Comparison of the stability of CYP105A1 and its variants engineered for production of active forms of vitamin D.

CYP105A1 from Streptomyces griseolus converts vitamin D3 to its biologically active form, 1α,25-dihydroxy vitamin D3. R73A/R84A mutation enhanced the 1α- and 25-hydroxylation activity for vitamin D3, while M239A mutation generated the 1α-hydroxylation activity for vitamin D2. In this study, the stability of six CYP105A1 enzymes, including 5 variants (R73A/R84A, M239A, R73A/R84A/M239A (=TriA), TriA/E90A, and TriA/E90D), was examined. Circular dichroism analysis revealed that M239A markedly reduces the enzyme stability. Protein fluorescence analysis disclosed that these mutations, especially M239A, induce large changes in the local conformation around Trp residues. Strong stabilizing effect of glycerol was observed. Nondenaturing PAGE analysis showed that CYP105A1 enzymes are prone to self-association. Fluorescence analysis using a hydrophobic probe 8-anilino-1-naphthalenesulfonic acid suggested that M239A mutation enhances self-association and that E90A and E90D mutations, in cooperation with M239A, accelerate self-association with little effect on the stability.

Comparative studies on the activities of collagenases from Grimontia hollisae and Clostridium hystoliticum in the hydrolysis of synthetic substrates.

The collagenase produced by a gram-negative bacterium Grimontia hollisae strain 1706B (Ghcol) degrades collagen more efficiently than that produced by a gram-positive bacterium Clostridium histolyticum (Chcol), which is currently the most widely used collagenase in industry [Teramura et al. (Cloning of a novel collagenase gene from the gram-negative bacterium Grimotia (Vibrio) hollisae 1706B and its efficient expression in Brevibacillus choshinensis. J Bacteriol 2011;193:3049-3056)]. Here, we compared the Ghcol and Chcol activities using two synthetic substrates. In the hydrolysis of (7-methoxycoumarin-4-yl)acetyl-L-Lys-L-Pro-L-Leu-Gly-L-Leu-[N3-(2, 4-dinitrophenyl)-L-2, 3-diaminopropioyl]-L-Ala-L-Arg-NH2, Ghcol exhibited 350-fold higher activity than Chcol in the absence of CaCl2 and NaCl. The Ghcol activity markedly decreased with increasing concentrations of buffer, CaCl2 or NaCl, while the Chcol activity did not, suggesting that the Ghcol activity was sensitive to solvent components. In the hydrolysis of N-[3-(2-furyl)acryloyl]-L-Leu-Gly-L-Pro-Ala, Ghcol exhibited 16-fold higher activity than Chcol in the absence of CaCl2 and NaCl, and both enzyme activities did not decrease with increasing concentrations of buffer, CaCl2 or NaCl. pH dependences of activity revealed that the ionizable group responsible for acidic pKe may be Glu for Ghcol and Chcol, while that for alkaline pKe may be His for Ghcol and Tyr for Chcol. These striking differences suggest that the catalytic mechanism of Ghcol might be considerably different from that of clostridial collagenases.

Cultivation of denitrifying anaerobic methane-oxidizing microorganisms in a continuous-flow sponge bioreactor.

Anaerobic treatment of sewage has many advantages; however, the effluent contains high levels of dissolved methane. In this study, we investigated the use of a closed-type downflow hanging sponge (DHS) reactor for application of the denitrifying anaerobic methane oxidation (DAMO) reaction for nitrogen and dissolved methane removal. When using nitrate, the DAMO reaction achieved a denitrification rate of 84.4 g N m-3 day-1, which is close to that required for practical application of denitrification to anaerobic sewage treatment. The microbial community that developed in the DHS was investigated using16S rRNA, and novel species of DAMO bacteria affiliated with Group b of NC10 phylum were enriched. This contrasted with the results of previous studies in which the Candidatus Methylomirabilis oxyfera affiliated with Group a was enriched. The results obtained herein suggest that a post-treatment system for anaerobically treated sewage using a closed-type DHS reactor may become practical in the near future.