Johanna Mansfeld

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A low-molecular weight phospholipase A2 from Arabidopsis thaliana, isoform phospholipase A2-alpha, has been expressed in Escherichia coli in the form of inclusion bodies, refolded, and purified to homogeneity to yield the active mature enzyme. The enzyme was characterized with respect to pH, temperature optimum, and Ca2+ ion requirement. The enzyme has been(More)
Ala-Phe-Ile-Gly-Leu-Met has been disengaged by cyclization of H-Leu-Met-Ala-Phe-Ile-Gly-OH by means of dicyclohexylcarbodiimide/N-Hydroxysuccinimide or the adequate p-nitrophenylester. Acc. to various strategic variants, the design of the linear precursor has been performed by condensation of the segments of Boc-Leu-Met-OH and H-Ala-Phe-Ile-Gly-OH. The(More)
The thermal inactivation of broad specificity proteases such as thermolysin and subtilisin is initiated by partial unfolding processes that render the enzyme susceptible to autolysis. Previous studies have revealed that a surface-located region in the N-terminal domain of the thermolysin-like protease produced by Bacillus stearothermophilus is crucial for(More)
Boilysin (BLN) is an engineered, highly thermostable neutral protease from Bacillus stearothermophilus. Its high resistance is based on the stabilization of a surface loop (amino acid residues 55-69) by eight amino acid exchanges, including the introduction of a disulphide bond. In the present study, BLN was compared with the well-known and structurally(More)
Although highly stable toward unfolding, native ribonuclease A is known to be cleaved by unspecific proteases in the flexible loop region near Ala20. With the aim to create a protease-resistant ribonuclease A, Ala20 was substituted for Pro by site-directed mutagenesis. The resulting mutant enzyme was nearly identical to the wild-type enzyme in the near-UV(More)
Engineered extremely thermostable variants of the thermolysin-like protease from Bacillus stearothermophilus possessing an introduced disulfide bond G8C/N60C (double mutant, DM) and six additional amino acid substitutions in the exposed loop region 56-69 (Boilysin, BLN) have been probed with respect to stability toward water-miscible organic solvents and(More)
Protein stabilization by immobilization has been proposed to be most effective if the protein is attached to the carrier at that region where unfolding is initiated. To probe this hypothesis, we have studied the effects of site-specific immobilization on the thermal stability of mutants of the thermolysin-like protease from Bacillus stearothermophilus(More)
The recent progress in knowledge on biochemical properties and functions of phospholipases A(2) in plants paved the way for approving the suitability of these enzymes for commercial use now. The secreted phospholipases A(2), representing one type of phospholipases A(2) occurring in plants, show distinct differences in substrate specificities with respect to(More)
Most phospholipases D (PLDs) occurring in microorganisms, plants and animals belong to a superfamily which is characterized by several conserved regions of amino acid sequence including the two HKD motifs necessary for catalytic activity. Most eukaryotic PLDs possess additional regulatory structures such as the Phox and Pleckstrin homology domains in(More)
Disulfide bonds are known to be crucial for protein stability. To probe the contribution of each of the five disulfide bonds (C9-C31, C30-C70, C37-C63, C61-C95, and C105-C113) in bee venom phospholipase A(2) to stability, variants with deleted disulfide bonds were produced by substituting two serine residues for each pair of cysteine residues. The mutations(More)