Rational Engineering of the Substrate Specificity of a Thermostable D-Hydantoinase (Dihydropyrimidinase)

D-hydantoinases catalyze an enantioselective opening of 5- and 6-membered cyclic structures and therefore can be used for the production of optically pure precursors for biomedical applications. The thermostable D-hydantoinase from Geobacillus stearothermophilus ATCC 31783 is a manganese-dependent enzyme and exhibits low activity towards bulky hydantoin derivatives. Homology modeling with a known 3D structure (PDB code: 1K1D) allowed us to identify the amino acids to be mutated at the substrate binding site and in its immediate vicinity to modulate the substrate specificity. Both single and double substituted mutants were generated by site-directed mutagenesis at appropriate sites located inside and outside of the stereochemistry gate loops (SGL) involved in the substrate binding. Substrate specificity and kinetic constant data demonstrate that the replacement of Phe159 and Trp287 with alanine leads to an increase in the enzyme activity towards D,L-5-benzyl and D,L-5-indolylmethyl hydantoins. The length of the side chain and the hydrophobicity of substrates are essential parameters to consider when designing the substrate binding pocket for bulky hydantoins. Our data highlight that D-hydantoinase is the authentic dihydropyrimidinase involved in the pyrimidine reductive catabolic pathway in moderate thermophiles.

An XPS and XANES Study on the Bioleaching of Arsenopyrite with or without Pyrite

In the present study, we investigated the bioleaching of arsenopyrite with or without pyrite by moderate thermophiles. In both chemical leaching and bioleaching, the addition of pyrite decreased the leaching rate of arsenopyrite. The arsenic speciation and minerology changes in the residues were analysed by X-ray Absorption Near Edge Structure (XANES) Spectroscopy, X-ray Photoelectron Spectroscopy (XPS) and powder X-ray Diffraction (XRD). The XANES analysis showed no detectable arsenopyrite in the final residues from the experiments without pyrite. However, there was still 21.7% of arsenic species presented as arsenopyrite after bioleaching, when the initial arsenopyrite/pyrite ratio was 1:5. The XPS analysis revealed there was only As(V) on the surface of most of the residues, except on one chemically leached sample where As(III) was found.

Complete Genome Sequences of Two Interactive Moderate Thermophiles,Paenibacillus napthalenovorans32O-Y andPaenibacillussp. 32O-W

Microorganisms with the capability to desulfurize petroleum are in high demand with escalating restrictions currently placed on fuel purity. Thermophilic desulfurizers are particularly valuable in high-temperature industrial applications. We report the whole-genome sequences ofPaenibacillus napthalenovorans32O-Y andPaenibacillussp. 32O-W, which can and cannot, respectively, metabolize dibenzothiophene.

Evolution of Leaching Products on the Surface of Chalcopyrite by Mesophiles and Thermophiles Based on SR-XRD and XANES Spectroscopy

The bioleaching experiments of chalcopyrite were conducted with single and mixed mesophiles (30 °C) and moderate thermophiles (45 °C) and extreme thermophile (65 °C), respectively, and analyzed by synchrotron radiation (SR) based X-ray diffraction (XRD) and X-ray absorption near edge structure (XANES) spectroscopy. The results showed that the copper extraction of chalcopyrite could be significantly promoted by bioleaching microorganisms, and the promotion effects for both the mixed cultures grown at different temperature and the different cells grown at the same temperature were significantly different. The surface of chalcopyrite after bioleached by the mixed or sole cultures are serious corroded and became complicated. More S0 was found to form in the sole cultures of specific iron-oxidizing microorganism L. ferrooxidans and L. ferriphilum and sulfur-oxidizing microorganisms A. thiooxidans and A. caldus cultures. Jarosite and secondary minerals (chalcocite and covellite) were detected for the mixed cultures and sole cultures of iron/sulfur-oxidizing microorganisms. The evolution of chalcocite and covellite were just relevant to the potential of leaching solution, no matter which cultures were used, where chalcocite could be formed at Eh value less than 500 mV and then converted to covellite at Eh value ~550 mV.