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1.
Partially purified glucose isomerase from Streptomyces thermonitrificans when coupled to glutaraldehyde-activated Indion 48-R, retained 30–40% activity of the soluble enzyme. However, an approximately
twofold increase in the activity could be achieved by binding the enzyme in the presence of glucose. Binding the enzyme to
matrices presaturated with either glucose or fructose and influence of lysine modification on the activity of the soluble
enzyme revealed that the comparatively low activity observed in case of the enzyme bound in the absence of substrate is the
result of the nonspecific binding of either substrate or product to the matrix. Immobilization did not affect the pH and temperature
optima of the enzyme, but it lowered the temperature stability. Immobilization resulted in a marginal increase in the K
m
and a threefold decrease in the V
max
. Substrate concentrations as high as 36% glucose could be converted to 18.5% fructose in 5 h, at pH 7.0 and 70‡C. The bound
enzyme, however, showed inferior stability to repeated use and lost approx 40% of its initial activity after five cycles of
use. Indion 48-R bound glucose isomerase could be stored, in wet state, for 30 d without any apparent loss in its initial
activity. 相似文献
2.
The gpdA-promoter-controlled exocellular production of glucose oxidase (GOD) by recombinant Aspergillus niger NRRL-3 (GOD3-18) during growth on glucose and nonglucose carbon sources was investigated. Screening of various carbon substrates
in shake-flask cultures revealed that exocellular GOD activities were not only obtained on glucose but also during growth
on mannose, fructose, and xylose. The performance of A. niger NRRL-3 (GOD3-18) using glucose, fructose, or xylose as carbon substrate was compared in more detail in bioreactor cultures.
These studies revealed that gpdA-promoter-controlled GOD synthesis was strictly coupled to cell growth. The gpdA-promoter was most active during growth on glucose. However, the unfavorable rapid GOD-catalyzed transformation of glucose
into gluconic acid, a carbon source not supporting further cell growth and GOD production, resulted in low biomass yields
and, therefore, reduced the advantageous properties of glucose. The total (endo- and exocellular) specific GOD activities
were lowest when growth occurred on fructose (only a third of the activity that was obtained on glucose), whereas utilization
of xylose resulted in total specific GOD activities nearly as high as reached during growth on glucose. Also, the portion
of GOD excreted into the culture fluid reached similar high levels (≅ 90%) by using either glucose or xylose as substrate,
whereas growth on fructose resulted in a more pelleted morphology with more than half the total GOD activity retained in the
fungal biomass. Finally, growth on xylose resulted in the highest biomass yield and, consequently, the highest total volumetric
GOD activity. These results show that xylose is the most favorable carbon substrate for gpdA-promoter-controlled production of exocellular GOD. 相似文献
3.
Thielavia terrestris is a soil-borne thermophilic fungus whose molecular/cellular biology is poorly understood. Only a few genes have been cloned
from the Thielavia genus. We detected an extracellular glucoamylase in culture filtrates of T. terrestris and cloned the corresponding glaA gene. The coding region contains five introns. Based on the amino acid sequence, the glucoamylase was 65% identical to Neurospora crassa glucoamylase. Sequence comparisons suggested that the enzyme belongs to the glycosyl hydrolase family 15. The T. terrestris glaA gene was expressed in Aspergillus oryzae under the control of an A. oryzae α-amylase promoter and an Aspergillus niger glucoamylase terminator. The 75-kDa recombinant glucoamylase showed a specific activity of 2.8 μmol/(min·mg) with maltose
as substrate. With maltotriose as a substrate, the enzyme had an optimum pH of 4.0 and an optimum temperature of 60°C. The
enzyme was stable at 60°C for 30 min. The K
m
and k
cat
of the enzyme for maltotriose were determined at various pHs and temperatures. At 20°C and pH 4.0, the enzyme had a K
m
of 0.33±0.07 m M and a k
cat
of (5.5±0.5)×10 3 min −1 for maltotriose. The temperature dependence of k
cat
/ K
m
indicated an activation free energy of 2.8 kJ/mol across the range of 20–70°C. Overall, the enzyme derived from the thermophilic
fungus exhibited properties comparable with that of its homolog derived from mesophilic fungi. 相似文献
4.
Glucoamylase production by Aspergillus niger in solid-state fermentation was optimized using factorial design and response surface techniques. The variables evaluated
were pH and bed thickness in tray, having as response enzyme production and productivity. The bed thickness in tray was the
most significant variable for both responses. The highest values for glucoamylase production occurred using pH 4.5 and bed
thickness in the inferior limits at 2.0–4.2 cm. For productivity, the optimal conditions were at pH 4.5 as well and bed thickness
from 4.4 to 7.5 cm. The optimal conditions for glucoamylase production while obtaining high activity without loss of productivity
were pH 4.5 and bed thickness in tray from 4.0 to 4.5 cm, which resulted in an enzyme production of 695 U/g and productivity
of 5791 U/h. 相似文献
5.
The acetone-butanol production by simultaneous saccharification and extractive fermentation (SSEF) was investigated. In the
SSEF employing cellulase enzymes and Clostridium acetobutylicum, both glucan and xylan fractions of pretreated aspen are concurrently converted into acetone and butanol. Continuous removal
of the fermentation products from the bioreactor by extraction was an important factor that allowed long-term fed-batch operation.
The use of membrane extraction prevented the problems of phase separation and extractant loss. Increase in substrate feeding
as well as reduction of nutrient supply was found to be beneficial in suppressing the acid production, thereby improving the
solvent yield. Because of prolonged low growth conditions prevalent in the fed-batch operation, the butanol-to-acetone ratio
in the product was significantly higher at 2.6–2.8 compared to the typical value of two. 相似文献
6.
The effect of nutrients on L(+)-lactic acid production from glucose was investigated using Rhizopus oryzae ATCC 523 11. From the shake-flask experiments, the optimal medium composition was defined for improved lactic-acid production.
In order to enhance lactic-acid production rate and product yield, controlled aeration in a bubble column was conducted under
optimal conditions. Results showed a maximum lactic-acid production rate of 2.58 g/L/h was obtained with an initial glucose
concentration of 94 g/L. Finallactic-acid concentration of 83 g/L was achieved after 32 h of fermentation with a weight of
0.88 glactic acid/g glucose consumed. 相似文献
7.
The effect of culture conditions such as medium composition and shear stress on the fungal pellet morphology in shake-flask
cultures and its relation to glucose oxidase (GOD) excretion by recombinant Aspergillus niger NRRL 3 (GOD 3–18) was investigated. It was shown that culture conditions resulting in the formation of smaller fungal pellets
with an increased mycelial density result in higher yields of exocellular GOD. The pellets obtained in shake-flask cultures
showed distinct layers of mycelial density with only the thin outer layer consisting of a dense mycelial network. The performance
of the recombinant strain and the process of pellet formation was also analyzed during batch cultivation in a stirred-tank
bioreactor. It was shown that the process of pellet formation occurred in two steps: (1) aggregation of free spores to spore
clusters with subsequent germination and formation of small aggregates surrounded by a loose hyphal network, and (2) aggregation
of the primary aggregates to the final full-size pellets. The fungal pellets formed during bioreactor cultivation were smaller,
did not show large differences in mycelial density, and were more efficient with respect to the production of exocellular
GOD. The decreasing pellet size also correlated with an increased mycelial density, indicating an improvement of the transport
of nutrients to the inner parts of the pellet. 相似文献
8.
Aspergillus niger NCIM 1207 produces high levels of extracellular beta-glucosidase and xylanase activities in submerged fermentation. Among the nitrogen sources, ammonium sulfate, ammonium dihydrogen orthophosphate, and corn-steep liquor were the best for the production of cellulolytic enzymes by A. niger. The optimum pH and temperature for cellulase production were 3.0-5.5 and 28 degrees C, respectively. The cellulase complex of this strain was found to undergo catabolite repression in the presence of high concentrations of glucose. Glycerol at all concentrations caused catabolite repression of cellulase production. The addition of glucose (up to 1% concentration) enhanced the production of cellulolytic enzymes, but a higher concentration of glucose effected the pronounced repression of enzymes. Generally the growth on glucose- or glycerol-containing medium was accompanied by a sudden drop in the pH of the fermentation medium to 2.0. 相似文献
9.
The five-carbon sugar d-xylose is a major component of hemicellulose and accounts for roughly one-third of the carbohydrate content of many lignocellulosic
materials. The efficient fermentation of xylose-rich hemicellulose hydrolyzates (prehydrolyzates) represents an opportunity
to improve significantly the economics of large-scale fuel ethanol production from lignocellulosic feedstocks. The National
Renewable Energy Laboratory (NREL) is currently investigating a simultaneous saccharification and cofermentation (SSCF) process
for ethanol production from biomass that uses a dilute-acid pretreatment and a metabolically engineered strain of Zymomonas mobilis that can coferment glucose and xylose. The objective of this study was to establish optimal conditions for cost-effective
seed production that are compatible with the SSCF process design.
Two-level and three-level full factorial experimental designs were employed to characterize efficiently the growth performance
of recombinant Z. mobilis CP4:pZB5 as a function of nutrient level, pH, and acetic acid concentration using a synthetic hardwood hemicellulose hydrolyzate
containing 4% (w/v) xylose and 0.8% (w/v) glucose. Fermentations were run batchwise and were pH-controlled at low levels of
clarified corn steep liquor (cCSL, 1-2% v/v), which were used as the sole source of nutrients. For the purpose of assessing
comparative fermentation performance, seed production was also carried out using a “benchmark” yeast extract-based laboratory
medium. Analysis of variance (ANOVA) of experimental results was performed to determine the main effects and possible interactive
effects of nutrient (cCSL) level, pH, and acetic acid concentration on the rate of xylose utilization and the extent of cell
mass production. Results indicate that the concentration of acetic acid is the most significant limiting factor for the xylose
utilization rate and the extent of cell mass production; nutrient level and pH exerted weaker, but statistically significant
effects. At pH 6.0, in the absence of acetic acid, the final cell mass concentration was 1.4 g dry cell mass/L (g DCM/L),
but decreased to 0.92 and 0.64 g DCM/L in the presence of 0.5 and 1.0% (w/v) acetic acid, respectively. At concentrations
of acetic acid of 0.75 (w/v) or lower, fermentation was complete within 1.5 d. In contrast, in the presence of 1.0% (w/v)
acetic acid, 25% of the xylose remained after 2 d. At a volumetric supplementation level of 1.5–2.0% (v/v), cCSL proved to
be a cost-effective single-source nutritional adjunct that can support growth and fermentation performance at levels comparable
to those achieved using the expensive yeast extract-based laboratory reference medium. 相似文献
10.
The interaction between the neutral 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphatidylcholine (POPC) liposomes and cell membrane of Streptomyces griseus induced by the heat treatment at specific temperature was investigated, focusing on the internalization of the neutral POPC liposomes with S. griseus cells. In an attempt to clarify the modes of liposome internalization, various kinds of inhibitors of endocytotic pathways were used to treat S. griseus cells. The efficiency of the heat treatment on liposome–cell membrane interactions was finally characterized based on the hydrophobic, electrostatic interactions and hydration effect. In fact, the internalization of the neutral liposomes into these cells was found to show higher rate and greater amount at higher temperatures. The kinetic study showed that the maximum amount of the internalized liposomes was, respectively, 469 × 10 5 and 643 × 10 5 liposomes/cell at 37 and 41 °C. The internalization of the neutral liposomes induced by the heat treatment was characterized, implying that the endocytosis occurred. The interactions involving the internalization, adsorption, and fusion of these liposomes with S. griseus cells were mainly contributed by the hydrophobic interaction and the unstable hydrogen bonds caused by the loss of water of surface hydration of cell membrane rather than the electrostatic interaction under the specific heat condition. 相似文献
11.
Protein disulfide isomerase (PDI) enzymes are eukaryotic oxidoreductases that catalyze the correct formation of disulfide bonds during protein folding. Structurally they are characterized by the presence of functional thioredoxin-like (Trx) domains. For the protozoan parasite causative of the human amebiasis (Entamoeba histolytica), the correct formation of disulfide bonds is important for an accurate folding of its proteins, including some virulence factors. However, little is known about the enzymes involved in this mechanism. We undertook a post-genomic approach to identify the PDI family of this parasite. The genome database survey revealed a set of 11 PDI-encoding sequences with predictable protein thiol/disulfide oxidoreductase activities. 相似文献
12.
In this work, the effect of the addition of different concentrations of Tween-80 and three different zeolite-like products
on enzymatic hydrolysis, ethanol fermentation, and simultaneous saccharification and fermentation (SSF) process has been investigated.
The ability of these products to enhance the effectiveness of the SSF process to ethanol of steam-exploded poplar biomass
using the thermotolerant strain Kluyveromyces marxianus EMS-26 has been tested.
Tween-80 (0.4 g/L) increased enzymatic hydrolysis yield by 20% when compared to results obtained in hydrolysis in absence
of the additive. Zeolite-like products (ZESEP-56 and ZECER-56) (2.5 g/L) improved rates of conversion and ethanol yields in
the fermentation of liquid fraction recovered from steam-exploded poplar. The periods required for the completion of fermentation
were approx 10 h in the presence of zeolite-like products and 24 h in the absence of additives. The probable mode of action
is through lowered levels of inhibitory substances because of adsorption by the additive. 相似文献
13.
The gene encoding xylose isomerase ( xylA) was cloned from Thermus flavus AT62 and the DNA sequence was determined. The xylA gene encodes the enzyme xylose isomerase (XI or xylA) consisting of 387 amino acids (calculated Mr of 44,941). Also, there was a partial xylulose kinase gene that was 4 bp overlapped
in the end of XI gene. The XI gene was stably expressed in E. coli under the control of tac promoter. XI produced in E. coli was simply purified by heat treatment at 90°C for 10 min and column chromatography of DEAE-Sephacel. The Mr of the purified
enzyme was estimated to be 45 kDa on SDS-polyacrylamide gel electrophoresis. However, Mr of the cloned XI was 185 kDa on native
condition, indicating that the XI consists of homomeric tetramer. The enzyme has an optimum temperature at 90°C. Thermostability
tests revealed that half life at 85°C was 2 mo and 2 h at 95°C. The optimum pH is around 7.0, close to where by-product formation
is minimal. The isomerization yield of the cloned XI was about 55% from glucose, indicating that the yield is higher than
those of reported enzymes. The Km values for various sugar substrates were calculated as 106 mM for glucose. Divalent cations
such as Mn 2+, Co 2+, and Mg 2+ are required for the enzyme activity and 100 mM EDTA completely inhibited the enzyme activity. 相似文献
14.
A series of bifunctional chemical modification reagents, presenting variations in both the chemistry of the functional groups
and in the length of the spacer between the two reactive groups, have been evaluated as agents for enhancing the thermal stability
of purified Aspergillus niger amyloglucosidase by means of intramolecular cross-linking. Several chemical modifiers (e.g., diimidoesters) were identified
that more than double the half-life of this industrially important enzyme during incubation at 65°C in the absence of substrate.
The increased stability of the modified enzymes has been correlated with changes in the fluorescence-monitored thermal denaturation
curves of the modified enzymes, relative to that of the native enzyme. 相似文献
15.
A study on preparation of industrial glucoamylase irradiated by 60Co gamma-ray showed that an abvious change took place in the extraviolet absorption spectrum and the pH - value of the enzyme solution and the activity of the enzyme preparation was much more resistant to irradiation than that of microbes contained in the preparation. The result indicates that irradiated glucoamylase preparation is not only favourable to activity preservation but also is improved in the hygienic quality of products. A comparison of the various properties of irradiated enzyme preparation with those of unirradiated one has proved that irradiation does not change the normal application of glucoamylase preparation. 相似文献
16.
The yeast Candida lipolytica IA 1055 produced an inducible extracellular emulsification activity while utilizing glucose at different concentrations as
carbon source during batch fermentation at 27°C. In all glucose concentrations studied, maximum production of emulsification
activity was detected in the stationary phase of growth, after pH reached minimal values. The bioemulsifier isolated was a
complex biopolymer constituting proteins, carbohydrates, and lipids. The results obtained in this work show that the biosynthesis
of a bioemulsifier is not simply a prerequisite for the degradation of extracellular hydrocarbon. 相似文献
17.
Hexokinase (HK) and glucose 6-phosphate dehydrogenase (G6PDH) are important enzymes used in biochemical studies and in analytical
methods. The stability of the enzymes can be affected by several variables, pH being one of them. The effect of pH on the
stability of HK and G6PDH was evaluated in this work. Baker’s yeast cells were suspended in 50 m M Tris-HCl buffer (pH 7.5) containing 5.0 m M MgCl2, and submitted to disruption by agitation with glass beads and in the presence of protease inhibitors. The cell-free
extract was obtained by centrifugation (2880 g; 10 min), followed by dilution into the buffers: 0.1 M acetate-acetic acid (pH: 4.0, 4.5, 5.0, or 5.5), 0.1 M phosphate buffer (pH: 6.0, 6.5, or 7.0), and 0.1 M Tris-HCl buffer (pH: 7.5, 8.0, 8.5, 9.0 or 9.5). The residual activity of HK and G6PDH, expressed as μmol of NADPH formed
per min, were measured through a period of buffer-enzyme contact from 0 to 51 h at 4°C. It was observed that up to 4 h both
enzymes were stable in all buffers used. However, after 51 h HK was stable at pH 6.0 and 7.5, whereas G6PDH was stable at
pH 7.0, 9.5, and between 4.5 and 5.5. 相似文献
18.
Glucose dehydrogenase (GDH) fromBacillus megaterium was immobilized using aminopropyl controlled-pore silica (CPS, average pore sizes of 170 and 500 Å) as a support and glutaraldehyde as a bifunctional crosslinking agent. The CPS-immobilized enzyme could be reused 12 times and the best results were obtained using aminopropyl CPS-500 and bovine serum albumin as a feeder for stabilizing the protein layer on the support. DEAE-Sephadex (A-25 and A-50) was also used as a support for immobilizing GDH, with yields of around 42% for A-25 and 25–30% for A-50. The effect of pH on the immobilization procedure showed pH 6.5 to be better than pH 7.5 with respect to the recovery of enzyme activity. Both preparations of DEAE-Sephadex immobilized GDH could be reused several times and were thermostable at 40°C for 7 h. The kinetic parameters as Michaelis constant and maximum rate were determined for the immobilized enzyme and compared with those for the freeform. 相似文献
19.
An enantioselective synthesis of (+)-prelactone B 1 has been achieved on a multigram scale starting from a known bicyclic precursor 2. The key feature of the strategy is the generation of 3-stereogenic centres from a single bicyclic precursor, which has been utilized as a chiral building block for the synthesis of various natural products. 相似文献
20.
The morphologic and physiologic effects of vitamin E, a powerful antioxidant, on the autolysis and sporulation of Aspergillus nidulans FGSC26 were studied. In carbon-depleted submerged cultures, reactive oxygen species (ROS) accumulated in the cells and, concomitantly,
progressing autolysis was observed, which was characterized by decreasing dry cell masses and pellet diameters as well as
by increasing extracellular chitinase activities. Vitamin E supplemented at a concentration of 1 g/L hindered effectively
the intracellular accumulation of ROS, the autolytic loss of biomass, the disintegration of pellets, and the release of chitinase
activities. In surface cultures, vitamin E inhibited autolysis of both A. nidulans FGSC26 and a loss-of-function FlbA autolytic phenotype mutant. In addition, supplementation of the culture medium with this antioxidant also had a negative
effect on the sporulation of strain FGSC26 and the FadA
G203R
hypersporulating phenotype mutant. These results suggest that accumulation of ROS was involved in the initiation of both
sporulation and autolysis in this filamentous fungus, but that FadA/FlbA signaling was not involved in this vitamin E-dependent
regulation. Vitamin E can be recommended as a supplement in fermentations in which the disintegration of pellets and gross
autolysis should be avoided. 相似文献
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