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1.
Sene Luciane Felipe Maria G. A. Silva Silvio S. Vitolo Michele 《Applied biochemistry and biotechnology》2001,91(1-9):671-680
Candida guilliermondii FTI 20037 was cultured in sugarcane bagasse hydrolysate supplemented with 2.0 g/L of (NH4)2SO4, 0.1 g/L of CaCl2·2H2O, and 20.0 g/L of rice bran at 35°C; pH 4.0; agitation of 300 rpm; and aeration of 0.4, 0.6, or 0.8 vvm. The high xylitol
production (20.0 g/L) and xylose reductase (XR) activity (658.8 U/mg of protein) occurred at an aeration of 0.4 vvm. Under
this condition, the xylitol dehydrogenase (XD) activity was low. The apparent K
M for XR and XD against substrates and cofactors were as follows: for XR, 6.4×10−2
M (xylose) and 9.5×10−3 mM (NADPH); for XD, 1.6×10−1
M (xylitol) and 9.9×10−2 mM (NAD+). Because XR requires about 10-fold less xylose and cofactor than XD for the condition in which the reaction rate is
half of the V
max, some interference on the overall xylitol production by the yeast could be expected. 相似文献
2.
Min Hyung Kang Haiying Ni Thomas W. Jeffries 《Applied biochemistry and biotechnology》2003,106(1-3):265-276
Candida boidinii produces significant amounts of xylitol from xylose, and assays of crude homogenates for aldose (xylose) reductase (XYL1p)
have been reported to show relatively high activity with NADH as a cofactor even though XYL1p purified from this yeast does
not have such activity. A gene coding for XYL1p from C. boidinii (CbXYL1) was isolated by amplifying the central region using primers to conserved domains and by genome walking. CbXYL1 has an open reading frame of 966 bp encoding 321 amino acids. The C. boidinii XYL1p is highly similar to other known yeast aldose reductases and is most closely related to the NAD(P)H-linked XYL1p of
Kluyveromyces lactis. Cell homogenates from C. boidinii and recombinant Saccharomyces cerevisiae were tested for XYL1p activity to confirm the previously reported high ratio of NADH:NADPH linked activity. C. boidinii grown under fully aerobic conditions showed an NADH:NADPH activity ratio of 0.76, which was similar to that observed with
the XYL1p from Pichia stipitis XYL1, but which is much lower than what was previously reported. Cells grown under low aeration showed an NADH:NADPH activity
ratio of 2.13. Recombinant S. cerevisiae expressing CbXYL1 showed only NADH-linked activity in cell homogenates. Southern hybridization did not reveal additional bands. These results
imply that a second, unrelated gene for XYL1p is present in C. boidinii. 相似文献
3.
d-Xylose is a major constituent of hemicellulose, which makes up 20–30% of renewable biomass in nature.d-Xylose can be fermented by most yeasts, includingSaccharomyces cerevisiae, by a two-stage process. In this process, xylose is first converted to xylulose in vitro by the enzyme xylose (glucose) isomerase,
and the latter sugar is then fermented by yeast to ethanol. With the availability of an inexpensive source of xylose isomerase
produced by recombinantE. coli, this process of fermenting xylose to ethanol can become quite effective. In this paper, we report that yeast xylose and
xylulose fermentation can be further improved by cloning and overexpression of the xylulokinase gene. For instance, the level
of xylulokinase activity in S.cerevisiae can be increased 230fold by cloning its xylulokinase gene on a high copy-number plasmid, coupled with fusion of the gene
with an effective promoter. The resulting genetically-engineered yeasts can ferment xylose and xylulose more than twice as
fast as the parent yeast. 相似文献
4.
Mayerhoff Zea D. V. L. Roberto Inês C. Franco Telma T. 《Applied biochemistry and biotechnology》2001,91(1-9):729-737
Xylose reductase (XR) activity was evaluated in extracts of Candida mogii grown in media containing different concentrations of rice straw hydrolysate. Results of X Ractivity were compared to xylitol
production and a similar behavior was observed for these parameters. Highest values of specific production and productivity
were found for xylose reductase (35 U/g of cell and 0.97 U/[g of cell·h], respectively) and for xylitol (5.63 g/g of cell
and 0.13 g/[g of cell·h]) in fermentation conducted in medium containing 49.2 g of xylose/L. The maximum value of XR:XD ratio
(1.82) was also calculated under this initial xylose concentration with 60 h of fermentation. 相似文献
5.
Rodrigues Denise C. G. A. Da Silva Silvio S. Almeida E Silva J. B. Vitolo Michele 《Applied biochemistry and biotechnology》2002,98(1-9):875-883
Xylose reductase activity of Candida guilliermondii FTI 20037 was evaluated during xylitol production by fed-batch fermentation of sugarcane bagasse hydrolysate. A 24-1 fractional factorial design was used to select process variables. The xylose concentrations in the feeding solution (S
F
) and in the fermentor (S
0), the pH, and the aeration rate were selected for optimization of this process, which will be undertaken in the near future.
The best experimental result was achieved at S
F
=45 g/L, S
0=40 g/L, pH controlled at 6.0, and aeration rate of 1.2 vvm. Under these conditions, the xylose reductase activity was 0.81
U/mg of protein and xylitol production was 26.3 g/L, corresponding to a volumetric productivity of 0.55 g/(L·h) and a xylose
xylitol yield factor of 0.68 g/g. 相似文献
6.
Of the sugars recovered from lignocellulose, d-glucose can be readily converted into ethanol by baker’s or brewer’s yeast (Saccharomyces cerevisiae). However, xylose that is obtained by the hydrolysis of the hemicellulosic portion is not fermentable by the same species
of yeasts. Xylose fermentation by native yeasts can be achieved via isomerization of xylose to its ketose isomer, xylulose.
Isomerization with exogenous xylose isomerase (XI) occurs optimally at a pH of 7–8, whereas subsequent fermentation of xylulose
to ethanol occurs at a pH of 4–5. We present a novel scheme for efficient isomerization of xylose to xylulose at conditions
suitable for the fermentation by using an immobilized enzyme system capable of sustaining two different pH microenvironments
in a single vessel. The proof-of-concept of the two-enzyme pellet is presented, showing conversion of xylose to xylulose even
when the immobilized enzyme pellets are suspended in a bulk solution whose pH is sub-optimal for XI activity. The co-immobilized
enzyme pellets may prove extremely valuable in effectively conducting “simultaneous isomerization and fermentation” (SIF)
of xylose. To help further shift the equilibrium in favor of xylulose formation, sodium tetraborate (borax) was added to the
isomerization solution. Binding of tetrahydroxyborate ions to xylulose effectively reduces the concentration of xylulose and
leads to increased xylose isomerization. The formation of tetrahydroxyborate ions and the enhancement in xylulose production
resulting from the complexation was studied at two different bulk pH values. The addition of 0.05 M borax to the isomerization
solution containing our co-immobilized enzyme pellets resulted in xylose to xylulose conversion as high as 86% under pH conditions
that are suboptimal for XI activity. These initial findings, which can be optimized for industrial conditions, have significant
potential for increasing the yield of ethanol from xylose in an SIF approach. 相似文献
7.
Gao Qiang Zhang Min McMillan James D. Kompala Dhinakar S. 《Applied biochemistry and biotechnology》2002,98(1-9):341-355
Zymomonas mobilis has been metabolically engineered to broaden its substrate utilization range to include d-xylose and l-arabinose. Both genomically integrated and plasmid-bearing Z. mobilis strains that are capable of fermenting the pentose d-xylose have been created by incorporating four genes: two genes encoding xylose utilization metabolic enzymes (xylA/xylB) and two genes encoding pentose phosphate pathway enzymes (talB/tktA). We have characterized the activities of the four newly introduced enzymes for xylose metabolism, along with those of three
native glycolytic enzymes, in two different xylose-fermenting Z. mobilis strains. These strains were grown on glucose-xylose mixtures in computer-controlled fermentors. Samples were collected and
analyzed to determine extracellular metabolite concentrations as well as the activities of several intracellular enzymes in
the xylose and glucose uptake and catabolism pathways. These measurements provide new insights on the possible bottlenecks
in the engineered metabolic pathways and suggest methods for further improving the efficiency of xylose fermentation. 相似文献
8.
Byoung Chul Park Sukhoon Koh Changsoo Chang Se Won Suh Dae-Sil Lee Si Myung Byun 《Applied biochemistry and biotechnology》1997,62(1):15-27
The gene encoding xylose isomerase (xylA) was cloned fromThermus flavus AT62 and the DNA sequence was determined. ThexylA gene encodes the enzyme xylose isomerase (XI orxylA) 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 inE. coli under the control oftac promoter. XI produced inE. 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 Mn2+, Co2+, and Mg2+ are required for the enzyme activity and 100 mM EDTA completely inhibited the enzyme activity. 相似文献
9.
Recent studies have proven ethanol to be the idael liquid fuel for transportation, and renewable ligno cellulosic materials
to be the attractive feed stocks for ethanol fuel production by fermentation. The major fermentable sugars from hydrolysis
of most cellulosic biomass are D-glucose and D-xylose. The naturally occurring Saccharomyces yeasts that are used by industry to produce ethanol from starches and cane sugar cannot metabolize xylose. Our group at Purdue
University succeded in developing genetically engineered Saccharomyces yeasts capable of effectively cofermenting glucose and xylose to ethanol, which was accomplished by cloning three xylose-metabolizing
genes into the yeast. In this study, we demonstrated that our stable recombinant Sacharomyces yeast, 424A (LNH-ST), which contains the cloned xylose-metabolizing genes stably integrated into the yeast chromosome in
high copy numbers, can efficiently ferment glucose and xylose present in hydrolysates from different cellulosic biomass to
ethanol. 相似文献
10.
Glycerol production from xylose fermentation usingRhizopus javanicus (ATCC 22581) has been investigated in shake flasks. The medium composition (xylose concentration, nitrogen sources), aeration
rate, and temperature have been found to affect the accumulation and yield of glycerol. Some of these effects are explained
in terms of the critical parameters, osmotic pressure, and dissolved oxygen levels in the medium. Relatively high glycerol
yields and concentrations have been obtained at high sugar concentration with high level of aeration at room temperature.
The addition of polyethylene glycol or sulfite can improve the yield and accumulation of glycerol. 相似文献
11.
Dahn Kristine M. Davis Brian P. Pittman Paul E. Kenealy William R. Jeffries Thomas W. 《Applied biochemistry and biotechnology》1996,57(1):267-276
Applied Biochemistry and Biotechnology - ThePichia stipitis xylose reductase gene (XYL1) was inserted into an autonomous plasmid thatP. stipitis maintains in multicopy. The plasmid pXOR with... 相似文献
12.
Rosa S. M. A. Felipe M. G. A. Silva S. S. Vitolo M. 《Applied biochemistry and biotechnology》1998,(1):127-135
The effect of pH, time of fermentation, and xylose and glucose concentration on xylitol production, cell growth, xylose reductase
(XR), and xylitol dehydrogenase (XD) activities ofCandida guilliermondii FTI 20037 were determined. For attaining XR and XD activities of 129-2190 U/mg of protein and 24-917 U/mg of protein, respectively,
the cited parameters could vary as follows: initial pH: 3.0-5.0; xylose: 15-60 g/L; glucose: 0-5 g/L; and fermentation time:
12-24 h. Moreover, the high XR and XD activities occurred when the xylitol production by the yeast was less than 19.0 g/L. 相似文献
13.
14.
Gene XYL1 from Candida shehatae and gene XYL2 from Pichia stipitis were amplified by polymerase chain reaction (PCR), and the two genes were both placed under the strong promoter of alcohol dehydrogenase (ADH) of plasmid pAD2 to produce the recombinant expression vector pAD2-P12. Because the amplified XYL1 fragment lacks the stop codon UAA, the polypeptide expressed in yeast cells should be a fusion protein, which is a fusion of xylose reductase and xylitol dehydrogenase. Subsequently, the pAD2-P12 vector was transformed into Saccharomyces cerevisiae YS58 to produce a recombinant S. cerevisiae YS58-12. It was indicated that S. cerevisiae YS58-12 has the ability of metabolizing xylose to produce ethanol by fermentation experiment. The result of cofermentation of glucose and xylose by using this recombinant S. cerevisiae YS58-12 showed a relatively satisfactory result. The highest percentage of xylose consumption rate reached 81.3% and the ethanol yield was equal to 67.14% of the ideal value. 相似文献
15.
Débora Danielle Virgínio da Silva Maria das Graças de Almeida Felipe Ismael Maciel de Mancilha Sílvio Silvério da Silva 《Applied biochemistry and biotechnology》2005,121(1-3):427-437
The effect of glucose on xylose-xylitol metabolism in fermentation medium consisting of sugarcane bagasse hydrolysate was
evaluated by employing an inoculum of Candida guilliermondii grown in synthetic media containing, as carbon sources, glucose (30 g/L), xylose (30 g/L), or a mixture of glucose (2 g/L)
and xylose (30 g/L). The inoculum medium containing glucose promoted a 2.5-fold increase in xylose reductase activity (0.582
IU/mgprot) and a 2-fold increase in xylitol dehydrogenase activity (0.203 IU/mgprot) when compared with an inoculum-grown medium containing only xylose. The improvement in enzyme activities resulted in higher
values of xylitol yield (0.56 g/g) and productivity (0.46 g/[L·h]) after 48 h of fermentation. 相似文献
16.
Alves Lourdes A. Vitolo Michele Felipe Maria das Graças A. de Almeida e Silva João Batista 《Applied biochemistry and biotechnology》2002,98(1-9):403-413
The sugarcane bagasse hydrolysate, which is rich in xylose, can be used as culture medium for Candida guilliermondii in xylitol production. However, the hydrolysate obtained from bagasse by acid hydrolysis at 120°C for 20 min has by-products
(acetic acid and furfural, among others), which are toxic to the yeast over certain concentrations. So, the hydrolysate must
be pretreated before using in fermentation. The pretreatment variables considered were: adsorption time (15,37.5, and 60 min),
type of acid used (H2So4 and H3Po4), hydrolysate concentration (original, twofold, and fourfold. concentrated), and active
charcoal (0.5, 1.75 and 3.0%). The suitability of the pretreatment was followed by measuring the xylose reductase (XR) and
xylitol dehydrogenase (XD) activity of yeast grown in each treated hydrolysate. The response surface methodology (24 full factorial design with a centered face) indicated that the hydrolysate might be concentrated fourfold and the pH adjusted
to 7.0 with CaO, followed by reduction to 5.5 with H3PO4. After that it was treated with active charcoal (3.0%) by 60 min. This pretreated hydrolysate attained the high XR/XD ratio
of 4.5. 相似文献
17.
Sene L. Vitolo M. Felipe M. G. A. Silva S. S. 《Applied biochemistry and biotechnology》2000,84(1-9):371-380
The effects of environmental conditions, namely initial pH (2.5–7.0) and temperature (25 and 35°C), on xylose reductase and
xylitol dehydrogenase levels, as well as on xylitol production, were evaluated. Although the fermentative parameter values
increased with an increase in pH and temperature (the maximum YP/s and Q
p
were 0.75 g/g and 0.95 g/[L·h], respectively, both attained at pH 6.0, 35°C), the highest xylose reductase activities (nearly
900 1U/mg of protein) were observed at an initial pH varying from 4.0 to 6.0. Xylitol dehydrogenase was favored by an increase
in both initial pH and temperature of the medium. The highest xylitol dehydrogenase specific activity was attained at pH 6.5
and 35°C (577 1U/mg of protein). 相似文献
18.
M. Lappalainen I. Pitkänen H. Heikkilä J. Nurmi 《Journal of Thermal Analysis and Calorimetry》2006,84(2):367-376
Two
enantiomeric forms of xylose were identified as α-D-xylopyranose
and α-L-xylopyranose by powder diffraction.
Their melting behaviour was studied with conventional DSC and StepScan DSC
method, the decomposition was studied with TG and evolved gases were analyzed
with combined TG-FTIR technique. The measurements were performed at different
heating rates. The decomposition of xylose samples took place in four steps
and the main evolved gases were H2O, CO2
and furans. The initial temperature of TG measurements and the onset and peak
temperatures of DSC measurements were moved to higher temperatures as heating
rates were increased. The decomposition of L-xylose
started at slightly higher temperatures than that of D-xylose
and L-xylose melted at higher temperatures
than D-xylose. The differences were more
obvious at low heating rates. There were also differences in the melting temperatures
among different samples of the same sugar. The StepScan measurements showed
that the kinetic part of melting was considerable. The melting of xylose was
anomalous because, besides the melting, also partial thermal decomposition
and mutarotation occurred. The melting points are affected by both the method
of determination and the origin and quality of samples. Melting point analysis
with a standardized method appears to be a good measure of the quality of
crystalline xylose. However, the melting point alone cannot be used for the
identification of xylose samples in all cases. 相似文献
19.
Xylitol dehydrogenase (EC1.1.1.9) from xylose-grown cells ofCandida shehatae was purified 215-fold by sequential chromatography on NAD-C8 affinity, Superose-12, and Cibacron blue columns, and a single
band was observed by SDS gel electrophoresis. The purified enzyme had a native molecular weight of 82 kDa and a denatured
molecular weight of 40 kDa following SDS gel electrophoresis, indicating that it was composed of two subunits. Alcohol dehydrogenase
copurified on the NAD-C8 but was substantially removed by Superose-12 and was not detected following Cibacron blue chromatography.
The kinetic properties of the C.shehatae xylitol dehydrogenase differed considerably from those described previously for thePachysolen tannophilus enzyme. The Km of the C.shehatae enzyme for xylitol was 3.8 times smaller, whereas the Km for xylulose was 1.7-fold bigger. These factors could account for the lower xylitol production by C.shehatae. 相似文献
20.
Neurospora crassa XI was found to ferment xylose and glucose simultaneously. Xylose was the appropriate inducer for the production of xylose
reductase that had two isoenzymes designated as EI and EII. Both EI and EII, which were purified by affinity chromatography,
had NADPH-dependent xylose reductase activities. EII also had NADH-dependent activity, and EI is the only xylose reductase
found so far without any NADH-dependent activity. EI and EII had MWs of 30 kDa and 27 kDa, and pIs of 5.6 and 5.2, respectively. The specifities of EI and EII against triose, pentoses, and hexoses were studied. The Kms against xylose for EI and EII were 2.3 mM and 1.1 mM respectively, which were much lower than those of the xylose reductase from yeast. 相似文献