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1.
Summary A study was made of a β-fructosidase, which is produced extracellularly and intracellularly bySaccharomyces fragilis. The enzyme catalyzes the hydrolysis of inulin, bacterial levans, sucrose, and the fructose portion of raffinose, by splitting
off terminal fructosyl units. It attacks β-2,1 as well as β-2,6 linkages. The enzyme content of inulin-grown cells is sufficient
to allow fermentation of inulin at the same rate as glucose. The ratio of hydrolysis rates with sucrose and inulin was about
25 for the β-fructosidase ofS. fragilis and about 14,000 for invertase.S. fragilis does not contain significant amounts of invertase and it ferments inulin, sucrose and raffinose with the aid of a related,
but different enzyme, inulinase.
Conditions of growth were established which favor inulinase synthesis. Highest yields were obtained with inulin as the carbon
source, and somewhat lower yields with raffinose. Glucose, fructose and sucrose were poor inducers of inulinase. The pH of
the medium during growth on inulin had to be in the range where inulinase could act, otherwise growth was tardy and poor.
In an inulin containing medium aeration favored enzyme production as a result of stimulation of growth. The inulinase content
of the cells in a unit volume was generally greater than that in the culture medium. The intracellular inulinase could be
solubilized quantitatively by autolysis. The intra-and extracellular inulinases were concentrated and purified to the same
extent. Comparison of the two preparations with respect to substrate specificity, rate of inactivation by heat, pH optima
with sucrose (4.2) and with inulin (5.0), and elution patterns from a column of diethylaminoethyl cellulose, indicated that
the intra-and extracellular enzymes were identical. 相似文献
2.
Ph. Looten D. Blanchet J. P. Vandecasteele 《Applied microbiology and biotechnology》1987,25(5):419-425
Summary The -fructofuranosidase activities of a strain of Clostridium acetobutylicum, selected for its capacity to grow on inulinic substrates, were investigated. When grown on inulin, this strain produced extracellular and intracellular -fructofuranosidases, both of which hydrolysed inulin (inulinase activity) and sucrose (invertase activity). Inulinase activity was higher than invertase activity in the extracellular preparation, the opposite being observed for the cellular preparation. The effects of pH and temperature, substrate specificity and the kinetic constants for inulin and sucrose were studied on both preparations, as well as induction by inulin and repression by glucose and fructose of inulinase and invertase activities. The overall results were consistent with the existence of a least one inulinase, (EC 3.2.1.7), mainly but not entirely released in the extracellular medium, and an invertase (3.2.1.26) localized within the cell.Time course hydrolysis experiments of dalhia inulin and Jerusalem artichoke inulofructans by extracellular inulinase showed that this preparation had a remarkably high specificity for hydrolysis of long chain inulofructans. 相似文献
3.
To date, all of microbial inulinases reported showed optimal activity at pH values ranging from 3.5 to 7.0. A bacterial strain,
Marinimicrobium sp. LS-A18, showing high extracellular inulinolytic activity was isolated from a marine solar saltern of the Yellow Sea in
China. Maximum enzyme activity was obtained at 55°C and pH 9.0, respectively. The inulinase activity was induced by inulin,
but not by the other carbon sources employed. Under the optimal medium and culture condition, the highest inulinase activity,
14.6 U/ml, was obtained after 96 h of incubation at shake flask level. The optimal medium for inulinase production was MHI
medium containing 4% inulin, 1% peptone and 5% NaCl, while the optimal culture condition for inulinase production were pH
7.5, temperature 37°C, agitation speed 210 rpm, medium volume 40 ml in 250 ml shake flask, and incubation time 96 h. A large
amount of monosaccharides was released after inulin hydrolysis by the inulinase from strain LS-A18. This is the first report
on alkaline inulinase production from microorganism. 相似文献
4.
Identification of Catalytically Active Groups in Inulinase from Bacillus polymyxa 722 总被引:2,自引:0,他引:2
Zherebtsov N. A. Abramova I. N. Shelamova S. A. Popova T. N. 《Applied Biochemistry and Microbiology》2003,39(6):544-548
Inulinase from Bacillus polymyxa 722, hydrolyzing a polyfructosan inulin, was studied. The dependence of inulinase activity on pH, measurements of pK value, calculation of the ionization heat, photoinactivation with methylene blue, and inhibition with p-chloromercuribenzoate suggest that the active center of this enzyme contains imidazole and sulfhydryl groups. A possible mechanism underlying the cleavage of -2,1-fructoside bonds in the inulin molecule by inulinase is considered. 相似文献
5.
VariousSaccharomyces cerevisiae strains were transformed with a 2 μ-based multicopy expression plasmid, pYIGP, carryingKluyveromyces marxianus inulinase gene under the control ofGAPDH promoter. Among them two strains, SEY2102 and 2805, showed high levels of cell growth and inulinase expression, and were
selected to study their fermentation properties on inulin. Jerusalem artichoke inulin was more effective for cell growth (10∼11
g-dry wt./L at 48 hr) and inulinase expression (1.0 units/mL with SEY2102/pYIGP and 2.5 units/mL with 2805/pYIGP) than other
inulin sources such as dahlia and chicory. It was also found that maximal ethanol production of 9 g/L was obtained from Jerusalem
artichoke inulin at the early stationary phase (around 30 hr), indicating that recombinantS. cerevisiae cells secreting exoinulinase could be used for the simultaneous saccharification of inulin and ethanol fermentation. 相似文献
6.
This is the first report describing the gene structure and the enzymatic properties of a β-fructosidase of a hyperthermophilic
organism. The bfrA gene of the ancestral bacterium Thermotoga maritima MSB8 codes for a 432-residue, polypeptide of about 50 kDa, with significant sequence similarity to other β-fructosidases.
On the basis of its primary structure, BfrA can be assigned to glycosyl hydrolase family 32. The bfrA gene was expressed in Escherichia coli and the recombinant enzyme was purified and characterised. BfrA was specific for the fructose moiety and the β-anomeric configuration
of the glycosidic linkages of its substrates. The enzyme released fructose from sucrose and raffinose, and the fructose polymer
inulin was hydrolysed quantitatively in an exo-type fashion. BfrA displayed similar catalytic efficiencies for the hydrolysis
of sucrose and inulin with k
cat/K
m values (at 75 °C, pH 5.5) of about 4.1 × 104 M−1s−1 and 3.1 × 104 M−1s−1 respectively. BfrA had an optimum temperature of 90–95 °C (10-min assay) and was extremely insensitive to thermo-inactivation.
During 5 h at temperatures up to 80 °C at pH 7, the enzyme retained at least 85% of its initial activity. Thus, BfrA is the
most thermostable β-fructosidase and also the most thermostable inulinase described to date. In conclusion, the T. maritima enzyme can be classified as an exo-β-d-fructofuranosidase (EC 3.2.1.26) with invertase and inulinase activity. Its catalytic properties along with the extreme thermostability
recommend it for use in biotechnology.
Received: 28 August 1997 / Received revision: 19 January 1998 / Accepted: 24 January 1998 相似文献
7.
The optimization of process parameters for high inulinase production by the marine yeast strain Cryptococcus aureus G7a in solid-state fermentation (SSF) was carried out using central composite design (CCD), one of the response surface methodologies
(RSMs). We found that moisture, inoculation size, the amount ratio of wheat bran to rice husk, temperature and pH had great
influence on inulinase production by strain G7a. Therefore, the CCD was used to evaluate the influence of the five factors
on the inulinase production by strain G7a. Then, five levels of the five factors above were further optimized using the CCD.
Finally, the optimal parameters obtained with the RSM were the initial moisture 61.5%, inoculum 2.75%, the amount ratio of
wheat bran to rice husk 0.42, temperature 29 °C, pH 5.5. Under the optimized conditions, 420.9 U g−1 of dry substrate of inulinase activity was reached in the solid-state fermentation culture of strain G7a within 120 h whereas
the predicted maximum inulinase activity of 436.2 U g−1 of inulinase activity of 436.2 U g−1 of dry weight was derived from the RSM regression. This is the highest inulinase activity produced by the yeast strain reported
so far. A large amount of monosaccharides and oligosaccharides were detected after inulin hydrolysis by the crude inulinase. 相似文献
8.
Raba’atun Adawiyah S. Shuhaimi M. Mohd Yazid A. M. Abdul Manaf A. Rosli N. Sreeramanan S. 《World journal of microbiology & biotechnology》2011,27(9):2173-2185
Selected endophytic fungi have been report to be inulin degraders to produce fructose or other oligosaccharides. In this study,
the Aspergillus sp. producing inulinase were isolated from selected plant species at Serdang area in Malaysia. Fungal isolates were screened
solely based on inulin degrading enzymes production and two isolates named Asf1 and Onf1 were selected as the best inulinase
enzyme producers. Genomic DNA of these two isolates were extracted and amplified by polymerase chain reaction (PCR). A 1,341 bp
DNA fragment containing inulinase gene was successfully amplified from Asf1 fungal isolate and was named as inu2 gene in this study. Based on the morphological characteristics, rDNA and neighbour-joining phylogenetic analysis, Asf1 fungal
isolate could display closely-related to the genus of Aspergillus. The complete sequence designated Asf1 Inu2 gene was successfully obtained via rapid-amplification of cDNA ends-polymerase chain reaction (RACE-PCR). A 2.3 kb DNA fragment
encoding endoinulinase, inu2, from Asf1 fungal isolate includes an open reading frame of 1,552 bp with calculated molecular weight of 55,954.1 Da and
signal peptide sequence of 23 amino acids. The deduced amino acid sequence of the Asf1 inu2 displayed 97, 96, 69 and 22% identities
to that of A. ficuum inu2, A. niger inuB, P. purpurogenum and K. marxianus, respectively. Phylogenetic analysis showed that fungal endo- and exo-inulinases have indepently evolved with the respective
hydrolytic activities toward terminal and internal β-(2 → 1)-fructofuranosidic linkages in inulin. 相似文献
9.
Inulinase production by a marine yeast <Emphasis Type="Italic">Pichia guilliermondii</Emphasis> and inulin hydrolysis by the crude inulinase 总被引:1,自引:0,他引:1
Marine yeast strain 1, isolated from the surface of a marine alga, was found to secrete a large amount of inulinase into the
medium. This marine yeast was identified as a strain of Pichia guilliermondii according to the results of routine yeast identification and molecular methods. The crude inulinase produced by this marine
yeast worked optimally at pH 6.0 and 60°C. The optimal medium for inulinase production was seawater containing 4.0% (w/v)
inulin and 0.5% (w/v) yeast extract, while the optimal cultivation conditions for inulinase production were pH 8.0, 28°C and
170 rpm. Under the optimal conditions, over 60 U ml−1 of inulinase activity was produced within 48 h of fermentation in shake flasks. A large amount of monosaccharides and a trace
amount of oligosaccharides were detected after the hydrolysis, indicating that the crude inulinase had a high exoinulinase
activity. 相似文献
10.
Two new effective microbial producers of inulinases were isolated from Jerusalem artichoke tubers grown in Thailand and identified
as Aspergillus niger TISTR 3570 and Candida
guilliermondii TISTR 5844. The inulinases produced by both these microorganisms were appropriate for hydrolysing inulin to fructose as the
principal product. An initial inulin concentration of ∼100 g l−1 and the enzyme concentration of 0.2 U g−1 of substrate, yielded 37.5 g l−1 of fructose in 20 h at 40°C when A. niger TISTR 3570 inulinase was the biocatalyst. The yield of fructose on inulin was 0.39 g g−1. Under identical conditions, the yeast inulinase afforded 35.3 g l−1 of fructose in 25 h. The fructose yield was 0.35 g g−1 of substrate. The fructose productivities were 1.9 g l−1 h−1 and 1.4 g l−1 h−1 for the mold and yeast enzymes, respectively. After 20 h of reaction, the mold enzyme hydrolysate contained 53% fructose
and more than 41% of initial inulin had been hydrolysed. Using the yeast enzymes, the hydrolysate contained nearly 38% fructose
at 25 h and nearly 36% of initial inulin had been hydrolysed. The A. niger TISTR 3570 inulinases exhibited both endo-inulinase and exo-inulinase activities. In contrast, the yeast inulinases displayed
mainly exo-inulinase activity. The mold and yeast crude inulinases mixed in the activity ratio of 5:1 proved superior to individual
crude inulinases in hydrolysing inulin to fructose. The enzyme mixture provided a better combination of endo- and exo-inulinase
activities than did the crude extracts of either the mold or the yeast individually. 相似文献
11.
Irène Efstathiou Gilles Reysset Nicole Truffaut 《Applied microbiology and biotechnology》1986,25(2):143-149
Summary Several strains ofClostridium acetobutylicum, isolated from sugar beet pulps or Jerusalem artichokes, are able to utilize inulin, a -polyfructosane polymer of fructose with glucose as the terminal residue. Inulin-degrading activity, which was detected in cultures of one such strain, ABKn8, grown in Basol-medium containing inulin, reached a maximum at the end of exponential phase. Most of the enzyme activity was detected in the supernatant. It was stably maintained in 0.1 M acetate buffer pH 5.0, and was optimal at pH 4.6. The enzyme, inulinase was induced by inulin, but not by xylose, fructose or sucrose and was repressed by glucose. Inulinase was active against inulin, sucrose and raffinose, but not melezitose. It had a higher affinity for inulin (K
m
: 1.2×10-2 mM) than all the other known inulinases. 相似文献
12.
Kluyveromyces marxianus NRRL Y-1196 produced the highest inulinase activity (38 U/mg protein) of six yeasts examined after 24 h growth in sauerkraut brine in shaking flasks at 30°C with 0.3% inulin as an enzyme inducer. The enzyme was recovered by acetone fractionation, with a yield of 81%. It had maximum activity at pH 4.4 and 55°C with K
m values for inulin and sucrose of 3.92 mm and 11.9 mm, respectively. The yeast raised the pH from 3.4 to above 7.0, using all the lactic acid in the brine. Growth of K. marxianus in sauerkraut brine with a small amount of inulin may usefully decrease the BOD and concomitantly produce inulinase.The authors are with the Department of Food Science and Technology, Cornell University, Geneva, New York 14456, USA 相似文献
13.
G. Pratap Kumar A. Kunamneni T. Prabhakar P. Ellaiah 《World journal of microbiology & biotechnology》2005,21(8-9):1359-1361
Summary Fifty strains were isolated from different soil samples on synthetic medium containing inulin as a sole carbon source for
the production of extracellular inulinase. Of them, five isolates showed high inulinase activity and one of them was selected
for identification and medium optimization studies. The isolate was identified as Aspergillus niger. Various physical and chemical parameters were optimized for inulinase production. Maximum productivity of inulinase (176 U ml−1) was achieved by employing medium containing 5% (w/v) inulin, galactose as additional carbon source, corn steep liquor and
(NH4)H2PO4 as nitrogen sources, incubation period of 72 h, incubation temperature of 28 °C, pH 6.5, inoculum load at 10% (v/v) level
and medium volume to flask volume ratio of 1:20 (v/v) with indented flasks. 相似文献
14.
Expression of exo‐inulinase gene from Aspergillus niger 12 in E. coli strain Rosetta‐gami B (DE3) and its characterization 下载免费PDF全文
Inulin is a linear carbohydrate polymer of fructose subunits (2‐60) with terminal glucose units, produced as carbon storage in selected plants. It cannot directly be taken up by most microorganisms due to its large size, unless prior hydrolysis through inulinase enzymes occurs. The hydrolyzed inulin can be taken up by microbes and/or recovered and used industrially for the production of high fructose syrup, inulo‐oligosaccharides, biofuel, and nutraceuticals. Cell‐free enzymatic hydrolysis would be desirable for industrial applications, hence the recombinant expression, purification and characterization of an Aspergillus niger derived exo‐inulinase was investigated in this study. The eukaroyototic exo‐inulinase of Aspergillus niger 12 has been expressed, for the first time, in an E. coli strain [Rosetta‐gami B (DE3)]. The molecular weight of recombinant exo‐inulinase was estimated to be ~81 kDa. The values of Km and Vmax of the recombinant exo‐inulinase toward inulin were 5.3 ± 1.1 mM and 402.1 ± 53.1 µmol min?1 mg?1 protein, respectively. Towards sucrose the corresponding values were 12.20 ± 1.6 mM and 902.8 ± 40.2 µmol min?1 mg?1 protein towards sucrose. The S/I ratio was 2.24 ± 0.7, which is in the range of native inulinase. The optimum temperature and pH of the recombinant exo‐inulinase towards inulin was 55°C and 5.0, while they were 50°C and 5.5 towards sucrose. The recombinant exo‐inulinase activity towards inulin was enhanced by Cu2+ and reduced by Fe2+, while its activity towards sucrose was enhanced by Co2+ and reduced by Zn2+. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:629–637, 2016 相似文献
15.
The yeast Kluyveromyces marxianus var. bulgaricus produced large amounts of extracellular inulinase activity when grown on inulin, sucrose, fructose and glucose as carbon
source. This protein has been purified to homogeneity by using successive DEAE-Trisacryl Plus and Superose 6HR 10/30 columns.
The purified enzyme showed a relative molecular weight of 57 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE) and 77 kDa by gel filtration in Superose 6 HR 10/30. Analysis by SDS-PAGE showed a unique polypeptide band with
Coomassie Blue stain and nondenaturing PAGE of the purified enzyme obtained from media with different carbon sources showed
the band, too, when stained for glucose oxidase activity. The optimal hydrolysis temperature for sucrose, raffinose and inulin
was 55°C and the optimal pH for sucrose was 4.75. The apparent K
m values for sucrose, raffinose and inulin are 4.58, 7.41 and 86.9 mg/ml, respectively. Thin layer chromatography showed that
inulinase from K. marxianus var. bulgaricus was capable of hydrolyzing different substrates (sucrose, raffinose and inulin), releasing monosaccharides and oligosaccharides.
The results obtained suggest the hypothesis that enzyme production was constitutive. Journal of Industrial Microbiology & Biotechnology (2000) 25, 63–69.
Received 17 November 1999/ Accepted in revised form 30 May 2000 相似文献
16.
17.
Arun Dev Sharma Prabhjot Kaur Gill Sukhdev Singh Bhullar Prabhjeet Singh 《World journal of microbiology & biotechnology》2005,21(6-7):929-932
Summary An extracellular inulinase (fructanase)-producing strain of Penicillium purpurogenum was isolated from the rhizosphere soil of chicory. Conidia of this selected strain were subjected to simultaneous treatment
with NTG–UV (N-methyl-N′-nitro-N-nitrosoguanidine and ultraviolet radiation) and EtBr–UV (Ethidium bromide–ultraviolet radiation). After mutagenesis, colonies
were screened and among them a few were selected to carry out the inulinase study, which showed a significantly higher inulinase
activity with higher I/S (inulin/sucrose) ratio in all the selected colonies, indicating enhancement of inulinase production
after mutagenic treatments in all the selected mutants. 相似文献
18.
G. C. Passador-Gurgel S. A. Furlan J. K. Melier R. Jonas 《Applied microbiology and biotechnology》1996,45(1-2):158-161
Fourteen strains of yeast from genera Kluyveromyces, Candida, Debaryomyces and Schizosaccharomyces were investigated for inulinase production. In the first stage, the microtitre reader system SLT was used for the determination
of enzyme activity and the evaluation of cellular growth. Different culture conditions were tested and four strains of Kluyveromyces were selected on the basis of enzyme activity and growth capacity at low pH and high temperature: K. marxianus CBS 6397, DSM 70792, ATCC 36907 and IZ 619. These strains were tested in greater volume using pH 4.0, 45°C and inulin (10 g/l) as selection conditions. On the basis of results obtained, the strain K. marxianus ATCC 36907 was selected for inulinase production. Enzyme stability at low pH (4.0) as well as high temperature (50°C) for 10, 30 and 60 min was also evaluated, but no significant difference in enzyme activity was observed. It could be demonstrated
that the microtitre reader system is an excellent method for the screening of microorganisms.
Received: 31 May 1995/Received revision: 20 September 1995/Accepted: 29 September 1995 相似文献
19.
Production, Distribution, and Kinetic Properties of Inulinase in Continuous Cultures of Kluyveromyces marxianus CBS 6556 总被引:5,自引:3,他引:2 下载免费PDF全文
Robert J. Rouwenhorst Leo E. Visser Adriaan A. Van Der Baan W. Alexander Scheffers Johannes P. Van Dijken 《Applied microbiology》1988,54(5):1131-1137
From a screening of several Kluyveromyces strains, the yeast Kluyveromyces marxianus CBS 6556 was selected for a study of the parameters relevant to the commercial production of inulinase (EC 3.2.1.7). This yeast exhibited superior properties with respect to growth at elevated temperatures (40 to 45°C), substrate specificity, and inulinase production. In sucrose-limited chemostat cultures growing on mineral medium, the amount of enzyme decreased from 52 U mg of cell dry weight−1 at D = 0.1 h−1 to 2 U mg of cell dry weight−1 at D = 0.8 h−1. Experiments with nitrogen-limited cultures further confirmed that synthesis of the enzyme is negatively controlled by the residual sugar concentration in the culture. High enzyme activities were observed during growth on nonsugar substrates, indicating that synthesis of the enzyme is a result of a derepression/repression mechanism. A substantial part of the inulinase produced by K. marxianus was associated with the cell wall. The enzyme could be released from the cell wall via a simple chemical treatment of cells. Results are presented on the effect of cultivation conditions on the distribution of the enzyme. Inulinase was active with sucrose, raffinose, stachyose, and inulin as substrates and exhibited an S/I ratio (relative activities with sucrose and inulin) of 15 under standard assay conditions. The enzyme activity decreased with increasing chain length of the substrate. 相似文献
20.
Enzymatic activity and substrate specificity of recombinant tomato β-galactosidases 4 and 5 总被引:1,自引:0,他引:1
The open reading frames of tomato β-galactosidase (TBG) 4 and 5 cDNAs were expressed in yeast, and the enzymes properties
and substrate specificities were investigated. The two enzymes had peak activities between pH 4–4.5 and 37–45°C. TBG4 specifically
hydrolyzed β-(1→4) and 4-linked galactooligosaccharides. TBG5 had a strong preference to hydrolyze β-(1→3) and β-(1→6)-linked
galactooligosaccharides. Exo-β-galactanase activity of the TBG enzymes was measured by determining the release of galactosyl
residues from native tomato cell wall fractions throughout fruit development and ripening. Both TBGs released galactose from
all of the fractions and stages tested. TBG4 activity was highest using chelator soluble pectin and alkali soluble pectin
at the turning stage of ripening. Using aminopyrene trisulfonate labeled substrates, TBG4 was the only enzyme with strong
exo-β-(1→4)-galactanase activity on 5 mer or greater galactans. TBG4 and TBG5 were both able to degrade galactosylated rhamnogalacturonan.
Neither enzyme was able to degrade galactosylated xyloglucan. 相似文献