Location of cellulolytic enzyme activity in the marine fungus Trichocladium achrasporum

1985 ◽  
Vol 31 (2) ◽  
pp. 145-148 ◽  
Author(s):  
Malcolm J. MacDonald ◽  
Donna L. Hartley ◽  
Marilyn K. Speedie
Keyword(s):  
Cellulase Activity ◽  
Marine Fungus ◽  
Particulate Fraction ◽  
Cellulolytic Enzymes ◽  
Crystalline Cellulose ◽  
Cellulolytic Enzyme ◽  
Active Growth ◽  
Endoglucanase Activity ◽  
Produce Cell ◽  
Soluble Cell

Cellulase activity in Trichocladium achrasporum was demonstrated by its ability to produce cell-associated and extracellular cellulolytic enzymes when grown on a crystalline cellulose substrate. In addition, azure dye was solubilized from dyed crystalline cellulose, appearing in the growth medium during the phase of cell lysis. Exoglucanase activity was highest in the culture filtrate, with slight activity in the cell fractions, while endoglucanase was associated only with the mycelium. No desorbable exoglucanase nor endoglucanase activity could be released by sonication of residual cellulose particles removed from actively growing cultures. β-Glucosidase activity was located only in the cell-associated fractions during active growth. All enzymes had optimal activity at 50 °C; in the particulate fraction β-glucosidase exhibited a second optimum at 30 °C. In the filtrate, soluble intracellular and particulate fractions optimal exoglucanase activity occurred at pH 6.4, 7.0, and 5.8, respectively. Endoglucanase activity was optimal at pH 5.8 in the soluble cell fraction, and at pH 5.4 in the particulate fraction.

Cell-associated and extracellular cellulolytic enzyme activity in the marine fungus Dendryphiella arenaria

1982 ◽  
Vol 60 (6) ◽  
pp. 838-844 ◽  
Author(s):  
Malcolm J. MacDonald ◽  
Marilyn K. Speedie
Keyword(s):  
Extracellular Enzymes ◽  
Marine Fungus ◽  
Crystalline Cellulose ◽  
Cellulolytic Enzyme ◽  
Cellulolytic Activity ◽  
Endoglucanase Activity ◽  
Produce Cell ◽  
Intracellular Fraction ◽  
Glucosidase Activity ◽  
Cell Fractions

Cellulolytic activity in Dendryphiella arenaria was demonstrated by its ability to release dye from dyed crystalline cellulose and to produce cell-associated and extracellular enzymes when grown on a similar substrate. Exoglucanase was found only in an insoluble form, associated with the hyphae and cellulose particles following sonic disruption of the cells. Endoglucanase was predominantly cell free but with a small amount associated with the cell fractions. High β-glucosidase activity was seen in the filtrate and cell fractions, although in the soluble intracellular fraction its activity disappeared with time. Optimal pH activities for exoglucanase and endoglucanase were demonstrated to be 7.0 and 5.8, respectively. Temperature optima of the enzyme components also differed; maximum exoglucanase activity in the cell-associated fractions and endoglucanase activity in the cell-associated and filtrate fractions occurred at 65 and 60 °C, respectively. Maximum β-glucosidase activity in the filtrate and soluble intracellular fractions occurred at 55 °C and that in the particulate fraction occurred at 60 °C.

scholarly journals Development of a powerful synthetic hybrid promoter to improve the cellulase system of Trichoderma reesei for efficient saccharification of corncob residues

2022 ◽  
Vol 21 (1) ◽  
Author(s):  
Yifan Wang ◽  
Ruiyan Liu ◽  
Hong Liu ◽  
Xihai Li ◽  
Linjing Shen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Trichoderma Reesei ◽  
Translation Initiation ◽  
Cellulase Activity ◽  
Initiation Codon ◽  
Cellulolytic Enzymes ◽  
Cellulolytic Enzyme ◽  
Activity Increase ◽  
Hybrid Promoter ◽  
Translation Initiation Codon

Abstract Background The filamentous fungus Trichoderma reesei is a widely used workhorse for cellulase production in industry due to its prominent secretion capacity of extracellular cellulolytic enzymes. However, some key components are not always sufficient in this cellulase cocktail, making the conversion of cellulose-based biomass costly on the industrial scale. Development of strong and efficient promoters would enable cellulase cocktail to be optimized for bioconversion of biomass. Results In this study, a synthetic hybrid promoter was constructed and applied to optimize the cellulolytic system of T. reesei for efficient saccharification towards corncob residues. Firstly, a series of 5’ truncated promoters in different lengths were established based on the strong constitutive promoter Pcdna1. The strongest promoter amongst them was Pcdna1-3 (− 640 to − 1 bp upstream of the translation initiation codon ATG), exhibiting a 1.4-fold higher activity than that of the native cdna1 promoter. Meanwhile, the activation region (− 821 to − 622 bp upstream of the translation initiation codon ATG and devoid of the Cre1-binding sites) of the strong inducible promoter Pcbh1 was cloned and identified to be an amplifier in initiating gene expression. Finally, this activation region was fused to the strongest promoter Pcdna1-3, generating the novel synthetic hybrid promoter Pcc. This engineered promoter Pcc drove strong gene expression by displaying 1.6- and 1.8-fold stronger fluorescence intensity than Pcbh1 and Pcdna1 under the inducible condition using egfp as the reporter gene, respectively. Furthermore, Pcc was applied to overexpress the Aspergillus niger β-glucosidase BGLA coding gene bglA and the native endoglucanase EG2 coding gene eg2, achieving 43.5-fold BGL activity and 1.2-fold EG activity increase, respectively. Ultimately, to overcome the defects of the native cellulase system in T. reesei, the bglA and eg2 were co-overexpressed under the control of Pcc promoter. The bglA-eg2 double expression strain QPEB70 exhibited a 178% increase in total cellulase activity, whose cellulase system displayed 2.3- and 2.4-fold higher saccharification efficiency towards acid-pretreated and delignified corncob residues than the parental strain, respectively. Conclusions The synthetic hybrid promoter Pcc was generated and employed to improve the cellulase system of T. reesei by expressing specific components. Therefore, construction of synthetic hybrid promoters would allow particular cellulase genes to be expressed at desired levels, which is a viable strategy to optimize the cellulolytic enzyme system for efficient biomass bioconversion.

Cellulolytic enzyme system of Acetivibrio cellulolyticus

1981 ◽  
Vol 27 (3) ◽  
pp. 288-294 ◽  
Author(s):  
J. N. Saddler ◽  
A. W. Khan
Keyword(s):  
Molecular Weight ◽  
Polyacrylamide Gel Electrophoresis ◽  
Cellulolytic Enzymes ◽  
Cellulolytic Enzyme ◽  
Endoglucanase Activity ◽  
Molecular Weights ◽  
Reversible Dissociation ◽  
Electrophoretic Mobilities ◽  
Novel Method ◽  
Culture Supernate

Polyacrylamide gel electrophoresis of the cellulolytic system from culture supernates of Acetivibrio cellulolyticus showed the presence of four major enzymes: a β-glucosidase, an exoglucanase, and two endoglucanases. The relative proportions of these enzymes in the culture supernate were affected by the nature of the cellulosic substrate and by the length of the incubation period. The molecular weights of the cellulolytic enzymes were β-glucosidase, 81 000; exoglucanase, 38 000; endoglucanase C2, 33 000; and endoglucanase C3, 10 400, as estimated by their electrophoretic mobilities relative to proteins of known molecular weight. Treatment of the high molecular weight endoglucanase with SDS–mercaptoethanol led to reversible dissociation of the enzyme into polypeptide subunits similar to the low molecular weight endoglucanase. Endoglucanase activity could be assayed for directly using a novel method of incorporating carboxymethyl cellulose in the polyacrylamide gels. The molecular weights and functions of these enzymes are compared with those detected in culture filtrates of various fungi.

scholarly journals Isolation, Cellulase Activity Test and Molecular Identification of Selected Cellulolytic Bacteria Indigenous Rice Bran

2018 ◽  
Vol 18 (3) ◽  
pp. 514 ◽  
Author(s):  
Akyunul Jannah ◽  
Aulanni`am Aulanni`am ◽  
Tri Ardyati ◽  
Suharjono Suharjono
Keyword(s):  
Rice Bran ◽  
Waste Product ◽  
Cellulase Activity ◽  
Raw Material ◽  
Cellulolytic Enzymes ◽  
Cellulolytic Activity ◽  
Cellulolytic Bacteria ◽  
Endoglucanase Activity ◽  
Activity Test ◽  
Rice Milling

Rice bran is the waste product of rice milling which is abundant in Indonesia, it can be used as a raw material for the manufacture of bioethanol by fermentation. Before being fermented, rice bran must be hydrolyzed into glucose by biomass degrading. This study was aimed to isolate indigenous cellulolytic bacteria from rice bran as producer of cellulolytic enzymes and resulted in 22 bacterial isolates that demonstrated cellulolytic activity being identified. Among them, BE 8 and BE 14 isolates showed the highest endoglucanase activity at 2.16 and 1.31 U/mL respectively. Identification of the 16S rDNA showed that BE 8 belongs to Bacillus subtilis and BE 14 in Bacillus cereus.

scholarly journals Development of a Powerful Synthetic Hybrid Promoter to Improve the Cellulase System of Trichoderma Reesei for Efficient Saccharification of Corncob Residues

2021 ◽  
Author(s):  
Yifan Wang ◽  
Ruiyan Liu ◽  
Hong Liu ◽  
Xihai Li ◽  
Linjing Shen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Trichoderma Reesei ◽  
Cellulase Activity ◽  
Inducible Promoter ◽  
Cellulase Production ◽  
Cellulolytic Enzymes ◽  
Cellulolytic Enzyme ◽  
Activity Increase ◽  
Hybrid Promoter ◽  
Saccharification Efficiency

Abstract Background: The filamentous fungus Trichoderma reesei is a widely used workhorse for cellulase production in industry due to its prominent secretion capacity of extracellular cellulolytic enzymes. However, some key components are not always sufficient in this cellulase cocktail, making the conversion of cellulose-based biomass costly on the industrial scale. Development of strong and efficient promoters would enable cellulase cocktail to be optimized for bioconversion of biomass.Results: In this study, a synthetic hybrid promoter was constructed and applied to optimize the cellulolytic system of T. reesei for efficient saccharification towards corncob residues. Firstly, a promoter library was established by sequence truncation based on the strong constitutive promoter Pcdna1. The strongest promoter amongst them was Pcdna1-3, exhibiting a 1.4-fold higher activity than that of the native cdna1 promoter. Meanwhile, the activation region (-821 to -622 bp upstream of the translation initiation codon ATG and devoid of the Cre1-binding sites) of the strong inducible promoter Pcbh1 was cloned and identified to be an amplifier in initiating gene expression. Finally, this activation region was fused to the strongest promoter Pcdna1-3, generating the novel synthetic hybrid promoter Pcc. This engineered promoter Pcc drove strong gene expression by displaying 1.6- and 1.8-fold stronger fluorescence intensity than Pcbh1 and Pcdna1 under the inducible condition using egfp as the reporter gene, respectively. Furthermore, Pcc was applied to overexpress the Aspergillus niger β-glucosidase BGLA coding gene bglA and the native endoglucanase EG2 coding gene eg2, achieving a 43.5-fold BGL activity and 1.2-fold EG activity increase, respectively. Ultimately, to overcome the defects of the native cellulase system in T. reesei, the bglA and eg2 were co-overexpressed under the control of Pcc promoter. The bglA-eg2 double expression strain QPEB70 exhibited a 178% increase in total cellulase activity, whose cellulase system displayed 2.3- and 2.4-fold higher saccharification efficiency towards acid-pretreated and delignified corncob residues than the parental strain, respectively.Conclusions: The synthetic hybrid promoter Pcc was generated and employed to improve the cellulase system of T. reesei by expressing specific components. Therefore, construction of synthetic hybrid promoters would allow particular cellulase genes to be expressed at desired levels, which is a viable strategy to optimize the cellulolytic enzyme system for efficient biomass bioconversion.

scholarly journals Defining the Frontiers of Synergism between Cellulolytic Enzymes for Improved Hydrolysis of Lignocellulosic Feedstocks

Catalysts ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 1343
Author(s):  
Mpho S. Mafa ◽  
Brett I. Pletschke ◽  
Samkelo Malgas
Keyword(s):  
Value Added ◽  
Product Yield ◽  
Cellulose Hydrolysis ◽  
Cellulolytic Enzymes ◽  
Cellulolytic Enzyme ◽  
Key Factors ◽  
Cellulose Conversion ◽  
Lignocellulosic Feedstocks ◽  
Polysaccharide Monooxygenases ◽  
Hydrolysis Of

Lignocellulose has economic potential as a bio-resource for the production of value-added products (VAPs) and biofuels. The commercialization of biofuels and VAPs requires efficient enzyme cocktail activities that can lower their costs. However, the basis of the synergism between enzymes that compose cellulolytic enzyme cocktails for depolymerizing lignocellulose is not understood. This review aims to address the degree of synergism (DS) thresholds between the cellulolytic enzymes and how this can be used in the formulation of effective cellulolytic enzyme cocktails. DS is a powerful tool that distinguishes between enzymes’ synergism and anti-synergism during the hydrolysis of biomass. It has been established that cellulases, or cellulases and lytic polysaccharide monooxygenases (LPMOs), always synergize during cellulose hydrolysis. However, recent evidence suggests that this is not always the case, as synergism depends on the specific mechanism of action of each enzyme in the combination. Additionally, expansins, nonenzymatic proteins responsible for loosening cell wall fibers, seem to also synergize with cellulases during biomass depolymerization. This review highlighted the following four key factors linked to DS: (1) a DS threshold at which the enzymes synergize and produce a higher product yield than their theoretical sum, (2) a DS threshold at which the enzymes display synergism, but not a higher product yield, (3) a DS threshold at which enzymes do not synergize, and (4) a DS threshold that displays anti-synergy. This review deconvolutes the DS concept for cellulolytic enzymes, to postulate an experimental design approach for achieving higher synergism and cellulose conversion yields.

scholarly journals Supercharged Cellulases Show Reduced Non-Productive Binding, But Enhanced Activity, on Pretreated Lignocellulosic Biomass

2021 ◽  
Author(s):  
Bhargava Nemmaru ◽  
Jenna Douglass ◽  
John M Yarbrough ◽  
Antonio De Chellis ◽  
Srivatsan Shankar ◽  
...  
Keyword(s):  
Cell Wall ◽  
Corn Stover ◽  
Lignocellulosic Biomass ◽  
Hydrolytic Activity ◽  
Fine Tuning ◽  
Cellulolytic Enzymes ◽  
Crystalline Cellulose ◽  
Cell Wall Components ◽  
Carbohydrate Binding Modules ◽  
Wall Components

Non-productive adsorption of cellulolytic enzymes to various plant cell wall components, such as lignin and cellulose, necessitates high enzyme loadings to achieve efficient conversion of pretreated lignocellulosic biomass to fermentable sugars. Carbohydrate-binding modules (CBMs), appended to various catalytic domains (CDs), promote lignocellulose deconstruction by increasing targeted substrate-bound CD concentration but often at the cost of increased non-productive enzyme binding. Here, we demonstrate how a computational protein design strategy can be applied to a model endocellulase enzyme (Cel5A) from Thermobifida fusca to allow fine-tuning its CBM surface charge, which led to increased hydrolytic activity towards pretreated lignocellulosic biomass (e.g., corn stover) by up to ~330% versus the wild-type Cel5A control. We established that the mechanistic basis for this improvement arises from reduced non-productive binding of supercharged Cel5A mutants to cell wall components such as crystalline cellulose (up to 1.7-fold) and lignin (up to 1.8-fold). Interestingly, supercharged Cel5A mutants that showed improved activity on various forms of pretreated corn stover showed increased reversible binding to lignin (up to 2.2-fold) while showing no change in overall thermal stability remarkably. In general, negative supercharging led to increase hydrolytic activity towards both pretreated lignocellulosic biomass and crystalline cellulose whereas positive supercharging led to a reduction of hydrolytic activity. Overall, selective supercharging of protein surfaces was shown to be an effective strategy for improving hydrolytic performance of cellulolytic enzymes for saccharification of real-world pretreated lignocellulosic biomass substrates. Future work should address the implications of supercharging cellulases from various families on inter-enzyme interactions and synergism.

scholarly journals Isolation of Cellulose Degrading Fungi from Decaying Banana Pseudostem and Strelitzia alba

2019 ◽  
Vol 2019 ◽  
pp. 1-10 ◽  
Author(s):  
L. M. Legodi ◽  
D. La Grange ◽  
E. L. Jansen van Rensburg ◽  
I. Ncube
Keyword(s):  
Aspergillus Fumigatus ◽  
Hydrolytic Enzymes ◽  
Cellulase Activity ◽  
Screening Process ◽  
Endoglucanase Activity ◽  
Trichoderma Longibrachiatum ◽  
Fungal Isolates ◽  
Initial Ph ◽  
Synthetic Media ◽  
Initial Selection

Cellulases are a group of hydrolytic enzymes that break down cellulose to glucose units. These enzymes are used in the food, beverage, textile, pulp, and paper and the biofuel industries. The aim of this study was to isolate fungi from natural compost and produce cellulases in submerged fermentation (SmF). Initial selection was based on the ability of the fungi to grow on agar containing Avicel followed by cellulase activity determination in the form of endoglucanase and total cellulase activity. Ten fungal isolates obtained from the screening process showed good endoglucanase activity on carboxymethyl cellulose-Congo Red agar plates. Six of the fungal isolates were selected based on high total cellulase activity and identified as belonging to the genera Trichoderma and Aspergillus. In SmF of synthetic media with an initial pH of 6.5 at 30°C Trichoderma longibrachiatum LMLSAUL 14-1 produced total cellulase activity of 8 FPU/mL and endoglucanase activity of 23 U/mL whilst Trichoderma harzianum LMLBP07 13-5 produced 6 FPU/mL and endoglucanase activity of 16 U/mL. The produced levels of both cellulases and endoglucanase by Trichoderma species were higher than the levels for the Aspergillus fumigatus strains. Aspergillus fumigatus LMLPS 13-4 produced higher β-glucosidase 38 U/mL activity than Trichoderma species.

scholarly journals Gene Integration and Expression and Extracellular Secretion of Erwinia chrysanthemi Endoglucanase CelY (celY) and CelZ (celZ) in EthanologenicKlebsiella oxytoca P2

2001 ◽  
Vol 67 (1) ◽  
pp. 6-14 ◽  
Author(s):  
Shengde Zhou ◽  
F. C. Davis ◽  
L. O. Ingram
Keyword(s):  
Fermentation Broth ◽  
Klebsiella Oxytoca ◽  
Cellulase Activity ◽  
Erwinia Chrysanthemi ◽  
Crystalline Cellulose ◽  
Fungal Enzymes ◽  
Extracellular Milieu ◽  
Extracellular Secretion ◽  
Genes Encoding ◽  
Fuels And Chemicals

ABSTRACT The development of methods to reduce costs associated with the solubilization of cellulose is essential for the utilization of lignocellulose as a renewable feedstock for fuels and chemicals. One promising approach is the genetic engineering of ethanol-producing microorganisms that also produce cellulase enzymes during fermentation. By starting with an ethanologenic derivative (strain P2) ofKlebsiella oxytoca M5A1 with the native ability to metabolize cellobiose, the need for supplemental β-glucosidase was previously eliminated. In the current study, this approach has been extended by adding genes encoding endoglucanase activities. GenescelY and celZ from Erwinia chrysanthemi have been functionally integrated into the chromosome of P2 using surrogate promoters from Zymomonas mobilis for expression. Both were secreted into the extracellular milieu, producing more than 20,000 endoglucanase units (carboxymethyl cellulase activity) per liter of fermentation broth. During the fermentation of crystalline cellulose with low levels of commercial cellulases of fungal origin, these new strains produced up to 22% more ethanol than unmodified P2. Most of the beneficial contribution was attributed to CelY rather than to CelZ. These results suggest that fungal enzymes with substrate profiles resembling CelY (preference for long-chain polymers and lack of activity on soluble cello-oligosaccharides of two to five glucosyl residues) may be limiting in commercial cellulase preparations.

Fractionation and identification of cellulases and other extracellular enzymes produced by Sporotrichum (Chrysosporium) thermophile during growth on cellulose or cellobiose

1983 ◽  
Vol 29 (9) ◽  
pp. 1071-1080 ◽  
Author(s):  
G. Canevascini ◽  
D. Fracheboud ◽  
H. Meier
Keyword(s):  
Extracellular Enzymes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Reducing Power ◽  
Extracellular Enzyme ◽  
Cellulolytic Enzymes ◽  
Crystalline Cellulose ◽  
Concomitant Increase ◽  
Amorphous Cellulose ◽  
Enzymatic Action ◽  
Isoelectric Points

The extracellular enzyme proteins secreted by Sporotrichum (Chrysosporium) thermophile, ATCC 42 464, upon growth on cellulose or cellobiose, were separated by polyacrylamide gel electrophoresis and electrofocusing into different fractions which were then analyzed with respect to their enzymatic character to identify the cellulolytic enzymes. A positive reaction against carboxymethylcellulose azure (CMC azure) was taken as evidence for an endo-acting cellulase, whereas the criterion for the presence of an exo-cellulase was a negative reaction with CMC azure and a concomitant increase in reducing power upon action of any kind of cellulose. With this procedure, four main cellulolytic enzymes were detected: three endo-cellulases, named endo-cellulases I, II, and III (with corresponding isoelectric points 5.1, 4.2, 5.7), and an exo-cellulase (isoelectric point 4.7). With respect to their enzymatic action on amorphous cellulose, endo-cellulases I and III were isofunctional, releasing cellobiose and cellodextrins as hydrolytic products, whereas endo-cellulase II was found to produce additionally some glucose. Endo-cellulases I and III were also able to attack native (crystalline) cellulose like filter paper or Avicel, but endo-cellulase II could not and thus behaved as a true carboxymethylcellulase. The rate of formation of endo-cellulase I during growth was distinctly superior from that of the other cellulases so that the proportion of the activity due to endo-cellulase. I compared with that due to the others constantly increased during the culture.

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