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1.
Either the natural biodegradation process or the industrial hydrolytic process requires synergistic interactions between various
cellulases. However, it is sometimes impeded by low hydrolytic rate of existing cellulases and the lack of accessory enzymes.
Herein, the ability of a commercial cellulase (Spezyme CP, from Genencor) to degrade steam explosion-pretreated corn stover
was significantly improved. Firstly, a fungal cellulase producer, Aspergillus fumigatus ECU0811, was isolated from hundreds of soil samples. A 96-deep-well microscale-based platform was developed here to reduce
the labor-intensive screening work and proved to be consistent with macroscale screening work. After optimization of fermentation,
3% corn cob could induce A. fumigatus ECU0811 to yield the highest cellulase production. Based on the high activities of β-glucosidase and xylanase by A. fumigatus ECU0811, 0.91 and 125 U/mg protein, respectively, an enzyme cocktail was composed with a fixed dosage of Spezyme CP (CPCel)
at 14.2 filter paper units (FPU)/g glucan and varied dosages of A. fumigatus cellulase (AFCel). Consequently, the glucan-to-glucose conversion of corn stover was increased from 25.6% in the presence
of CPCel at a dosage of 14.2 FPU/g glucan to 99.5% in the presence of the enzyme cocktail (14.2 FPU CPCel plus 1.21 FPU AFCel
per gram of glucan). On the other side, it reduced the total protein amount of CPCel by as much as tenfold, which extremely
improved the hydrolytic rate of Spezyme CP and reduced its dosage. 相似文献
2.
Plasma-assisted pretreated wheat straw was investigated for cellulase and xylanase production by Trichoderma reesei fermentation. Fermentations were conducted with media containing washed and unwashed plasma-assisted pretreated wheat straw
as carbon source which was sterilized by autoclavation. To account for any effects of autoclavation, a comparison was made
with unsterilized media containing antibiotics. It was found that unsterilized washed plasma-assisted pretreated wheat straw
(which contained antibiotics) was best suited for the production of xylanases (110 IU ml −1) and cellulases (0.5 filter paper units (FPU) ml −1). Addition of Avicel boosted enzyme titers with the highest cellulase titers (1.5 FPU ml −1) found with addition of 50 % w/ w Avicel and with the highest xylanase production (350 IU ml −1) reached in the presence of 10 % w/ w Avicel. Comparison with enzyme titers from other nonrefined feedstocks suggests that plasma pretreated wheat straw is a promising
and suitable substrate for cellulase and hemicellulase production. 相似文献
3.
The modified medium composed of the alkaline-pretreated oil palm empty fruit bunch (APEFB) and tuna condensate powder was used for cellulase and xylanase productions by Streptomyces thermocoprophilus strain TC13W. The APEFB contained 74.46% (w/w) cellulose, 15.72% (w/w) hemicellulose, and 6.40% (w/w) lignin. The tuna condensate powder contained 55.49% (w/w) protein and 11.05% (w/w) salt. In the modified medium with only 6.75 g/l tuna condensate powder, 10 g/l APEFB, and 0.5 g/l Tween 80, S. thermocoprophilus strain TC13W produced cellulase 4.9 U/ml and xylanase 9.0 U/ml. The enzyme productions in the modified medium were lower than cellulase (6.0 U/ml) and xylanase (12.0 U/ml) productions in the complex medium (CaCl2 0.1, MgSO4·7H2O 0.1, KH2PO4 0.5, K2HPO4 1.0, NaCl 0.2, yeast extract 5.0, NH4NO3 1.0, Tween 80 0.5). When tuna condensate powder in the modified medium was reduced to 5.0 g/l and Tween 80 was increased to 1.5 g/l, S. thermocoprophilus strain TC13W produced cellulase and xylanase activities of 9.1 and 12.1 U/ml, respectively. This study shows that the cost of enzyme production could be reduced by using pretreated EFB and tuna condensate as a carbon and a nitrogen source, respectively. 相似文献
4.
A highly thermostable alkaline xylanase was purified to homogeneity from culture supernatant of Bacillus sp. JB 99 using DEAE-Sepharose and Sephadex G-100 gel filtration with 25.7-fold increase in activity and 43.5% recovery.
The molecular weight of the purified xylanase was found to be 20 kDA by SDS-PAGE and zymogram analysis. The enzyme was optimally
active at 70 °C, pH 8.0 and stable over pH range of 6.0–10.0.The relative activity at 9.0 and 10.0 were 90% and 85% of that
of pH 8.0, respectively. The enzyme showed high thermal stability at 60 °C with 95% of its activity after 5 h. The K
m and V
max of enzyme for oat spelt xylan were 4.8 mg/ml and 218.6 μM min −1 mg −1, respectively. Analysis of N-terminal amino acid sequence revealed that the xylanase belongs to glycosyl hydrolase family
11 from thermoalkalophilic Bacillus sp. with basic p I. Substrate specificity showed a high activity on xylan-containing substrate and cellulase-free nature. The hydrolyzed product
pattern of oat spelt xylan on thin-layer chromatography suggested xylanase as an endoxylanase. Due to these properties, xylanase
from Bacillus sp. JB 99 was found to be highly compatible for paper and pulp industry. 相似文献
5.
An extracellular thermostable xylanase from a newly isolated thermophilic Actinomadura sp. strain Cpt20 was purified and characterized. Based on matrix-assisted laser desorption–ionization time-of-flight mass
spectrometry analysis, the purified enzyme is a monomer with a molecular mass of 20,110.13 Da. The 19 residue N-terminal sequence
of the enzyme showed 84% homology with those of actinomycete endoxylanases. The optimum pH and temperature values for xylanase
activity were pH 10 and 80 °C, respectively. This xylanase was stable within a pH range of 5–10 and up to a temperature of
90 °C. It showed high thermostability at 60 °C for 5 days and half-life times at 90 °C and 100 °C were 2 and 1 h, respectively.
The xylanase was specific for xylans, showing higher specific activity on soluble oat-spelt xylan followed by beechwood xylan.
This enzyme obeyed the Michaelis–Menten kinetics, with the K
m and k
cat values being 1.55 mg soluble oat-spelt xylan/ml and 388 min −1, respectively. While the xylanase from Actinomadura sp. Cpt20 was activated by Mn 2+, Ca 2+, and Cu 2+, it was, strongly inhibited by Hg 2+, Zn 2+, and Ba 2+. These properties make this enzyme a potential candidate for future use in biotechnological applications particularly in
the pulp and paper industry. 相似文献
6.
In this study, with combined carboxymethyl cellulose agar plate, xylan agar plate and filter paper hydrolysis assay, a novel cellulase and xylanase-producing strain identified as Bacillus sp. was isolated. Using lactose as the only carbon source, a complete and balanced lignocellulolytic enzyme system containing at least endoglucanase (9.6 U/ml), exoglucanase (0.8 U/ml), Fpase (1.4 U/ml), xylanase (3.8 U/ml) and β-glucosidase (1.2 U/ml) was produced. Interestingly, a zymogram of the crude culture supernatant displayed a multifunctional lignocellulolytic enzyme system including at least four bonds with both endoglucanase activity and xylanase activity at 21.2, 23.8, 28.9 and 31.2 kDa, respectively, indicating that these enzymes might be bifunctional. More gratifyingly, according to the binding affinity analysis and scanning electron microscopy, the crude enzyme complex produced by strain BS-5 was capable of hydrolyzing not only pure insoluble polysaccharides, but also agricultural residues such as corn cob. At 5% substrate concentration and 20 FPU/g enzyme loading, the reducing sugar was 350.8 mg/g of alkali-pretreated corn cob after 72 h enzymatic hydrolysis. These results suggested that this strain could be a good candidate for the development of a more cost-effective and efficient lignocellulolytic enzyme cocktail for the saccharification of lignocellulosic biomass. 相似文献
7.
A psychrotrophic fungus identified as Trichoderma sp. SC9 produced 36.7 U/ml of xylanase when grown on a medium containing corncob xylan at 20 °C for 6 days. The xylanase
was purified 37-fold with a recovery yield of 8.2%. The purified xylanase appeared as a single protein band on SDS-PAGE with
a molecular mass of approximately 20.5 kDa. The enzyme had an optimal pH of 6.0, and was stable over pH 3.5–9.0. The optimal
temperature of the xylanase was 42.5 °C and it was stable up to 35 °C at pH 6.0 for 30 min. The xylanase was thermolabile
with a half-life of 23.9 min at 45 °C. The apparent K
m
values of the xylanase for birchwood, beechwood, and oat-spelt xylans were found to be 3, 2.1, and 16 mg/ml respectively.
The xylanase hydrolyzed beechwood xylan and birchwood xylan to yield mainly xylobiose as end products. The enzyme-hydrolysed
xylotriose, xylotetraose, and xylopentose to produce xylobiose, but it hardly hydrolysed xylobiose. A xylanase gene ( xynA) with an open reading frame of 669 nucleotide base pairs (bp), encoding 222 amino acids, from the strain was cloned and sequenced.
The deduced amino acid sequence of XynA showed 85% homology with Xyn2 from a mesophilic strain of Trichoderma viride. 相似文献
8.
The efficient saccharification of lignocellulosic materials requires the cooperative actions of different cellulase enzyme
activities: exoglucanase, endoglucanase, β-glucosidase, and xylanase. Previous studies with the fungi strains Aureobasidium sp. CHTE-18, Penicillium sp. CH-TE-001, and Aspergillus terreus CH-TE-013, selected mainly because of their different cellulolytic and xylanolytic activities, have demonstrated the capacity
of culture filtrates of cross-synergistic action in the saccharification of native sugarcane bagasse pith. In an attempt to
improve the enzymatic hydrolysis of different cellulosic materials, we investigated a coculture fermentation with two of these
strains to enhance the production of cellulases and xylanases. The 48-h batch experimental results showed that the mixed culture
of Penicillium sp. CH-TE-001 and A. terreus CH-TE-013 produced culture filtrates with high protein content, cellulase (mainly β-glucosidase), and xylanase activities
compared with the individual culture of each strain. The same culture conditions were used in a simple medium with mineral
salts, corn syrup liquor, and sugarcane bagasse pith as the sole carbon source with moderate shaking at 29°C. Finally, we
compared the effect of the cell-free culture filtrates obtained from the mixed and single fermentations on the saccharification
of different kinds of cellulosic materials. 相似文献
9.
Dialdehyde starch (DAS) was used as a novel coupling agent to prepare chitosan carrier to immobilize the xylanase from Aspergillus niger A-25. Compared with glutaraldehyde-cross-linked chitosan (CS-GA) and pure chitosan beads, the DAS-cross-linked chitosan (CS-DAS)
beads exhibited the highest xylanase activity recovery. The DAS adding amount and cross-linking time in CS-DAS preparation
process were optimized with respect to activity recovery to the values of 1.0 g (6.7% w/ v concentration) and 16 h, respectively. The optimum temperature of both the CS-DAS- and CS-GA-immobilized xylanase was observed
to be 5 °C higher than that of free enzyme (50 °C). The CS-DAS-immobilized xylanase had the highest thermal and storage stability
as compared to the CS-GA-immobilized and free xylanase. The apparent K
m and V
max values of the CS-DAS-immobilized xylanase were estimated to be 1.29 mg/ml and 300.7 μmol/min/mg protein, respectively. The
CS-DAS-immobilized xylanase could produce from birchwood xylan high-quality xylo-oligosaccharides, mainly composed of xylotriose,
as free xylanase did. The proposed CS-DAS carrier was more advantageous over the CS-GA or pure chitosan carrier for xylanase
immobilization application. 相似文献
10.
A xylanase-encoding gene, xyn11F63, was isolated from Penicillium sp. F63 CGMCC1669 using degenerated polymerase chain reaction (PCR) and thermal asymmetric interlaced (TAIL)-PCR techniques.
The full-length chromosomal gene consists of 724 bp, including a 73-bp intron, and encodes a 217 amino acid polypeptide. The
deduced amino acid sequence of xyn11F63 shows the highest identity of 70% to the xylanase from Penicillium sp. strain 40, which belongs to glycosyl hydrolases family 11. The gene was overexpressed in Pichia pastoris, and its activity in the culture medium reached 516 U ml −1. After purification to electrophoretic homogeneity, the enzyme showed maximal activity at pH 4.5 and 40°C, was stable at
acidic buffers of pH 4.5–9.0, and was resistant to proteases (proteinase K, trypsin, subtilisin A, and α-chymotrypsin). The
specific activity, K
m, and V
max for oat spelt xylan substrate was 7,988 U mg −1, 22.2 mg ml −1, and 15,105.7 μmol min −1 mg −1, respectively. These properties make XYN11F63 a potential economical candidate for use in feed and food industrial applications. 相似文献
11.
Cellulase-free xylanase has potential for its application in the selective removal of hemicellulose from kraft pulp to give
good strength to paper. In this study, a gene ( xyn) encoding cellulase activity–free xylanase enzyme (Xyn) was isolated from Paenibacillus polymyxa PPL-3. The xyn gene encoded a protein of 221 amino acids, and the purified Xyn was about 22.5 kDa measured by sodium dodecyl sulfate–polyacrylamide
gel electrophoresis. Moreover, the cellulase activity–free xylanase enzyme (Xyn) was displayed on the cell surface of Saccharomyces cerevisiae EBY100 using Aga2p as an anchor protein. Cell surface display of xylanase enzyme (Xyn) on S. cerevisiae EBY100 was confirmed by immunofluorescence microscopy. Optimum cell surface display of xylanase enzyme (Xyn) was observed
at pH 7 and 40 °C. Therefore, cell surface–displayed xylanase enzyme (Xyn) can be used in the paper industry. 相似文献
12.
A xylanase gene, xynBS27, was cloned from Streptomyces sp. S27 and consisted of 693 bp encoding a 230-residue protein, including a putative 41-residue signal peptide. Belonging
to the glycoside hydrolase family 11, XynBS27 exhibits the maximum identity (75.9%) to the xylanase from Streptomyces sp. zxy19. Recombinant XynBS27 was overexpressed in Pichia pastoris, and the xylanase activity was 7624.0 U/ml after high-cell-density fermentation in 3.7-L fermenter. The purified recombinant
XynBS27 had a high specific activity of 3272.0 U/mg. The optimum temperature and pH for XynBS27 activity was 65 °C and pH 6.5,
respectively. XynBS27 showed good pH stability and retained more than 80% of the maximum activity after incubation in buffers
with pH ranging between 4.0 and 12.0 at 37 °C for 1 h. The main hydrolysis product of xylan by XynBS27 was xylobiose (>75%),
which was good for human health derived from its ability to modulate the intestinal function. The attractive biochemical characteristics
of XynBS27 suggest that it may be a good candidate in a variety of industrial applications. 相似文献
13.
A cellulase-producing mesophilic fungal strain, named G5, was isolated from the acidic wastewater and mud of a tin mine and
identified as Phialophora sp. based on the internal transcribed spacer sequence. The volumetric activities and specific activities of cellulase induced
by different carbon sources (Avicel, corn cob, wheat bran and corn stover) were compared. The cellulase complex of Phialophora sp. G5 exhibited the optimal activities at 60–65 °C and pH 4.0–5.0, and had good long-term thermostability at 50 °C. Compared
with the commercial cellulase (Accellerase 1500, Genencor), the enzyme under study showed 60% and 80% of the capacity to hydrolyze
pure cellulose and natural cellulose, respectively. This is the first study to report that a cellulytic enzymes complex from
Phialophora genus, and the superior properties of this enzyme complex make strain G5 a potential microbial source to produce cellulase
for industrial applications, and the production ability could be improved by mutagenesis. 相似文献
14.
The aim of this work was to have cellulase activity and hemicellulase activity screenings of endophyte Acremonium species ( Acremonium zeae EA0802 and Acremonium sp. EA0810). Both fungi were cultivated in submerged culture (SC) containing l-arabinose, d-xylose, oat spelt xylan, sugarcane bagasse, or corn straw as carbon source. In solid-state fermentation, it was tested as
carbon source sugarcane bagasse or corn straw. The highest FPase, endoglucanase, and xylanase activities were produced by
Acremonium sp. EA0810 cultivated in SC containing sugarcane bagasse as a carbon source. The highest β-glucosidase activity was produced
by Acremonium sp. EA0810 cultivated in SC using d-xylose as carbon source. A. zeae EA0802 has highest α-arabinofuranosidase and α-galactosidase activities in SC using xylan as a carbon source. FPase, endoglucanase,
β-glucosidase, and xylanase from Acremonium sp. EA0810 has optimum pH and temperatures of 6.0, 55 °C; 5.0, 70 °C; 4.5, 60 °C; and 6.5, 50 °C, respectively. α-Arabinofuranosidase
and α-galactosidase from A. zeae EA0802 has optimum pH and temperatures of 5.0, 60 °C and 4.5, 45 °C, respectively. It was analyzed the application of Acremonium sp. EA0810 to hydrolyze sugarcane bagasse, and it was achieved 63% of conversion into reducing sugar and 42% of conversion
into glucose. 相似文献
15.
Water hyacinth ( Eichhornia crassipes), an aquatic weed common to the subtropic/tropical regions, was utilized as an inexpensive lignocellulosic substrate for
production of cellulase by Trichoderma reesei. The effects of process parameters like substrate pretreatment, substrate concentration, initial medium pH, mode of inoculation,
and incubation temperature on cellulase production were investigated. Under optimal conditions, a maximal cellulase activity
of 0.22 ± 0.04 IU/ml (approximately 73.3 IU/g cellulose) was recorded at the end of 15-day incubation period. Specific activity
of the enzyme was 6.25 IU/mg protein. Hydrolysis of 1% substrate (water hyacinth) using crude enzyme dosage of 1.2 IU/g water
hyacinth showed 28.7% saccharification in 1 h. The observations in present study indicate that saccharification of cellulose
from water hyacinth was significantly higher by laboratory-produced cellulase than the commercial blend. 相似文献
16.
Sugar cane bagasse consists of hemicellulose (24%) and cellulose (38%), and bioconversion of both fractions to ethanol should
be considered for a viable process. We have evaluated the hydrolysis of pretreated bagasse with combinations of cellulase,
β-glucosidase, and hemicellulase. Ground bagasse was pretreated either by the AFEX process (2NH 3: 1 biomass, 100 °C, 30 min) or with NH 4OH (0.5 g NH 4OH of a 28% [ v/ v] per gram dry biomass; 160 °C, 60 min), and composition analysis showed that the glucan and xylan fractions remained largely
intact. The enzyme activities of four commercial xylanase preparations and supernatants of four laboratory-grown fungi were
determined and evaluated for their ability to boost xylan hydrolysis when added to cellulase and β-glucosidase (10 filter
paper units [FPU]: 20 cellobiase units [CBU]/g glucan). At 1% glucan loading, the commercial enzyme preparations (added at
10% or 50% levels of total protein in the enzyme preparations) boosted xylan and glucan hydrolysis in both pretreated bagasse
samples. Xylanase addition at 10% protein level also improved hydrolysis of xylan and glucan fractions up to 10% glucan loading
(28% solids loading). Significant xylanase activity in enzyme cocktails appears to be required for improving hydrolysis of
both glucan and xylan fractions of ammonia pretreated sugar cane bagasse. 相似文献
17.
To enhance the conversion of the cellulose and hemicellulose, the corncob pretreated by aqueous ammonia soaking was hydrolyzed
by enzyme complexes. The saturation limit for cellulase (Spezyme CP) was determined as 15 mg protein/g glucan (50 filter paper
unit (FPU)/g glucan). The accessory enzymes (β-glucosidase, xylanase, and pectinase) were supplemented to hydrolyze cellobiose
(cellulase-inhibiting product), hemicellulose, and pectin (the component covering the fiber surfaces), respectively. It was
found that β-glucosidase (Novozyme 188) loading of 1.45 mg protein/g glucan [30 cellobiase units (CBU)/g glucan] was enough
to eliminate the cellobiose inhibitor, and 2.9 mg protein/g glucan (60 CBU/g glucan) was the saturation limit. The supplementation
of xylanase and pectinase can increase the conversion of cellulose and hemicellulose significantly. The yields of glucose
and xylose enhanced with the increasing enzyme loading, but the increasing trend became low at high loading. Compared with
xylanase, pectinase was more effective to promote the hydrolysis of cellulose and hemicellulose. The supplementation of pectinase
with 0.12 mg protein/g glucan could increase the yields of glucose and xylose by 7.5% and 29.3%, respectively. 相似文献
18.
A cellulase production process was developed by growing the fungi Trichoderma reesei and Aspergillus phoenicis on dairy manure. T. reesei produced a high total cellulase titer (1.7 filter paper units [FPU]/mL, filter paper activity) in medium containing 10 g/L
of manure (dry basis [w/w]), 2 g/L KH 2PO 4, 2 mL/L of Tween-80, and 2mg/L of CoCl 2. However, β-glucosidase activity in the T. reesei-enzyme system was very low. T. reesei was then cocultured with A. phoenicis to enhance the β-glucosidase level. The mixed culture resulted in a relatively high level of total cellulase (1.54 FPU/mL)
and β-glucosidase (0.64 IU/mL). The ratio of β-glucosidase activity to filter paper activity was 0.41, suitable for hydrolyzing
manure cellulose. The crude enzyme broth from the mixed culture was used for hydrolyzing the manure cellulose, and the produced
glucose was significantly ( p<0.01) higher than levels obtained by using the commercial enzyme or the enzyme broth of the pure culture T. reesei. 相似文献
19.
The aim of this work was to evaluate the biochemical features of the white-rot fungi Pycnoporus sanguineus cellulolytic complex and its utilization to sugarcane bagasse hydrolysis. When cultivated under submerged fermentation using
corn cobs as carbon source, P. sanguineus produced high FPase, endoglucanase, β-glucosidase, xylanase, mannanase, α-galactosidase, α-arabinofuranosidase, and polygalacturonase
activities. Cellulase activities were characterized in relation to pH and temperature. β-Glucosidase and FPase activities
were higher at 55 °C, pH 4.5, and endoglucanase activity was higher at 60 °C, in a pH range of 3.5–4.0. All cellulase activities
were highly stable at 40 and 50 °C through 48 h of pre-incubation. Crude enzymatic extract from P. sanguineus was applied in a saccharification experiment using acid-treated and alkali-treated sugarcane bagasse as substrate, and the
hydrolysis yields were compared to that obtained by a commercial cellulase preparation. Reducing sugar yields of 60.4% and
64.0% were reached when alkali-treated bagasse was hydrolyzed by P. sanguineus extract and commercial cellulase, respectively. Considering the glucose production, it was observed that P. sanguineus extract and commercial cellulase ensured yields of 22.6% and 36.5%, respectively. The saccharification of acid-treated bagasse
was lower than that of alkali-treated bagasse regardless of the cellulolytic extract. The present work showed that P. sanguineus has a great potential as an enzyme producer for biomass saccharification. 相似文献
20.
Alcaligenes sp. ECU0401 has been isolated from soil samples with high nitrilase activity against glycolonitrile using the enrichment
culture technique. The preferred carbon/nitrogen sources and metal ions were sodium acetate, a composite of peptone and yeast
extract, and Cu 2+, respectively. Glycolic acid was obtained in a yield of 96.5% after 14 h of biotransformation from a total of 200 mM glycolonitrile
in the mode of sequential addition during the cultivation of Alcaligenes sp. ECU0401 in a 5-L jar fermenter. Fifty micromolars of glycolonitrile could be hydrolyzed in a yield of 94.1% by resting
cells after 36 h. The microbial nitrilase system could hydrolyze various nitriles with high activity, and no amidase activity
and glycolic acid were observed in hydrolyzing glycolamide. It significantly exhibited high enantioselectivity in the hydrolysis
of mandelonitrile and 2-chloromandelonitrile (>99.9% e.e.
p
). Efficient biocatalyst recycling was achieved as a result of immobilization in glutaraldehyde/polyethylenimine cross-linked
carrageenan with immobilized cells exhibiting a biocatalyst productivity of 1,042.2 g glycolic acid per gram dry cell weight
after 29 batch recycles. 相似文献
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