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161.
162.
The recently identified benzoate oxidation (box) pathway in Burkholderia xenovorans LB400 (LB400 hereinafter) assimilates benzoate through a unique mechanism where each intermediate is processed as a coenzyme A (CoA) thioester. A key step in this process is the conversion of 3,4-dehydroadipyl-CoA semialdehyde into its corresponding CoA acid by a novel aldehyde dehydrogenase (ALDH) (EC 1.2.1.x). The goal of this study is to characterize the biochemical and structural properties of the chromosomally encoded form of this new class of ALDHs from LB400 (ALDHC) in order to better understand its role in benzoate degradation. To this end, we carried out kinetic studies with six structurally diverse aldehydes and nicotinamide adenine dinucleotide (phosphate) (NAD + and NADP +). Our data definitively show that ALDHC is more active in the presence of NADP + and selective for linear medium-chain to long-chain aldehydes. To elucidate the structural basis for these biochemical observations, we solved the 1.6-Å crystal structure of ALDHC in complex with NADPH bound in the cofactor-binding pocket and an ordered fragment of a polyethylene glycol molecule bound in the substrate tunnel. These data show that cofactor selectivity is governed by a complex network of hydrogen bonds between the oxygen atoms of the 2′-phosphoryl moiety of NADP + and a threonine/lysine pair on ALDHC. The catalytic preference of ALDHC for linear longer-chain substrates is mediated by a deep narrow configuration of the substrate tunnel. Comparative analysis reveals that reorientation of an extended loop (Asn478-Pro490) in ALDHC induces the constricted structure of the substrate tunnel, with the side chain of Asn478 imposing steric restrictions on branched-chain and aromatic aldehydes. Furthermore, a key glycine (Gly104) positioned at the mouth of the tunnel allows for maximum tunnel depth required to bind medium-chain to long-chain aldehydes. This study provides the first integrated biochemical and structural characterization of a box-pathway-encoded ALDH from any organism and offers insight into the catalytic role of ALDHC in benzoate degradation. 相似文献
163.
Attila Fábián Katalin Jäger Éva Darkó Beáta Barnabás 《Acta Physiologiae Plantarum》2008,30(5):737-744
A procedure has been developed for the cryopreservation of wheat female gametes. The procedure involves loading the cells with 25% concentrated vitrification solution consisting of 30% glycerol, 10% sucrose, 120 mM ascorbic acid (AA) and 5% propylene glycol (PG), dehydration in 80% concentrated vitrification solution, droplet vitrification and storage in liquid nitrogen, unloading and rehydration of the cells by gradual addition of isolation solution. Supplementation with AA significantly increased the proportion of viable egg cells after de- and rehydration. During the early phase of rehydration AA reduced the probability of membrane damage caused by rapid water uptake. Maintaining the temperature of the cells at 0°C during the de- and rehydration processes increased cell survival. Microscopic examination of the semi-thin sections of untreated and viable cryopreserved cells revealed that the vitrification process might cause changes in cell structure. 相似文献
164.
Zgoła-Grześkowiak A Grześkowiak T Zembrzuska J Frańska M Frański R Lukaszewski Z 《Biodegradation》2008,19(3):365-373
Aerobic biodegradation of tripropylene glycol (PG3) was investigated under the conditions of the OECD screening test 301E
and the Continuous Flow Activated Sludge Simulation test (CFAS). A modified two-chamber facility with a denitrification stage
was used for the CFAS test. Primary PG3 biodegradation was measured by the HPLC with fluorimetric detection and analyte derivatisation.
Metabolites were identified by LC-MS with electrospray ionisation and GC-MS with electron impact ionisation, as well as semiquantitatively
determined by the LC-MS technique. PG3 was found to be inherently biodegradable and it exhibits a strong poisonous effect
on activated sludge after exceeding the threshold concentration (10 mg l−1). Metabolite accumulation onto the activated sludge is probably responsible for this poisonous effect. Probable biotransformation
products of tripropylene glycol under the aerobic conditions include metabolites with a single terminal aldehyde or a ketone
group and metabolites with two terminal aldehyde or ketone groups. Their concentration rises at the end of the OECD screening
test. 相似文献
165.
166.
Yi Y. Zuo Ruud A.W. Veldhuizen A. Wilhelm Neumann Fred Possmayer 《生物化学与生物物理学报:生物膜》2008,1778(10):1947-1977
Pulmonary surfactant (PS) is a complicated mixture of approximately 90% lipids and 10% proteins. It plays an important role in maintaining normal respiratory mechanics by reducing alveolar surface tension to near-zero values. Supplementing exogenous surfactant to newborns suffering from respiratory distress syndrome (RDS), a leading cause of perinatal mortality, has completely altered neonatal care in industrialized countries. Surfactant therapy has also been applied to the acute respiratory distress syndrome (ARDS) but with only limited success. Biophysical studies suggest that surfactant inhibition is partially responsible for this unsatisfactory performance. This paper reviews the biophysical properties of functional and dysfunctional PS. The biophysical properties of PS are further limited to surface activity, i.e., properties related to highly dynamic and very low surface tensions. Three main perspectives are reviewed. (1) How does PS permit both rapid adsorption and the ability to reach very low surface tensions? (2) How is PS inactivated by different inhibitory substances and how can this inhibition be counteracted? A recent research focus of using water-soluble polymers as additives to enhance the surface activity of clinical PS and to overcome inhibition is extensively discussed. (3) Which in vivo, in situ, and in vitro methods are available for evaluating the surface activity of PS and what are their relative merits? A better understanding of the biophysical properties of functional and dysfunctional PS is important for the further development of surfactant therapy, especially for its potential application in ARDS. 相似文献
167.
Films of methylcellulose (MC), poly(ethylene glycol)400 (PEG400) plasticized MC, and MC gels (MC crosslinked with glutaraldehyde (GA)) were prepared by casting from aqueous solutions. The swelling test has shown that the MC gels were insoluble in water and that their crosslinking density increased with increasing GA and HCl concentrations. The effect of the addition of PEG400 or GA to MC was investigated through dynamic mechanical analysis (DMA). The DMA analysis of PEG400/MC blends has shown that PEG400 was compatible with MC and was an effective plasticizer since the curves of tan δ against temperature exhibited single peaks (corresponding to a single glass transition temperature), which were displaced to lower values with increasing PEG400 content. The thermogravimetric analysis (TGA) indicated that the thermal stability of MC was not affected by the chemical crosslinking. The tensile strength was slightly increased through crosslinking while the elongation was slightly decreased. The presence of moisture in MC hydrogels decreased the tensile strength and enhanced the elongation while the addition of PEG400 decreased the tensile strength but sharply increased the elongation. 相似文献
168.
A new extractive fermentation process using PEG and potassium phosphate aqueous two-phase system (ATPS) was developed for enhanced production of gellan-hydrolysing enzyme by Bacillus thuringiensis H14. Five different Bacillus sp. were tested for their ability to synthesize gellan-hydrolysing enzyme. Bacillus thuringiensis H14 was found to be the best organism for gellan-hydrolysing enzyme production. The enzyme showed maximum activity at pH 7.5 and 40 °C. The partition studies of gellan-hydrolysing enzyme in the system using PEG X (X = 9000, 6000, 4000) and potassium phosphate–water and PEG–sodium citrate–water system indicated at PEG (4000)– potassium phosphate–water is the best system for partitioning of gellan-hydrolysing enzyme into the PEG phase (K = 4.99). Gellan-hydrolysing enzyme production by Bacillus thuringiensis H14 was studied in ATPSs composed of PEG X (X = 9000, 6000, 4000) and potassium phosphate. The top phase is continuous and rich in PEG while the bottom phase is dispersed and is rich in phosphate, microbial cells being mainly retained in the bottom phase. The gellan-hydrolysing enzyme produced during fermentation partitioned into the upper PEG phase and total gellan-hydrolysing enzyme produced was 2.12, 2.29 and 2.40 times higher than that of homogeneous fermentation when the fermentations were carried out using PEG 9000–potassium phosphate–water, PEG 6000–potassium phosphate–water, PEG 4000–potassium phosphate–water systems respectively. 相似文献
169.
170.
Haeshin Lee Ji Hoon Jeong Je Hoon Lee Tae Gwan Park 《Biotechnology and Bioprocess Engineering》2001,6(4):269-273
This study presents a new formulation method for improving DNA transfection efficiency using a fusogenic peptide and polyethylene
glycol grafted polyethylenimine. Succinimidyl succinate polyethylene glycol (PEG-SSA) was conjugated with polyethylenimine
(PEI). PEI is well known for a good endosomal escaping and DNA condensing agent. The positively charged synthetic fusogenic
peptide, KALA, was coated on the negatively charged PEG-g-PEI/DNA and PEI/DNA complexes. The KALA/PEI/DNA complexes exhibited
aggregation behavior at higher KALA coating amounts with an effective diameter of around 1,000 nm. However, the KALA/PEG-g-PEI/DNA
complexes were 100–300 nm in size with a surface zeta-potential (ζ) value of about +20 mV. The conjugated PEG molecules suppressed
any KALA-mediated inter-particle aggregation, and thereby improved the transfection efficiency. Consequently, the transfection
efficiency of the KALA/PEG-g-PEI/DNA complexes was obtained by utilizing both the fusogenic activity of KALA and the steric
repulsion effect of PEC. 相似文献