首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 46 毫秒
1.
Human α1-antitrypsin (AAT) was produced in the recombinant yeast Saccharomyces cerevisiae ATCC 20699 grown in batch and fed-batch culture. The final biomass concentration and antitrypsin concentration attained were 55 g·L−1 and 1.23 g·L−1, respectively, in the fed-batch. The maximum productivities of biomass and antitrypsin were 1.6 and > 0.04 g L−1h−1, respectively, or substantially greater than the highest productivity values reported in the past. For recovering the antitrypsin, the cell slurry was concentrated 4-fold (231 g·L−1 biomass, 122 min of processing) by cross-flow microfiltration and the cells were disrupted by bead milling (3 passes of 3 min total retention time). The cell homogenate was treated with aluminum chloride or PBS (pH 7) to aid separation of the cell debris by flocculation and sedimentation. The clarified cell homogenate was subjected to ammonium sulfate fractionation to precipitate the recombinant antitrypsin. The AAT precipitated at 45–75% saturation of ammonium sulfate, depending on the age of the homogenate. The crude AAT in the homogenate degraded at room temperature (25°C), with a zero order deactivation rate of 1.815 × 10−3 ± 3.43 × 10−4 g AAT L−1h−1.  相似文献   

2.
The inversion of sucrose with β- -fructofuranosidase (EC 3.2.1.26) immobilized by an ionic bond on bead cellulose containing weak basic N,N-diethylamino-2-hydroxypropyl groups has been investigated. The immobilized enzyme is strongly bound at an ionic strength up to 0.1 M in the pH range 3–6. The amount adsorbed is proportional to porosity and to the exchange capacity of the ion exchange cellulose, reaching values up to 200 mg/g dry carrier, with an activity in 10% sucrose solution at 30°C, pH 5, >8000 μmol min−1 g−1. The inversion of sucrose with immobilized β- -fructofuranosidase was carried out in a stirred reactor. The dependence of activity on pH (3–7), temperature (0–70°C) and concentration of the substrate (2–64 wt%) were determined, and the inversion was compared with that obtained using non-immobilized enzyme under similar conditions. The rate of inversion at low substrate concentration (2–19 wt%) was described by Michaelis-Menten kinetics.  相似文献   

3.
1. The fat mouse Steatomys pratensis natalensis (mean body mass 37.4±0.43 (se)) has a low euthermic body temperature Tb=30.1–33.8 °C and a low basal metabolic rate (BMR)=0.50 ml O2 g−1 h−1.
2. Below an ambient temperature (Ta)=15 °C, the mice were hypothermic.
3. The lowest survivable Ta=10 °C.
4. Torpor is efficient in conserving energy between Ta=15–30 °C, below Ta=15 °C, the mice arouse.
5. Euthermic and torpid mice were hyperthermic at Ta=35 °C.
6. Thermal conductance was 0.159 ml O2 g−1 h−1 °C−1, 98.8% of the expected value.
7. Non-shivering thermogenesis (NST) was 2.196 ml O2 g−1 h−1 (3.69×BMR).
8. Maximal oxygen consumption, however, was 3.83 ml O2 g−1 h−1 (6.44×BMR), indicating that other methods of heat production are additive.
9. Because fat mice conserve energy by torpor only between Ta=15–30 °C, we suggest that torpor may be a more important mechanism for surviving food shortages than for surviving cold weather.
Keywords: Steatomys pratensis natalensis; Metabolism; Torpor; Fat mouse  相似文献   

4.
Process strategies for production of recombinant rhamnulose 1-phosphate aldolase (RhuA) in Escherichia coli were found to have an important impact on downstream processing when preparing the enzyme for its use as immobilized biocatalyst. First, a continuous inducer feed was implemented in substrate limited fed-batch cultures to overexpress RhuA with a hexa histidine-tag (6xHis-tag) at its N-terminus. The final specific RhuA level was 180 mg g−1 DCW, but the final specific enzyme activity (1.7 AU mg−1 RhuA) was considerably lower than expected. Only 55% of immobilization yield was achieved when immobilized metal affinity chromatography (IMAC) was used to purify and immobilize RhuA from cellular lysate in a single step. Western blot analyses showed that only 20% of overexpressed RhuA kept the whole 6xHis-tag at the end of the culture due to partial proteolysis. Two different growth strategies improved protein quality and immobilization yield:
(i) Temperature reduction to 28 °C in substrate limited operation decreased proteolysis and allowed higher specific activities, 210 mg g−1 DCW. The enzyme activity increased to 4 AU mg−1 RhuA and purification-immobilization yield to 93%.
(ii) A novel fed-batch operational procedure, working at high glucose concentration was implemented. High aldolase levels, 233 mg g−1 DCW, were reached at the end of the culture. The final enzyme activity was also higher than 4 AU mg−1 RhuA, and 95% of immobilization yield was achieved.
For both cases, Western blot analyses showed that 80–100% of overexpressed RhuA kept the whole 6xHis-tag at the end of the culture, confirming that recombinant protein quality had been improved.  相似文献   

5.
In this study, the hydraulic conductivity (Lp), Me2SO permeability ( Me2SO), and the reflection coefficients (ς) and their activation energies were determined for Metaphase II (MII) mouse oocytes by exposing them to 1.5 M Me2SO at temperatures of 30, 20, 10, 3, 0, and −3°C. These data were then used to calculate the intracellular concentration of Me2SO at given temperatures. Individual oocytes were immobilized using a holding pipette in 5 μl of an isosmotic PBS solution and perfused with precooled or prewarmed 1.5 M Me2SO solutions. Oocyte images were video recorded. The cell volume changes were calculated from the measurement of the diameter of the oocytes, assuming a spherical shape. The initial volume of the oocytes in the isoosmotic solution was considered 100%, and relative changes in the volume of the oocytes after exposure to the Me2SO were plotted against time. Mean (means ± SEM) Lpvalues in the presence of Me2SO ( Me2SOp) at 30, 20, 10, 3, 0, and −3°C were determined to be 1.07 ± 0.03, 0.40 ± 0.02, 0.18 ± 0.01, 7.60 × 10−2± 0.60 × 10−2, 5.29 × 10−2± 0.40 × 10−2, and 3.69 × 10−2± 0.30 × 10−2μm/min/atm, respectively. The Me2SOvalues were 3.69 × 10−3± 0.3 × 10−3, 1.07 × 10−3± 0.1 × 10−3, 2.75 × 10−4± 0.15 × 10−4, 7.83 × 10−5± 0.50 × 10−5, 5.24 × 10−5± 0.50 × 10−5, and 3.69 × 10−5± 0.40 × 10−5cm/min, respectively. The ς values were 0.70 ± 0.03, 0.77 ± 0.04, 0.81 ± 0.06, 0.91 ± 0.05, 0.97 ± 0.03, and 1 ± 0.04, respectively. The estimated activation energies (Ea) for Me2SOp, Me2SO, and ς were 16.39, 23.24, and −1.75 Kcal/mol, respectively. These data may provide the fundamental basis for the development of more optimal cryopreservation protocols for MII mouse oocytes.  相似文献   

6.
The kinetics of fungal peroxidase-catalyzed phenolic compounds (PCs) oxidation was investigated in presence of acetylenic-based surfactant Dynol 604 at pH 5.5 and 25 °C. It was shown that the presence of ppm concentrations of surfactant did not influence initial rate of PCs oxidation. The calculated apparent bimolecular rate constants were (1.8 ± 0.2) × 105 M−1 s−1, (1.4 ± 0.4) × 107 M−1 s−1, (1.30 ± 0.06) × 107 M−1 s−1 and 1.1 × 108 M−1 s−1 for phenol, 1-naphthol, 2-naphthol and 1-hydroxypyrene, respectively.During an extensive substrates conversion Dynol 604 showed diverse action for different PCs. The oxidation of phenol practically did not change, whereas the surfactant enhanced the conversion of 1- and 2-naphthol and 1-hydroxypyrene in dose response manner. The results accounted by a scheme, which contains a stadium of enzyme inhibition by oligomeric PC oxidation products. The action of the surfactant was explained by avoidance the enzyme active center clothing with the oligomers. The results acquired demonstrate a remarkable increase of substrates conversion in the presence of Dynol 604.  相似文献   

7.
Thyroid hormone (T3) has been demonstrated to inhibit the action of aldosterone on sodium transport in toad urinary bladder and rat kidney. We have exammined the effect of T3 on aldosterone action and specific nuclear binding in cultured epithelial cells derived from toad urinary bladder. In cell line TB6-C, addition of 5·10−8 M T3 to culture media for up to 3 days results in no change in short-circuit current or transepithelial resistance. This concentration of T3 completely inhibits the maximal increase in short-circuit current in response to 1·10−7 M aldosterone. The inhibition can be demonstrated with 18 h preincubation or with simultaneous addition of T3 and aldosterone. The half-maximal concentration for the inhibition of the aldosterone effect is approx. 5·10−9 M T3. T3 has no effect on cyclic AMP-stimulated short-circuit current in these cells. The effect of T3 on nuclear binding of [3H]aldosterone was examined using a filtration assay with data analysis by at least-squares curve-fitting program. Best fit was obtained with a model for two binding sites. The dissociation constants for the binding were Kd1 = (0.82 ± 0.36)·10−10 M and Kd2 = (3.2±0.60)·10−8 M.The half-maximal concentration for aldosterone-stimulated sodium transport in these cells is approx. 1·10−8 M. Analysis of nuclear aldosterone binding in cells preincubated for 18 h with 5·10−8 M T3 showed a Kd1 = (0.15 ± 0.10)·10−10 M and Kd2 = (3.5 ± 0.10)·10−8 M. We conclude that T3 i action of aldosterone on sodium transport at a site after receptor binding in the nucleus.  相似文献   

8.
A vasoactive intestinal peptide-sensitive adenylate cyclase in intestinal epithelial cell membranes was characterized. Stimulation of adenylate cyclase activity was a function of vasoactive intestinal peptide concentration over a range of 1 · 10−10−1 · 10−7 M and was increased six-times by a maximally stimulating concentration of vasoactive intestinal peptide. Half-maximal stimulation was observed with 4.1 ± 0.7 nM vasoactive intestinal peptide. Fluoride ion stimulated adenylate cyclase activity to a higher extent than did vasoactive intestinal peptide. Under standard assay conditions, basal, vasoactive inteetinal peptide- and fluoride-stimulated adenylate cyclase activities were proportional to time of incubation up to 15 min and to membrane concentration up to 60 μg protein per assay. The vasoactive intestinal peptide-sensitive enzyme required 5–10 mM Mg2+ and was inhibited by 1 · 10−5 M Ca2+. At sufficiently high concentrations, both ATP (3 mM) and Mg2+ (40 mM) inhibited the enzyme.Secretin also stimulated the adenylate cyclase activity from intestinal epithelial cell membranes but its effectiveness was 1/1000 that of vasoactive intestinal peptide. Prostaglandins E1 and E2 at 1 · 10−5 M induced a two-fold increase of cyclic AMP production. Vasoactive intestinal peptide was the most potent stimulator of adenylate cyclase activity, suggesting an important physiological role of this peptide in the cyclic AMP-dependent regulation of the intestinal epithelial cell function.  相似文献   

9.
A novel raw starch degrading α-cyclodextrin glycosyltransferase (CGTase; E.C. 2.4.1.19), produced by Klebsiella pneumoniae AS-22, was purified to homogeneity by ultrafiltration, affinity and gel filtration chromatography. The specific cyclization activity of the pure enzyme preparation was 523 U/mg of protein. No hydrolysis activity was detected when soluble starch was used as the substrate. The molecular weight of the pure protein was estimated to be 75 kDa with SDS-PAGE and gel filtration. The isoelectric point of the pure enzyme was 7.3. The enzyme was most active in the pH range 5.5–9.0 whereas it was most stable in the pH range 6–9. The CGTase was most active in the temperature range 35–50°C. This CGTase is inherently temperature labile and rapidly loses activity above 30°C. However, presence of soluble starch and calcium chloride improved the temperature stability of the enzyme up to 40°C. In presence of 30% (v/v) glycerol, this enzyme was almost 100% stable at 30°C for a month. The Km and kcat values for the pure enzyme were 1.35 mg ml−1 and 249 μM mg−1 min−1, respectively, with soluble starch as the substrate. The enzyme predominantly produced α-cyclodextrin without addition of any complexing agents. The conditions employed for maximum α-cyclodextrin production were 100 g l−1 gelatinized soluble starch or 125 g l−1 raw wheat starch at an enzyme concentration of 10 U g−1 of starch. The α:β:γ-cyclodextrins were produced in the ratios of 81:12:7 and 89:9:2 from gelatinized soluble starch and raw wheat starch, respectively.  相似文献   

10.
A hydrogen peroxide permselective membrane with asymmetric structure was prepared and -glucose oxidase (EC 1.1.3.4) was immobilized onto the porous layer. The activity of the immobilized -glucose oxidase membrane was 0.34 units cm−2 and the activity yield was 6.8% of that of the native enzyme. Optimum pH, optimum temperature, pH stability and temperature stability were found to be pH 5.0, 30–40°C, pH 4.0–7.0 and below 55°C, respectively. The apparent Michaelis constant of the immobilized -glucose oxidase membrane was 1.6 × 10−3 mol l−1 and that of free enzyme was 4.8 × 10−2 mol l−1. An enzyme electrode was constructed by combination of a hydrogen peroxide electrode with the immobilized -glucose oxidase membrane. The enzyme electrode responded linearly to -glucose over the concentration 0–1000 mg dl−1 within 10 s. When the enzyme electrode was applied to the determination of -glucose in human serum, within day precision (CV) was 1.29% for -glucose concentration with a mean value of 106.8 mg dl−1. The correlation coefficient between the enzyme electrode method and the conventional colorimetric method using a free enzyme was 0.984. The immobilized -glucose oxidase membrane was sufficiently stable to perform 1000 assays (2 to 4 weeks operation) for the determination of -glucose in human whole blood. The dried membrane retained 77% of its initial activity after storage at 4°C for 16 months.  相似文献   

11.
Active transport of Cl accounts for 90% of the short-circuit current (s.c.c.) in the isolated frog cornea. 1·10−5 M furosemide produced a 50% reversible inhibition of this s.c.c. 1·10−4 M ethacrynic acid reduced the corneal s.c.c. to 32% of the control. In the isolated frog skin epithelium furosemide had no effect on the s.c. at a concentration of 1·10−4 M and a small stimulation at a concentration of 1·10−3 M. The furosemide inhibitory effects seems to be specific for Cl, as it also inhibits Cl transport in the ascending limb of the loop of Henle (Burg, M.B. (1972) Proc. 5th Int. Congr. Nephrol., p. 50, Abstr.).  相似文献   

12.
Dispersed acini from dog pancreas were used to examine the ability of dopamine to increase cyclic AMP cellular content and the binding of [3H]dopamine. Cyclic AMP accumulation caused by dopamine was detected at 1·10−8 M and was half-maximal at 7.9±3.4·10−7M. The increase at 1·10−5 M, (7.5-fold) was equal to the half-maximal increase caused by secretin at 1·10−9 M. Haloperidol, a dopaminergic receptor antagonist inhibited cyclic AMP accumulation caused by dopamine. The IC50 value for haloperidol, calculated from the inhibition of cyclic AMP increase caused by 1·10−5 M dopamine was 2.3±0.9·10−6M. Haloperidol did not alter basal or secretin-stimulated cyclic AMP content. [3H]Dopamine binding was studied on the same batch of cells as cyclic AMP accumulation. At 37°C, it was rapid, reversible, saturable and stereospecific. The Kd value for high affinity binding sites was 0.43±0.1·10−7M and 4.7±1.6·10−7M for low affinity binding sites. The concentration of drugs necessary to inhibit specific binding of dopamine by 50% was 1.2±0.4·10/t-7M noradrenaline, 2·10/t-7 M epinine, 4.1±1.8·10/t-6M fluphenazine, 8.0±1.6·10/t-6M haloperidol, 4.2±1.2·10−6Mcis-flupenthixol, 2.7±0.4·10−5Mtrans-flupenthixol, >1·10−5M apomorphine, sulpiride, naloxone and isoproterenol.  相似文献   

13.
Chromosome set manipulation was used to produce rainbow trout, Oncorhynchus mykiss, with identical nuclear backgrounds, but different maternal backgrounds to determine mitochondrial effects on development rate and oxygen consumption. Significant differences in development rate and oxygen consumption were observed between groups from different females. Development rates ranged from a mean of 317.97 degree days (°d) to 335.25 °d in progeny from different females. Mean oxygen consumption rates ranged from 3.31 μmol O2 g− 1 wet mass h− 1 to 9.66 μmol O2 g− 1 wet mass h− 1. Oxygen consumption and development rate analysis revealed the two slowest developing groups had the highest oxygen consumption rates. Development rate differences between second generation clonal females indicate that mitochondrial genomes play a significant role on early development and are comparable to development rate differences between clonal lines of rainbow trout. These results indicate that selection for mitochondrial genomes could increase growth rates and possibly food conversion ratios in aquaculture species.  相似文献   

14.
The kinetics of substitution reactions of [η-CpFe(CO)3]PF6 with PPh3 in the presence of R-PyOs have been studied. For all the R-PyOs (R = 4-OMe, 4-Me, 3,4-(CH)4, 4-Ph, 3-Me, 2,3-(CH)4, 2,6-Me2, 2-Me), the reactions yeild the same product [η5-CpFe(CO)2PPh3]PF6, according to a second-order rate law that is first order in concentrations of [η5-CpFe(CO)3]PF6 and of R-PyO but zero order in PPh3 concentration. These results, along with the dependence of the reaction rate on the nature of R-PyO, are consistent with an associative mechanism. Activation parameters further support the bimmolecular nature of the reactions: ΔH = 13.4 ± 0.4 kcal mol−1, ΔS = −19.1 ± 1.3 cal k−1 mol−1 for 4-PhPyO; ΔH = 12.3 ± 0.3 kcal mol−1, ΔS = 24.7 ±1.0 cal K−1 mol−1 for 2-MePyO. For the various substituted pyridine N-oxides studied in this paper, the rates of reaction increase with the increasing electron-donating abilities of the substituents on the pyridine ring or N-oxide basicities, but decrease with increasing 17O chemical shifts of the N-oxides. Electronic and steric factors contributing to the reactivity of pyridine N-oxides have been quantitatively assessed.  相似文献   

15.
Hairy root cultures of Salvia sclarea were grown in shake flasks and 10 L nutrient sprinkle bioreactor, running for 30 days and the effects of methyl jasmonate (MJ) on their growth and capacity to accumulate diterpenoids were measured. We found that MJ concentration and exposure time to the elicitor were factors that strongly affected the diterpenoid production. The highest diterpenoid accumulation (67.5 ± 7.1 mg g−1 dry weight, calculated as a sum of ferruginol, salvipisone, aethiopinone and 1-oxoaethiopinone) without reduction of biomass, was achieved when the 23-day-old hairy roots in bioreactor culture were exposed to 125 μM MJ for 7 days. The roots produced 9 and 3.8 times as much aethiopinone (40 ± 5.9 mg g−1 dry weight) and salvipisone (12.6 ± 0.4 mg g−1 dry weight), respectively, as roots cultured in shake flasks. Our results imply that cultivation of S. sclarea hairy roots in sprinkle bioreactor after elicitation with MJ may be valuable to enhance production of the bioactive diterpenoids.  相似文献   

16.
1. 1. The Michaelis-Menten parameters of labelled d-glucose exit from human erythrocytes at 2°C into external solution containing 50 mM d-galactose were obtained. The Km is 3.4 ± 0.4 mM, V 17.3 ± 1.4 mmol · 1−1 cell water · min−1 for this infinite-trans exit procedure.
2. 2. The kinetic parameters of equilibrium exchange of d-glucose at 2°C are Km = 25 ± 3.4 mM, V 30 ± 4.1 mmol · 1−1 cell water · min−1.
3. 3. The Km for net exit of d-glucose into solutions containing zero sugar is 15.8 ± 1.7 mM, V 9.3 ± 3.3 mol 9.3 ± 3.3 mol · 1−1 cell water · min−1.
4. 4. This experimental evidence corroborates the previous finding of Hankin, B.L., Lieb, W.R. and Stein, W.D. [(1972) Biochim. Biophys. Acta 255, 126–132] that there are sites with both high and low operational affinities for d-glucose at the inner surface of the human erythrocyte membrane. This result is inconsistent with current asymmetric carrier models of sugar transport.
Keywords: d-Glucose transport; Asymmetric carrier; Pore kinetics; (Erythrocyte)  相似文献   

17.
1H and 31P continuous wave and spin-echo NMR measurements have been made on lecithin-water mixtures as a function of water content at 20 °C. An analysis of the data demonstrates the existence of two water environments, lecithin bound and free. Conclusions are presented concerning the stoichiometry and kinetics of the binding. The results indicate that six molecules of water are bound to one lecithin molecule and the lifetime of the bound water is 6·10−5±3·10−5 s.  相似文献   

18.
The kinetics of formation of the complex ion, μ-carbonato-di-μ-hydroxo-bis((1,5-diamino-3-aza-pentane) cobalt(III), from the tri-μ-hydroxo-bis((1,5-diamino-3-aza-pentane(III)cobalt(III)) ion in aqueous buffered carbonate solution have been studied spectrophotometrically at 295 nm over the ranges 20.0θ°C34.8, 8.03pH9.44, 5 mM [CO32−35 mM and at an ionic strength of 0.1 M (LiClO4). On the basis of the kinetic results a mechanism, involving rapid cleavage of an hydroxo bridge followed by carbon dioxide uptake with subsequent bridge formation, has been proposed. At 25 °C, the rate of the carbon dioxide uptake is 0.58 M−1 s−1 with ΔH≠ = (13.2±0.7) kcal mol−1 and ΔS≠ = (−15.1 ± 0.7) cal deg−1 mol−1. The results are composed with those obtained for several mononuclear cobalt(III) and one dinuclear cobalt(III) complexes.  相似文献   

19.
The diffusion translational coefficient DT of core particles in monodisperse solutions has been measured by the quasielastic light scattering method in a large scale of salinities over the range 6.10−4 to 2M Na+ or K+. The observed values of DT are independent of particle concentration in the range 0.1–2 mg/ml and do not vary with the scattering vector q corresponding to scattering angles between 40°–120°. When the salinity is progressively raised an increase of DT from 1.9.10−7 cm2s−1 to 3.2.10−7 cm2s−1 was observed at about 2.10−3 M NaCl followed by a decrease of DT beyond 0.6 M NaCl.The various possible causes of the changes of DT such as interactions between particles or between particles and salt ions are discussed. We show that the single low ionic strength change is due to a conformational transition of the core particles, while the second variation of DT accompanies the disorganization of the core particles.  相似文献   

20.
Jerome Lavergne 《BBA》1982,682(3):345-353
The kinetics and concentration dependence of the binding of dichlorophenyldimethylurea (DCMU) to Photosystem II (PS II) were monitored through fluorescence measurements. According to whether the acceptor system is in the ‘odd’ state (QB ag QB) or ‘even’ state (QB), very different results are obtained. The binding to centers in the even state is rapid ( at [DCMU] = 10−5 M and [chlorophyll] = 10 μg/ml), with a pH-independent rate. The concentration curve of the bound inhibitor (at equilibrium) corresponds to an association constant of about 3.3·107 M−1·1. The binding of the inhibitor to centers in the odd state is slow ( at pH 7, same DCMU and chlorophyll concentrations as above), and depends on pH. In the pH range 6–8, the lower the pH, the slower the kinetics. The association constant is also diminished by a factor of approx. 20 (at pH 7) compared to the even state centers. It is shown that these effects are in good agreement with predictions from Velthuys' hypothesis (Velthuys, B.R. (1981) FEBS Lett. 126, 277–281) that the mode of action of DCMU is a competition with plastoquinone for the binding to the secondary acceptor site. A large part of PS II photochemical quenching corresponds to acceptors which seem to possess a secondary acceptor distinct from B. They were called ‘non-B-type acceptors’ (Lavergne, J. (1982) Photobiochem. Photobiophys. 3, 257–285) and may be identified with Joliot's ‘Q2’ (Joliot P. and Joliot, A. (1977) Biochim. Biophys. Acta 462, 559–574). However, the rate at which the inhibition affects these non-B-type acceptors is similar to the rate of DCMU binding on the B site (i.e., slow in the odd state, fast in the even state).  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号