几种理化因子对海洋硝化细菌去除氨氮效果的影响

研究pH、温度、供氧状况、投菌量、氨氮初始浓度等理化因子对海洋硝化细菌去除氨氮效果的影响。结果表明,各种理化因子对硝化细菌去除氨氮效果均有明显影响,海洋硝化细菌的最佳作用条件是pH 8.5,25℃,好氧,对氨氮的去除效果随着投菌量的增加而增强。在适宜条件下,海洋硝化细菌对海水中氨氮具有较好的去除效果。研究海洋硝化细菌在去除氨氮过程中D IN之间相互转化关系发现,在处理系统中随着NH4 -N含量的降低,NO2--N、NO3--N含量持续增加,大约有66.57%的D IN在海洋硝化细菌作用过程中以其他形式脱离了处理系统。     

Endothelial nitric oxide production is tightly coupled to the citrulline–NO cycle

Nitric oxide (NO) is an important vasorelaxant produced along with L-citrulline from L-arginine in a reaction catalyzed by endothelial nitric oxide synthase (eNOS). Previous studies suggested that the recycling of L-citrulline to L-arginine is essential for NO production in endothelial cells. However, there is no direct evidence demonstrating the degree to which the recycling of L-citrulline to L-arginine is coupled to NO production. We hypothesized that the amount of NO formed would be significantly higher than the amount of L-citrulline formed due to the efficiency of L-citrulline recycling via the citrulline-NO cycle. To test this hypothesis, endothelial cells were incubated with [14C]-L-arginine and stimulated by various agents to produce NO. The extent of NO and [14C]-L-citrulline formation were simultaneously determined. NO production exceeded apparent L-citrulline formation of the order of 8 to 1, under both basal and stimulated conditions. As further support, alpha-methyl-DL-aspartate, an inhibitor of argininosuccinate synthase (AS), a component of the citrulline-NO cycle, inhibited NO production in a dose-dependent manner. The results of this study provide evidence for the essential and efficient coupling of L-citrulline recycling, via the citrulline-NO cycle, to endothelial NO production.     

Anaerobic treatment of highly concentrated aniline wastewater using packed-bed biofilm reactor

The performance of packed-bed biofilm reactor (PBBR) with self-floating bio-carriers was investigated to treat highly concentrated organic nitrogenous aniline wastewater with a COD value as high as 24,000 mg/L. With 45 vol% of carrier charge inside the reactor, the aniline wastewater can be effectively treated with 94% of COD removal efficiency at a low organic loading rate (OLR) of 0.9 kg COD/(m³ d). The removal efficiency decreased gradually down to 75% when OLR increased to 12.27 kg COD/(m³ d) that corresponded to 1 day of HRT. Separate tests with biofilm alone showed that the conversion contribution of the biofilm was about half of the overall COD conversion by the biofilm plus sludge system at the same OLRs of 3–4 kg COD/(m³ d), and that the biofilm had higher activity than suspended sludge. Ammonium released from decomposed aniline was increased gradually from 500 to 1700 mg/L with the OLR increase from 0.9 to 12.27 kg COD/(m³ d), which resulted in inhibitory effect to the microorganism due to the toxicity of free ammonia. Batch anaerobic toxicity tests showed that the biofilm was less sensitive to toxic compounds than suspended sludge and could tolerate higher concentration of free ammonia.     

Stereoselective hexobarbital 3'-hydroxylation by CYP2C19 expressed in yeast cells and the roles of amino acid residues at positions 300 and 476

We examined the enzymatic function of recombinant CYP2C19 in enantiomeric hexobarbital (HB) 3'-hydroxylation, and searched the roles of amino acid residues, such as Phe-100, Phe-114, Asp-293, Glu-300, and Phe-476 of CYP2C19 in the stereoselective HB 3'-hydroxylation, using a yeast cell expression system and site-directed mutagenesis method. CYP2C19 wild-type exerted substrate enantioselectivity of (R)-HB>(S)-HB and metabolite diastereoselectivity of 3'(R)<3'(S) in 3'-hydroxylation of HB enantiomers. The substitution of Asp-293 by alanine failed to yield an observable peak at 450 nm in its reduced carbon monoxide-difference spectrum. CYP2C19-E300A and CYP2C19-E300V with alanine and valine, respectively, in place of Glu-300 exerted total HB 3'-hydroxylation activities of 45 and 108%, respectively, that of the wild-type. Interestingly, these two mutants showed substrate enantioselectivity of (R)-HB<(S)-HB, which is opposite to that of the wild-type, while metabolite diasteroselectivity remained unchanged. The replacement of Phe-476 by alanine increased total HB 3'-hydroxylation activity to approximately 3-fold that of the wild-type. Particularly, 3'(S)-OH-(S)-HB-forming activity elevated to 7-fold that of the wild-type, resulting in the reversal of the substrate enantioselectivity. In contrast, the substitution of phenylalanine at positions 100 and 114 by alanine did not produce a remarkable change in the total activity or the substrate enantioselectivity. These results indicate that Glu-300 and Phe-476 are important in stereoselective oxidation of HB enantiomers by CYP2C19.     

Crystal structures of halohydrin hydrogen‐halide‐lyases from Corynebacterium sp. N‐1074

Halohydrin hydrogen‐halide‐lyase (H‐Lyase) is a bacterial enzyme that is involved in the degradation of halohydrins. This enzyme catalyzes the intramolecular nucleophilic displacement of a halogen by a vicinal hydroxyl group in halohydrins to produce the corresponding epoxides. The epoxide products are subsequently hydrolyzed by an epoxide hydrolase, yielding the corresponding 1, 2‐diol. Until now, six different H‐Lyases have been studied. These H‐Lyases are grouped into three subtypes (A, B, and C) based on amino acid sequence similarities and exhibit different enantioselectivity. Corynebacterium sp. strain N‐1074 has two different isozymes of H‐Lyase, HheA (A‐type) and HheB (B‐type). We have determined their crystal structures to elucidate the differences in enantioselectivity among them. All three groups share a similar structure, including catalytic sites. The lack of enantioselectivity of HheA seems to be due to the relatively wide size of the substrate tunnel compared to that of other H‐Lyases. Among the B‐type H‐Lyases, HheB shows relatively high enantioselectivity compared to that of HheB_GP1. This difference seems to be due to amino acid replacements at the active site tunnel. The binding mode of 1, 3‐dicyano‐2‐propanol at the catalytic site in the crystal structure of the HheB‐DiCN complex suggests that the product should be (R)‐epichlorohydrin, which agrees with the enantioselectivity of HheB. Comparison with the structure of HheC provides a clue for the difference in their enantioselectivity. Proteins 2015; 83:2230–2239. © 2015 Wiley Periodicals, Inc.     

Macroecological patterns of archaeal ammonia oxidizers in the Atlantic Ocean

Macroecological patterns are found in animals and plants, but also in micro‐organisms. Macroecological and biogeographic distribution patterns in marine Archaea, however, have not been studied yet. Ammonia‐oxidizing Archaea (AOA) show a bipolar distribution (i.e. similar communities in the northernmost and the southernmost locations, separated by distinct communities in the tropical and gyral regions) throughout the Atlantic, detectable from epipelagic to upper bathypelagic layers (<2000 m depth). This tentatively suggests an influence of the epipelagic conditions of organic matter production on bathypelagic AOA communities. The AOA communities below 2000 m depth showed a less pronounced biogeographic distribution pattern than the upper 2000 m water column. Overall, AOA in the surface and deep Atlantic waters exhibit distance–decay relationships and follow the Rapoport rule in a similar way as bacterial communities and macroorganisms. This indicates a major role of environmental conditions in shaping the community composition and assembly (species sorting) and no, or only weak limits for dispersal in the oceanic thaumarchaeal communities. However, there is indication of a different strength of these relationships between AOA and Bacteria, linked to the intrinsic differences between these two domains.     

Human SLC4A11 Is a Novel NH3/H+ Co-transporter

SLC4A11 has been proposed to be an electrogenic membrane transporter, permeable to Na⁺, H⁺ (OH⁻), bicarbonate, borate, and NH₄⁺. Recent studies indicate, however, that neither bicarbonate or borate is a substrate. Here, we examined potential NH₄⁺, Na⁺, and H⁺ contributions to electrogenic ion transport through SLC4A11 stably expressed in Na⁺/H⁺ exchanger-deficient PS120 fibroblasts. Inward currents observed during exposure to NH₄Cl were determined by the [NH₃]_o, not [NH₄⁺]_o, and current amplitudes varied with the [H⁺] gradient. These currents were relatively unaffected by removal of Na⁺, K⁺, or Cl⁻ from the bath but could be reduced by inclusion of NH₄Cl in the pipette solution. Bath pH changes alone did not generate significant currents through SLC4A11, except immediately following exposure to NH₄Cl. Reversal potential shifts in response to changing [NH₃]_o and pH_o suggested an NH₃/H⁺-coupled transport mode for SLC4A11. Proton flux through SLC4A11 in the absence of ammonia was relatively small, suggesting that ammonia transport is of more physiological relevance. Methylammonia produced currents similar to NH₃ but with reduced amplitude. Estimated stoichiometry of SLC4A11 transport was 1:2 (NH₃/H⁺). NH₃-dependent currents were insensitive to 10 μm ethyl-isopropyl amiloride or 100 μm 4,4′- diisothiocyanatostilbene-2,2′-disulfonic acid. We propose that SLC4A11 is an NH₃/2H⁺ co-transporter exhibiting unique characteristics.     

Production of mesaconate in Escherichia coli by engineered glutamate mutase pathway

Mesaconate is an intermediate in the glutamate degradation pathway of microorganisms such as Clostridium tetanomorphum. However, metabolic engineering to produce mesaconate has not been reported previously. In this work, two enzymes involved in mesaconate production, glutamate mutase and 3-methylaspartate ammonia lyase from C. tetanomorphum, were recombinantly expressed in Escherichia coli. To improve mesaconate production, reactivatase of glutamate mutase was discovered and adenosylcobalamin availability was increased. In addition, glutamate mutase was engineered to improve the in vivo activity. These efforts led to efficient mesaconate production at a titer of 7.81 g/L in shake flask with glutamate feeding. Then a full biosynthetic pathway was constructed to produce mesaconate at a titer of 6.96 g/L directly from glucose. In summary, we have engineered an efficient system in E. coli for the biosynthesis of mesaconate.