The expression and purification of melittin (MET) in microbials are difficult because of its antibacterial activities. In this work, MET was fused with a glutathione-S-transferase (GST) tag and expressed in Escherichia coli to overcome its lethality to host cells. The fusion protein GST-MET was highly expressed and then purified by glutathione sepharose high-performance affinity chromatography, digested with prescission protease, and further purified by Superdex Peptide 10/300 GL chromatography. Finally, 3.5 mg/L recombinant melittin (rMET) with a purity of >90% was obtained; its antibacterial activities against Gram-positive Bacillus pumilus and Staphylococcus pasteuri were similar to those of commercial MET. A circular dichroism spectroscopic assay showed that the rMET peptide secondary structure was similar to those of the commercial form. To our knowledge, this is the report of the preparation of active pure rMET with no tags. The successful expression and purification of rMET will enable large-scale, industrial biosynthesis of MET.

Background: (S)-1-phenyl-1,2-ethanediol is an important chiral intermediate in the synthesis of liquid crystals and chiral biphosphines. (S)-carbonyl reductase II from Candida parapsilosis catalyzes the conversion of 2-hydroxyacetophenone to (S)-1-phenyl-1,2-ethanediol with NADPH as a cofactor. Glucose dehydrogenase with a Ala258Phe mutation is able to catalyze the oxidation of xylose with concomitant reduction of NADP + to NADPH, while endo-β-1,4-xylanase 2 catalyzes the conversion of xylan to xylose. In the present work, the Ala258Phe glucose dehydrogenase mutant and endo-β-1,4-xylanase 2 were introduced into the (S)-carbonyl reductase II-mediated chiral pathway to strengthen cofactor regeneration by using xylan as a naturally abundant co-substrate. Results: We constructed several coupled multi-enzyme systems by introducing (S)-carbonyl reductase II, the A258F glucose dehydrogenase mutant and endo-β-1,4-xylanase 2 into Escherichia coli. Different strains were produced by altering the location of the encoding genes on the plasmid. Only recombinant E. coli/pET-G-S-2 expressed all three enzymes, and this strain produced (S)-1-phenyl-1,2-ethanediol from 2-hydroxyacetophenone as a substrate and xylan as a co-substrate. The optical purity was 100% and the yield was 98.3% (6 g/L 2-HAP) under optimal conditions of 35 °C, pH 6.5 and a 2:1 substrate-co-substrate ratio. The introduction of A258F glucose dehydrogenase and endo-β-1,4-xylanase 2 into the (S)-carbonyl reductase II-mediated chiral pathway caused a 54.6% increase in yield, and simultaneously reduced the reaction time from 48 to 28 h. Conclusions: This study demonstrates efficient chiral synthesis using a pentose as a co-substrate to enhance cofactor regeneration. This provides a new approach for enantiomeric catalysis through the inclusion of naturally abundant materials.

No abstract

The GdmC1-induced equilibrium unfolding of firefly luciferase was investigated by detection of activity, fluorescence, ANS fluorescence and circular dichroism. These Studies revealed that the unfolding process presented multiple intermediates. Low refolding efficiency was observed when luciferase was denatured by moderate concentration of GdmC1, however, the enzyme with slight alteration in tertiary structure or complete destruction in secondary structure could be partially or completely renaturated. These results also implied that there may exist intermediates that differ from N and U State. The large exposed hydrophobic surface of intermediate states had great tendency to form aggregate so as to prevent luciferase from refolding properly. Three of six mAbs, 4G3 S2G10, N2E3 could increase the yield of reactivation dramatically, they might bind to the exposed hydrophobic surface of the intermediates and prevent it from the aggregation formation. Epitope mapping showed that the epitopes of all the three mAbs lie in A subdomain of the N-terminal domain of luciferase. These results have shed light on the structural features of the intermediate and its interface involved in formation of aggregation. 1502 Equilibrium association of unfolding intermediate of designed A Cro-repressor variants.The Cro-repressor from the A phage is a small homodimer protein. It was previously shown that at equilibrium conditions this protein and its S-S-bridged mutant, CroV55C, can adopt an alternative partially folded conformation stabilized by intermolecular association [1,2]. Here we show that a designed monomeric variant of Cro, Cro.mDG [3], and some BrCN-fragments of CroV55C are also able to form that complex, which allows us t o localize the sequence participating in the association. By differential scanning calorimetry it is also shown that increasing ionic strength stabilises the complex but does not change the Gibbs energy of the native state. This, together with a sharp pH-dependence, suggests that some charge clusters/salt bridges are specifically formed within the complex. 1.Folding is believed to start the formation of the folding initiation nuclei. This hypothesis suffers from the scarcity of experimental evidence. Folding nuclei are hard to find in the unfolded state ensemble because these are most probably relatively unstable species. In unfolded protein only a small fraction of molecules adopts a conformation of a folding nucleus, which nevertheless promotes further folding of the entire population of the molecules. We used double mutant strategy and disulphide formation kinetics measurements in different conditions of temperature and solvent composition to study the impact on folding of the residual structure in unfolded bovine pancreatic trypsin inhibitor BPTI (Zdanowski, K., Dadlez, M. (1999) J. Mol. Biol. 287,433-445).Our results indicate that folding initiation site is stabilised by an enthalpy driven interaction. The most probable candidate to account for such an interaction is a network of hydrogen bonds. Our data ...

Background: ( S )-1-phenyl-1,2-ethanediol is an important chiral intermediate in the synthesis of liquid crystals and chiral biphosphines.(S)-carbonyl reductase II from Candida parapsilosis catalyzes the conversion of 2-hydroxyacetophenone to ( S )-1-phenyl-1,2-ethanediol with NADPH as a cofactor. Glucose dehydrogenase with a Ala258Phe mutation is able to catalyze the oxidation of xylose with concomitant reduction of NADP + to NADPH, while endo-β-1,4-xylanase 2 catalyzes the conversion of xylan to xylose. In the present work, the Ala258Phe glucose dehydrogenase mutant and endo-β-1,4-xylanase 2 were introduced into the ( S )-carbonyl reductase II-mediated chiral pathway to strengthen cofactor regeneration by using xylan as a naturally abundant co-substrate. Results: We constructed several coupled multi-enzyme systems by introducing ( S )-carbonyl reductase II, the A258F glucose dehydrogenase mutant and endo-β-1,4-xylanase 2 into Escherichia coli . Different strains were produced by altering the location of the encoding genes on the plasmid. Only recombinant E. coli /pET-G-S-2 expressed all three enzymes, and this strain produced ( S )-1-phenyl-1,2-ethanediol from 2-hydroxyacetophenone as a substrate and xylan as a co-substrate. The optical purity was 100% and the yield was 98.3% (6 g/L 2-HAP) under optimal conditions of 35°C, pH 6.5 and a 2:1 substrate-co-substrate ratio. The introduction of A258F glucose dehydrogenase and endo-β-1,4-xylanase 2 into the ( S )-carbonyl reductase II-mediated chiral pathway caused a 54.6% increase in yield, and simultaneously reduced the reaction time from 48 h to 28 h. Conclusions: This study demonstrates efficient chiral synthesis using a pentose as a co-substrate to enhance cofactor regeneration. This provides a new approach for enantiomeric catalysis through the inclusion of naturally abundant materials.