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1.
Synergistic Saccharification, and Direct Fermentation to Ethanol, of Amorphous Cellulose by Use of an Engineered Yeast Strain Codisplaying Three Types of Cellulolytic Enzyme 总被引:12,自引:2,他引:12
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Yasuya Fujita Junji Ito Mitsuyoshi Ueda Hideki Fukuda Akihiko Kondo 《Applied microbiology》2004,70(2):1207-1212
A whole-cell biocatalyst with the ability to induce synergistic and sequential cellulose-degradation reaction was constructed through codisplay of three types of cellulolytic enzyme on the cell surface of the yeast Saccharomyces cerevisiae. When a cell surface display system based on α-agglutinin was used, Trichoderma reesei endoglucanase II and cellobiohydrolase II and Aspergillus aculeatus β-glucosidase 1 were simultaneously codisplayed as individual fusion proteins with the C-terminal-half region of α-agglutinin. Codisplay of the three enzymes on the cell surface was confirmed by observation of immunofluorescence-labeled cells with a fluorescence microscope. A yeast strain codisplaying endoglucanase II and cellobiohydrolase II showed significantly higher hydrolytic activity with amorphous cellulose (phosphoric acid-swollen cellulose) than one displaying only endoglucanase II, and its main product was cellobiose; codisplay of β-glucosidase 1, endoglucanase II, and cellobiohydrolase II enabled the yeast strain to directly produce ethanol from the amorphous cellulose (which a yeast strain codisplaying β-glucosidase 1 and endoglucanase II could not), with a yield of approximately 3 g per liter from 10 g per liter within 40 h. The yield (in grams of ethanol produced per gram of carbohydrate consumed) was 0.45 g/g, which corresponds to 88.5% of the theoretical yield. This indicates that simultaneous and synergistic saccharification and fermentation of amorphous cellulose to ethanol can be efficiently accomplished using a yeast strain codisplaying the three cellulolytic enzymes. 相似文献
2.
3.
A whole-cell biocatalyst with the ability to induce synergistic and sequential cellulose-degradation reaction was constructed through codisplay of three types of cellulolytic enzyme on the cell surface of the yeast Saccharomyces cerevisiae. When a cell surface display system based on alpha-agglutinin was used, Trichoderma reesei endoglucanase II and cellobiohydrolase II and Aspergillus aculeatus beta-glucosidase 1 were simultaneously codisplayed as individual fusion proteins with the C-terminal-half region of alpha-agglutinin. Codisplay of the three enzymes on the cell surface was confirmed by observation of immunofluorescence-labeled cells with a fluorescence microscope. A yeast strain codisplaying endoglucanase II and cellobiohydrolase II showed significantly higher hydrolytic activity with amorphous cellulose (phosphoric acid-swollen cellulose) than one displaying only endoglucanase II, and its main product was cellobiose; codisplay of beta-glucosidase 1, endoglucanase II, and cellobiohydrolase II enabled the yeast strain to directly produce ethanol from the amorphous cellulose (which a yeast strain codisplaying beta-glucosidase 1 and endoglucanase II could not), with a yield of approximately 3 g per liter from 10 g per liter within 40 h. The yield (in grams of ethanol produced per gram of carbohydrate consumed) was 0.45 g/g, which corresponds to 88.5% of the theoretical yield. This indicates that simultaneous and synergistic saccharification and fermentation of amorphous cellulose to ethanol can be efficiently accomplished using a yeast strain codisplaying the three cellulolytic enzymes. 相似文献
4.
Ryosuke Yamada Naho Taniguchi Tsutomu Tanaka Chiaki Ogino Hideki Fukuda Akihiko Kondo 《Microbial cell factories》2010,9(1):32
Background
The filamentous fungus T. reesei effectively degrades cellulose and is known to produce various cellulolytic enzymes such as β-glucosidase, endoglucanase, and cellobiohydrolase. The expression levels of each cellulase are controlled simultaneously, and their ratios and synergetic effects are important for effective cellulose degradation. However, in recombinant Saccharomyces cerevisiae, it is difficult to simultaneously control many different enzymes. To construct engineered yeast with efficient cellulose degradation, we developed a simple method to optimize cellulase expression levels, named cocktail δ-integration. 相似文献5.
Ryosuke Yamada Naho Taniguchi Tsutomu Tanaka Chiaki Ogino Hideki Fukuda Akihiko Kondo 《Biotechnology for biofuels》2011,4(1):8
Background
Hydrolysis of cellulose requires the action of the cellulolytic enzymes endoglucanase, cellobiohydrolase and β-glucosidase. The expression ratios and synergetic effects of these enzymes significantly influence the extent and specific rate of cellulose degradation. In this study, using our previously developed method to optimize cellulase-expression levels in yeast, we constructed a diploid Saccharomyces cerevisiae strain optimized for expression of cellulolytic enzymes, and attempted to improve the cellulose-degradation activity and enable direct ethanol production from rice straw, one of the most abundant sources of lignocellulosic biomass. 相似文献6.
K. Jatinder B.S. Chadha H.S. Saini 《World journal of microbiology & biotechnology》2006,22(2):169-176
Summary
Scytalidium thermophilum type culture Humicola insolens MTCC 4520 isolated from composting soil was optimized for production of cellulolytic and hemicellulolytic enzymes (endoglucanase, Avicel-adsorbable endoglucanase, FPase, β-glucosidase, xylanase and mannanase) by solid-state fermentation (SSF). Initial experiments showed that culture medium containing rice straw and wheat bran (1:3) as carbon source prepared in a synthetic basal medium supported maximal enzyme production at 45 °C. Further optimization of enzyme production was carried out using Box-Behnken design of experiments to study the influence of process variables (inoculum level, (NH4)2SO4 and pH) on enzyme production. The response surface plots revealed the conditions for obtaining optimal enzyme levels. The models computed for R
2
value ranged between 95% and 98.7% indicating they are appropriate and can be useful to predict the effect of inoculum level, (NH4)2SO4 and pH on enzyme production. Under optimized conditions 62.5 ± 0.50, 23.0 ± 0.58, 3.0 ± 0.50, 151.00 ± 8.194, 196 ± 5.033 and 4.9 ± 0.32 (units/g substrate) of endoglucanase (EG), Avicel-adsorbable endoglucanase (AAEG), FPase, β-glucosidase, xylanase and mannanase were produced, respectively. Isoelectric focusing (IEF) of the crude extract showed that S. thermophilum produced six different EG isoforms, of which the EG corresponding to pI values of 8.4, 7.9 and 6.5 showed affinity for Avicel, thereby indicating the presence of a cellulose-binding domain (CBD). Furthermore, seven isoforms of β-glucosidase and ten multiple forms of xylanase distributed over a wide range of pI were also detected. 相似文献
7.
The wood-decomposing fungal species Antrodia macra, A. pulvinascens, Ceriporiopsis aneirina, C. resinascens and Dichomitus albidofuscus were determined for production of laccase (LAC), Mn peroxidase (MnP), lignin peroxidase (LiP), endo-l,4-P-β-glucanase, endo-l,4-β-xylanase,
cellobiohydrolase, 1,4-β-glucosidase and 1,4-β-xylosidase. The results confirmed the brown-rot mode of Antrodia spp. which did not produce the activity of LAC and MnP. The remaining species performed detectable activity of both enzymes
while no strain produced LiP. Significant inhibition of LAC production by high nitrogen was found in all white-rot species
while only MnP of D. albidofuscus was regulated in the same way. The endoglucanase and endoxylanase activities of white-rotting species were inhibited by glucose
in the medium while those of Antrodia spp. were not influenced by glucose concentration. The regulation of enzyme activity and bio-mass production can vary even
within a single fungal genus. 相似文献
8.
The anaerobic fungus Anaeromyces mucronatus KF8 grown in batch culture on M10 medium with rumen fluid and microcrystalline cellulose as carbon source produced a broad
range of enzymes requisite for degradation of plant structural and storage saccharides including cellulase, endoglucanase,
xylanase, α-xylosidase, β-xylosidase, α-glucosidase, β-glucosidase, β-galactosidase, mannosidase, cellobiohydrolase, amylase,
laminarinase, pectinase and pectate lyase. These enzymes were detected in both the intra- and extracellular fractions, but
production into the medium was prevalent with the exception of intracellular β-xylosidase, chitinases, N-acetylglucosaminidase, and lipase. Xylanase activity was predominant among the polysaccharide hydrolases. Extracellular production
of xylanase was stimulated by the presence of cellobiose and oat spelt xylan. Zymogram of xylanases of strain KF8 grown on
different carbon sources revealed several isoforms of xylanases with approximate molar masses ranging from 26 to 130 kDa. 相似文献
9.
K. Jatinder B. S. Chadha H. S. Saini 《World journal of microbiology & biotechnology》2006,22(1):15-22
Summary The medium components for the production of extracellular cellulases by Melanocarpus sp. MTCC 3922 were optimized using solid-state fermentation. Melanocarpus sp. cultured in optimized medium containing 1.5% urea, and 0.12% KH2PO4 along with a trace element solution and surfactant (Tween 20), produced endoglucanase (142.4 U/g of substrate), Avicel-adsorbable
endoglucanase (27.0 U/g of substrate), Avicelase (0.65 U/ g of substrate), FPase (39.9 U/g of substrate) and β-glucosidase (109.0 U/g of substrate) activities. The presence of sulphate ions in traces stimulated endoglucanase yields.
The IEF fractionation of the crude proteins from Melanocarpus sp. showed the expression of 3, 1 and 11 isoforms of endoglucanase, β-glucosidase and xylanase, respectively. 相似文献
10.
M. S. Umikalsom A. B. Ariff Z. H. Shamsuddin C. C. Tong M. A. Hassan M. I. A. Karim 《Applied microbiology and biotechnology》1997,47(5):590-595
Studies on the feasibility of using delignified oil palm empty-fruit-bunch (OPEFB) fibres as a substrate for cellulase production
by Chaetomium globosum strain 414 were carried out in shake-flask cultures containing different types and concentrations of nitrogen source. Peptone,
as nitrogen source, gave maximum production of all the three main components of the cellulase complex (endoglucanase or carboxymethylcellulase,
cellobiohydrolase or filter-paper-hydrolysing enzyme and β-glucosidase), followed by yeast extract, urea, KNO3 and (NH4)2SO4. The maximum specific growth rate (μmax) of C. globosum strain 414 grown in medium containing OPEFB and peptone was 0.038 h−1. In all the fermentations, the fungus was able to produce all the three cellulases with significant amounts of β-glucosidase,
except when using (NH4)2SO4 as nitrogen source, where β-glucosidase was not produced. With 6 g/l peptone and 10 g/l delignified OPEFB fibres, the fungus
produced maximum concentrations of FPase, carboxymethylcellulase and β-glucosidase: 1.4, 30.8 and 9.8 U/ml, giving productivities
of 10, 214 and 24 U l−1h−1, respectively. The cellulase mixture, partially purified by ammonium sulphate precipitation, was able to hydrolyse delignified
OPEFB fibres, converting about 68 % of the cellulosics to reducing sugars after 5 days.
Received: 17 June 1996 / Received revision: 18 November 1996 / Accepted: 23 November 1996 相似文献
11.
Swangkeaw Jutaporn Vichitphan Sukanda Butzke Christian E. Vichitphan Kanit 《World journal of microbiology & biotechnology》2011,27(2):423-430
The properties of intracellular β-glucosidases produced from two yeast isolates identified as Hanseniaspora sp. BC9 and Pichia anomala MDD24 were characterized. β-Glucosidase from Hanseniaspora sp. BC9 was not inhibited by both 20% w/v fructose and 20% w/v sucrose and was slightly inhibited by glucose (> 40% relative
β-glucosidase activity with 10% w/v glucose). β-Glucosidase from P. anomala MDD24 was inhibited by glucose, fructose and sucrose. In the presence of 4–12% v/v ethanol, β-glucosidase from P. anomala MDD24 was stimulated in range 110–130% relative activity whereas β-glucosidase from Hanseniaspora sp. BC9 was substantially inhibited in the presence of ethanol. Finally, juice and wine of the Muscat-type grape variety,
Traminette, were selected to determine sugar-bound volatile aroma release, particularly terpenes, by the activity of those
β-glucosidases. The results showed that high concentration of free aroma compounds were detected from Traminette juice treated
with β-glucosidase from Hanseniaspora sp. BC9 and Traminette wine treated with β-glucosidase from P. anomala MDD24. The preliminary results with proposed an application of these enzymes in commercial wine production lead to more efficient
of β-glucosidase from Hanseniaspora sp. BC9 in releasing desirable aromas during an early stage of alcoholic fermentation while β-glucosidase from P. anomala MDD24 is suitable at the final stage of alcoholic fermentation. 相似文献
12.
Tsuchidate T Tateno T Okai N Tanaka T Ogino C Kondo A 《Applied microbiology and biotechnology》2011,90(3):895-901
We demonstrate glutamate production from β-glucan using endoglucanase (EG)-expressing Corynebacterium glutamicum. The signal sequence torA derived from Escherichia coli K12, which belongs to the Tat pathway, was suitable for secreting EG of Clostridium thermocellum using C. glutamicum as a host. Using the torA signal sequence, endoglucanase from Clostridium cellulovorans 743B was successfully expressed, and the secreted EG produced 123 mg of reducing sugar from 5 g of β-glucan at 30 °C for
72 h, which is the optimal condition for C. glutamicum growth. Subsequently, glutamate fermentation from β-glucan was carried out with the addition of Aspergillus aculeatus β-glucosidase produced by recombinant Aspergillus oryzae. Using EG-secreting C. glutamicum, 178 mg/l of glutamate was produced from 15 g of β-glucan. This is the first report of glutamate fermentation from β-glucan
using endoglucanase-secreting C. glutamicum. 相似文献
13.
Shuhei Yanase Tomohisa Hasunuma Ryosuke Yamada Tsutomu Tanaka Chiaki Ogino Hideki Fukuda Akihiko Kondo 《Applied microbiology and biotechnology》2010,88(1):381-388
To exploit cellulosic materials for fuel ethanol production, a microorganism capable of high temperature and simultaneous
saccharification–fermentation has been required. However, a major drawback is the optimum temperature for the saccharification
and fermentation. Most ethanol-fermenting microbes have an optimum temperature for ethanol fermentation ranging between 28 °C
and 37 °C, while the activity of cellulolytic enzymes is highest at around 50 °C and significantly decreases with a decrease
in temperature. Therefore, in the present study, a thermotolerant yeast, Kluyveromyces marxianus, which has high growth and fermentation at elevated temperatures, was used as a producer of ethanol from cellulose. The strain
was genetically engineered to display Trichoderma reesei endoglucanase and Aspergillus aculeatus β-glucosidase on the cell surface, which successfully converts a cellulosic β-glucan to ethanol directly at 48 °C with a
yield of 4.24 g/l from 10 g/l within 12 h. The yield (in grams of ethanol produced per gram of β-glucan consumed) was 0.47 g/g,
which corresponds to 92.2% of the theoretical yield. This indicates that high-temperature cellulose fermentation to ethanol
can be efficiently accomplished using a recombinant K. marxianus strain displaying thermostable cellulolytic enzymes on the cell surface. 相似文献
14.
Xylanase, β-glucosidase, β-xylosidase, endoglucanase and polygalacturonase production fromCurvularia inaequalis was carried out by means of solid-state and submerged fermentation using different carbon sources. β-Glucosidase. β-xylosidase,
polygalacturonase and xylanase produced by the microorganisms were characterized. β-Glucosidase presented optimum activity
at pH 5.5 whereas xylanase, poly-galacturonase and β-xylosidase activities were optimal at pH 5.0. Maximal activity of β-glucosidase
was determined at 60°C, β-xylosidase at 70°C, and polygalacturonase and xylanase at 55°C. These enzymes were stable at acidic
to neutral pH and at 40–45 °C. The crude enzyme solution was studied for the hydrolysis of agricultural residues. 相似文献
15.
The culture-medium composition was optimised, on a shake-flask scale, for simultaneous production of high activities of endoglucanase
and β-glucosidase by Thermoascus aurantiacus using statistical factorial designs. The optimised medium containing 40.2 g l−1 Solka Floc as the carbon source and 9 g l−1 soymeal as the organic nitrogen source yielded 1130 nkat ml−1 endoglucanase and 116 nkat ml−1β-glucosidase activities after 264 h as shake cultures. In addition, good levels of β-xylanase (3479 nkat ml−1) and low levels of filter-paper cellulase, β-xylosidase, α-l-arabinofuranosidase, β-mannanase, β-mannosidase, α-galactosidase and β-galactosidase were detected. Batch fermentation in
a 5-l laboratory fermentor using the optimised medium allowed the production of 940 nkat ml−1 endoglucanase and 102 nkat ml−1β-glucosidase in 192 h. Endoglucanase and β-glucosidase showed optimum activity at pH 4.5 and pH 5, respectively, and they
displayed optimum activity at 75 °C. Endoglucanase and β-glucosidase showed good stability at pH values 4–8 and 4–7, respectively,
after a prolonged incubation (48 h at 50 °C). Endoglucanase had half-lives of 98 h at 70 °C and 4.1 h at 75 °C, while β-glucosidase
had half-lives of 23.5 h at 70 °C and 1.7 h at 75 °C. Alkali-treated bagasse, steam-treated wheat straw, Solka floc and Sigmacell
50 were 66, 48.5, 33.5 and 14.4% hydrolysed by a crude enzyme complex of T. aurantiacus in 50 h.
Received: 12 November 1999 / Accepted: 14 November 1999 相似文献
16.
We constructed a recombinant industrial Saccharomyces cerevisiae yeast strain OC2-AXYL2-ABGL2-Xyl2 by inserting two copies of the β-glucosidase (BGL) and β-xylosidase (XYL) genes, and a
gene cassette for xylose assimilation in the genome of yeast strain OC-2HUT. Both BGL and XYL were expressed on the yeast
cell surface with high enzyme activities. Using OC2-AXYL2-ABGL2-Xyl2, we performed ethanol fermentation from a mixture of
powdered cellulose (KC-flock) and Birchwood xylan, with the additional supplementation of a 30-g/l Trichoderma reesei cellulase complex mixture. The ethanol yield (gram per gram of added cellulases) of the strain OC2-AXYL2-ABGL2-Xyl2 increased
approximately 2.5-fold compared to that of strain OC2-Xyl2, which lacked β-glucosidase and β-xylosidase activities. Notably,
the concentration of additional T. reesei cellulase was reduced from 30 to 24 g/l without affecting ethanol production. The BGL- and XYL-displaying industrial yeast
of the strain OC2-AXYL2-ABGL2-Xyl2 represents a promising yeast for reducing cellulase consumption of ethanol fermentation
from lignocellulosic biomass by compensating for the inherent weak BGL and XYL activities of T. reesei cellulase complexes. 相似文献
17.
Yongling Qin Haiyan He Nan Li Min Ling Zhiqun Liang 《World journal of microbiology & biotechnology》2010,26(11):1991-1997
A thermostable cellulase-producing fungus, HML 0278, was identified as Fusarium chlamydosporum by morphological characteristics and ITS rDNA sequence analysis. HML 0278 produced extracellular cellulases in solid-state
fermentation using sugar cane bassage as the carbon source. Native-PAGE analysis demonstrated that this fungus strain was
capable of producing the three major components of cellulases and xylanase, with a yield of 281.8 IU/g for CMCase, 182.4 IU/g
for cellobiohydrolase, 135.2 IU/g for β-glucosidase, 95.2 IU/g for filter paper activity, and 4,720 IU/g for xylanase. More
importantly, the CMCase and β-glucosidase produced by HML 0278 showed stable enzymatic activities within pH 4–9 and pH 4–10,
and at temperatures below 70 and 60°C, respectively. 相似文献
18.
The extracellular enzymes of seven fungal strains isolated from koala faeces have been comprehensively characterised for the
first time, revealing potential for biotechnological applications. The fungal isolates were grown in a hydrolase-inducing
liquid medium and the supernatants were analysed using enzyme assays and zymogram gels. Temperature and pH profiles were established
for xylanase (EC 3.2.1.8 endo-1,4-β-xylanase), mannanase (EC 3.2.1.78 mannan endo-1,4-β-mannosidase), endoglucanase (EC 3.2.1.4
cellulase), β-glucosidase (EC 3.2.1.21 β-glucosidase), amylase (EC 3.2.1.1 α-amylase), lipase (EC 3.1.1.3 triacylglycerol
lipase) and protease (EC 3.4 peptidase) activities. Comparisons were made to the high-secreting hypercellulolytic mutant strain
Trichoderma reesei RUT-C30 and the wild-type T. reesei QM6a. The isolates from koala faeces Gelasinospora cratophora A10 and Trichoderma atroviride A2 were good secretors of total protein and heat-tolerant enzymes. Doratomyces stemonitis C8 secreted hemicellulase(s), endoglucanase(s) and β-glucosidase(s) with neutral to alkaline pH optimums. A cold-tolerant
lipase was secreted by Mariannaea camptospora A11. The characteristics displayed by the enzymes are highly sought after for industrial processes such as the manufacture
of paper, detergents and food products. Furthermore, the enzymes were produced at good starting levels that could be increased
further by strain improvement programs. 相似文献
19.
Kenji Okano Qiao Zhang Shogo Yoshida Tsutomu Tanaka Chiaki Ogino Hideki Fukuda Akihiko Kondo 《Applied microbiology and biotechnology》2010,85(3):643-650
In order to achieve direct fermentation of an optically pure d-lactic acid from cellulosic materials, an endoglucanase from a Clostridium thermocellum (CelA)-secreting plasmid was introduced into an l-lactate dehydrogenase gene (ldhL1)-deficient Lactobacillus plantarum (∆ldhL1) bacterial strain. CelA expression and its degradation of β-glucan was confirmed by western blot analysis and enzyme assay,
respectively. Although the CelA-secreting ∆ldhL1 assimilated cellooligosaccharides up to cellohexaose (although not cellotetraose), the main end product was acetic acid,
not lactic acid, due to the conversion of lactic acid to acetic acid. Cultivation under anaerobic conditions partially suppressed
this conversion resulting in the production of 1.27 g/l of D-lactic acid with a high optical purity of 99.5% from a medium containing 2 g/l of cellohexaose. Subsequently, D-lactic acid fermentation from barley β-glucan was carried out with the addition of Aspergillus aculeatus β-glucosidase produced by recombinant Aspergillus oryzae and 1.47 g/l of D-lactic was produced with a high optical purity of 99.7%. This is the first report of direct lactic acid fermentation from
β-glucan and a cellooligosaccharide that is a more highly polymerized sugar than cellotriose. 相似文献
20.
Solid-state fermentation conditions for cellulases production by a newly isolated Penicillium chrysogenum QML-2 were investigated using statistical methods. At first, significant variables for cellulases production including (NH4)2SO4, initial pH and inoculum size were screened by using Plackett-Burman Design. Then the optimal regions of the significant
variables were investigated by using the method of steepest ascent. Finally, central composite design and response surface
analysis were adopted to determine the optimal values of the significant variables and investigate the combined effects of
each variable’s pair on cellulases production. The results showed that the optimal ranges of (NH4)2SO4 concentration, initial pH and inoculum size for three types of cellulases activities were 1.97–2.15 g, pH 4.32–4.41 and 13.3–13.7%
(v/w), respectively. Using the mixture of corn stover powder and wheat bran (CSP/WB, 1/1) as carbon source, the optimization
resulted in 370.15, 101.76 and 321.56 U/g for maximal endoglucanase activity, filter paper activity and β-glucosidase activity,
respectively. Compared with maximum values of cellulases activities (endoglucanase activity 85.21 U/g, filter paper activity
16.62 U/g and β-glucosidase activity 67.68 U/g) obtained under unoptimized conditions, the optimization resulted in 3.34,
5.12 and 3.75 folds improvement for endoglucanase activity, filter paper activity and β-glucosidase activity, respectively.
For chitosan hydrolysis, the crude cellulases had the optimal temperature of 55°C, pH of 4.4 and exhibited Michaelis constant
(K
m) value of 8.34 mg/ml and maximum velocity (V
max) of 2.21 μmol glucosamine/min by 1 ml of the crude cellulases. 相似文献