共查询到20条相似文献,搜索用时 46 毫秒
2.
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.
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
7.
Trichoderma reesei is the preferred organism for producing industrial cellulases. However, cellulases derived from T. reesei have their highest activity at acidic pH. When the pH value increased above 7, the enzyme activities almost disappeared, thereby limiting the application of fungal cellulases under neutral or alkaline conditions. A lot of heterologous alkaline cellulases have been successfully expressed in T. reesei to improve its cellulolytic profile. To our knowledge, there are few reports describing the co-expression of two or more heterologous cellulases in T. reesei. We designed and constructed a promoter collection for gene expression and co-expression in T. reesei. Taking alkaline cellulase as a reporter gene, we assessed our promoters with strengths ranging from 4 to 106 % as compared to the pWEF31 expression vector (Lv D, Wang W, Wei D (2012) Construction of two vectors for gene expression in Trichoderma reesei. Plasmid 67(1):67–71). The promoter collection was used in a proof-of-principle approach to achieve the co-expression of an alkaline endoglucanase and an alkaline cellobiohydrolase. We observed higher activities of both cellulose degradation and biostoning by the co-expression of an endoglucanase and a cellobiohydrolase than the activities obtained by the expression of only endoglucanase or cellobiohydrolase. This study makes the process of engineering expression of multiple genes easier in T. reesei. 相似文献
8.
A laboratory scale study to evaluate the potentiality of filamentous fungi for the production of cellulolytic enzymes using
palm oil mill effluent (POME) as a basal medium was initiated. A total of 25 filamentous fungi in which 16 filamentous fungi
were isolated and purified from oil palm industrial residues and 9 strains from laboratory stock were screened using POME
with 1% total suspended solids. Trichoderma reesei RUT C-30 was identified as a potential strain for cellulolytic enzyme production as compared to other genera of Aspergillus, Penicillum, Rhizopus, Phanerochaete, Trichoderma and basidiomycete groups. The results showed that T. reesei RUT C-30 gave the highest filter paper cellulase and carboxy methyl cellulase activity of 0.917 and 2.51 U/ml respectively
at day 5 of fermentation. Other parameters such as growth formation, pH, filterability and total biosolids were observed to
evaluate the bioconversion process. 相似文献
9.
Summary The cellulolytic fungus Trichoderma reesei QM9414 can be cultivated on spent brewery grains for the production of cellulases. The levels of the cellulase components endoglucanase and exoglucanase synthesized, and the complexes filter paper cellulase and grain-hydrolyzing cellulase synthesized by the organism on spent grains were as high as 287, 182, 187, and 449 units per g available cellulose, respectively. Scaling up the spent grains fermentation system by up to 40-fold (200g dry substrate/tray) demonstrated that cellulase production was comparable to laboratory scale (5g dry substrate/flask) yields. Cultivation of the fungus was feasible on spent grains without pretreatment or further adjustment, although the enzyme yield was somewhat lower than that on dried grains moistened with water or Trichoderma medium. This suggested the possible reutilization of spent grains, with minimal pretreatment, in the cultivation of T.reesei QM9414 for cellulase synthesis and for future incorporation into animal feed. 相似文献
12.
Trichoderma reesei represents an important workhorse for industrial production of cellulases as well as other proteins. The molecular mechanism underlying the regulation of cellulase production as well as other physiological processes in T. reesei is still insufficiently understood. We constructed a P
tcu1
-based promoter substitution cassette that allowed one-step replacement of the endogenous promoter for controlling the target gene expression with copper. We then showed that copper repression of the histone acetyltransferase gene gcn5 phenocopied the gcn5 deletion strain. Using the same strategy, we further characterized the function of another putative Spt-Ada-Gcn5 acetyltransferase (SAGA) complex subunit encoding gene, ada2, in T. reesei. Similar to the repression of gcn5, the addition of copper to the P
tcu1
-ada2 strain not only drastically reduced the vegetative growth and conidiation in T. reesei but also severely compromised the induced cellulase gene expression. The developed strategy will thus be potentially useful to probe the biological function of the large fraction of T. reesei genes with unknown functions including those essential genes in the genome to expand its extraordinary biotechnological potential. 相似文献
13.
Methods for the production and analysis of cellulase and hemicellulase enzyme preparations of various compositions based on the Penicillium verruculosum carbohydrase complex and intended for the effective hydrolysis of different types of cellulose-containing materials (CCMs) have been developed. New recombinant strains of P. verruculosum producing multienzyme carbohydrase complexes with increased activities of cellulases (due to the expression of endo-β-1,4-glucanases I and IV and cellobiohydrolase II from Trichoderma reesei) and hemicellulases (due to the expression of endo-β-1,4-xylanases from P. canescens and T. reesei and endo-β-1,4-mannanase from T. reesei) were constructed. The hydrolytic efficiency of the enzyme preparations (EPs) produced by the new recombinant strains during continuous hydrolysis of three CCM types (milled aspen, depitched pine wood, and milled bagasse) was studied. It was shown that new EPs containing recombinant proteins and retaining their own basic cellulase complex are characterized by the highest hydrolytic ability, exceeding that of the EP based on the original P. verruculosum strain. The recombinant enzyme preparations were highly stable; the optimal pH and temperature values for cellulase, xylanase and mannanase activities were in the range of 3.5–5.5 and 50–80°C, respectively. 相似文献
14.
Agrobacterium-mediated T-DNA transfer has been proven to be an efficient strategy for insertional mutagenesis and elucidation of gene function
in filamentous fungi. The implementation of large-scale T-DNA insertional mutagenesis requires the development of high-efficient
transformation and high-throughput screening procedures. Here, using green fluorescent protein (GFP) as a vital marker, a
highly efficient T-DNA-based mutagenesis and screening system was developed in Trichoderma reesei. The uridine auxotrophic T. reesei M23 as the host was transformed with A. tumefaciens EH105 strain harboring a binary vector pC-OEP, which beared the pyrG gene for primary selection on minimal medium without uridine and the egfp gene for fluorescence-based rapid screening of the mitotically stable transformants. The efficiency of transformation was
up to 10–20 transformants per 10 5 target conidia. Microscopic examination revealed strong GFP expression and fluorescence emission in conidia, growing hyphae
and mycelia. An effective and convenient screening procedure using 96-well plates and multilabel counter for fluorescence
intensity counting was developed to rapidly identify the T-DNA tagged T. reesei mutants. Furthermore, the presence of T-DNA integration at random sites in the genome was confirmed by Southern blot analysis.
This report of the T-DNA-based mutagenesis and rapid screening system using GFP as a vital reporter provides a promising strategy
to speeding up the genome-scale T-DNA insertional mutagenesis and functional genomics analysis of this cellulolytic fungus
T. reesei. 相似文献
15.
The cre1 genes of the filamentous fungi Trichoderma reesei and T. harzianum were isolated and characterized. The deduced CREI proteins are 46% identical to the product of the glucose repressor gene creA of Aspergillus nidulans, encoding a DNA-binding protein with zinc fingers of the C 2H 2 type. The cre1 promoters contain several sequence elements that are identical to the previously identified binding sites for A. nidulans CREA. Steady-state mRNA levels for cre1 of the T. reesei strain QM9414 varied depending on the carbon source, being low on glucose-containing media. These observations suggest that cre1 expression may be autoregulated. The T. reesei strain Rut-C30, a hyperproducer of cellulolytic enzymes, was found to express a truncated form of the cre1 gene ( cre1-1) with an ORF corresponding to a protein of 95 amino acids with only one zinc finger. Unlike QM9414 the strain Rut-C30 produced cellulase mRNAs on glucose-containing medium and transformation of the full-length cre1 gene into this strain caused glucose repression of cbh1 expression, demonstrating that cre1 regulates cellulase expression. 相似文献
16.
microRNAs (miRNAs) are non-coding small RNAs (sRNAs) capable of negatively regulating gene expression. Recently, microRNA-like small RNAs (milRNAs) were discovered in several filamentous fungi but not yet in Trichoderma reesei, an industrial filamentous fungus that can secrete abundant hydrolases. To explore the presence of milRNA in T. reesei and evaluate their expression under induction of cellulose, two T. reesei sRNA libraries of cellulose induction (IN) and non-induction (CON) were generated and sequenced using Solexa sequencing technology. A total of 726 and 631 sRNAs were obtained from the IN and CON samples, respectively. Global expression analysis showed an extensively differential expression of sRNAs in T. reesei under the two conditions. Thirteen predicted milRNAs were identified in T. reesei based on the short hairpin structure analysis. The milRNA profiles obtained in deep sequencing were further validated by RT-qPCR assay. Computational analysis predicted a number of potential targets relating to many processes including regulation of enzyme expression. The presence and differential expression of T. reesei milRNAs imply that milRNA might play a role in T. reesei growth and cellulase induction. This work lays foundation for further functional study of fungal milRNAs and their industrial application. 相似文献
17.
Eighteen strains of xylariaceous fungi have been screened for higher activities of cellulolytic enzymes, Trichoderma reesei QM 9414 was also examined for comparison. Strains of Xylaria anisopleura and X. regalis had higher endocellulase (CMCase) and exocellulase (Avicelase) activities after 2 weeks' incubation. Hypoxylon stygium produced the highest activity of -glucosidase 3 days after inoculation. The optimum pH for these cellulolytic enzymes was approx. 5.0 and the optimum temperatures ranged from 37 to 50°C. A mixed culture process using T. reesei QM 9414 and H. stygium was developed to obtain enhanced synthesis of cellulase. -Glucosidase activities in the mixed culture increased within 48h when H. stygium was introduced after 24h. 相似文献
18.
Four fungal strains— Trichoderma viride, Aspergillus niger, Trichoderma koningii, and Trichoderma reesei—were selected for cellulase production using furfural residues and microcrystalline cellulose (MCC) as the substrates. The
filter paper activity (FPA) of the supernatant from each fungus was measured, and the performance of the enzymes from different
fungal strains was compared. Moreover, the individual activities of the three components of the cellulase system, i.e., β-glucosidase,
endoglucanase, and exoglucanase were evaluated. T. koningii showed the highest activity (27.81 FPU/ml) on furfural residues, while T. viride showed an activity of 21.61 FPU/ml on MCC. The FPA of the crude enzyme supernatant from T. koningii was 30% higher on furfural residues than on MCC. T. koningii and T. viride exhibited high stability and productivity and were chosen for cellulases production. The crystallinity index (CrI) of the
furfural residues varied after digested by the fungi. The results indicated differences in the functioning of the cellulase
system from each fungus. In the case of T. koningii, T. reesei and T. viride, furfural residues supported a better environment for cellulase production than MCC. Moreover, the CrI of the furfural residues
decreased, indicating that this material was largely digested by the fungi. Thus, our results suggest that it may be possible
to use the cellulases produced from these fungi for the simultaneous saccharification and fermentation of lignocellulosic
materials in ethanol production. 相似文献
19.
Trichoderma reesei is a well-known cellulase producer and widely applied in enzyme industry. To increase its ability to efficiently decompose cellulose, the beta-glucosidase activity of its enzyme cocktail needs to be enhanced. In this study, a beta-glucosidase I coding sequence from Penicillium decumbens was ligated with the cellobiohydrolase I ( cbh1) promoter of T. reesei and introduced into the genome of T. reesei strain Rut-C30 by Agrobacterium-mediated transformation. In comparison to that from the parent strain, the beta-glucosidase activity of the enzyme complexes from two selected transformants increased 6- to 8-fold and their filter paper activity (FPAs) was enhanced by 30% on average. The transformant's saccharifying ability towards pretreated cornstalk was also significantly enhanced. To further confirm the effect of heterologous beta-glucosidase on the cellulase activity of T. reesei, the heterologously expressed pBGL1 was purified and added to the enzyme complex produced by T. reesei Rut-C30. Supplementation of the Rut-C30 enzyme complex with pBGL1 brought about 80% increase of glucose yield during the saccharification of pretreated cornstalk. Our results indicated that the heterologous expression of a beta-glucosidase gene in T. reesei might produce balanced cellulase preparation. 相似文献
20.
Due to its elevated cellulolytic activity, the filamentous fungus Trichoderma harzianum ( T. harzianum) has considerable potential in biomass hydrolysis application. Cellulases from Trichoderma reesei have been widely used in studies of cellulose breakdown. However, cellulases from T. harzianum are less-studied enzymes that have not been characterized biophysically and biochemically as yet. Here, we examined the effects
of pH and temperature on the secondary and tertiary structures, compactness, and enzymatic activity of cellobiohydrolase Cel7A
from T. harzianum ( Th Cel7A) using a number of biophysical and biochemical techniques. Our results show that pH and temperature perturbations affect
Th Cel7A stability by two different mechanisms. Variations in pH modify protonation of the enzyme residues, directly affecting
its activity, while leading to structural destabilization only at extreme pH limits. Temperature, on the other hand, has direct
influence on mobility, fold, and compactness of the enzyme, causing unfolding of Th Cel7A just above the optimum temperature limit. Finally, we demonstrated that incubation with cellobiose, the product of
the reaction and a competitive inhibitor, significantly increased the thermal stability of Th Cel7A. Our studies might provide insights into understanding, at a molecular level, the interplay between structure and activity
of Th Cel7A at different pH and temperature conditions. 相似文献
|