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.

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.

scholarly journals Expression of a Cellobiose Phosphorylase fromThermotoga maritimainCaldicellulosiruptor besciiImproves the Phosphorolytic Pathway and Results in a Dramatic Increase in Cellulolytic Activity

2017 ◽  
Vol 84 (3) ◽  
Author(s):  
Sun-Ki Kim ◽  
Michael E. Himmel ◽  
Yannick J. Bomble ◽  
Janet Westpheling
Keyword(s):  
Extracellular Enzymes ◽  
Plant Biomass ◽  
Rational Approach ◽  
Crystalline Cellulose ◽  
Synergistic Activity ◽  
Cellulolytic Activity ◽  
Glycosyl Hydrolases ◽  
Content Type ◽  
Biomass Deconstruction ◽  
Cellobiose Phosphorylase

ABSTRACTMembers of the genusCaldicellulosiruptorhave the ability to deconstruct and grow on lignocellulosic biomass without conventional pretreatment. A genetically tractable species,Caldicellulosiruptor bescii, was recently engineered to produce ethanol directly from switchgrass.C. besciicontains more than 50 glycosyl hydrolases and a suite of extracellular enzymes for biomass deconstruction, most prominently CelA, a multidomain cellulase that uses a novel mechanism to deconstruct plant biomass. Accumulation of cellobiose, a product of CelA during growth on biomass, inhibits cellulase activity. Here, we show that heterologous expression of a cellobiose phosphorylase fromThermotoga maritimaimproves the phosphorolytic pathway inC. besciiand results in synergistic activity with endogenous enzymes, including CelA, to increase cellulolytic activity and growth on crystalline cellulose.IMPORTANCECelA is the only known cellulase to function well on highly crystalline cellulose and it uses a mechanism distinct from those of other cellulases, including fungal cellulases. Also unlike fungal cellulases, it functions at high temperature and, in fact, outperforms commercial cellulase cocktails. Factors that inhibit CelA during biomass deconstruction are significantly different than those that impact the performance of fungal cellulases and commercial mixtures. This work contributes to understanding of cellulase inhibition and enzyme function and will suggest a rational approach to engineering optimal activity.

scholarly journals Biomolecular Characterization of Bacillus tequilensis A1C1 Isolated from Soil and Chromatographic Analysis of D-serine in its Cellular Fraction

2021 ◽  
Author(s):  
Shikha Kapil ◽  
Monika Bhattu ◽  
Tarun Kumar ◽  
Vipasha Sharma
Keyword(s):  
Extracellular Enzymes ◽  
Growth Parameters ◽  
Maximum Growth ◽  
Bacterial Cells ◽  
Standard Curve ◽  
Intracellular Fraction ◽  
Cellular Fraction ◽  
Lactose Fermentation ◽  
Bacillus Tequilensis ◽  
Soil Site

Abstract The current work was carried out to investigate serine enantiomers in bacterial cells. The bacteria isolated from the pomace dumping soil site (bacteria id A1C1) showed maximum growth (O.D600 = 1.97±0.4 X 109cells/ml) within 48h in the minimal salt media supplemented with L-serine. The isolated strain was identified as ‘Bacillus tequilensis’ through 16sRNA sequencing. The study’s peculiarity reflects the fact that the isolated strain was explored for the first time to detect the presence of serine enantiomers. The strain was quantified for D-serine content by using RP-HPLC. The D-serine concentration was calculated as 0.919±0.02 nM in the bacterial cellular fraction by using a standard curve plot and linear curve equation. Further, recovery % was also calculated for the spiked samples which vary from 85-90%. The optimum growth parameters were recorded as 37℃±0.5, 150±0.5 RPM, and 7±0.5pH. The strain was Gram-positive, rod shape, large, irregular, off-white-coloured, and synthesized endospores. A1C1 showed positive results (within 14±2h of incubation) for indole production, lactose fermentation, and protease (0.9 mm, clear zone). The antibacterial assay showed 5% and 2% efficacy of the extracellular fraction against MTCC 40 and MTCC 11949 respectively within 12h of incubation. These results demonstrate that Bacillus tequilensis A1C1 has antibacterial activity, the potential to secrete extracellular enzymes, and D-serine content in the intracellular fraction of the cultivated cells. Given results demonstrate the industrial significance and implication of the isolated strain for the synthesis of commercially valuable products.

Growth and cellulolytic activity of Cellulomonas flavigena

1981 ◽  
Vol 27 (12) ◽  
pp. 1260-1266 ◽  
Author(s):  
B. H. Kim ◽  
J. W. T. Wimpenny
Keyword(s):  
Free Enzyme ◽  
Crystalline Cellulose ◽  
Exponential Growth Phase ◽  
Cellulolytic Bacterium ◽  
Cellulolytic Activity ◽  
Mineral Salts ◽  
Cellulomonas Flavigena ◽  
Rate Limiting Step ◽  
Rate Limiting ◽  
Hydrolysis Of

Growth factor requirements, growth kinetics, and the ability to produce the enzyme cellulase were examined in the cellulolytic bacterium Cellulomonas flavigena KIST 321. The organism was found to require only thiamine for growth in mineral salts medium containing simple sugars or cellulose. Growth rates on various carbohydrates suggested that disruption of the crystalline structure was the rate-limiting step in the utilization of crystalline cellulose, and hydrolysis of the polymer itself was as rapid as the uptake of the hydrolytic product. When the organism was grown on cellulose the cellulolytic activity appeared to be bound to the cell at the beginning of the exponential growth phase: only after this did cell-free enzyme activity appear. The cell-free enzyme appeared to be unstable, and its activity decreased at the beginning of the stationary phase.

In vitro cellulolytic activity of the plant pathogen Clavibacter michiganensis subsp. sepedonicus

1995 ◽  
Vol 41 (10) ◽  
pp. 877-888 ◽  
Author(s):  
Debra Baer ◽  
Neil C. Gudmestad
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein Band ◽  
Cellulolytic Activity ◽  
Clavibacter Michiganensis ◽  
Endoglucanase Activity ◽  
Clavibacter Michiganensis Subsp ◽  
Cellulose Substrates ◽  
Key Words Cellulase

The activity of four Clavibacter michiganensis subsp. sepedonicus strains against various cellulose substrates was investigated. Sixty-seven Clavibacter michiganensis subsp. sepedonicus strains grew well on media amended with carboxymethylcellulose, 64 strains produced zones of hydrolysis. Endoglucanase activity was optimal at 37 °C and pH 6.0 against carboxymethylcellulose incorporated in plate assays. Zymogram and sodium dodecyl sulfate – polyacrylamide gel electrophoresis revealed the presence of a protein band corresponding to the cellulolytic activity in the molecular weight (MW) range of approximately 28 000. Protein bands in the same range were detected in five Clavibacter michiganensis subsp. sepedonicus strains. Studies on crude enzyme extracts of Clavibacter michiganensis subsp. sepedonicus strain N-1-1 revealed that p-nitrophenyl β-D-cellobioside (pNPC) was hydrolyzed, with optimal activity at 37 °C and pH 7.0.Key words: cellulase, endo-1,4-β-glucanase (EC 3.2.1.4), Corynebacterium sepedonicum, Solanum tuberosum.

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.

scholarly journals Biomolecular Characterization of Bacillus tequilensis A1C1 Isolated from Soil and Chromatographic Analysis of D-serine in its Cellular Fraction

2021 ◽  
Author(s):  
Shikha Kapil ◽  
Monika Bhattu ◽  
Tarun Kumar ◽  
Vipasha Sharma
Keyword(s):  
Extracellular Enzymes ◽  
Growth Parameters ◽  
Maximum Growth ◽  
Standard Curve ◽  
Intracellular Fraction ◽  
Cellular Fraction ◽  
Negative Results ◽  
Lactose Fermentation ◽  
Bacillus Tequilensis ◽  
Soil Site

Abstract The bacteria isolated from the pomace dumping soil site (bacteria id A1C1) showed maximum growth (O.D600 = 1.97±0.4 X 109 cells/ml) within 48h in the minimal salt media supplemented with L-serine. The isolated strain was identified as ‘Bacillus tequilensis’ through 16sRNA sequencing. The strain was quantified for D-serine content by using RP-HPLC. The D-serine concentration was calculated as 0.919±0.02 nM in the bacterial cellular fraction by using a standard curve plot and linear curve equation. Further, recovery % was also calculated for the spiked samples which vary from 85-90%. The study’s peculiarity reflects the fact that the isolated strain was explored for the first time to detect the presence of serine enantiomers. The biochemical features also showed 70% similarity to the standard strain Bacillus tequilensis 10bT. The optimum growth parameters were recorded as 37℃±0.5, 150±0.5 RPM, and 7±0.5pH. The strain was Gram-positive and synthesized endospores. Morphological results showed its rod shape, large, irregular, and off-white-coloured colonies. A1C1 was also tested for the production of secondary metabolites and enzymes. A1C1 showed positive results for indole production, lactose fermentation, protease, and gelatinase whereas, negative results for catalase, MR-VP, citrate utilization, cellulase, amylase, and pectinase. Further, the strain was assayed for PGPR attributes and showed a negative phosphate solubilization index and IAA production. The antibacterial assay showed 5% and 2% efficacy of the extracellular fraction against MTCC 40 and MTCC 11949 respectively. These results demonstrate that Bacillus tequilensis A1C1 has antibacterial activity, the potential to secrete extracellular enzymes, and D-serine content in the intracellular fraction of the cultivated cells.

scholarly journals Biomolecular Characterization of Bacillus tequilensis A1C1 Isolated from Soil and Chromatographic Analysis of D-serine in its Cellular Fraction

2021 ◽  
Author(s):  
Shikha Kapil ◽  
Monika Bhattu ◽  
Tarun Kumar ◽  
Vipasha Sharma
Keyword(s):  
Extracellular Enzymes ◽  
Growth Parameters ◽  
Maximum Growth ◽  
Standard Curve ◽  
Intracellular Fraction ◽  
Cellular Fraction ◽  
Negative Results ◽  
Lactose Fermentation ◽  
Bacillus Tequilensis ◽  
Soil Site

Abstract The bacteria isolated from the pomace dumping soil site (bacteria id A1C1) showed maximum growth (O.D600 = 1.97 ± 0.4 X 109 cells/ml) within 48h in the minimal salt media supplemented with L-serine. The isolated strain was identified as ‘Bacillus tequilensis’ through 16sRNA sequencing. The strain was quantified for D-serine content by using RP-HPLC. The D-serine concentration was calculated as 0.919 ± 0.02 nM in the bacterial cellular fraction by using a standard curve plot and linear curve equation. Further, recovery % was also calculated for the spiked samples which vary from 85–90%. The study’s peculiarity reflects the fact that the isolated strain was explored for the first time to detect the presence of serine enantiomers. The biochemical features also showed 70% similarity to the standard strain Bacillus tequilensis 10bT. The optimum growth parameters were recorded as 37℃±0.5, 150 ± 0.5 RPM, and 7 ± 0.5pH. The strain was Gram-positive and synthesized endospores. Morphological results showed its rod shape, large, irregular, and off-white-coloured colonies. A1C1 was also tested for the production of secondary metabolites and enzymes. A1C1 showed positive results for indole production, lactose fermentation, protease, and gelatinase whereas, negative results for catalase, MR-VP, citrate utilization, cellulase, amylase, and pectinase. Further, the strain was assayed for PGPR attributes and showed a negative phosphate solubilization index and IAA production. The antibacterial assay showed 5% and 2% efficacy of the extracellular fraction against MTCC 40 and MTCC 11949 respectively. These results demonstrate that Bacillus tequilensis A1C1 has antibacterial activity, the potential to secrete extracellular enzymes, and D-serine content in the intracellular fraction of the cultivated cells.

Cellulosomes localise on the surface of membrane vesicles from the cellulolytic bacterium Clostridium thermocellum

2019 ◽  
Vol 366 (12) ◽  
Author(s):  
Shunsuke Ichikawa ◽  
Satoru Ogawa ◽  
Ayami Nishida ◽  
Yuzuki Kobayashi ◽  
Toshihito Kurosawa ◽  
...  
Keyword(s):  
Clostridium Thermocellum ◽  
Cell Communication ◽  
Membrane Vesicles ◽  
Cellulolytic Enzymes ◽  
Crystalline Cellulose ◽  
Cellulolytic Bacterium ◽  
Cellulolytic Activity ◽  
Cellulolytic Bacteria ◽  
Degradation Activity ◽  
Enzyme Complexes

ABSTRACTMembrane vesicles released from bacteria contribute to cell–cell communication by carrying various cargos such as proteins, nucleic acids and signaling molecules. Cellulolytic bacteria have been isolated from many environments, yet the function of membrane vesicles for cellulolytic ability has been rarely described. Here, we show that a Gram-positive cellulolytic bacterium Clostridium thermocellum released membrane vesicles, each approximately 50–300 nm in diameter, into the broth. The observations with immunoelectron microscopy also revealed that cellulosomes, which are carbohydrate-active enzyme complexes that give C. thermocellum high cellulolytic activity, localized on the surface of the membrane vesicles. The membrane vesicles collected by ultracentrifugation maintained the cellulolytic activity. Supplementation with the biosurfactant surfactin or sonication treatment disrupted the membrane vesicles in the exoproteome of C. thermocellum and significantly decreased the degradation activity of the exoproteome for microcrystalline cellulose. However, these did not affect the degradation activity for soluble carboxymethyl cellulose. These results suggest a novel function of membrane vesicles: C. thermocellum releases cellulolytic enzymes on the surface of membrane vesicles to enhance the cellulolytic activity of C. thermocellum for crystalline cellulose.

Predominant secretion of cellobiohydrolases and endo-β-1,4-glucanases in nutrient-limited medium by Aspergillus spp. isolated from subtropical field

2020 ◽  
Vol 168 (3) ◽  
pp. 243-256 ◽  
Author(s):  
May Thin Kyu ◽  
Shunsuke Nishio ◽  
Koki Noda ◽  
Bay Dar ◽  
San San Aye ◽  
...  
Keyword(s):  
Rice Straw ◽  
Plant Biomass ◽  
Cellulose Degradation ◽  
Industrial Wastes ◽  
Added Value ◽  
Crystalline Cellulose ◽  
Cellulolytic Activity ◽  
Biological Degradation ◽  
Industry Waste ◽  
Degradation Intermediates

Abstract Biological degradation of cellulose from dead plants in nature and plant biomass from agricultural and food-industry waste is important for sustainable carbon recirculation. This study aimed at searching diverse cellulose-degrading systems of wild filamentous fungi and obtaining fungal lines useful for cellooligosaccharide production from agro-industrial wastes. Fungal lines with cellulolytic activity were screened and isolated from stacked rice straw and soil in subtropical fields. Among 13 isolated lines, in liquid culture with a nutrition-limited cellulose-containing medium, four lines of Aspergillus spp. secreted 50–60 kDa proteins as markedly dominant components and gave clear activity bands of possible endo-β-1,4-glucanase in zymography. Mass spectroscopy (MS) analysis of the dominant components identified three endo-β-1,4-glucanases (GH5, GH7 and GH12) and two cellobiohydrolases (GH6 and GH7). Cellulose degradation by the secreted proteins was analysed by LC-MS-based measurement of derivatized reducing sugars. The enzymes from the four Aspergillus spp. produced cellobiose from crystalline cellulose and cellotriose at a low level compared with cellobiose. Moreover, though smaller than that from crystalline cellulose, the enzymes of two representative lines degraded powdered rice straw and produced cellobiose. These fungal lines and enzymes would be effective for production of cellooligosaccharides as cellulose degradation-intermediates with added value other than glucose.

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