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1.
Summary The production of extracellular alkaline proteases from Aspergillus clavatus was evaluated in a culture filtrate medium, with different carbon and nitrogen sources. The fungus was cultivated at three different temperatures during 10 days. The proteolytic activity was determined on casein pH 9.5 at 37 °C. The highest alkaline proteolytic activity (38 U/ml) was verified for culture medium containing glucose and casein at 1% (w/v) as substrates, obtained from cultures developed at 25 °C for 6 days. Cultures developed in Vogel medium with glucose at 2% (w/v) and 0.2% (w/v) NH4NO3 showed higher proteolytic activity (27 U/ml) when compared to the cultures with 1% of the same sugar. Optimum temperature was 40 °C and the half-lives at 40, 45 and 50 °C were 90, 25 and 18 min, respectively. Optimum pH of enzymatic activity was 9.5 and the enzyme was stable from pH 6.0 to 12.0. 相似文献
2.
A moderately halophilic alkalitolerant Bacillus sp. Strain TSCVKK, with an ability to produce extracellular halophilic, alkalitolerant, surfactant, and detergent-stable alpha-amylase was
isolated from soil samples obtained from a salt-manufacturing industry in Chennai. The culture conditions for higher amylase
production were optimized with respect to NaCl, substrate, pH, and temperature. Maximum amylase production of 592 mU/ml was
achieved in the medium at 48 h with 10% NaCl, 1% dextrin, 0.4% yeast extract, 0.2% tryptone, and 0.2% CaCl2 at pH 8.0 at 30 °C. The enzyme activity in the culture supernatant was highest with 10% NaCl at pH 7.5 and 55 °C. The amylase
that was partially purified by acetone precipitation was highly stable in various surfactants and detergents. Glucose, maltose,
and maltooligosaccharides were the main end products of starch hydrolysis indicating that it is an alpha-amylase. 相似文献
3.
Fusarium sp. BLB, which produces a strongly fibrinolytic enzyme, was isolated from plant leaf (Hibiscus). Fibrinolytic alkaline protease was purified from a culture filtrate of Fusarium sp. BLB by precipitation with (NH4)2SO4 and column chromatography with CM-Toyopearl 650M and Superdex 75. The purified enzyme was homogeneous on sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight was 27,000 by SDS-PAGE. Maximum activity of protease was
observed at pH 9.5 and 50°C. Purified protease was active between pH 2.5 and 11.5 and was found to be stable up to 50°C. The
enzyme derived from Fusarium sp. BLB is useful for thrombolytic therapy because this enzyme showed pH resistance. The activity was inhibited by diisopropylfluorophosphate
and phenylmethylsulfonyl fluoride. The N-terminal amino acid sequence of the enzyme showed a similarity to those of proteases
from Fusarium sp., Streptomyces griseus, Bos taurus bovine, Katsuwo pelamis digestive tract, and Lumbricus rubellus. 相似文献
4.
The increasing CO2 concentration in Earths atmosphere is expected to cause a greater decline in the nutritional quality of C3 than C4 plants. As a compensatory response, herbivorous insects may increase their feeding disproportionately on C3 plants. These hypotheses were tested by growing the grasses Lolium multiflorum C3) and Bouteloua curtipendula C4) at ambient (370 ppm) and elevated (740 ppm) CO2 levels in open top chambers in the field, and comparing the growth and digestive efficiencies of the generalist grasshopper Melanoplus sanguinipes on each of the four plant × CO2 treatment combinations. As expected, the nutritional quality of the C3 grass declined to a greater extent than did that of the C4 grass at elevated CO2; protein levels declined in the C3 grass, while levels of carbohydrates (sugar, fructan and starch) increased. However, M. sanguinipes did not significantly increase its consumption rate to compensate for the lower nutritional quality of the C3 grass grown under elevated CO2. Instead, these grasshoppers appear to use post-ingestive mechanisms to maintain their growth rates on the C3 grass under elevated CO2. Consumption rates of the C3 and C4 grasses were also similar, demonstrating a lack of compensatory feeding on the C4 grass. We also examined the relative efficiencies of nutrient utilization from a C3 and C4 grass by M. sanguinipes to test the basis for the C4 plant avoidance hypothesis. Contrary to this hypothesis, neither protein nor sugar was digested with a lower efficiency from the C4 grass than from the C3 grass. A novel finding of this study is that fructan, a potentially large carbohydrate source in C3 grasses, is utilized by grasshoppers. Based on the higher nutrient levels in the C3 grass and the better growth performance of M. sanguinipes on this grass at both CO2 levels, we conclude that C3 grasses are likely to remain better host plants than C4 grasses in future CO2 conditions. 相似文献
5.
Kiffer-Moreira T Pinheiro AA Pinto MR Esteves FF Souto-Padrón T Barreto-Bergter E Meyer-Fernandes JR 《Archives of microbiology》2007,188(2):159-166
Phosphatase activities were characterized in intact mycelial forms of Pseudallescheria boydii, which are able to hydrolyze the artificial substrate p-nitrophenylphosphate (p-NPP) to p-nitrophenol (p-NP) at a rate of 41.41 ± 2.33 nmol p-NP per h per mg dry weight, linearly with increasing time and with increasing cell density.
MgCl2, MnCl2 and ZnCl2 were able to increase the (p-NPP) hydrolysis while CdCl2 and CuCl2 inhibited it. The (p-NPP) hydrolysis was enhanced by increasing pH values (2.5-8.5) over an approximately 5-fold range. High sensitivity to specific
inhibitors of alkaline and acid phosphatases suggests the presence of both acid and alkaline phosphatase activities on P. boydii mycelia surface. Cytochemical localization of the acid and alkaline phosphatase showed electron-dense cerium phosphate deposits
on the cell wall, as visualized by electron microscopy. The product of p-NPP hydrolysis, inorganic phosphate (Pi), and different inhibitors for phosphatase activities inhibited p-NPP hydrolysis in a dose-dependent manner, but only the inhibition promoted by sodium orthovanadate and ammonium molybdate
is irreversible. Intact mycelial forms of P. boydii are also able to hydrolyze phosphoaminoacids with different specificity. 相似文献
6.
Jorge CD Sampaio MM Hreggvidsson GO Kristjánson JK Santos H 《Extremophiles : life under extreme conditions》2007,11(1):115-122
Trehalases play a central role in the metabolism of trehalose and can be found in a wide variety of organisms. A periplasmic
trehalase (α,α-trehalose glucohydrolase, EC 3.2.1.28) from the thermophilic bacterium Rhodothermus marinus was purified and the respective encoding gene was identified, cloned and overexpressed in Escherichia coli. The recombinant trehalase is a monomeric protein with a molecular mass of 59 kDa. Maximum activity was observed at 88°C
and pH 6.5. The recombinant trehalase exhibited a K
m
of 0.16 mM and a V
max of 81 μmol of trehalose (min)−1 (mg of protein)−1 at the optimal temperature for growth of R. marinus (65°C) and pH 6.5. The enzyme was highly specific for trehalose and was inhibited by glucose with a K
i
of 7 mM. This is the most thermostable trehalase ever characterized. Moreover, this is the first report on the identification
and characterization of a trehalase from a thermophilic bacterium. 相似文献
7.
Energy status of the novel alkalitolerant Yarrowia lipolytica yeast strain grown at alkaline conditions (pH 9.7) was examined. Cells grown under such severe conditions were found to preserve high respiratory activity. The oxidative phosphorylation system dominated in the energy budget of the cell. A procedure was specially design to isolate tightly coupled mitochondria from yeast cells grown at alkaline conditions. The isolated mitochondrial preparations met known criteria of physiological intactness, as inferred from their ability to maintain distinctive state 4–3 respiration transition upon addition of ADP, high respiratory rates, good respiratory control values, and ADP/O ratios close to the theoretically expected maxima for the substrates used. 相似文献
8.
Mortierella alpina was grown in a fed-batch culture using a 12-l jar fermenter with an initial 8-l working volume containing 20 g glucose l−1 and 10 g corn-steep powder l−1. Glucose was intermittently fed to give 32 g l−1 at each time. The pH of culture was maintained using 14% (v/v) NH4OH, which also acted as a nitrogen source. A final cell density of 72.5 g l−1 was reached after 12.5 days with a content of arachidonic acid (ARA) at 18.8 g l−1. These values were 4 and 1.8 times higher than the respective values in batch culture. Our results suggest that the combined feeding of glucose and NH4+ to the growth of M. alpina could be applied for the industrial scale production of ARA. 相似文献
9.
Trchounian K Pinske C Sawers RG Trchounian A 《Journal of bioenergetics and biomembranes》2011,43(6):645-650
Escherichia coli has four [NiFe]-hydrogenases (Hyd); three of these, Hyd-1, Hyd-2 and Hyd-3 have been characterized well. In this study the
requirement for the F0F1-ATP synthase for the activities of the hydrogen-oxidizing hydrogenases Hyd-1 and Hyd-2 was examined. During fermentative
growth on glucose at pH 7.5 an E. coli F0F1-ATP synthase mutant (DK8) lacked hydrogenase activity. At pH 5.5 hydrogenase activity was only 20% that of the wild type.
Using in-gel activity staining, it could be demonstrated that both Hyd-1 and Hyd-2 were essentially inactive at these pHs,
indicating that the residual activity at pH 5.5 was due to the hydrogen-evolving Hyd-3 enzyme. During fermentative growth
in the presence of glycerol, hydrogenase activity in the mutant was highest at pH 7.5 attaining a value of 0.76 U/mg, or ~50%
of wild type activity, and Hyd-2 was only partially active at this pH, while Hyd-1 was inactive. Essentially no hydrogenase
activity was measured at pH 5.5 during growth with glycerol. At this pH the mutant had a hydrogenase activity that was maximally
only ~10% of wild type activity with either carbon substrate but a weak activity of both Hyd-1 and Hyd-2 could be detected.
Taken together, these results demonstrate for the first time that the activity of the hydrogen-oxidizing hydrogenases in E. coli depends on an active F0F1-ATP synthase during growth at high and low pH. 相似文献
10.
Xylanase A of Thermotoga neapolitana contains binding domains both at the N- and C-terminal ends of the catalytic domain. In the N-terminal position it contains
two carbohydrate-binding modules (CBM) which belong to family 22. These CBMs bind xylan but not to cellulose. The gene encoding
the mature peptide of these CBMs was fused with an alkaline active GH10 xylanase from Bacillus halodurans S7 and expressed in Escherichia coli. The (His)6 tagged hybrid protein was purified by immobilized metal affinity chromatography and characterized. Xylan binding by the chimeric
protein was influenced by NaCl concentration and pH of the binding medium. Binding increased with increasing salt concentration
up to 200 mM. Higher extent of binding was observed under acidic conditions. The fusion of the CBM structures enhanced the
hydrolytic efficiency of the xylanase against insoluble xylan, but decreased the stability of the enzyme. The optimum temperature
and pH for the activity of the xylanase did not change. 相似文献
11.
Summary A locally isolated strain of Aspergillus foetidus MTCC 4898 was studied for xylanase (EC 3.2.1.8) production using lignocellulosic substrates under solid state fermentation. Corncobs were found as the best substrates for high yield of xylanases with poor cellulase production. The influence of various parameters such as temperature, pH, moistening agents, moisture level, nitrogen sources and pretreatment of substrates were evaluated with respect to xylanase yield, specific activity and cellulase production. Influence of nitrogen sources on protease secretion was also examined. Maximum xylanase production (3065 U/g) was obtained on untreated corncobs moistened with modified Mandels and Strenberg medium, pH 5.0 at 1 5 moisture levels at 30 °C in 4 days of cultivation. Submerged fermentation under the same conditions gave higher yield (3300 U/g) in 5 days of cultivation, but productivity was less. Ammonium sulphate fractionation yielded 3.56-fold purified xylanase with 76% recovery. Optimum pH and temperature for xylanase activity were found to be 5.3 and 50 °C respectively. Kinetic parameters like Km and Vmax were found to be 3.58 mg/ml and 570 μmol/mg/min. Activity of the enzyme was found to be enhanced by cystiene hydrochloride, CoCl2, xylose and Tween 80, while significantly inhibited by Hg++, Cu++ and glucose. The enzyme was found to be stable at 40 °C. The half life at 50 °C was 57.53 min. However thermostability was enhanced by glycerol, trehalose and Ca++. The crude enzyme was stable during lyophilization and could be stored at less than 0 °C. 相似文献
12.
Frank W. Koopman Johannes H. de Winde Harald J. Ruijssenaars 《Applied microbiology and biotechnology》2009,83(4):705-713
The solvent-tolerant bacterium Pseudomonas putida S12 was engineered to efficiently utilize the C1 compounds methanol and formaldehyde as auxiliary substrate. The hps and phi genes of Bacillus brevis, encoding two key steps of the ribulose monophosphate (RuMP) pathway, were introduced to construct a pathway for the metabolism
of the toxic methanol oxidation intermediate formaldehyde. This approach resulted in a remarkably increased biomass yield
on the primary substrate glucose when cultured in C-limited chemostats fed with a mixture of glucose and formaldehyde. With
increasing relative formaldehyde feed concentrations, the biomass yield increased from 35% (C-mol biomass/C-mol glucose) without
formaldehyde to 91% at 60% relative formaldehyde concentration. The RuMP-pathway expressing strain was also capable of growing
to higher relative formaldehyde concentrations than the control strain. The presence of an endogenous methanol oxidizing enzyme
activity in P. putida S12 allowed the replacement of formaldehyde with the less toxic methanol, resulting in an 84% (C-mol/C-mol) biomass yield.
Thus, by introducing two enzymes of the RuMP pathway, co-utilization of the cheap and renewable substrate methanol was achieved,
making an important contribution to the efficient use of P. putida S12 as a bioconversion platform host. 相似文献
13.
Osório H Moradas-Ferreira P Günther Sillero MA Sillero A 《Archives of microbiology》2004,181(3):231-236
As has been previously shown, Saccharomyces cerevisiae grown in 2% or 0.025% glucose uses this carbohydrate by the fermentative or oxidative pathways, respectively. Depending on the glucose concentration in the medium, the effect of the addition of H2O2 on the level of ATP and on glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity differed. In the presence of 2% glucose, ATP and GAPDH decreased sharply during the first few minutes of treatment, whereas in the presence of 0.025% glucose, GAPDH activity decreased similarly, but the ATP level remained practically unchanged. The addition of 3 mM glutathione to the culture media prevented the depletion of ATP levels and GAPDH activity in the presence of H2O2. Catalase and superoxide dismutase activities did not vary significantly when yeast cells were grown either in 2% or in 0.025% glucose. 相似文献
14.
Chu WH 《Journal of industrial microbiology & biotechnology》2007,34(3):241-245
Thirty-five strains capable of secreting extracellular alkaline proteases were isolated from the soil and waste water near
the milk processing plant, slaughterhouse. Strain APP1 with the highest-yield alkaline proteases was identified as Bacillus sp. The cultural conditions were optimized for maximum enzyme production. When the initial pH of the medium was 9.0, the
culture maintained maximum proteolytic activity for 2,560 U ml−1 at 50°C for 48 h under the optimized conditions containing (g−1): soyabean meal, 15; wheat flour, 30; K2HPO4, 4; Na2HPO4, 1; MgSO4·7H2O, 0.1; Na2CO3, 6. The alkaline protease showed extreme stability toward SDS and oxidizing agents, which retained its activity above 73
and 110% on treatment for 72 h with 5% SDS and 5% H2O2, respectively. 相似文献
15.
Erhard E. Pfündel 《Photosynthesis research》2009,100(3):163-177
The F
0 and F
M level fluorescence from a wild-type barley, a Chl b-less mutant barley, and a maize leaf was determined from 430 to 685 nm at 10 nm intervals using pulse amplitude-modulated
(PAM) fluorimetry. Variable wavelengths of the pulsed excitation light were achieved by passing the broadband emission of
a Xe flash lamp through a birefringent tunable optical filter. For the three leaf types, spectra of F
V/F
M (=(F
M − F
0)/F
M) have been derived: within each of the three spectra of F
V/F
M, statistically meaningful variations were detected. Also, at distinct wavelength regions, the F
V/F
M differed significantly between leaf types. From spectra of F
V/F
M, excitation spectra of PS I and PS II fluorescence were calculated using a model that considers PS I fluorescence to be constant
but variable PS II fluorescence. The photosystem spectra suggest that LHC II absorption results in high values of F
V/F
M between 470 and 490 nm in the two wild-type leaves but the absence of LHC II in the Chl b-less mutant barley leaf decreases the F
V/F
M at these wavelengths. All three leaves exhibited low values of F
V/F
M around 520 nm which was tentatively ascribed to light absorption by PS I-associated carotenoids. In the 550–650 nm region,
the F
V/F
M in the maize leaf was lower than in the barley wild-type leaf which is explained with higher light absorption by PS I in
maize, which is a NADP-ME C4 species, than in barley, a C3 species. Finally, low values of F
V/F
M at 685 in maize leaf and in the Chl b-less mutant barley leaf are in agreement with preferential PS I absorption at this wavelength. The potential use of spectra
of the F
V/F
M ratio to derive information on spectral absorption properties of PS I and PS II is discussed. 相似文献
16.
Nack-Shick Choi Jae Jun Song Dong-Min Chung Young Jae Kim Pil Jae Maeng Seung-Ho Kim 《Journal of industrial microbiology & biotechnology》2009,36(3):417-426
A novel fibrinolytic enzyme (AJ) was purified from Staphylococcus sp. strain AJ screened from Korean salt-fermented Anchovy-jeot. Relative molecular weight of AJ was determined as 26 kDa by using SDS-PAGE and fibrin zymography. Based on a 2D gel, AJ
was found to consist of three active isoforms (pI 5.5–6.0) with the same N-terminal amino acid sequence. AJ exhibited optimum pH and temperature at 2.5–3.0 and 85°C, respectively.
AJ kept 85% of the initial activity after heating at 100°C for 20 min on the zymogram gel. The Michaelis constant (K
m) and K
cat values of AJ towards α-casein were 0.38 mM and 19.73 s−1, respectively. AJ cleaved the Aα-chain of fibrinogen but did not affect the Bβ- and γ-chains, indicating that it is an α-fibrinogenase.
The fibrinolytic activity was inhibited by diisopropyl fluorophosphate, indicating AJ is a serine protease. Interestingly,
AJ was very stable at acidic condition, SDS, and heat (100°C), whereas it was easily degraded at neutral and alkaline conditions.
In particular, AJ formed an active homo-dimer in the pH range from 7.0 to 8.0. To our knowledge, a similar combination of
acid and heat stability has not yet been reported for other fibrinolytic enzymes. 相似文献
17.
B. R. Maricle R. W. Lee C. E. Hellquist O. Kiirats G. E. Edwards 《Photosynthetica》2007,45(3):433-440
The effects of salinity (sea water at 0 ‰ versus 30 ‰) on gross rates of O2 evolution (J
O2) and net rates of CO2 uptake (P
N) were measured in the halotolerant estuarine C4 grasses Spartina patens, S. alterniflora, S. densiflora, and Distichlis spicata in controlled growth environments. Under high irradiance, salinity had no significant effect on the intercellular to ambient
CO2 concentration ratio (C
i/C
a). However, during photosynthesis under limiting irradiance, the maximum quantum efficiency of CO2 fixation decreased under salinity across species, suggesting there is increased leakage of the CO2 delivered to the bundle sheath cells by the C4 pump. Growth under salinity did not affect the maximum intrinsic efficiency of photosystem 2, PS2 (FV/FM) in these species, suggesting salinity had no effect on photosynthesis by inactivation of PS2 reaction centers. Under saline
conditions and high irradiance, P
N was reduced by 75 % in Spartina patens and S. alterniflora, whereas salinity had no effect on P
N in S. densiflora or D. spicata. This inhibition of P
N in S. patens and S. alterniflora was not due to an effect on stomatal conductance since the ratio of C
i/C
a did not decrease under saline conditions. In growth with and without salt, P
N was saturated at ∼500 μmol(quantum) m−2 s−1 while J
O2 continued to increase up to full sunlight, indicating that carbon assimilation was not tightly coupled to photochemistry
in these halophytic species. This increase in alternative electron flow under high irradiance might be an inherent function
in these halophytes for dissipating excess energy. 相似文献
18.
This study examined the effects of carbon dioxide (CO2)-, ozone (O3)-, and genotype-mediated changes in quaking aspen (Populus tremuloides) chemistry on performance of the forest tent caterpillar (Malacosoma disstria) and its dipteran parasitoid (Compsilura concinnata) at the Aspen Free-Air CO2 Enrichment (FACE) site. Parasitized and non-parasitized forest tent caterpillars were reared on two aspen genotypes under elevated levels of CO2 and O3, alone and in combination. Foliage was collected for determination of the chemical composition of leaves fed upon by forest tent caterpillars during the period of endoparasitoid larval development. Elevated CO2 decreased nitrogen levels but had no effect on concentrations of carbon-based compounds. In contrast, elevated O3 decreased nitrogen and phenolic glycoside levels, but increased concentrations of starch and condensed tannins. Foliar chemistry also differed between aspen genotypes. CO2, O3, genotype, and their interactions altered forest tent caterpillar performance, and differentially so between sexes. In general, enriched CO2 had little effect on forest tent caterpillar performance under ambient O3, but reduced performance (for insects on one aspen genotype) under elevated O3. Conversely, elevated O3 improved forest tent caterpillar performance under ambient, but not elevated, CO2. Parasitoid larval survivorship decreased under elevated O3, depending upon levels of CO2 and aspen genotype. Additionally, larval performance and masses of mature female parasitoids differed between aspen genotypes. These results suggest that host-parasitoid interactions in forest systems may be altered by atmospheric conditions anticipated for the future, and that the degree of change may be influenced by plant genotype. 相似文献
19.
In Escherichia coli, the F1FO ATP synthase b subunits house a conserved arginine in the tether domain at position 36 where the subunit emerges from the membrane. Previous
experiments showed that substitution of isoleucine or glutamate result in a loss of enzyme activity. Double mutants have been
constructed in an attempt to achieve an intragenic suppressor of the b
arg36→ile and the b
arg36→glu mutations. The b
arg36→ile mutation could not be suppressed. In contrast, the phenotypic defect resulting from the b
arg36→glu mutation was largely suppressed in the b
arg36→glu,glu39→arg double mutant. E. coli expressing the b
arg36→glu,glu39→arg subunit grew well on succinate-based medium. F1FO ATP synthase complexes were more efficiently assembled and ATP driven proton pumping activity was improved. The evidence
suggests that efficient coupling in F1FO ATP synthase is dependent upon a basic amino acid located at the base of the peripheral stalk. 相似文献
20.
Isocitrate dehydrogenase [IDH; EC 1.1.1.42] from the thermoacidophilic archaeon Thermoplasma acidophilum (TaIDH) showed high thermal stability with an apparent melting temperature, T
m, of 82.2 and 84.5°C at pH 7.5 and 5.8, respectively. Based on structural alignment of TaIDH with IDH from Aeropyrum pernix (ApIDH) and Archaeoglobus
fulgidus (AfIDH) residues forming an aromatic cluster in the clasp-domain thought to strengthen the dimer interface in ApIDH and AfIDH were identified in the former enzyme. Moreover, TaIDH had a shortened N-terminus that may protect the enzyme from thermal denaturation. The enzyme activity of TaIDH was highest at 70°C. The pH-activity profile was bell-shaped with an optimum shifted to a lower pH compared to AfIDH. The activity of TaIDH was influenced by changes in pH with a three-fold reduction in activity when the pH was shifted from the pH-optimum at
7.5 to pH 5.8. However, the specific activity at pH 5.8 was still high when compared with AfIDH. The reduction in activity at pH 5.8 was not due to instability of the enzyme as the T
m of TaIDH was higher at pH 5.8 than at 7.5 and the enzyme retained 91% of its activity after incubation at 1 h at pH 5 and 60°C.
The difference in the pH-profile of TaIDH in comparison with AfIDH may thus be related to the pK
as of their catalytic residues involved in the initial proton abstraction and the final proton donation during the catalysis
of oxidative decarboxylation of isocitrate to 2-oxoglutarate and reduced coenzyme. 相似文献