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 Extracellular hydrolytic enzyme production by proteolytic bacteria from the Antarctic

2013 ◽  
Vol 34 (3) ◽  
pp. 253-267 ◽  
Author(s):  
Mauro Tropeano ◽  
Susana Vázquez ◽  
Silvia Coria ◽  
Adrián Turjanski ◽  
Daniel Cicero ◽  
...  
Keyword(s):  
Extracellular Enzymes ◽  
Restriction Analysis ◽  
Hydrolytic Enzyme ◽  
Hydrolytic Enzymes ◽  
Extracellular Enzyme ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Potter Cove ◽  
Starting Point ◽  
Dna Restriction

AbstractCold−adapted marine bacteria producing extracellular hydrolytic enzymes are important for their industrial application and play a key role in degradation of particulate organic matter in their natural environment. In this work, members of a previously−obtained protease−producing bacterial collection isolated from different marine sources from Potter Cove (King George Island, South Shetlands) were taxonomically identified and screened for their ability to produce other economically relevant enzymes. Eighty−eight proteolytic bacterial isolates were grouped into 25 phylotypes based on their Amplified Ribosomal DNA Restriction Analysis profiles. The sequencing of the 16S rRNA genes from representative isolates of the phylotypes showed that the predominant culturable protease−producing bacteria belonged to the class Gammaproteobacteria and were affiliated to the genera Pseudomonas, Shewanella, Colwellia, and Pseudoalteromonas, the latter being the predominant group (64% of isolates). In addition, members of the classes Actinobacteria, Bacilli and Flavobacteria were found. Among the 88 isolates screened we detected producers of amylases (21), pectinases (67), cellulases (53), CM−cellulases (68), xylanases (55) and agarases (57). More than 85% of the isolates showed at least one of the extracellular enzymatic activities tested, with some of them producing up to six extracellular enzymes. Our results confirmed that using selective conditions to isolate producers of one extracellular enzyme activity increases the probability of recovering bacteria that will also produce additional extracellular enzymes. This finding establishes a starting point for future programs oriented to the prospecting for biomolecules in Antarctica.

scholarly journals Purification and Characterization of 2,6-β-d-Fructan 6-Levanbiohydrolase fromStreptomyces exfoliatus F3-2

2000 ◽  
Vol 66 (1) ◽  
pp. 252-256 ◽  
Author(s):  
Katsuichi Saito ◽  
Kazuya Kondo ◽  
Ichiro Kojima ◽  
Atsushi Yokota ◽  
Fusao Tomita
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Extracellular Enzyme ◽  
Molecular Weights ◽  
Sds Page ◽  
Monomeric Structure ◽  
Ph Range ◽  
Hydrolysis Of

ABSTRACT Streptomyces exfoliatus F3-2 produced an extracellular enzyme that converted levan, a β-2,6-linked fructan, into levanbiose. The enzyme was purified 50-fold from culture supernatant to give a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weights of this enzyme were 54,000 by SDS-PAGE and 60,000 by gel filtration, suggesting the monomeric structure of the enzyme. The isoelectric point of the enzyme was determined to be 4.7. The optimal pH and temperature of the enzyme for levan degradation were pH 5.5 and 60°C, respectively. The enzyme was stable in the pH range 3.5 to 8.0 and also up to 50°C. The enzyme gave levanbiose as a major degradation product from levan in an exo-acting manner. It was also found that this enzyme catalyzed hydrolysis of such fructooligosaccharides as 1-kestose, nystose, and 1-fructosylnystose by liberating fructose. Thus, this enzyme appeared to hydrolyze not only β-2,6-linkage of levan, but also β-2,1-linkage of fructooligosaccharides. From these data, the enzyme from S. exfoliatus F3-2 was identified as a novel 2,6-β-d-fructan 6-levanbiohydrolase (EC 3.2.1.64 ).

scholarly journals Response of <i>Nodularia spumigena</i> to <i>p</i>CO<sub>2</sub> – Part 2: Exudation and extracellular enzyme activities

2013 ◽  
Vol 10 (1) ◽  
pp. 567-582 ◽  
Author(s):  
S. Endres ◽  
J. Unger ◽  
N. Wannicke ◽  
M. Nausch ◽  
M. Voss ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Organic Matter ◽  
Enzyme Activities ◽  
Extracellular Enzymes ◽  
Extracellular Enzyme ◽  
High Pco2 ◽  
Nodularia Spumigena ◽  
Extracellular Enzyme Activities ◽  
Pco2 Treatment ◽  
Co2 Concentrations

Abstract. The filamentous and diazotrophic cyanobacterium Nodularia spumigena plays a major role in the productivity of the Baltic Sea as it forms extensive blooms regularly. Under phosphorus limiting conditions Nodularia spumigena have a high enzyme affinity for dissolved organic phosphorus (DOP) by production and release of alkaline phosphatase. Additionally, they are able to degrade proteinaceous compounds by expressing the extracellular enzyme leucine aminopeptidase. As atmospheric CO2 concentrations are increasing, we expect marine phytoplankton to experience changes in several environmental parameters, including pH, temperature, and nutrient availability. The aim of this study was to investigate the combined effect of CO2-induced changes in seawater carbonate chemistry and of phosphate deficiency on the exudation of organic matter, and its subsequent recycling by extracellular enzymes in a Nodularia spumigena culture. Batch cultures of Nodularia spumigena were grown for 15 days under aeration with low (180 μatm), medium (380 μatm), and high (780 μatm) CO2 concentrations. Obtained pCO2 levels in the treatments were on median 315, 353, and 548 μatm CO2, respectively. Extracellular enzyme activities as well as changes in organic and inorganic compound concentrations were monitored. CO2 treatment–related effects were identified for cyanobacterial growth, which in turn influenced the concentration of mucinous substances and the recycling of organic matter by extracellular enzymes. Biomass production was increased by 56.5% and 90.7% in the medium and high pCO2 treatment, respectively, compared to the low pCO2 treatment. In total, significantly more mucinous substances accumulated in the high pCO2 treatment, reaching 363 μg Xeq L−1 compared to 269 μg Xeq L−1 in the low pCO2 treatment. However, cell-specific rates did not change. After phosphate depletion, the acquisition of P from DOP by alkaline phosphatase was significantly enhanced. Alkaline phosphatase activities were increased by factor 1.64 and 2.25, respectively, in the medium and high compared to the low pCO2 treatment. We hypothesise from our results that Nodularia spumigena can grow faster under elevated pCO2 by enhancing the recycling of organic matter to acquire nutrients.

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 Conditions of hydrolysis with a specific pair of endo- and exo-cellulases

2021 ◽  
Author(s):  
Yoshiki Kitano
Keyword(s):  
Synergistic Effect ◽  
Crystalline Cellulose ◽  
Carbohydrate Binding ◽  
Amorphous Cellulose ◽  
Potential Synergy ◽  
Hydrolysis Of Cellulose ◽  
Potential Alternative ◽  
Specific Pair ◽  
Hydrolysis Of ◽  
Increased Activity

Enzymatic hydrolysis of cellulose is a technology involved in the production of bioethanol, a potential alternative renewable energy. Many cellulases with endo- and exo- type of activity are known to hydrolyze cellulose synergistically. In this thesis, potential synergy between an endo-cellulase, Cel5B, with and without a carbohydrate- binding module (CBM6), and a new exo-cellulase, CBH1, from Trichoderma harzianum FP108 were examined during the hydrolysis of semi- crystalline cellulose (Avicel). Since CBM6 is recognized as having a high affinity for amorphous cellulose, it was hypothesized that this affinity could enhance the synergistic effect between the endo- and exo-cellulases by focusing the action to Cel5B+CBM6 on the amorphous regions of the Avicel substrate. The increased activity of Cel5B+CBM6 over Cel5B alone was confirmed. However, in contrast to our expectations, a synergistic effect was not observed between either endo- and exo-cellulase pairs. From the obtained hydrolysis yield, it was inferred that Cel5B+CBM6 may have exo-type activity that caused a competitive interaction with the exo-cellulase, which resulted in no synergy.

Temperature Sensitivity of Intracellular and Extracellular Soil Enzyme Activities

2021 ◽  
Author(s):  
Adetunji Alex Adekanmbi ◽  
Laurence Dale ◽  
Liz Shaw ◽  
Tom Sizmur
Keyword(s):  
Enzyme Activity ◽  
Soil Temperature ◽  
Temperature Sensitivity ◽  
Extracellular Enzymes ◽  
Extracellular Enzyme ◽  
Daily Maximum ◽  
Temperature Sensitive ◽  
Intracellular Enzyme ◽  
Temperature Changes ◽  
Som Decomposition

&lt;p&gt;Predicting the pattern of soil organic matter (SOM) decomposition as a feedback to climate change, via release of CO&lt;sub&gt;2&lt;/sub&gt;, is extremely complex and has received much attention. However, investigations often do not differentiate between the extracellular and intracellular processes involved and work is needed to identify their relative temperature sensitivities. Samples were collected from a grassland soil at Sonning, UK with average daily maximum and minimum soil temperature of 15 &amp;#176;C and 5 &amp;#176;C. We measured potential activities of &amp;#946;-glucosidase (BG) and chitinase (NAG) (extracellular enzymes) and glucose-induced CO&lt;sub&gt;2 &lt;/sub&gt;respiration (intracellular enzymes) at a range of assay temperatures (5 &amp;#176;C, 15 &amp;#176;C, 26 &amp;#176;C, 37&lt;sup&gt; &amp;#160;&lt;/sup&gt;&amp;#176;C, and 45 &amp;#176;C). The temperature coefficient Q&lt;sub&gt;10&lt;/sub&gt; (the increase in enzyme activity that occurs after a 10 &amp;#176;C increase in soil temperature) was calculated to assess the temperature sensitivity of intracellular and extracellular enzymes activities. Between 5 &amp;#176;C and 15 &amp;#176;C intracellular and extracellular enzyme activities had equal temperature sensitivity, but between 15 &amp;#176;C and 26&amp;#176;C intracellular enzyme activity was more temperature sensitive than extracellular enzyme activity and between 26 &amp;#176;C and 37 &amp;#176;C extracellular enzyme activity was more temperature sensitive than intracellular enzyme activity. This result implies that extracellular depolymerisation of higher molecular weight organic compounds is more sensitive to temperature changes at higher temperatures (e.g. changes to daily maximum summer temperature) but the intracellular respiration of the generated monomers is more sensitive to temperature changes at moderate temperatures (e.g. changes to daily mean summer temperature). We therefore conclude that the extracellular and intracellular steps of SOM mineralisation are not equally sensitive to changes in soil temperature. The finding is important because we have observed greater increases in average daily minimum temperatures than average daily mean or maximum temperatures due to increased cloud cover and sulphate aerosol emission. Accounting for this asymmetrical global warming may reduce the importance of extracellular depolymerisation and increase the importance of intracellular catalytic activities as the rate limiting step of SOM decomposition.&lt;/p&gt;

scholarly journals Extraction, Characterization and Antioxidant Activity in vitro of Proteins from Semen Allii Fistulosi

Molecules ◽  
2018 ◽  
Vol 23 (12) ◽  
pp. 3235
Author(s):  
Min Zuo ◽  
Xiao-xiao Liu ◽  
Di Liu ◽  
Hang-yun Zhao ◽  
Lu-lu Xuan ◽  
...  
Keyword(s):  
Antioxidant Activity ◽  
High Performance ◽  
Orthogonal Design ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Reducing Power ◽  
Raw Material ◽  
Extraction Solvent ◽  
Sds Page

Semen Allii Fistulosi (PSAF) is the seed of Allium fistulosum L. of the Liliaceae family. The purpose of this study was to extract, characterize, and evaluate the antioxidant activity in vitro of proteins. Using single factor and orthogonal design, the optimum conditions of extraction were determined to be as follows: extraction time 150 min, pH 8.5, temperature 60 °C, and ratio (v/w, mL/g) of extraction solvent to raw material 35. The isoelectric point of the pH was determined to be about 4.4 and 10.2, by measuring the protein content of PSAF solutions at different pH values. The amino acid composition of PSAF was determined by high performance liquid chromatography (HPLC), and the results suggested that the species of amino acids contained in the PSAF was complete. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE) analysis showed the molecular weight was mainly between 40 and 55 kDa, and Fourier-transform infrared spectroscopy (FTIR) characterized prevalent protein absorption peaks. PSAF exhibited potent scavenging activities against DPPH assays, via targeting of hydroxyl and superoxide radicals, while chelating Fe2+ activity and demonstrating weak reducing power. This work revealed that PSAF possessed potential antioxidant activity in vitro, suggesting potential for use of PSAF as a natural antioxidant.

scholarly journals Patterns and Drivers of Extracellular Enzyme Activity in New Zealand Glacier-Fed Streams

2020 ◽  
Vol 11 ◽  
Author(s):  
Tyler J. Kohler ◽  
Hannes Peter ◽  
Stilianos Fodelianakis ◽  
Paraskevi Pramateftaki ◽  
Michail Styllas ◽  
...  
Keyword(s):  
Climate Change ◽  
Extracellular Polymeric Substances ◽  
Extracellular Enzymes ◽  
Inorganic Nitrogen ◽  
Extracellular Enzyme ◽  
Microbial Life ◽  
Mountain Glaciers ◽  
P Limitation ◽  
Habitat Template ◽  
N Demand

Glacier-fed streams (GFSs) exhibit near-freezing temperatures, variable flows, and often high turbidities. Currently, the rapid shrinkage of mountain glaciers is altering the delivery of meltwater, solutes, and particulate matter to GFSs, with unknown consequences for their ecology. Benthic biofilms dominate microbial life in GFSs, and play a major role in their biogeochemical cycling. Mineralization is likely an important process for microbes to meet elemental budgets in these systems due to commonly oligotrophic conditions, and extracellular enzymes retained within the biofilm enable the degradation of organic matter and acquisition of carbon (C), nitrogen (N), and phosphorus (P). The measurement and comparison of these extracellular enzyme activities (EEA) can in turn provide insight into microbial elemental acquisition effort relative to environmental availability. To better understand how benthic biofilm communities meet resource demands, and how this might shift as glaciers vanish under climate change, we investigated biofilm EEA in 20 GFSs varying in glacier influence from New Zealand’s Southern Alps. Using turbidity and distance to the glacier snout normalized for glacier size as proxies for glacier influence, we found that bacterial abundance (BA), chlorophyll a (Chl a), extracellular polymeric substances (EPS), and total EEA per gram of sediment increased with decreasing glacier influence. Yet, when normalized by BA, EPS decreased with decreasing glacier influence, Chl a still increased, and there was no relationship with total EEA. Based on EEA ratios, we found that the majority of GFS microbial communities were N-limited, with a few streams of different underlying bedrock geology exhibiting P-limitation. Cell-specific C-acquiring EEA was positively related to the ratio of Chl a to BA, presumably reflecting the utilization of algal exudates. Meanwhile, cell-specific N-acquiring EEA were positively correlated with the concentration of dissolved inorganic nitrogen (DIN), and both N- and P-acquiring EEA increased with greater cell-specific EPS. Overall, our results reveal greater glacier influence to be negatively related to GFS biofilm biomass parameters, and generally associated with greater microbial N demand. These results help to illuminate the ecology of GFS biofilms, along with their biogeochemical response to a shifting habitat template with ongoing climate change.

scholarly journals A Model of Extracellular Enzymes in Free-Living Microbes: Which Strategy Pays Off?

2015 ◽  
Vol 81 (21) ◽  
pp. 7385-7393 ◽  
Author(s):  
Sachia J. Traving ◽  
Uffe H. Thygesen ◽  
Lasse Riemann ◽  
Colin A. Stedmon
Keyword(s):  
Extracellular Enzymes ◽  
Extracellular Enzyme ◽  
Threshold Concentration ◽  
Free Living ◽  
Deployment Strategy ◽  
Solitary Cell ◽  
Low Concentrations ◽  
Refractory State ◽  
Ocean Carbon ◽  
Cost Efficient

ABSTRACTAn initial modeling approach was applied to analyze how a single, nonmotile, free-living, heterotrophic bacterial cell may optimize the deployment of its extracellular enzymes. Free-living cells live in a dilute and complex substrate field, and to gain enough substrate, their extracellular enzymes must be utilized efficiently. The model revealed that surface-attached and free enzymes generate unique enzyme and substrate fields, and each deployment strategy has distinctive advantages. For a solitary cell, surface-attached enzymes are suggested to be the most cost-efficient strategy. This strategy entails potential substrates being reduced to very low concentrations. Free enzymes, on the other hand, generate a radically different substrate field, which suggests significant benefits for the strategy if free cells engage in social foraging or experience high substrate concentrations. Swimming has a slight positive effect for the attached-enzyme strategy, while the effect is negative for the free-enzyme strategy. The results of this study suggest that specific dissolved organic compounds in the ocean likely persist below a threshold concentration impervious to biological utilization. This could help explain the persistence and apparent refractory state of oceanic dissolved organic matter (DOM). Microbial extracellular enzyme strategies, therefore, have important implications for larger-scale processes, such as shaping the role of DOM in ocean carbon sequestration.

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