2; renaturation; Enzyme; Escherichia coli; Esterases; Fermentation; Phospholipase A; Carboxylic ester hydrolases; Variant; Production; bee venom; Bacteria; Hydrolases; Inclusion body; high cell density fermentation inclusion bodies; Enterobacteriaceae
• ISSN: 0006-3592; 1097-0290
• DOI: 10.1002/bit.21392

Phospholipases A2 (PLA2) 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 PLA2 to industrial application, synthetic genes encoding PLA2 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‐PLA2, I1V‐PLA2, His6‐tagged PLA2 and PLA2 still containing the start methionine) were successfully expressed. In all cases, the PLA2 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 PLA2 variants showed activities, far‐UV CD and fluorescence spectra similar to the glycosylated PLA2 isolated from the venom glands of honey bee (bv‐PLA2). 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‐PLA2. For the variant I1A‐PLA2 high‐cell density fermentation in 10 L‐scale using mineral salt medium was shown to increase the volumetric enzyme yield considerably. Biotechnol. Bioeng. 2007; 98: 48–59. © 2007 Wiley Periodicals, Inc.