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991.
Mei-Jin Guo Ying-Ping Zhuang Ju Chu Si-Liang Zhang Ai-Sheng Xiong Ri-He Peng Quan-Hong Yao 《Process Biochemistry》2007,42(12):1660-1665
A genetically engineered Pichia pastoris FPHY34 strain containing a 1.3 kb thermostable phytase gene (fphy) evolved by DNA shuffling was constructed and screened. Expression and purification conditions for the recombinant phytase were developed in this study. The effect of Pi on recombinant phytase expression and cell growth of P. pastoris FPHY34 was tested in shake flask culture. Optimization of carbon sources for cell growth and methanol feeding strategies for phytase expression in P. pastoris FPHY34 was carried out in a 50-L fermenter by fed-batch fermentation. The purification of phytase was investigated by micro-filtration and ultra-filtration followed by desalting, ion-exchange chromatography, and gel filtration in the ÄKTA system. It showed that the optimum inorganic phosphorus is 13.6 g L−1 and that glucose can be used as a substrate for P. pastoris cell growth instead of glycerol; the biomass yield of glycerol (YX/S) is slightly higher than that of glucose. Different profiles of lag phase and respiratory quotient (RQ) displayed between glucose and glycerol as the sole carbon source. The maximum phytase activity in per millimetre reached 2508 U mL−1 at a methanol feed rate of 3.0 mL L−1 h−1 after 80 h period of induction. A purification factor of 41.1 with a 32% yield was achieved after chromatographic purification. The specific enzyme activity was 80 U mg−1 and 3281 U mg−1 in that supernatant fraction and after gel filtration purification, respectively. The strain P. pastoris FPHY34 showed a promising application in phytase industrial production. 相似文献
992.
The concentration of free Mg2+ in the matrix of isolated heart mitochondria has been monitored by using the fluorescent probe furaptra (mag-fura-2). Beef heart mitochondria respiring in a KCl medium in the absence of external Mg2+ maintain free matrix Mg2+ near 0.50 mM. Addition of Pi under these conditions decreases free Mg2+ by 0.12-0.17 mM depending on the substrate. This decrease in free Mg2+ appears to reflect changing ligand availability in the matrix. The decrease is prevented when the Pi transporter is blocked by mersalyl. Addition of ADP to initiate state 3 respiration causes a marked increase in free matrix Mg2+ (0.1-0.2 mM) that persists as long as ATP formation is taking place; free Mg2+ then returns to the base level. This cyclic change is blocked by oligomycin and carboxyatractyloside and appears to reflect to a large extent the decrease in matrix Pi that accompanies oxidative phosphorylation. Exchange of external ADP for matrix ATP may also contribute to the increase in free matrix Mg2+. Addition of an uncoupler promotes anion efflux and increases free matrix Mg2+. Similar changes in free Mg2+ on addition of Pi, ADP, or uncoupler are seen when extramitochondrial Mg2+ is buffered from 0.5 to 2 mM, but the basal free matrix Mg2+ increases as external Mg2+ concentration increases in this range. Free matrix Mg2+ also increases when total mitochondrial Mg2+ is increased by respiration-dependent uptake in the presence of Pi. It is concluded that matrix free Mg2+ changes significantly with changing ligand availability and that such changes may contribute to the regulation of Mg2(+)-sensitive matrix enzymes and membrane transporters. 相似文献
993.
The trans-aconitate methyltransferase from the bacterium Escherichia coli catalyzes the monomethyl esterification of trans-aconitate and related compounds. Using two-dimensional (1)H/(13)C nuclear magnetic resonance spectroscopy, we show that the methylation is specific to one of the three carboxyl groups and further demonstrate that the product is the 6-methyl ester of trans-aconitate (E-3-carboxy-2-pentenedioate 6-methyl ester). A similar enzymatic activity is present in the yeast Saccharomyces cerevisiae. Although we find that yeast trans-aconitate methyltransferase also catalyzes the monomethyl esterification of trans-aconitate, we identify that the methylation product of yeast is the 5-methyl ester (E-3-carboxyl-2-pentenedioate 5-methyl ester). The difference in the reaction catalyzed by the two enzymes may explain why a close homologue of the E. coli methyltransferase gene is not found in the yeast genome and furthermore suggests that these two enzymes may play distinct roles. However, we demonstrate here that the conversion of trans-aconitate to each of these products can mitigate its inhibitory effect on aconitase, a key enzyme of the citric acid cycle, suggesting that these methyltransferases may achieve the same physiological function with distinct chemistries. 相似文献
994.
Alexey Rivkin Sean P. Ahearn Stephanie M. Chichetti Christopher L. Hamblett Yudith Garcia Michelle Martinez Jed L. Hubbs Michael H. Reutershan Matthew H. Daniels Phieng Siliphaivanh Karin M. Otte Chaomin Li Andrew Rosenau Laura M. Surdi Joon Jung Bethany L. Hughes Jamie L. Crispino George N. Nikov Richard E. Middleton Christopher M. Moxham Mark S. Shearman 《Bioorganic & medicinal chemistry letters》2010,20(7):2279-2282
The development of a novel series of purines as γ-secretase modulators for potential use in the treatment of Alzheimer’s disease is disclosed herein. Optimization of a previously disclosed pyrimidine series afforded a series of potent purine-based γ-secretase modulators with 300- to 2000-fold in vitro selectivity over inhibition of Notch cleavage and that selectively reduces Αβ42 in an APP-YAC transgenic mouse model. 相似文献
995.
The Cucumber mosaic virus (CMV)-encoded 1a protein has been implicated to play a role in replication of the viral genome along with 2a and one or more host factors. To identify the host cell factors interacting with CMV 1a, we used the yeast two-hybrid system using tobacco cDNA library. One of the cDNA clones encoded a protein homologous to the Arabidopsis putative protein kinase and was designated as Tcoi2 (Tobacco CMV 1a interacting protein 2). Tcoi2 specifically interacted with methyltransferase (MT) domain of CMV 1a protein in yeast cell. In vitro analyses using recombinant proteins showed that Tcoi2 also specifically interacted with CMV 1a MT domain. Tcoi2 did not have autophosphorylation activity but phosphorylated CMV 1a MT domain. Analysis of the subcellular localization of the Tcoi2 fused to GFP demonstrated that it is targeted to the endoplasmic reticulum. These results suggest Tcoi2 as a novel host factor that is capable of interacting and phosphorylating MT domain of CMV 1a protein. 相似文献
996.
The Korean species of the genus Eudorylas Aczél have been studied. A total of 16 species are arranged herein. Among them, E. paraappendiculatus sp. nov. is new to science, and E. nomurai Morakote et Yano, 1990 is newly recorded in the Korean fauna. 相似文献
997.
998.
999.
Sugiura M Ogami S Kusumi M Un S Rappaport F Boussac A 《The Journal of biological chemistry》2012,287(16):13336-13347
The main cofactors that determine the photosystem II (PSII) oxygen evolution
activity are borne by the D1 and D2 subunits. In the cyanobacterium
Thermosynechococcus elongatus, there are three
psbA genes coding for D1. Among the 344 residues
constituting D1, there are 21 substitutions between PsbA1 and PsbA3, 31 between
PsbA1 and PsbA2, and 27 between PsbA2 and PsbA3. Here, we present the first
study of PsbA2-PSII. Using EPR and UV-visible time-resolved absorption
spectroscopy, we show that: (i) the time-resolved EPR spectrum of TyrZ• in the
(S3TyrZ•)′ is slightly modified; (ii) the split EPR signal
arising from TyrZ• in the (S2TyrZ•)′ state induced by near-infrared
illumination at 4.2 K of the S3TyrZ state is significantly
modified; and (iii) the slow phases of P680+⋅ reduction by TyrZ are
slowed down from the hundreds of μs time range to the ms time range,
whereas both the S1TyrZ• → S2TyrZ and
the S3TyrZ• → S0TyrZ + O2
transition kinetics remained similar to those in PsbA(1/3)-PSII. These results
show that the geometry of the TyrZ phenol and its environment, likely
the Tyr-O···H···Nϵ-His bonding,
are modified in PsbA2-PSII when compared with PsbA(1/3)-PSII. They also point to
the dynamics of the proton-coupled electron transfer processes associated with
the oxidation of TyrZ being affected. From sequence comparison, we
propose that the C144P and P173M substitutions in PsbA2-PSII
versus PsbA(1/3)-PSII, respectively located upstream of the
α-helix bearing TyrZ and between the two α-helices
bearing TyrZ and its hydrogen-bonded partner, His-190, are
responsible for these changes. 相似文献
1000.
A genetic mutation in the C9orf72 gene causes the most common forms of neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The C9orf72 protein, predicted to be a DENN-family protein, is reduced in ALS and FTD, but its functions remain poorly understood. Using a 3110043O21Rik/C9orf72 knockout mouse model, as well as cellular analysis, we have found that loss of C9orf72 causes alterations in the signaling states of central autophagy regulators. In particular, C9orf72 depletion leads to reduced activity of MTOR, a negative regulator of macroautophagy/autophagy, and concomitantly increased TFEB levels and nuclear translocation. Consistent with these alterations, cells exhibit enlarged lysosomal compartments and enhanced autophagic flux. Loss of the C9orf72 interaction partner SMCR8 results in similar phenotypes. Our findings suggest that C9orf72 functions as a potent negative regulator of autophagy, with a central role in coupling the cellular metabolic state with autophagy regulation. We thus propose C9orf72 as a fundamental component of autophagy signaling with implications in basic cell physiology and pathophysiology, including neurodegeneration. 相似文献