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1.
Niel van Wyk Riaan den Haan Willem H. van Zyl 《Applied microbiology and biotechnology》2010,87(5):1813-1820
The processive endoglucanase Cel9A of the moderately thermophilic actinomycete Thermobifida fusca was functionally produced in Saccharomyces cerevisiae. Recombinant Cel9A displayed activity on both soluble (carboxymethylcellulose) and insoluble (Avicel) cellulose substrates
confirming its processive endoglucanase activity. High-performance anionic exchange chromatography analyses of soluble sugars
released from Avicel revealed a cellobiose/glucose ratio of 2.5 ± 0.1. Growth by the recombinant strain on amorphous cellulose
was possible due to the sufficient amount of glucose cleaved from the cellulose chain. This is the first confirmed report
of S. cerevisiae growing on a cellulosic substrate as sole carbohydrate source while only expressing one recombinant gene. To improve the
cellulolytic capability of S. cerevisiae and to investigate the level of synergy among cellulases produced by a recombinant host, the cel9A gene was co-expressed with four cellulase-coding genes of Trichoderma reesei: two endoglucanases cel5A (egII) and cel7B (egI), and two cellobiohydrolases cel6A (cbhII) and cel7A (cbhI). Synergy, especially between the Cel9A and the two cellobiohydrolases, resulted in a higher cellulolytic capability of the
recombinant host. 相似文献
2.
Enzymatic Properties and Intracellular Localization of the Novel Trichoderma reesei β-Glucosidase BGLII (Cel1A)
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Markku Saloheimo Juha Kuja-Panula Erkko Ylsmki Michael Ward Merja Penttil 《Applied microbiology》2002,68(9):4546-4553
This paper describes the characterization of an intracellular β-glucosidase enzyme BGLII (Cel1a) and its gene (bgl2) from the cellulolytic fungus Trichoderma reesei (Hypocrea jecorina). The expression pattern of bgl2 is similar to that of other cellulase genes known from this fungus, and the gene would appear to be under the control of carbon catabolite repression mediated by the cre1 gene. The BGLII protein was produced in Escherichia coli, and its enzymatic properties were analyzed. It was shown to be a specific β-glucosidase, having no β-galactosidase side activity. It hydrolyzed both cellotriose and cellotetraose. BGLII exhibited transglycosylation activity, producing mainly cellotriose from cellobiose and sophorose and cellobiose from glucose. Antibodies raised against BGLII showed the presence of the enzyme in T. reesei cell lysates but not in the culture supernatant. Activity measurements and Western blot analysis of T. reesei strains expressing bgl2 from a constitutive promoter further confirmed the intracellular localization of this β-glucosidase. 相似文献
3.
Nakazawa H Okada K Kobayashi R Kubota T Onodera T Ochiai N Omata N Ogasawara W Okada H Morikawa Y 《Applied microbiology and biotechnology》2008,81(4):681-689
The genes encoding the catalytic domains (CD) of the three endoglucanases (EG I; Cel7B, EG II; Cel5A, and EG III; Cel12A)
from Trichoderma reesei QM9414 were expressed in Escherichia coli strains Rosetta-gami B (DE3) pLacI or Origami B (DE3) pLacI and were found to produce functional intracellular proteins.
Protein production by the three endoglucanase transformants was evaluated as a function of growth temperature. Maximal productivity
of EG I-CD at 15°C, EG II-CD at 20°C and EG III at 37°C resulted in yields of 6.9, 72, and 50 mg/l, respectively. The endoglucanases
were purified using a simple purification method based on removing E. coli proteins by isoelectric point precipitation. Specific activity toward carboxymethyl cellulose was found to be 65, 49, and
15 U/mg for EG I-CD, EG II-CD, and EG III, respectively. EG II-CD was able to cleave 1,3–1,4-β-d-glucan and soluble cellulose derivatives. EG III was found to be active against cellulose, 1,3–1,4-β-d-glucan and xyloglucan, while EG I-CD was active against cellulose, 1,3–1,4-β-d-glucan, xyloglucan, xylan, and mannan. 相似文献
4.
L. Zhang Z. -P. Guo Z. -Y. Ding Z. -X. Wang G. -Y. Shi 《Applied Biochemistry and Microbiology》2012,48(2):216-221
The gene mel1, encoding α-galactosidase in Schizosaccharomyces pombe, and the gene bgl2, encoding and α-glucosidase in Trichoderma reesei, were isolated and co-expressed in the industrial ethanolproducing strain of Saccharomyces cerevisiae. The resulting strains were able to grow on cellobiose and melibiose through simultaneous production of sufficient extracellular
α-galactosidase and β-glucosidase activity. Under aerobic conditions, the growth rate of the recombinant strain GC1 co-expressing
2 genes could achieve 0.29 OD600 h−1 and a biomass yield up to 7.8 g l−1 dry cell weight on medium containing 10.0 g l−1 cellobiose and 10.0 g l−1 melibiose as sole carbohydrate source. Meanwhile, the new strain of S. cerevisiae CG1 demonstrated the ability to directly produce ethanol from microcrystalline cellulose during simultaneous saccharification
and fermentation process. Approximately 36.5 g l−1 ethanol was produced from 100 g of cellulose supplied with 5 g l−1 melibose within 60 h. The yield (g of ethanol produced/g of carbohydrate consumed) was 0.44 g/g, which corresponds to 88.0%
of the theoretical yield. 相似文献
5.
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6.
7.
The synergism between the endo/exocellulase, Cel9A, and β-glucosidase (βgl) of Thermobifida
fusca was investigated. Wild type βgl or S319C, a βgl mutant with significantly improved cellobiase activity, were added to Cel9A.
Both wild type and mutant βgl enhanced the Cel9A hydrolysis of carboxymethyl cellulose (CMC) and filter paper by 50–100% compared
to Cel9A alone. No enhancement occurred with addition of E388A, an inactive form of βgl. HPLC analysis showed that, with Cel9A
alone, the resulting hydrolysate of glucose and cellobiose contained about half glucose; after addition of equimolar amounts
of either wild type βgl or mutant S319C to Cel9A, the hydrolysate contained more than 85% glucose. βgl thus acted synergistically
with Cel9A by converting cello-oligomers to glucose; this reduced the soluble sugar accumulation during hydrolysis of cellulose. 相似文献
8.
Robert L. Mach Bernhard Seiboth rey Myasnikov Ramon Gonzalez † Josef Strauss Anu M. Harkki Christian P. Kubicek 《Molecular microbiology》1995,16(4):687-697
We have investigated the effect of disruption of the bgl1-(β-glucosidase l-encoding) gene of Trichoderma reesei on the formation of other β-glucosidase activities and on the induction of cellulases. To this end the bgl1 locus was disrupted by insertion of the Aspergillus nidulans amdS (acetamidase-encoding) gene. The bgl1-disrupted strain did not produce the 75kDa extracellular β-glucosidase on cellulose or lactose, but still formed β-glucosidase activity on glucose, cellobiose, xylan or β-1,3-glucan, suggesting that the enzyme(s) exhibiting this β-glucosidase activity is (are) not encoded by bgl1. The cellulose-inducer sophorose induced the bgl1-encoded β-glucosidase, whereas the remaining β-glucosidase activity was induced by methyl-β-D-glucoside. The bgl1-gene product was mainly secreted into the medium, whereas the other β-glucosidase activity was mainly associated with the cells. A bgl1-multicopy strain formed higher amounts of cellulases than the parent strain. Nonsaturating concentrations of sophorose efficiently induced cellobiohydrolase I formation in the bgl1-multicopy strain, but less efficiently in the bgl1-disrupted strain. The multicopy strain and the parent strain were comparably efficient at saturating sophorose concentrations. The β-glucosidase inhibitor nojirimycin strongly inhibited induction in all strains. These data suggest that the bgl1-encoded β-glucosidase is not identical to the plasma-membrane-bound, constitutive, methyl-β-glucoside inducible β-glucosidase, but represents an extracellular cellulose-induced enzyme. Both enzymes contribute to rapid induction of cellulases by modifying the inducer sophorose. 相似文献
9.
The brown-rot basidiomycete Fomitopsis palustris is known to degrade crystalline cellulose (Avicel) and produce three major cellulases, exoglucanases, endoglucanases, and
β-glucosidases. A novel β-glucosidase designated as Cel3A was identified from F. palustris grown at the expense of Avicel. The deduced amino acid sequence of Cel3A showed high homology with those of other fungal
β-glucosidases that belong to glycosyl hydrolase (GH) family 3. The sequence analysis also indicated that Cel3A contains the
N- and C-terminal domains of GH family 3 and Asp-209 was conserved as a catalytic nucleophile. The cloned gene was successfully
expressed in the yeast Pichia pastoris and the recombinant protein exhibited β-glucosidase activity with cellobiose and some degree of thermostability. Considering
the size and sequence of the protein, the β-glucosidase identified in this study is different from the protein purified directly
from F. palustris in the previous study. Our results suggest that the fungus possesses at least two β-glucosidase genes. 相似文献
10.
Summary From cellulose and cellobiose the formation of sophorose, laminaribiose, and gentiobiose was catalyzed byTrichoderma reesei culture filtrate containing exo- and endoglucanase and -glucosidase activity and from cellobiose by a broken cell suspension fromT.reesei with -glucosidase activity. The results indicate that -glucosidase is the component responsible for transglycosylation reaction catalyzed byT.reesei cellulase enzyme complex. 相似文献
11.
A β-glucosidase gene (bglI) from Trichoderma reesei was cloned into the pPIC9 vector and integrated into the genome of Pichia pastoris GS115. Under the control of the methanol-inducible alcohol oxidase (AOX) promoter and using Saccharomyces
cerevisiae secretory signal peptide (α-factor), the recombinant β-glucosidase was expressed and secreted into the culture medium. The
maximum recombinant β-glucosidase activity achieved was 60 U/ml, and β-glucosidase expression reached 0.3 mg/ml. The recombinant
76 kDa β-glucosidase was purified 1.8-fold with 26% yield and a specific activity of 197 U/mg. It was optimally active at
70°C and pH 5.0. 相似文献
12.
Klebsiella oxytoca P2 was developed as a biocatalyst for the simultaneous saccharification and fermentation (SSF) of cellulose by chromosomally
integrating Zymomonas mobilis genes (pdc, adhB) encoding the ethanol pathway. This strain contains the native ability to transport and metabolize cellobiose, eliminating
the need to supplement with β-glucosidase during SSF. To increase the utility of this biocatalyst, we have now chromosomally
integrated the celZ gene encoding the primary endoglucanase from Erwinia chrysanthemi. This gene was expressed at high levels by replacing the native promoter with a surrogate promoter derived from Z. mobilis DNA. With the addition of out genes encoding the type II protein secretion system from E. chrysanthemi, over half of the active endoglucanase (EGZ) was secreted into the extracellular environment. The two most active strains,
SZ2(pCPP2006) and SZ6(pCPP2006), produced approximately 24 000 IU L−1 of CMCase activity, equivalent to 5% of total cellular protein. Recombinant EGZ partially depolymerized acid-swollen cellulose
and allowed the production of small amounts of ethanol by SZ6(pCPP2006) without the addition of fungal cellulase. However,
additional endoglucanase activities will be required to complete the depolymerization of cellulose into small soluble products
which can be efficiently metabolized to ethanol.
Received 14 December 1998/ Accepted in revised form 04 March 1999 相似文献
13.
Summary As a step towards constructing strains of Z. mobilis capable of converting cellulose to ethanol, DNA fragments encoding endoglucanase (from Xanthomonas albilineans) and -glucosidase (from either X.albilineans or Pseudomonas sp.) were linked on the same vector and transferred to Z. mobilis. All clones expressed endoglucanase. -Glucosidase was only produced by clones containing the Xanthomonas gene, and when two copies of this gene were present the -glucosidase activity was higher. 相似文献
14.
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15.
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
16.
Gundllapalli SB Pretorius IS Cordero Otero RR 《Journal of industrial microbiology & biotechnology》2007,34(6):413-421
Enzyme engineering was performed to link the β-glucosidase enzyme (BGL1) from Saccharomycopsis fibuligera to the cellulose-binding domain (CBD2) of Trichoderma reesei cellobiohydrolase (CBHII) to investigate the effect of a fungal CBD on the enzymatic characteristics of this non-cellulolytic
yeast enzyme. Recombinant enzymes were constructed with single and double copies of CBD2 fused at the N-terminus of BGL1 to
mimic the two-domain organization displayed by cellulolytic enzymes in nature. The engineered S. fibuligera β-glucosidases were expressed in Saccharomyces cerevisiae under the control of phosphoglycerate-kinase-1 promoter (PGK1
P
) and terminator (PGK1
T
) and yeast mating pheromone α-factor secretion signal (MFα1
S
). The secreted enzymes were purified and characterized using a range of cellulosic and non-cellulosic substrates to illustrate
the effect of the CBD on their enzymatic activity. The results indicated that the recombinant enzymes of BGL1 displayed a
2–4-fold increase in their hydrolytic activity toward cellulosic substrates like avicel, amorphous cellulose, bacterial microcrystalline
cellulose, and carboxy methyl cellulose in comparison with the native enzyme. The organization of the CBD in these recombinant
enzymes also resulted in enhanced substrate affinity, molecular flexibility and synergistic activity, thereby improving the
ability of the enzymes to act on and hydrolyze cellulosic substrates, as characterized by adsorption, kinetics, thermal stability,
and scanning electron microscopic analyses. 相似文献
17.
Sporotrichum thermophile Growth, Cellulose Degradation, and Cellulase Activity 总被引:3,自引:1,他引:2
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The activity of components of the extracellular cellulase system of the thermophilic fungus Sporotrichum thermophile showed appreciable differences between strains; β-glucosidase (EC 3.2.1.21) was the most variable component. Although its endoglucanase (EC 3.2.1.4) and exoglucanase (EC 3.2.1.91) activities were markedly lower, S. thermophile degraded cellulose faster than Trichoderma reesei. The production of β-glucosidase lagged behind that of endoglucanase and exoglucanase. The latter activities were produced during active growth. When growth was inhibited by cycloheximide treatment, the hydrolysis of cellulose was lower than in the control in spite of the presence of both endoglucanase and exoglucanase activities in the culture medium. Degradation of cellulose was a growth-associated process, with cellulase preparations hydrolyzing cellulose only to a limited extent. The growth rate and cell density of S. thermophile were similar in media containing cellulose or glucose. A distinctive feature of fungal development in media incorporating cellulose or lactose (inducers of cellulase activity) was the rapid differentiation of reproductive units and autolysis of hyphal cells to liberate propagules which were capable of renewing growth immediately. 相似文献
18.
Cesar Vanderlei Nascimento Flávio Henrique Moreira Souza Douglas Chodi Masui Francisco Assis Leone Rosane Marina Peralta João Atílio Jorge Rosa Prazeres Melo Furriel 《Journal of microbiology (Seoul, Korea)》2010,48(1):53-62
The effect of several carbon sources on the production of mycelial-bound β-glucosidase by Humicola grisea var. thermoidea in submerged fermentation was investigated. Maximum production occurred when cellulose was present in the culture medium,
but higher specific activities were achieved with cellobiose or sugarcane bagasse. Xylose or glucose (1%) in the reaction
medium stimulated β-glucosidase activity by about 2-fold in crude extracts from mycelia grown in sugarcane bagasse. The enzyme
was purified by ammonium sulfate precipitation, followed by Sephadex G-200 and DEAE-cellulose chromatography, showing a single
band in PAGE and SDS-PAGE. The β-glucosidase had a carbohydrate content of 43% and showed apparent molecular masses of 57
and 60 kDa, as estimated by SDS-PAGE and gel filtration, respectively. The optimal pH and temperature were 6.0 and 50°C, respectively.
The purified enzyme was thermostable up to 60 min in water at 55°C and showed half-lives of 7 and 14 min when incubated in
the absence or presence of 50 mM glucose, respectively, at 60°C. The enzyme hydrolyzed p-nitrophenyl-β-D-glucopyranoside, p-nitrophenyl-β-Dgalactopyranoside, p-nitrophenyl-β-D-fucopyranoside, p-nitrophenyl-β-D-xylopyranoside, o-nitrophenyl-β-Dgalactopyranoside, lactose, and cellobiose. The best synthetic and natural substrates were p-nitrophenyl-β-Dfucopyranoside and cellobiose, respectively. Purified enzyme activity was stimulated up to 2-fold by glucose
or xylose at concentrations from 25 to 200 mM. The addition of purified or crude β-glucosidase to a reaction medium containing
Trichoderma reesei cellulases increased the saccharification of sugarcane bagasse by about 50%. These findings suggest that H. grisea var. thermoidea β-glucosidase has a potential for biotechnological applications in the bioconversion of lignocellulosic materials. 相似文献
19.
Eugene Jeon Jeong-eun Hyeon Dong Jin Suh Young-Woong Suh Seoung Wook Kim Kwang Ho Song Sung Ok Han 《Molecules and cells》2009,28(4):369-373
Heterologous secretory expression of endoglucanase E (Clostridium thermocellum) and β-glucosidase 1 (Saccharomycopsis fibuligera) was achieved in Saccharomyces cerevisiae fermentation cultures as an α-mating factor signal peptide fusion, based on the native enzyme coding sequence. Ethanol production depends on simultaneous saccharification of cellulose to glucose and fermentation of glucose to ethanol by a recombinant yeast strain as a microbial biocatalyst. Recombinant yeast strain expressing endoglucanase and β-glucosidase was able to produce ethanol from β-glucan, CMC and acid swollen cellulose. This indicates that the resultant yeast strain of this study acts efficiently as a whole cell biocatalyst. 相似文献
20.
Yosuke?Shida Kaori?Yamaguchi Mikiko?Nitta Ayana?Nakamura Machiko?Takahashi Shun-ichi?Kidokoro Kazuki?Mori Kosuke?Tashiro Satoru?Kuhara Tomohiko?Matsuzawa Katsuro?Yaoi Yasumitsu?Sakamoto Nobutada?Tanaka Yasushi?Morikawa Wataru?Ogasawara