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1.
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
2.
Thermomucor indicae-seudaticae, a glucoamylase-producing thermophilic mould, was mutagenised using nitrous acid and gamma (60Co) irradiation in a sequential manner to isolate deregulated mutants for enhanced production of glucoamylase. The mutants
were isolated on Emerson YpSs agar containing a non-metabolisable glucose analogue 2-deoxy-d-glucose (2-DG) for selection. The preliminary screening for glucoamylase production using starch–iodine plate assay followed
by quantitative confirmation in submerged fermentation permitted the isolation of several variants showing varying levels
of derepression and glucoamylase secretion. The mutant strain T. indicae-seudaticae CR19 was able to grow in the presence of 0.5 g l−1 2-DG and produced 1.8-fold higher glucoamylase. As with the parent strain, glucoamylase production by T. indicae-seudaticae CR19 in 250-ml Erlenmeyer flasks attained a peak in 48 h of fermentation, showing higher glucoamylase productivity (0.67 U
ml−1 h−1) than the former (0.375 U ml−1 h−1). A large-scale cultivation in 5-l laboratory bioreactor confirmed similar fermentation profiles, though the glucoamylase
production peak was attained within 36 h attributable to the better control of process parameters. Although the mutant grew
slightly slow in the presence of 2-DG and exhibited less sporulation, it showed faster growth on normal Emerson medium with
a higher specific growth rate (0.138 h−1) compared to the parent strain (0.123 h−1). The glucoamylase produced by both strains was optimally active at 60 °C and pH 7.0 and displayed broad substrate specificity
by cleaving α-1,4- and α-1,6-glycosidic linkages in starch, amylopectin, amylose and pullulan. Improved productivity and higher
specific growth rate make T. indicae-seudaticae CR19 a useful strain for glucoamylase production. 相似文献
3.
George Bajszár Jon Croonenberghs Irina L. Karnushina Sun Y. Lee James R. Mattoon 《Applied biochemistry and biotechnology》1994,44(2):187-204
A new allelic variant of theSTA2 gene ofS. diastaticus, designated asSTA2
K, was cloned and characterized (1; accompanying paper). An application-oriented analysis of the promoter region ofSTA2
K is described, with an emphasis on its peculiar structural feature: A 1.1-kb natural deletion located 189 nucleotides upstream
of the translation start codon.
The strength of theSTA2
K promoter was found comparable to that of known strong constitutive yeast promoters(ADH1, GAPDH). Regulated glucoamylase expression was demonstrated by chimeric promoters, which were constructed by placing theSTA2
K promoter under the control of either thePH05 orCYC1 upstream regulatory sequences. On high-copy-number vectors, induction of the UASpho5-STA2K chimeric promoter by phosphate depletion resulted in a destructive overexpression of the secreted glucoamylase, which completely
halted cell growth, and promoted cell decay. In contrast, UAScyc1 was shown to mediate a fine-tuned regulation both by glucose
concentration and, indirectly, by starch, the substrate for the glucoamylase to produce glucose. 相似文献
4.
Takahashi CM Takahashi DF Carvalhal ML Alterthum F 《Applied biochemistry and biotechnology》1999,80(3):193-203
Escherichia coli KO11, in which the genes pdc (pyruvate decarboxylase) and adh (alcohol dehydrogenase) encoding the ethanolpathway from Zymomonas mobili were inserted into the chromosome, has been shown to metabolize all major sugars that are consituents of hemicellulosic hydrolysates
to ethanol, in anaerobic conditions. However, the growth and fermentation performance of this recombinant bacteria may be
affected by acetic acid a potential inhibitor present in hemicellulose hydrolysates in a range of 2.0–15.0 g/L. It was observed
that acetate affected the growth of E. coli KO11, prolonging the lag phase and inducing loss of biomass production and reduction of growth rate. At lower pH levels,
the sensitivity to acetic acid was enhanced owing to the increased concentration of the protonated species. On the other hand,
the recombinant bacteria showed a high tolerance to acetic acid regarding fermentative performance. In Luria broth medium
with glucose or xylose as a single sugar source, it was observed that neither yield nor productivity was affected by the addition
of acetate in a range of 2.0–12.0 g/L, suggesting some uncoupling of the growth vs ethanol production. 相似文献
5.
Rey MW Brown KM Golightly EJ Fuglsang CC Nielsen BR Hendriksen HV Butterworth A Xu F 《Applied biochemistry and biotechnology》2003,111(3):153-166
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 mM and a k
cat
of (5.5±0.5)×103 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
6.
The possibility of producing the biologically active material of the skin, ceramide, was studied using yeasts. The yeast strain
that produced the most ceramide, Saccharomyces cerevisiae (KCCM 50515), was selected, and the optimal conditions for ceramide production were determined using shakeflask culture and
batch fermentation. By measuring the production rate of ceramide at various pH values and temperatures, the optimal conditions
for ceramide production were found to be pH 6.0 and 30°C. When heat shock was applied to the cells for 1 h by increasing the
culture temperature from 30 to 40°C after cell growth, the amount of ceramide produced was increased 5.9-fold. A cell growth
and ceramide production model was developed with Monod kinetics and the Leudecking-Piret model. It showed that ceramide production
was increased when the cells were in the stationary phase. 相似文献
7.
Ji-Hee Yu Dae-Hee Lee Yong-Joo Oh Ki-Cheol Han Yeon-Woo Ryu Jin-Ho Seo 《Applied biochemistry and biotechnology》1996,131(1-3):870-879
Candida magnoliae isolated from honeycomb is an industrially important yeast with high erythritol-producing ability. Erythritol has been used
as functional sugar substitute for various foods. In order to analyze the physiological properties of C. magnoliae, a study on sugar utilization pattern was carried out. The fermentation kinetics of glucose and fructose revealed that C. magnoliae has the discrepancy in glucose and fructose utilization when it produces erythritol. In contrast to most yeasts, C. magnoliae showed preference for fructose to glucose as a carbon source, deserving the designation of fructophilic yeast. Such a peculiar
pattern of sugar utilization in C. magnoliae seems to be related to the evolutionary environment. 相似文献
8.
Methylotrophic yeast Pichia pastoris is convenient for the expression of eukaryotic foreign proteins owing to its potential for posttranslational modifications,
protein folding, and facile culturing. In this work, human interleukin (hIL)-2 was successfully produced as a secreted fusion
form in recombinant P. pastoris. By employing green fluorescent protein (GFP) as a monitoring fusion partner, clear identification of fusion protein expression
and quantification of intracellular hIL-2 were possible even though there was no correlation between culture supernatant fluorescence
and secreted hIL-2 owing to high media interference. Importantly, by the addition of casamino acids in basal medium, we were
able to enhance threefold amount of secreted hIL-2, which was present both as a fusion and as a clipped fragment. 相似文献
9.
10.
Eva Joachimsthal Kevin D. Haggett Peter L. Rogers 《Applied biochemistry and biotechnology》1999,77(1-3):147-157
The fermentation characteristics of two recombinant strains of Zymomonas mobilis, viz. CP4 (pZB5) and ZM4 (pZB5), capable of converting both glucose and xylose to ethanol, have been characterized in batch
and continuous culture studies. The strain ZM4 (pZB5) was found to be capable of converting a mixture of 65 g/L glucose and
65 g/L xylose to 62 g/L ethanol in 48h with a yield of 0.46 g/g. Higher sugar concentrations resulted in incompletexylose
utilization (80h) presumably owing to ethanol inhibition of xylose assimilation or metabolism. The fermentation results with
ZM4 (pZB5) show a significant improvement over results published previously for recombinant yeasts and other bacteria capable
of glucose and xylose utilization. 相似文献
11.
Among physical and nutritional parameters optimized by “one variable at a time” approach, four cultural variables (sucrose,
MgSO4
.7H2O, inoculum size, and incubation period) significantly affected glucoamylase production. These variables were, therefore,
selected for optimization using response surface methodology. The p-values of the coefficients for linear effect of sucrose and inoculum size were less than 0.0001, suggesting them to be the
key experimental variables in glucoamylase production. The enzyme production (34 U/ml) attained under optimized conditions
(sucrose, 2%; MgSO4
.7H2O, 0.13%; yeast extract, 0.1%; inoculum size, 5 × 106 spores per 50 ml production medium; incubation time, 48 h; temperature, 40°C; and pH 7.0) was comparable with the value predicted
by polynomial model (34.2 U/ml). An over all 3.1-fold higher enzyme titers were attained due to response surface optimization.
The experimental model was validated by carrying out glucoamylase production in shake flasks of increasing capacity (0.25–2.0 l)
and 22-l laboratory bioreactors (stirred tank and airlift), where the enzyme production was sustainable. Furthermore, the
fermentation time was reduced from 48 h in shake flasks to 32 h in bioreactors. 相似文献
12.
Julia Maria Naumann Gabriele Küttner Matthias Bureik 《Applied biochemistry and biotechnology》2011,163(1):80-89
There is a rapidly growing demand for fluorescent single-chain Fv (scFv) antibody fragments for many applications. Yeasts
have developed into attractive hosts for recombinant production of these functionalized proteins because they provide several
advantages over prokaryotes and higher eukaryotes as expression systems, e.g., being capable of high-level secretion of heterologous
proteins. In this study, we report Schizosaccharomyces pombe as a new host organism for secretory production of scFv-green fluorescent protein (GFP) fusions and compare it with previously
described yeast expression systems. We cloned a plasmid for the expression and secretion of the anti-p24 (human immunodeficiency
virus 1) CB4-1 scFv fused to GFP. After expression of the scFv–GFP fused to an N-terminal Cpy1 secretion signal sequence,
fluorescence microscopy of living yeast cells indicated that the heterologous protein entered the secretory pathway. Western
blot analysis of cell-free culture supernatants confirmed that the scFv–GFP was efficiently secreted with yields up to 5 mg/L.
In addition, fluorescence measurements of culture supernatants demonstrated that the GFP moiety of the scFv–GFP protein is
fully functional after secretion. Our data suggest that S. pombe has the potential for being used as alternative expression host in recombinant antibody fragment production by ensuring efficient
protein processing and secretion. 相似文献
13.
The endoglucanase I (EGI) from fungus Trichoderma reesei was cloned, expressed, and secreted from Yarrowia lipolytica using the XPR2 promoter. The signal sequence of EGI transferred from T. reesei was efficiently processed in the Y. lipolytica secretory pathway and directed the secretion of active EGI into the culture medium. However, the recombinant EGI produced
from YLCSIn strain was hyperglycosylated and significantly larger than the native enzyme produced by the parent strain. The
expression of EGI using XPR2 preproregion has caused secretion of modified proteins that still retained cellulase activity. This resulted from imprecise
processing of the N-terminus of recombinant protein. While the batch culture produced 5 mg EGI/L from YLCSIn strain, the EGI
yield was increased approx 20-fold when the fed-batch fermentation process strategy in combination with the high-cell density
cultivation technique was employed. These results showed that the Y. lipolytica is a useful host organism for production of a large amount of large size heterologous proteins, especially when used in combination
with high-cell density and fed-batch culture techniques. 相似文献
14.
Production of C-terminal amidated recombinant salmon calcitonin in Streptomyces lividans 总被引:4,自引:0,他引:4
Salmon calcitonin (sCT) is one of the many bioactive peptides that require C-terminal amidation for full biologic activity.
To produce fully bioactive sCT in large scale, we constructed Streptomyces lividans [pMSA], an engineering Streptomyces strain. In the expression vector, glycine-extended sCT, the substrate for amidation, and rat α-amidating enzyme cDNA were
cloned under the control of the strong constitutive promoter from the Streptomyces fradiae aph gene in pIJ680. Both were expressed in a secretory manner by the recombinant strain using the expression and secretion signals
of melC1. Extracellularly expressed recombinant sCT was purified to near homogeneity and characterized by enzyme immunoassay, followed
by direct amino-terminal sequencing. High-performance liquid chromatography, matrixassisted laser desorption ionization-time-of-flight
mass spectrometry, and bioassay in vivo demonstrated purified product to be equivalent to synthetic standard. Thus, the engineered
Streptomyces strain can produce bioactive, C-terminal amidated recombinant sCT in the culture supernatant directly. The ease of the recombinant
process, as well as its potential for scale-up, makes it adaptable to production demands for sCT, and it may be applied to
other bioactive peptides that need C-terminal amidation. 相似文献
15.
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
16.
Ethanol production was studied in simultaneous saccharification and fermentation (SSF) of steam-pretreated spruce at 42°C,
using a thermotolerant yeast. Three yeast strains of Kluyveromyces marxianus were compared in test fermentations. SSF experiments were performed with the best of these on 5% (w/w) of substrate at a
cellulase loading of 37 filter paper units/g of cellulose, and a β-glucosidase loading of 38 IU/gof cellulose. The detoxification
of the substrate and the lack of pH control in the experiments increased the final ethanol concentration. The final ethanol
yield was 15% lower compared to SSF with Saccharomyces cerevisiae at 37°C, owing to the cessation of ethanol fermentation after the first 10 h. 相似文献
17.
Ethanol production from lignocellulose by recombinant yeast with high level expression of heterologous cellulase genes has
been a major anticipation. The native secretion signal sequence of the cellulase endoglucanase I (eg1) gene was replaced by Saccharomyces cerevisiae mating factor α prepro-leader sequence (MFα). The transformants containing native secretion signal (Y
1) and MFα secretion signal (Y
2) were characterized with respect to gene expression and growth on cellulose substrate. Increased enzyme activity and cellulose
utilization were observed. The enzyme activity of Y
2 was 0.084 U/ml, 61.5% higher than Y
1 (0.052 U/ml). The sufficiency parameter (S value) was raised from 0.6 to 0.84. MFα signal peptide was more efficient than the native signal peptide of eg1 gene, suggesting that signal peptide replacement is an efficient way to enhance the cellulase expression level in yeast,
for cellulose-derived ethanol production. 相似文献
18.
An efficient system for the production of (R)-hydroxyalkanoicacids (RHAs) was developed in natural polyhydroxyalkanoate (PHA)-producing bacteria and recombinant Escherichia coli. Acidic alcoholysis of purified PHA and in vivo depolymerization of PHA accumulated in the cells allowed the production of
RHAs. In recombinant E. coli, RHA production was achieved by removing CoA from (R)-3-hydroxyacyl-CoA and by in vivo depolymerization of PHA. When the recombinant E. coli harboring the Ralstonia eutropha PHA biosynthesis genes and the depolymerase gene was cultured in a complex or a chemically defined medium containing glucose,
(R)-3-hydroxybutyric acid (R3HB) was produced as monomers and dimers. R3HB dimers could be efficiently converted to monomers
by mild alkaline heat treatment. A stable recombinant E. coli strain in which the R. eutropha PHA biosynthesis genes were integrated into the chromosome disrupting the pta gene was constructed and examined for the production of R3HB. When the R. eutropha intracellular depolymerase gene was expressed by using a stable plasmid containing the hok/sok locus of plasmid R1, R3HB could be efficiently produced. 相似文献
19.
Pereira Daniela Gerevini Kilikian Beatriz Vahan 《Applied biochemistry and biotechnology》2001,91(1-9):311-316
Growth kinetics and red pigment production of Monascus purpureus CCT 3802 was studied. A reproducible inoculum with extremely dispersed hyphae for bioreactor runs was obtained through a
two-step cultivation in a shaker. First, the spores were cultivated in a complex medium rendering a suspension of vegetative
cells. In the second step these cells were grown in a semisynthetic medium. Two types of media were employed in the bioreactor
runs: a semisynthetic (glucose, salts, and yeast extract), and a synthetic, without yeast extract. The inclusion of yeast
extract, caused an increase in cell yield on glucose (Ys/s) as high as 40%. Also, yeast extract probably yielded a higher proportion of red pigment associated with the cell, relative
to the synthetic medium. On the other hand, cells grown on the synthetic medium were slightly higher producers of red soluble
pigments. 相似文献
20.
pH and temperature play critical roles in multistep enzymatic conversions. In such conversions, the optimal pH for individual
steps differs greatly. In this article, we describe the production of glucoamylase (from Aspergillus oryzae MTCC152 in solid-state fermentation) and glucose isomerase (from Streptomyces griseus NCIM2020 in submerged fermentation), used in industries for producing high-fructose syrup. Optimum pH for glucoamylase was
found to be 5.0. For glucose isomerase, the optimum pH ranged between 7.0 and 8.5, depending on the type of buffer used. Optimum
temperature for glucoamylase and glucose isomerase was 50 and 60°C, respectively. When both the enzymatic conversions were
performed simultaneously at a compromised pH of 6.5, both the enzymes showed lowered activity. We also studied the kinetics
at different pHs, which allows the two-step reaction to take place simultaneously. This was done by separating two steps by
a thin layer of urease. Ammonia generated by the hydrolysis of urea consumed the hydrogen ions, thereby allowing optimal activity
of glucose isomerase at an acidic pH of 5.0. 相似文献