Chloroperoxidase (CPO) from the fungus Caldariomyces fumago is undoubtedly the most versatile member of the heme protein family. In addition to functioning as a halogenating enzyme and a classical peroxidase, CPO catalyzes the dismutation of peroxides in a catalase‐type reaction and carries out cytochrome P450 oxygen insertion reactions. From the viewpoint of biocatalysis the most important CPO reactions are chiral epoxidations, hydroxylations, and sulfoxidations. CPO catalyzes a variety of chiral epoxidation reactions with high yields and high enantioselectivities. However, the industrial use of native CPO for the synthesis of chiral epoxides is limited because of its relatively low epoxidation rates in comparison to its high catalase activity, which robs the epoxidation reaction of oxidant. The use of CPO is also restricted by its poor reactivities in organic solvents. Directed evolution technology has been used to address these problems. After three
rounds of PCR‐based random mutagenesis, we have isolated mutants of chloroperoxidase having greatly enhanced epoxidation activity compared to the wild‐type enzyme. In addition, in the screening of a first generation library of random mutation transformants, we have isolated three CPO mutant clones having improved chlorination activity and enhanced stability in a ternary solvent microemulsion comprised of toluene, isopropanol and water. Surprisingly, all three recombinant variants carry a single mutation in the cysteine residue that functions as the proximal heme ligand in the native enzyme. Two of these mutant clones are identical, having the proximal cysteine heme‐ligand replaced with a tyrosine residue. The third mutant has the cysteine‐29 replaced with a histidine residue. The cysteine mutation in the three mutants is the only amino acid replacement. All other mutations in the three clones were silent mutations. These data suggest that ‘‘directed
evolution’’ can be successfully applied to the engineering of chloroperoxidase in the quest for a better industrial biocatalyst.
The Cry1Ab ␦-endotoxin V171C mutant protein exhibits a 25-fold increase in toxicity against Lymantria dispar, which correlates with a faster rate of partitioning into the midgut membrane and slightly decreased protein stability. This is an insect-specific mechanism; similar results were not observed in Manduca sexta, another Cry1Ab ␦-endotoxin-susceptible insect.
BackgroundThe Cry toxins, or δ-endotoxins, are a diverse group of proteins produced by Bacillus thuringiensis. While DNA secondary structures are biologically relevant, it is unknown if such structures are formed in regions encoding conserved domains of Cry toxins under shuffling conditions. We analyzed 5 holotypes that encode Cry toxins and that grouped into 4 clusters according to their phylogenetic closeness. The mean number of DNA secondary structures that formed and the mean Gibbs free energy were determined by an in silico analysis using different experimental DNA shuffling scenarios. In terms of spontaneity, shuffling efficiency was directly proportional to the formation of secondary structures but inversely proportional to ∆G.ResultsThe results showed a shared thermodynamic pattern for each cluster and relationships among sequences that are phylogenetically close at the protein level. The regions of the cry11Aa, Ba and Bb genes that encode domain I showed more spontaneity and thus a greater tendency to form secondary structures (<∆G). In the region of domain III; this tendency was lower (>∆G) in the cry11Ba and Bb genes. Proteins that are phylogenetically closer to Cry11Ba and Cry11Bb, such as Cry2Aa and Cry18Aa, maintained the same thermodynamic pattern. More distant proteins, such as Cry1Aa, Cry1Ab, Cry30Aa and Cry30Ca, featured different thermodynamic patterns in their DNA.ConclusionThese results suggest the presence of thermodynamic variations associated to the formation of secondary structures and an evolutionary relationship with regions that encode highly conserved domains in Cry proteins. The findings of this study may have a role in the in silico design of cry gene assembly by DNA shuffling techniques.
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