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The Beauty Queen of Leenane Irony
by Martin McDonagh
About The Beauty Queen of Leenane The Beauty Queen of Leenane Summary Character List Glossary Themes Quotes Analysis Symbols, Allegory and Motifs Metaphors and Similes Irony Imagery Literary Elements Essay Questions
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The irony of insanity
One's perception is all they have to make sense of the world, so what is life even like if you can't trust your own ability to perceive reality? And yet, a significant portion of the population deals with that problem. There are those who adjust to their condition and lead happy, healthy lives, and then there are some sad cases like Maureen where without a commitment to figuring things out, the person feels entitled to hurt others in their insanity. The entire premise is ironic, because Maureen, like everyone, naturally trusts her perception, regardless of what other people say.
The act of killing one's mother is ironic because instead of a mother giving life, it's a child taking life away. It's especially ironic here because Mag was allegedly taking care of Maureen. Who knows if Maureen even killed her, because after all, she isn't charged for the murder.
Pato's role
The sexual encounter between Maureen and Pato seems dubious. Why wasn't he aware that Mag would be there? Is he still really there when they're fighting? Because later in the novel, it seems that Maureen sometimes hallucinates Pato, so technically, he could be a figment of her imagination. Perhaps the fight in the kitchen was actually about Maureen imagining things again.
Maureen in Mag's chair
The end of the novel shows Maureen in Mag's chair, a replacement perhaps. It's strange that Maureen would feel comfortable in the chair of her own mother who she murdered. But yet, somehow, this is what feels right for her, perhaps a statement of her own inability to distinguish people's roles.
No charges pressed
Just when the reader begins to feel as if they finally understand the truth of the novel, there is an interesting, silent detail. Why didn't Maureen go to jail for murder? The novel depicts a gruesome death with plenty of evidence, and there's a funeral, so the authorities know about the death, so why didn't anyone say anything about the fact that the death was obviously homicidal? This leaves the reader with the sense that there's still so much we are not being told about Maureen and her strange experience of life.
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About The Beauty Queen of Leenane
The Beauty Queen of Leenane Summary
Martin McDonagh Biography
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Study Guide for The Beauty Queen of Leenane
The Beauty Queen of Leenane study guide contains a biography of Martin McDonagh, literature essays, quiz questions, major themes, characters, and a full summary and analysis.
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The Beauty Queen of Leenane essays are academic essays for citation. These papers were written primarily by students and provide critical analysis of The Beauty Queen of Leenane by Martin McDonagh.
Caught in a Snare Trap: The Modern Irish Experience in "The Beauty Queen of Leenane" and "Six Shooter"
The Place of Violence in Martin McDonagh's The Lieutenant of Inishmore, The Beauty Queen of Leenane, and Three Billboards Outside Ebbing, Missouri
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redpajama_set_name: RedPajamaCommonCrawl


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About PRC
By admin 3d format, 3D Model, 3d pdf, PDF3D, pmi metadata, PRC, PRC Encoding, prc file, U3D Comments Off on About PRC
PRC 3D Format
PRC (Product Representation Compact) is a 3D data structure and file format, one of two systems used in 3D PDF documents. Along side U3D, PRC is now the preferred and most feature-rich method to embed 3D interactive data and models into a PDF file. A PDF file containing PRC based 3D model can be viewed in using the Adobe Reader as well as other tools. PRC is the recommended encoding method for PDF/E documents.
PRC is a compact representation for 3D models and assemblies, for the display and visual representation of various kinds of 3D data. It includes structure, geometry and product manufacturing information ( PMI ) metadata.
The format allows for either highly compressed geometric and visual representations, with varying levels of tessellated surface geometry spatial tolerance, or an exact form of representation including precise BREP data types. Dimensional and associated metadata may also be included as PMI annotations within the PRC encoding.
PRC is Used For
The PRC encoding method in 3D PDF is used for most 3D PDF application areas, including technical reports used in engineering, manufacturing, numerical modelling, medical, aerospace, geology, geophysics, oil & gas exploration, civil engineering, environmental surveys, and as an interoperable file format for Computer-Aided Manufacturing (CAM), Computer-Aided Engineering (CAE), and Product Life-Cycle Management (PLM) for supply-chain, training, marketing, archiving and engineering reports.
PRC Benefits over U3D
Although both U3D and PRC are available as 3D systems in a PDF, PRC is generally preferable due to:
More geometric shape definitions, including NURBS
Much better triangle mesh compression
Annotations, PMI, Markup (absent from U3D)
Internationally Agreed ISO Standard
Scales to Complex Assemblies
PRC as File Format
PRC can be used as a 3D file type for storage and interchange. Such 3D binary files can be interchanged with other PRC compatible applications. PRC files may be "wrapped" into a PDF file for display using the Adobe Reader by tools such as media9 and Acrobat Pro. Today, PRC is primarily used for 3D PDF internal encoding, rather than an interchange format.
PRC Generation Tools
PDF3D products include PRC for highly effective compression and 3D representations in 3D technical documents. Note that aspects of the PDF3D implementation of PRC generation are the subject of granted and pending patents. Products including PRC encoding include:
ParaView PDF3D Plug-in
PDF3D XML Server, and the
PDF3D-SDK development toolkit.
Examples of 3D PDF files with PRC encoding are available on the sample Gallery page.
PRC ISO Standard
PRC is referenced in three related ISO standards. First and foremost, PRC is defined by ISO-14739 as a stand-alone data structure and file definition. The PRC format definition is not owned by any one commercial entity, although was established and submitted to ISO by Adobe.
PRC is an explicitly allowed annotation & multimedia type in the core PDF file standard ISO-32000.
In addition, the PRC encoding scheme is part of the ISO specification PDF/Engineering ISO-24517 International Standard for how PRC plays a part of a PDF document under a strict subset for engineering archive purposes.
The ISO standardization process, committee work, publications and the process of moving these standards forward is an active and ongoing process. Visual Technology Services is actively involved in the PRC standardization effort, with representation through the ISO-TC171-SC2-WG7 committee.
PRC Technical Summary
The technical specification for the internal structure of PRC is defined in ~300 page ISO-14739, s o a brief technical summary hardly does it justice. The data structure is composed of a collection of linked tables, in a machine and language independent binary structure or file. Various geometric types may be specified, from points, lines, polygons to curved surfaces. Line curves, smooth curved surfaces including trimming curves using a lattice of control points and weights are included. A small collection of pre-defined shapes such as spheres and cylinders are included for convenience.
The structure includes a full model tree, part assembly or graphical scene graph hierarchy, supporting thousands of parts. Repeated (instanced) structures are well supported, with material types including optional texture images on any tessellated 3D part.
Annotations such as PMI, part labels, dimensions may also be specified within the PRC structure and associated with 3D parts. This allows for a rich set of 3D reporting options to include quantitative and meaningful annotations where needed.
Both full-resolution precise model definitions and high compression modes are available. In the case of tessellated geometry highly compressed modes, a very compact triangle mesh structure is used, with a cascade of efficient compression methods including run-length encoding, adaptive Lempel-Ziv, and frequency table Huffman coding compression schemes. In high compression, the model is accurate to a specific geometric vertex location tolerance, while still preserving every vertex and face without re-meshing or simplification.
PRC Format History
The PRC was first announced by Trade and Technologies France (TTF) in 2002. In 2006, Adobe Systems Inc. acquired TTF including rights to the PRC technology. The PRC format was introduced into Acrobat and the Adobe Reader at version 8, supplied as "Acrobat 3D" and "Acrobat Pro Extended 9".
In 2009, the PRC Format was submitted to ISO as an open 3D data structure standard for 3D PDF, no longer controlled by a single vendor as a proprietary format. The PRC format ISO-14739 is now accepted and in the publication process.
In 2010, Adobe transferred responsibility to several 3rd party organizations, whereby at Acrobat versions 10 and 11, PRC generation was no longer integrated within Acrobat; the capability is packaged as plugins and external file converters from 3rd party vendors.
Visual Technology Services developed independent PRC generation, released in 2010, not based on TTF, Adobe or any 3rd party software.
The ISO Standards effort for PDF/E for engineering archive applications is progressing with full reference to PRC as the preferred 3D embedding mechanism.
PRC – External Links
PRC File Format definition at Wikipedia
PRC File Format Introduction at 3D PDF Consortium
PRC FAQ published by AIIM.ORG
ISO-14739 PRC Standard as published by ISO
U3D File Format definition at Wikipedia


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La nazionale di hockey su pista di Macao è la selezione maschile di hockey su pista che rappresenta Macao in ambito internazionale.
Attiva dal 1984, opera sotto la giurisdizione della Federazione di pattinaggio di Macao.
Al 31 dicembre 2015 occupa il 29º posto nel ranking  FIRS.

Palmares
 (record)
  1º posto: 1987, 1991, 1997, 2004, 2005, 2007, 2009, 2012, 2014
  2º posto: 1989, 1995, 2001
  3º posto: 1999

Risultati

Campionato del mondo

Campionato del mondo B

Roller Hockey Asia Cup

Note

Voci correlate
 Fédération Internationale de Roller Sports
 Confederation of Asia Roller Sports
 Federazione di pattinaggio di Macao
 Macao

Collegamenti esterni

 Macao ai mondiali del 2005
 Macao ai mondiali B del 1984
 Macao ai mondiali B del 1988
 Macao ai mondiali B del 1990
 Macao ai mondiali B del 1992
 Macao ai mondiali B del 1994
 Macao ai mondiali B del 1996
 Macao ai mondiali B del 1998
 Macao ai mondiali B del 2000
 Macao ai mondiali B del 2002
 Macao ai mondiali B del 2004
 Macao ai mondiali B del 2006
 Macao ai mondiali B del 2008
 Macao ai mondiali B del 2010
 Macao ai mondiali B del 2012
 Macao ai mondiali B del 2014
 Macao alla Roller Hockey Asia Cup 1987
 Macao alla Roller Hockey Asia Cup 1991
 Macao alla Roller Hockey Asia Cup 1995
 Macao alla Roller Hockey Asia Cup 2004
 Macao alla Roller Hockey Asia Cup 2005

Hockey su pista a Macao
Macao
Hockey su pista

redpajama_set_name: RedPajamaWikipedia


text:
`Sublime` like multiple cursors in `vim`

Default keys:

- `<Ctrl-n>`: Pressing the key in Normal mode highlights the current word under the cursor in Visual mode and
places a virtual cursor at the end of it. Pressing it again finds the next occurrence and places
another virtual cursor at the end of the visual selection. If you select multiple lines in Visual
mode, pressing the key puts a virtual cursor at every line and leaves you in Normal mode.
- `<Ctrl-p>`: in Visual mode will remove the current virtual cursor and go back to the previous virtual
cursor location. This is useful if you are trigger happy with Ctrl-n and accidentally went too far.
- `<Ctrl-x>`: in Visual mode will remove the current virtual cursor and skip to the next virtual cursor
location. This is useful if you don't want the current selection to be a candidate to operate on
later.

## [vim-easymotion]

[vim-easymption]: https://github.com/easymotion/vim-easymotion
[vim-multiple-cursors]: https://github.com/terryma/vim-multiple-cursors

## vim-numbertoggle

**Repo:**
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**Performance issues:**
https://github.com/jeffkreeftmeijer/vim-numbertoggle/issues/33

Tricks to improve the speed:

~~~~
:set ttyfast
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~~~~

redpajama_set_name: RedPajamaGithub


text:
Enzyme Research
Enzyme Research / 2011 / Article
AbstractIntroductionMaterials and MethodsResultsDiscussionConclusionAbbreviationsAcknowledgmentsReferencesCopyright
Microbial Enzyme: Applications in Industry and in Bioremediation
View this Special Issue
Alexandre Arsenault, Hubert Cabana, J. Peter Jones, "Laccase-Based CLEAs: Chitosan as a Novel Cross-Linking Agent", Enzyme Research, vol. 2011, Article ID 376015, 10 pages, 2011. https://doi.org/10.4061/2011/376015
Laccase-Based CLEAs: Chitosan as a Novel Cross-Linking Agent
Alexandre Arsenault,1,2 Hubert Cabana,1,3 and J. Peter Jones2
1Environmental Engineering Laboratory, Department of Civil Engineering, University of Sherbrooke, 2 500 Boulevard de l'Université, Sherbrooke, QC, Canada
2Department of Chemical and Biotechnological Engineering, University of Sherbrooke, 2 500 Boulevard de l'Université, Sherbrooke, QC, Canada
3Étienne-Le Bel Centre de Recherche Clinique, Centre Hospitalier Universitaire de Sherbrooke, 3001, 12e Avenue Nord, Sherbrooke, QC, Canada
Academic Editor: J. Guisan
Accepted18 May 2011
Laccase from Coriolopsis Polyzona was insolubilized as cross-linked enzyme aggregates (CLEAs) for the first time with chitosan as the cross-linking agent. Concentrations between 0.01 and 1.867 g/L of chitosan were used and between 0.05 and 600 mM of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride. The laccase was precipitated using ammonium sulphate and cross-linked simultaneously. Specific activity and thermal stability of these biocatalysts were measured. Activities of up to 737 U/g were obtained when 2,2-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) was used as a substrate. Moreover, the stability of these biocatalysts was improved with regards to thermal degradation compared to free laccase when exposed to denaturing conditions of high temperature and low pH. The CLEAs stability against chemical denaturants was also tested but no significant improvement was detected. The total amount of ABTS to be oxidized during thermal degradation by CLEAs and free laccase was calculated and the insolubilized enzymes were reported to oxidize more substrate than free laccase. The formation conditions were analyzed by response surface methodology in order to determine an optimal environment for the production of efficient laccase-based CLEAs using chitosan as the cross-linking agent. After 24 hours of formation at pH 3 and at 4°C without agitation, the CLEAs exhibit the best specific activity.
There is growing interest in the use of enzymes in industrial bioprocesses dedicated to bioremediation purposes [1, 2]. Over the last years, laccases (polyphenoloxidase, EC 1.10.3.2) have gained attention due to their ability to convert a wide range of pollutants present in different environmental matrices [2–6]. Laccases are produced by fungi, higher plants, bacteria, and insects. These multicopper oxidases catalyze the oxidation of various phenol-like compounds, aromatic amines, and some inorganic compounds. They have received a growing attention due to their intrinsic properties such as relatively low substrate specificity, stability, and the simple and inexpensive culture media that could be used to produce them [7].
However, two major obstacles hamper the use of laccases in industrial bioprocesses: (1) their sensitivity to various environmental denaturants such as salts, solvents, and proteolytic enzymes [8] and (2) the difficulty of retaining the enzyme in a continuous flow bioreactor. These obstacles make the use of laccases a costly alternative to conventional environmental remediation alternatives.
In the interest of enhancing the industrial applicability of laccase, including the improvement of its stability and its repeated utilization, substantial efforts have been made to immobilize this enzyme with or without a solid support [9]. A well-known strategy to immobilize enzyme is to bind them covalently or through ionic interactions to a solid support or by trapping them in a matrix made of (bio)polymer [10]. These methods produce stable and reusable biocatalysts but can reduce considerably their specific activity [11]. The formation of cross-linked enzyme aggregates (CLEAs) can overcome this drawback. Insolubilization of enzyme as CLEAs is a simple technique to produce a biocatalyst with high enzyme activity per unit volume. Since it does not use a support to insolubilize the enzyme, it increases the specific activity of the biocatalyst formed [10]. An industrial process using CLEAs can make use of them in smaller reactors than the enzymes immobilized on a solid support.
CLEAs of laccase secreted by the white rot fungus (WRF) Coriolopsis polyzona have been prepared by Cabana et al. [5] using glutaraldehyde (GLU) as the cross-linking agent. These CLEAs have shown high enzyme activity and higher stability than free laccase against physical, chemical, and biological denaturants and good kinetics of reaction. These biocatalysts have been successfully used for the continuous treatment of water contaminated by the endocrine disrupting chemicals bisphenol A, nonylphenol, and triclosan [11]. In addition, Matijošyte et al. [12] have produced CLEAs with laccases from the WRF Trametes versicolor, Trametes villosa, and Agaricus bisporus. Their laccase CLEAs have also shown higher stability than the free enzymes and have been used in a laccase/mediator system for the successful oxidation of C5–C10 aliphatic alcohols.
The formation of CLEAs requires the use of a cross-linking agent. Generally, GLU is chosen for this purpose due to its low cost, ease of manipulation, and its ability to generate covalent bonds with most enzymes [10]. Even if this chemical is used in several applications, it presents adverse effects on the aquatic environment and on the health of the workers [13, 14]. GLU is suspected of reducing the hatching rate of some aquatic species eggs [14, 15]. It also has many effects on human health: it can provoke asthma, eczema, and respiratory tract and skin irritation [13]. The utilization of GLU for the formation of CLEAs dedicated to environmental applications can pose a problem because this cross-linking agent can potentially leach from the biocatalysts to the receiving environment where it can cause adverse effects to the aquatic ecosystems. To overcome this potential issue, an alternative must be found to cross-link the aggregated enzymes destined for environmental processes.
The renewable biopolymer chitosan represents an attractive candidate for the cross-linking of the enzyme aggregates. Chitosan is obtained from the deacetylation of the naturally occurring polymer chitin. The use of this biopolymer is favored by: (1) its high amino group content which favors link formation with enzymes, (2) its good mechanical strength and its resistance to chemical degradation, and (3) its biocompatibility and biodegradability [16]. Furthermore, its production is of low cost and ecologically interesting. The amino groups present on its chain can react with activated carboxylic group present in nonessential amino acid of the enzyme and form amide bonds [17]. Moreover, one molecule of chitosan can react with more than one carboxylic group and therefore more than one enzyme since it has many amino groups on its chain. This way, laccase aggregates can be covalently attached and permanently insolubilized. The activation of the carboxylic group is done through carbodiimide chemistry. 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDAC) is perhaps one of the most popular chemicals used for the binding of an enzyme to chitosan. Some investigations have used this strategy for the conjugation of free laccases to chitosan [18, 19]. However, the objective of this study is to use chitosan and EDAC as a cross-linking complex rather than a binding support.
The first objective of this study was to produce CLEAs of laccase from the WRF C. polyzona by using chitosan as the cross-linking agent and characterize them. The second objective was to determine the effects of the conditions of formation (pH, temperature, reaction time, and shaking speed) on the characteristics of the CLEAs produced by this new approach.
2.1. Materials
The WRF strain C. polyzona (MUCL 38443) was provided by the Belgian Coordinated Collections of Microorganisms (BCCM/MUCL). Cellulose membranes for dialysis came from Fisher Scientific (Pittsburgh, PA). All other reactants used came from Sigma-Aldrich (St. Louis, MO) and were of analytical grade or the highest grade available.
2.2. Laccase Production
The inoculum was grown in a rotary shaker at 150 rpm and 27°C in 250-mL Erlenmeyers containing 100 mL of standard medium: 10 g/L glucose, 2 g/L NH4NO3, 0.8 g/L KH2PO4, 0.4 g/L Na2HPO4, 0.5 g/L MgSO47H2O, 2 g/L yeast extract. The medium was adjusted to pH 6.0 with 2 M NaOH prior to autoclaving. After 10 days of cultivation or after reaching a laccase activity over 2000 U/L in the broth, the biomass was filtered and the supernatant was conserved. Enzymes were precipitated using 600 g/L ammonium sulphate. The resulting solution was then centrifuged at 10000 ×g for 5 minutes, and the supernatant was removed. The precipitation steps were repeated until no laccase activity was detected in the supernatant. The precipitates were solubilized in deionized water. The resulting preparation was dialyzed against distilled water using a regenerated cellulose membrane with a molecular cut-off of 13 kDa and then used as the source of laccase.
2.3. Enzyme Assay
Laccase activity was determined by monitoring the oxidation of 2,2-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) to its radical cation (ABTS•+) [20]. The assay mixture contained 0.5 mM ABTS. The pH was adjusted to 3 using 60 mM citric acid/disodium hydrogen phosphate buffer at room temperature. One unit of activity was defined as the amount of enzyme forming 1 mmol of ABTS•+ per min.
2.4. Chitosan Solution Preparation
Chitosan (mean molecular weight of 750 kDa and 64% deacetylated) was solubilized in HCl (0.1 M) solution to a final concentration of 5 g/L. The solution was shaken in a sonification bath during one hour.
2.5. CLEAs Production
CLEAs were prepared by simultaneously aggregating and cross-linking the laccases [5]. In 10 mL of solution, 550 g/L of ammonium sulphate, 10 units of laccase, chitosan and EDAC were added to a 500 mM phosphate buffer at pH 5. This reaction solution was stored at 4°C for 48 h. These formation conditions were used unless other conditions are cited. Subsequently, the solution was centrifuged at 10000 ×g for 5 minutes. The supernatant was discarded, and the precipitate was washed with 3 mL of 50 mM acetate buffer at pH 6.5 and centrifuged at 10000 ×g for 5 minutes. The washing and centrifugation steps were then repeated with deionised water until no laccase activity was detected in the supernatant. The washed precipitate was then suspended in 5 mL of deionised water. The CLEAs samples were identified as follows: CLEA-concentration of chitosan (in g/L), concentration of EDAC (in mM) (e.g., CLEA-1.0-200 for CLEA prepared with 1,0 g/L of chitosan and 200 mM of EDAC).
2.5.1. Concentrations Optimization
The impact of chitosan and EDAC concentrations on the performances of CLEAs was tested in two experimental designs. The concentrations used in these experiments are shown in Table 1. The center composite design was modified to have positive values of EDAC concentrations. The specific activity, the thermal stability, the half-life under thermal degradation, and the total amount of ABTS oxidized by the prepared CLEAs were evaluated. Design-Expert 6.0 (Stat-Ease, Minneapolis, MN) software was used to evaluate the influence of chitosan and EDAC concentrations on these parameters.
Experimental design type Sample names Chitosan concentration (g/L) EDAC concentration (mM)
32 CLEA-0.2-200 0.2 200
CLEA-0.2-400 0.2 400
Modified Central Composite CLEA-0.5-1 0.5 1
CLEA-1.5-1 1.5 1
CLEA-1.0-0.05 1.0 0.05
CLEA-1.0-50.5 1.0 50.5
CLEA-0.134-50.5 0.134 50.5
Chitosan and EDAC concentrations tested for the optimization of the CLEAs formation at 4°C during 48 hours.
2.5.2. Optimization of CLEAs Preparation Conditions
The physical conditions of CLEAs formation were also optimized to reach better specific activity and thermal stability. The different conditions tested are shown in Table 2. The concentrations of chitosan and EDAC were fixed at 1.87 g/L and 50.5 mM, respectively. Each condition was tested twice for a total of 72 samples. The specific activity and thermal stability of each sample were measured twice each, and an analysis of variance was conducted on the results with the Stat-Ease software Design-Expert 6.0 (Minneapolis, MN).
Condition Value
pH 3 and 5
Temperature 4, 20 and 30°C
Agitation 0 and 150 RPM
Reaction time 8, 16 and 24 hours
Conditions tested for the optimization of CLEAs characteristics.
2.6. CLEAs and Free Laccase Thermal Stability
The thermal stability was determined by monitoring free laccase and CLEAs activity through time when exposed to a temperature of 40°C and a pH of 3. CLEAs or laccase solution (400 μL) was incubated in 400 μL of 50 mM citric acid/sodium phosphate buffer. Activity was measured twice and at different moments during the degradation. The inactivation of free laccase and CLEAs was modeled using the 3-parameter phenomenological model proposed by Aymard and Belarbi [21]. This biexponential model is expressed by
The ratio represents the enzyme activity remaining after a time t compared to initial activity . The physical meaning of the parameters and their expressions as a function of individual rate constants differs according to the mechanism considered. This expression can be used irrespective of the thermal inactivation mechanism involved [21].The values of the different parameters of this model (, alpha, and beta) were obtained by curve fitting of the plot of the residual enzyme activity versus time using the Sigma Plot 7.0 software (SPSS Inc., Chicago, IL).
2.7. Total Amount of ABTS Oxidized under Denaturing Conditions
To consider both the initial activity of the biocatalysts formed and their thermal stability, the total amount of ABTS oxidized by the different biocatalysts under the thermal denaturing conditions was calculated. To do so the Aymard-Belarbi model was integrated to determine the theoretical total amount of ABTS oxidized under the thermal degradation conditions used:
The amount of ABTS oxidized ( (mmol)) was calculated on a 24-hour interval using the parameters , alpha, and beta obtained from the thermal degradation tests (see Section 2.7) and the initial activity of the biocatalyst ().
2.8. Enzyme Kinetics
Michaelis-Menten parameters of CLEAs and free laccase were determined using ABTS as a substrate at various concentrations (0.05, 0.1, 0.2, 0.4, 0.5, 0.75, 1.0, 1.5, and 2.0 mM). The activity with each substrate concentration was determined twice. The parameter values were obtained by curve fitting the plot of reaction rate versus substrate concentrations using the Sigma Plot 7.0 software.
2.9. CLEAs and Free Laccase Stability to Chemicals Denaturants
The CLEAs and free laccase were also exposed to chemicals to determine their stability against denaturing environments. Solutions of CaCl2 (10 μM), ZnCl2 (10 μM), ethylenediaminetetraacetic acid (EDTA) (10 μM), NaN3 (30 μM), acetone (25% (v/v)), and methanol (25% (v/v)) were prepared separately by dissolving the powders or diluting the solvents in a 50 mM citric acid/phosphate buffer at pH 3. CLEAs and free laccase duplicates (100 μL each) were exposed to each of these chemicals (400 μL) separately for 4 hours.
The stability of CLEAs and free laccase has also been tested after an exposition of 24 hours to an effluent of a wastewater treatment plant (WWTP). The effluent was taken from the Mont St-Grégoire (Québec, Canada) WWTP. As for the stability against chemical denaturants test, 100 μL of CLEAs or free laccase solution was mixed with 400 μL of denaturing medium. The methods used to characterize the wastewater samples were ICP-MS for total phosphorus content [22], infrared spectroscopy for COD [22], gravimetric for suspended particles [22], spectrophotometry for nitrogen-NH3 [22], HPLC for nitrites and nitrates [22], and hexane extraction and gravimetric method for oils and grease [22].
2.10. Scanning Electron Microscopy of CLEAs
Scanning electron micrographs (SEMs) of CLEAs were obtained on Hitachi S-4700 FESEM and S-3000N VPSEM (Tokyo, Japan) electron microscopes. The samples were previously dried at ambient temperature and then coated with platinum using an Emitech K550 (Ashford, UK) sputter coater.
2.11. Particle Size of CLEAs
The size of the CLEAs was measured by photon correlation spectroscopy (PCS) with a ZetaPlus zeta potential analyzer (Brookhaven Instruments Corporation, Holtsville, NY, USA). CLEAs suspension was diluted in a 1 : 1 ratio with deionised water in 2 mL cuvettes at 20°C. Each CLEAs particle size was measured twice.
3.1. Preliminary Screening
Table 3 shows the specific activities, the thermal stability, the half-life under thermal degradation, and the total amount of ABTS oxidized by CLEAs and free laccase. The specific activity of the CLEAs prepared was between 16 and 737 U/g while the free laccase used had a specific activity of 284 U/g.
Sample Specific activity* (U/g) Thermal stability*, a (%) Aymard-Belarbi parameters Half-life (h) Amount of ABTS oxidized in 24 h under denaturing conditions (mmol/U)
C α β
CLEA-0.2-200 298 ± 44 7.9 ± 1.2 0,71 0,15 1,78 2.36 0.305
CLEA-0.2-400 65.3 ± 1.9 13.5 ± 0.4 0,12 −0,01 0,19 5.79 0.135
CLEA-0.2-600 42.9 ± 3.5 17.7 ± 1.4 0,38 0,04 0,77 2.79 0.101
CLEA-0.6-200 18.6 ± 3.5 81.5 ± 15.3 0,79 0,006 183166 44.2 0.178
CLEA-0.6-400 103 ± 21 31.4 ± 6.4 0,74 0,025 49,72 0.84 0.094
CLEA-0.6-600 133 ± 29 34.4 ± 7.5 1,04 0,04 7,65 18.7 0.172
CLEA-1.0-200 77.1 ± 0.3 47.6 ± 0.2 19,3 0,34 0,34 20.8 0.159
CLEA-1.0-400 14.7 ± 3.2 68.6 ± 14.9 1,32 0,13 0,125 62.3 0.301
CLEA-1.0-600 24.4 ± 0.2 22.3 ± 0.2 0,76 0,027 41,2 16.5 0.123
CLEA-0.5-1 35.6 ± 4.1 13.6 ± 7.9 0,32 0,03 1,27 1.51 0.379
CLEA-0.5-100 156 ± 6 38.5 ± 4.6 0,76 0,026 56,8 5.84 0.334
CLEA-1.5-1 69 ± 14 82.9 ± 17.3 0,80 0,006 34,8 7.00 0.259
CLEA-1.5-100 39.5 ± 1.9 118.5 ± 1.8 1,58 0,03 0,03 78.48 1.103
CLEA1.0-0.05 21.2 ± 6.6 85.5 ± 28.6 13,4 0,025 0,025 2.77 ND
CLEA-1.0-50.5 78.8 ± 9.9 60.0 ± 4.8 0,51 0,027 15048 7.54 0.565
CLEA-1.0-136 737 ± 24 68.8 ± 2.7 0,465 0,024 2,62 1.29 0.539
CLEA-0.134-50.5 186 ± 10 37.2 ± 3.2 0,413 0,005 0,117 11.9 ND
CLEA-1.867-50.5 16.0 ± 2.9 91.8 ± 2.5 1,58 0,0264 0,0259 81.1 1.094
Free laccase 284 ± 33 21.3 ± 2.5 0,433 0,0189 0,0997 19.7 0.787
*The values represent means of duplicate experiments for the specific activity and thermal stability ± standard deviation.
aResidual activity after 24 hours at 40°C and pH 3.
ND: Not determined.
Specific activity, thermal stability, and total amount of ABTS oxidized by all the prepared samples.
Free laccase has a half-life of 19 hours while CLEA-1.867-50.5 and CLEA-1.5-100 have half-life of 81 and 78 hours, respectively. CLEA-0.5-1, CLEA1.0-136, and CLEA-0.6-400 all have a half-life under 2 hours. These half-lifes were calculated by using the kinetics of thermal degradation model proposed by Aymard and Belarbi [21].
In order to have a good basis to compare biocatalysts with different initial activities and stability, the total amount of ABTS theoretically oxidized during the thermal denaturation of the biocatalysts for 24 hours was calculated. After a 24-hour period of thermal denaturation, CLEA-1.5-100 and CLEA-1.867-50.5 both oxidized more ABTS than free laccase. CLEA-1.5-100 and CLEA-1.867-50.5, respectively, oxidize 1.103 mmol/U and 1.094 mmol/U of ABTS while free laccase oxidizes only 0.787 mmol/U. CLEA-1.0-136 and CLEA-1.0-50.5 have catalyzed a comparable number of reactions than free laccase after 24 hours (resp., 0.539 and 0.565 mmol/U).
From this point, only the four samples that oxidized the highest amount of ABTS in 24 h have been used to characterize the CLEAs. These samples are CLEA-1.0-136, CLEA-1.5-100, CLEA-1.867-50.5, and CLEA-1.0-50.5. They were the most promising samples for the preparation of efficient and stable biocatalysts dedicated to bioremediation purposes.
3.2. Optimization of CLEAs Preparation Conditions
Table 4 presents the results of the analysis of variance (ANOVA) performed on the results of specific activity for each of the prepared samples.
Source Sum of squares Degree of freedom Mean squares F Value P-value
Model 101.15 8 12.64 5.04 <0.0001
Agitation 5.21 1 5.21 2.07 0.1547
Temperature 27.36 2 13.68 5.45 0.0066
pH 7.09 1 7.09 2.82 0.0978
Reaction time 36.98 2 18.49 7.37 0.0013
Interaction Agitation/Reaction time 24.01 2 12.00 4.78 0.0116
Residues 158.07 63 2.51
Lack of Fit 86.03 27 3.19 1.59 0.0955
Pure error 72.03 36 2.00
Total 259.22 71
Results of the analysis of variance (ANOVA) performed on the specific activities of the CLEA-1.867-50.5.
The ANOVA performed show a significant effect of the temperature and reaction time on the specific activity of the CLEAs. Figures 1(a) and 1(b) show how these parameters influence the specific activity of the CLEAs while Figure 1(c) illustrates the interaction between the agitation and reaction time.
Influence of the temperature (a), the reaction time (b), and the interaction between agitation and reaction time (c) on the specific activity of the CLEA-1.867-50.5. On graph (c): Reaction time of 8 hours (black square), 16 hours (red triangle), and 24 hours (green diamond).
The specific activity of CLEAs prepared at 30°C is lower than those prepared at 20°C and 4°C. The longer the cross-linking reaction is, the more active the CLEAs will be. The reaction has to last more than 16 hours though according to Figure 1(b).
3.3. Michaelis-Menten Kinetic Parameters
Table 5 shows the enzyme kinetic parameters for the oxidation of ABTS by CLEAs and free laccase. They all follow Michaelis-Menten kinetics according to the correlation factor obtained by the curve-fitting analysis (results not shown).
Sample Km (mM) Kcat (μmol/s/mg) (L mg/s)
CLEA-1.0-136 0.083 ± 0,015 30.760 ± 0.900 0.369
CLEA-1.5-100 0.156 ± 0,026 2.524 ± 0.091 0.016
CLEA-1.867-50.5 0.259 ± 0,044 0.986 ± 0.046 0.004
CLEA-1.0-50.5 0.101 ± 0,027 5.295 ± 0.250 0.053
Free laccase 0.082 ± 0,010 2.694 ± 0.060 0.033
aResults are mean of triplicate measures ± standard deviation.
Michaelis-Menten kinetic constants of laccase CLEAs for the oxidation of ABTSa.
CLEA-1.0-136 and CLEA-1.0-50.5 have a comparable affinity for ABTS to free laccase according to the Michaelis-Menten constant (). Free laccase has a of 0.082 mM, and CLEA-1.0-136 and CLEA-1.0-50.5 are, respectively, 0.083 and 0.101 mM. The same CLEAs have a higher maximum rate of ABTS transformation than free laccase. CLEA-1.0-136 and CLEA-1.0-50.5 have of 30.760 and 5.295 μmol/s/mg, respectively, while free laccase has a of 2.694 μmol/s/mg. The biocatalytic efficiencies () of CLEAs are equivalent to free laccase except for CLEA-1.0-136 that has a 10 times higher than free laccase.
3.4. Stability against Chemical Denaturants
Figure 2 shows the resistance to chemical degradation of free laccase and CLEAs to various chemical denaturants. The resistance of CLEAs and free laccase to the chemical denaturants was similar except for the sodium azide and the organic solvents.
Residual activity of free laccase and CLEAs after 4 hours of incubation with various chemical denaturants at a pH of 3 and 20°C. From left to right: CLEA-1.0-136 (dark gray), CLEA-1.5-100 (light gray), CLEA-1.867-50.5 (gray), CLEA-1.0-50.5 (black), and free laccase (gray). Values represent means of triplicate results ± standard deviation.
When exposed to sodium azide, the residual activity of CLEAs is slightly lower than free laccase but not significantly. For the acetone, all CLEAs had a higher residual activity than free laccase except CLEA-1.867-50.5. And for the methanol, only CLEA-1.5-100 and CLEA-1.867-50.5 lost more apparent activity than free laccase and the other CLEAs.
3.5. Stability against Wastewater Effluent
To confirm the usability of our biocatalysts in a wastewater treatment bioprocess, the stability of CLEAs and free laccase has been determined by exposing the biocatalysts to a sample of WWTP effluent for which the characteristics are shown in Table 6.
Type of contaminant Concentration
Total phosphorus (mg/L) 1.8
Dissolved COD (mg/L) 36
Particles in suspension (mg/L)* 820
Nitrogen-NH3 (mg-N/L) 0.61
Nitrites (mg-N-NO2/L) 2.53
Nitrates (mg-N-NO3/L) 8.12
Oils and greases (mg/L) 24.1
*Before using this sample, it has been filtrated through a 0.02 μm filter so we assume there are no particles in suspension left.
Characteristics of the effluent taken at the Mont St-Grégoire WWTP after the settling basin.
The residual activities of free laccase and CLEAs after a 24-hour exposure to this wastewater effluent are shown in Figure 3.
Residual activity of free laccase and CLEAs after 24 hours of incubation in a wastewater effluent collected from the WWTP of Mont St-Grégoire (Qc, Canada).
Free laccase seems to be quite resistant to the wastewater effluent chosen (67.2% residual activity) but CLEA-1.5-100 and CLEA-1.867-50.5 have a residual activity of 107.5% and 93.8%, respectively. The other two samples are less stable to wastewater effluent than free laccase by 14% for CLEA-1.0-50.5 and 22% for CLEA-1.0-136.
3.6. SEMs
Figure 4 shows the SEMs of chitosan (Figure 4(a)) and CLEA-1.0-50.5 (Figure 4(b)). The chitosan is more than 50 μm long according to Figure 4(a) while the laccase-based CLEA is approximately 10 μm long (Figure 4(b)). The laccase aggregates seem to be attached to the chitosan backbone. The structure of the CLEA appears to be amorphous and relaxed rather than compact and uniform.
SEMs of (a) chitosan and (b) CLEA-1.0-50.5.
3.7. Particle Size
The size of the CLEAs produced has been determined by PCS, and the results are presented in Table 7. The CLEA-1.5-100 and CLEA-1.0-136 are approximately 35% smaller than CLEAs-1.867-50.5 and CLEA-1.0-50.5. The standard deviation is between 800 and 1200 nm.
CLEA-1.5-100 CLEA-1.0-136 CLEA-1.867-50.5 CLEA-1.0-50.5
Average (nm) 1694.3 1716.5 2313.7 2308.6
Standard deviation (nm) 900.9 835.9 1171.6 860.3
Particle sizes of CLEAs as determined by PCS.
It is important to determine good reaction conditions to produce efficient biocatalysts due to the possible denaturation of the enzyme or mass transfer limitations associated with the cross-linking agent [23]. The activator EDAC is known to react with carboxylic acids, like aspartic acid or glutamic acid found in laccase, to form O-acylisourea intermediates. These active intermediates can then react with a nucleophilic species such as a primary amine to form an amide bond [24]. In the present case, the primary amine is found in chitosan but also in the amino acid lysine present in laccase. A high concentration of the activator can reduce CLEAs activity according to the specific activities of CLEAs prepared with 200 mM or more of EDAC. Bindhu and Abraham [25] observed the same phenomenon when immobilizing horseradish peroxidase on chitosan. It is probably caused by the nonspecific activation of carboxyl groups provoking a perturbation in the tridimensional structure of the enzyme. Activity reduction is also observed when the amount of chitosan used is higher than 1.0 g/L. The dilution of laccase concentration by chitosan or a steric hindrance phenomena caused by the addition of the biopolymer can explain the loss of specific activity. The tridimensional structure of the CLEAs could also limit the diffusion of the substrate to the catalytic site therefore reducing the specific activity [26]. The specific activities of chitosan-based CLEAs obtained are lower than the specific activity of free laccase except for CLEA-1.0-136 and CLEA-0.2-200. D'Annibale et al. [26] used chitosan to immobilize laccase with GLU, and the biocatalysts retained 45% of the initial activity (specific activity of immobilized enzyme divided by the specific activity of free enzyme). Zhang et al. [23] produced laccase-chitosan biocatalysts that retained 52.2% of initial activity. The laccase-chitosan CLEAs retained up to 259% of initial specific activity. Considering that chitosan can account for an important mass in the biocatalyst, the retention of activity of 259% can indicate a hyperactivation of laccase when attached to chitosan [27]. Laccase attached to chitosan by Cabana et al. [27] also exhibited a hyperactivation of 265%.
An important factor to consider for specific activity optimization is the temperature of the formation of the CLEAs. The CLEAs prepared at 4°C had the highest activity, comparable to those prepared at 20°C. The CLEAs formed at 30°C however are less active. It is probably because the laccase was thermally degraded therefore yielding less active CLEAs. The optimal reaction time seems to be at 24 hours, but longer times have not yet been tested. Higher specific activity could be obtained for longer reaction time. The agitation seems to reduce the influence of reaction time. The CLEAs prepared under agitation at 150 RPM have a comparable specific activity for all the reaction times tested.
The thermal stability of CLEAs was significantly higher than the stability of free laccase. It can be explained by the formation of chemical bonds between chitosan and laccase. Bonds can also be formed between different molecules of laccase and inside the same enzyme. These decrease the mobility of the enzyme, therefore providing a greater resistance to thermal degradation, often caused by drastic conformational change [8, 28]. It can also be explained by the presence of chitosan which may somehow coat the enzyme. CLEAs prepared with GLU by Cabana et al. [5] had a residual activity between 20% and 40% after a 24-hour period under the same denaturing conditions.
The kinetics of thermal degradation of free laccase are accelerated, and these soluble enzymes present almost no activity after 24 hours of incubation under denaturing conditions while the CLEAs stayed active for a longer period of time. The experiment was run over 48 hours, and CLEAs were still active after two days of thermal degradation while laccase lost almost all its activity within the first 24 hours. The same tendency was observed by Cabana et al. [5] but the CLEAs prepared in that previous study were degraded faster than the chitosan-based CLEAs.
To our knowledge, no studies have determined the amount of substrate theoretically oxidized by laccase under thermal degradation. By using this approach, the thermal stability and the initial specific activity are integrated into one parameter describing the global performance of a biocatalyst. The model proposed by Aymard and Belarbi [21] is ideal for it describes well the kinetics of thermal degradation of our CLEAs. This model was integrated to obtain the total amount of substrate a laccase can oxidize when exposed to the denaturing conditions tested. It appears that, for each unit of laccase activity, the CLEAs can oxidize more molecules of ABTS than free laccase after 24 hours. The fast decay of free laccase activity can explain these results. The amount of substrate oxidized by the CLEAs prepared by Cabana et al. [5] with GLU was calculated on a 24-hour period and compared the chitosan-based CLEAs. The CLEAs made without a coprotein oxidized 0.642 mmol/U of ABTS while the CLEAs with 0.01, 0.1, and 1 mg/U of bovine serum albumin oxidized, respectively, 0.738, 0.792, and 0.954 mmol/U of ABTS. CLEA-1.867-50.5 and CLEA-1.5-100 oxidized more substrate (resp., 1.094 and 1.103 mmol/U of ABTS) while CLEA-1.0-136 and CLEA-1.0-50.5 oxidized less substrate than all the CLEAs from Cabana et al. [5].
The kinetic study, based on the Michaelis-Menten constant (), shows that the different CLEAs have almost the same affinity for the substrate (ABTS). However, the CLEAs are better biocatalysts when the turnover number is used for comparison (), and free laccase is better when it is the biocatalytic efficiency that is the base of the comparison (). In terms of kinetic properties, CLEA-1.867-50.5 is not as good as the others. CLEAs prepared with GLU in Cabana's group proved to have a turnover number 6 times higher than that of free laccase [5] but CLEA-1.0-136 has a 15 times higher than that of the free laccase. This can be explained by the hyperactivation of laccase when in contact with chitosan [27].
The stability of CLEAs and free laccase has been measured in solutions containing various chemicals known to denature enzymes. These chemicals inhibit or denature laccase in various ways. The chloride salts are known to inhibit laccase activity by raising the ionic strength of the solution [28]. The solvents lower the strength of hydrophobic interactions so the equilibrium is shifted to the denaturated state [28]. Sodium azide binds to the active site of laccase and modifies its structure [29], and EDTA is a chelator that can take out the copper ions present in the catalytic site of the laccase [30]. The amorphous structure of CLEAs reduces the mass transfer of EDTA to the catalytic site of the enzymes [5]. The CLEAs structure does not limit the diffusion of smaller molecules like chloride ions or sodium azide to the catalytic site but their rigidity reduces their denaturation by hydrophobic interactions. Therefore, the stability of CLEAs is not significantly higher than free laccase when exposed to salts or chelators but is slightly increased towards solvents.
Little research has been done to determine the stability of laccase in a real wastewater effluent. Auriol et al. [31, 32] used real wastewater effluents to test the capacity of commercial laccase from the WRF Trametes versicolor to transform the endocrine disrupting chemicals estrone, estriol, and estradiol, but the stability of the enzyme was not evaluated. Laccase can be used in wastewater effluents to eliminate phenolic compounds, and it is important to have a good stability in this complex medium. These results show that some CLEAs had a higher stability than free laccase probably because the denaturants present in the effluent cannot get to the catalytic site of the CLEAs due to their amorphous structure. The rigidity of the CLEAs is another factor in the enhancement of stability towards organic solvents. Since free laccase and CLEAs proved to be quite stable in a wastewater effluent, they are good candidate to eliminate phenolic compounds.
According to Schoevaart et al. [33], CLEAs can be separated in two types based on their structure. Type 1 aggregate has a uniform structure and has a diameter around 1 μm. Type 2 aggregates are usually smaller with a diameter around 0.1 μm. According to this classification, chitosan-based CLEAs have a structure similar to that of type 1 CLEAs. The results obtained concerning the particle size determined by PCS of the CLEAs confirm our observations with the SEM. The particles have a diameter of about 1700–2300 nm. An interesting fact is that the CLEAs prepared with more EDAC are smaller. This could be explained by a more important cross-linking level of the CLEAs formed. The small CLEAs have a higher surface/volume ratio that helps diffusion to the catalytic site of laccase and therefore makes them more active than the larger CLEAs (Table 6).
The results presented in this study demonstrate a novel method for making CLEAs with laccase by using chitosan as the cross-linking agent. The CLEAs formed are stable, active biocatalysts which are suitable for use in environmental and industrial bioprocesses. The method used should be applicable to many other enzymes although this was not demonstrated in this study. Chitosan used here for the first time as the cross-linking agent used to form CLEAs has considerable advantages from environmental and worker safety points of view over the traditional chemicals traditionally used. Because chitosan is obtained from aquatic organisms byproducts, its use is in good conformity with sustainable development. Its abundance and renewable nature make it an attractive candidate for the production of insolubilized enzyme, while its biocompatibility makes it an environmentally harmless reactant that does not put the health of the workers at risk.
CLEAs: Cross-linked enzyme aggregates
WRF: White rot fungus
GLU: Glutaraldehyde
EDAC: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
ABTS: 2,2_-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid)
SEMs: Scanning electron micrographs
PCS: Photon correlation spectroscopy.
This project was supported by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC). The authors are also thankful to Serge Bérubé, Maxime Sirois-Gosselin, and Catherine Beauregard-Paultre for their technical support.
S. K. Ahuja, G. M. Ferreira, and A. R. Moreira, "Utilization of enzymes for environmental applications," Critical Reviews in Biotechnology, vol. 24, no. 2-3, pp. 125–154, 2004. View at: Publisher Site | Google Scholar
E. Torres, I. Bustos-Jaimes, and S. Le Borgne, "Potential use of oxidative enzymes for the detoxification of organic pollutants," Applied Catalysis B, vol. 46, no. 1, pp. 1–15, 2003. View at: Publisher Site | Google Scholar
P. Giardina, V. Faraco, C. Pezzella, A. Piscitelli, S. Vanhulle, and G. Sannia, "Laccases: a never-ending story," Cellular and Molecular Life Sciences, vol. 67, no. 3, pp. 369–385, 2010. View at: Publisher Site | Google Scholar
A. Kunamneni, I. Ghazi, S. Camarero, A. Ballesteros, F. J. Plou, and M. Alcalde, "Decolorization of synthetic dyes by laccase immobilized on epoxy-activated carriers," Process Biochemistry, vol. 43, no. 2, pp. 169–178, 2008. View at: Publisher Site | Google Scholar
H. Cabana, J. P. Jones, and S. N. Agathos, "Preparation and characterization of cross-linked laccase aggregates and their application to the elimination of endocrine disrupting chemicals," Journal of Biotechnology, vol. 132, no. 1, pp. 23–31, 2007. View at: Publisher Site | Google Scholar
D. Wesenberg, I. Kyriakides, and S. N. Agathos, "White-rot fungi and their enzymes for the treatment of industrial dye effluents," Biotechnology Advances, vol. 22, no. 1-2, pp. 161–187, 2003. View at: Publisher Site | Google Scholar
V. Elisashvili, M. Penninckx, and E. et Kachlishvili, "White-rot basidiomycetes: lignocellulolytic enzymes production in bioconversion of plant raw materials," International Biodeterioration and Biodegradation, 2006. View at: Google Scholar
L. Lu, M. Zhao, and Y. Wang, "Immobilization of laccase by alginate-chitosan microcapsules and its use in dye decolorization," World Journal of Microbiology and Biotechnology, vol. 23, no. 2, pp. 159–166, 2007. View at: Publisher Site | Google Scholar
N. Durán, M. A. Rosa, A. D'Annibale, and L. Gianfreda, "Applications of laccases and tyrosinases (phenoloxidases) immobilized on different supports: a review," Enzyme and Microbial Technology, vol. 31, no. 7, pp. 907–931, 2002. View at: Publisher Site | Google Scholar
R. A. Sheldon, "Cross-linked enzyme aggregates (CLEA®s): stable and recyclable biocatalysts," Biochemical Society Transactions, vol. 35, no. 6, pp. 1583–1587, 2007. View at: Publisher Site | Google Scholar
H. Cabana, C. Alexandre, S. N. Agathos, and J. P. Jones, "Immobilization of laccase from the white rot fungus Coriolopsis polyzona and use of the immobilized biocatalyst for the continuous elimination of endocrine disrupting chemicals," Bioresource Technology, vol. 100, no. 14, pp. 3447–3458, 2009. View at: Publisher Site | Google Scholar
I. Matijošyte, I. W. C. E. Arends, S. de Vries, and R. A. Sheldon, "Preparation and use of cross-linked enzyme aggregates (CLEAs) of laccases," Journal of Molecular Catalysis B: Enzymatic, vol. 62, no. 2, pp. 142–148, 2010. View at: Publisher Site | Google Scholar
T. Takigawa and Y. Endo, "Effects of glutaraldehyde exposure on human health," Journal of Occupational Health, vol. 48, no. 2, pp. 75–87, 2006. View at: Publisher Site | Google Scholar
D. F. Raikow, P. F. Landrum, and D. F. Reid, "Aquatic invertebrate resting egg sensitivity to glutaraldehyde and sodium hypochlorite," Environmental Toxicology and Chemistry, vol. 26, no. 8, pp. 1770–1773, 2007. View at: Publisher Site | Google Scholar
E. Emmanuel, K. Hanna, C. Bazin, G. Keck, B. Clément, and Y. Perrodin, "Fate of glutaraldehyde in hospital wastewater and combined effects of glutaraldehyde and surfactants on aquatic organisms," Environment International, vol. 31, no. 3, pp. 399–406, 2005. View at: Publisher Site | Google Scholar
B. Krajewska, "Application of chitin- and chitosan-based materials for enzyme immobilizations: a review," Enzyme and Microbial Technology, vol. 35, no. 2-3, pp. 126–139, 2004. View at: Publisher Site | Google Scholar
A. Ghanem and A. Ghaly, "Immobilization of Glucose Oxidase in Chitosan Gel Beads," Journal of Applied Polymer Science, vol. 91, no. 2, pp. 861–866, 2004. View at: Publisher Site | Google Scholar
G. Delanoy, Q. Li, and J. Yu, "Activity and stability of laccase in conjugation with chitosan," International Journal of Biological Macromolecules, vol. 35, no. 1-2, pp. 89–95, 2005. View at: Publisher Site | Google Scholar
R. Vazquez-Duhalt, R. Tinoco, P. D'Antonio, L. D. Timmie Topoleski, and G. F. Payne, "Enzyme conjugation to the polysaccharide chitosan: smart biocatalysts and biocatalytic hydrogels," Bioconjugate Chemistry, vol. 12, no. 2, pp. 301–306, 2001. View at: Publisher Site | Google Scholar
R. Bourbonnais and M. G. Paice, "Oxidation of non-phenolic substrates. An expended role for laccase in lignin biodegradation," FEBS Letters, vol. 267, no. 1, pp. 99–102, 1990. View at: Publisher Site | Google Scholar
C. Aymard and A. Belarbi, "Kinetics of thermal deactivation of enzymes: a simple three parameters phenomenological model can describe the decay of enzyme activity, irrespectively of the mechanism," Enzyme and Microbial Technology, vol. 27, no. 8, pp. 612–618, 2000. View at: Publisher Site | Google Scholar
American Public Health Association (APHA), American Water Works Association (AWWA), and Water Environment Federation (WEF), Edited by A. E. Greenberg, L.S. Clesceri, A.D. Eaton, American Public Health Association (APHA), American Water Works Association (AWWA), Water Environment Federation (WEF), Baltimore, Md, USA.
J. Zhang, Z. Xu, H. Chen, and Y. Zong, "Removal of 2,4-dichlorophenol by chitosan-immobilized laccase from Coriolus versicolor," Biochemical Engineering Journal, vol. 45, no. 1, pp. 54–59, 2009. View at: Publisher Site | Google Scholar
Hermanson G. T, Ed., Bioconjugate Techniques, Academic Press, San Diego, Calif, USA, 1996.
L. V. Bindhu and E. T. Abraham, "Immobilization of horseradish peroxidase on chitosan for use in nonaqueous media," Journal of Applied Polymer Science, vol. 88, no. 6, pp. 1456–1464, 2003. View at: Publisher Site | Google Scholar
A. D'Annibale, S. Rita Stazi, V. Vinciguerra, E. Di Mattia, and G. Giovannozzi Sermanni, "Characterization of immobilized laccase from Lentinula edodes and its use in olive-mill wastewater treatment," Process Biochemistry, vol. 34, no. 6-7, pp. 697–706, 1999. View at: Publisher Site | Google Scholar
H. Cabana, A. Ahamed, and R. Leduc, "Conjugation of laccase from the white rot fungus Trametes versicolor to chitosan and its utilization for the elimination of triclosan," Bioresource Technology, vol. 102, no. 2, pp. 1656–1662, 2011. View at: Publisher Site | Google Scholar
S. R. Couto and J. L. Toca, "Inhibitors of laccases: a review," Current Enzyme Inhibition, vol. 2, no. 4, pp. 343–352, 2006. View at: Publisher Site | Google Scholar
G. Battistuzzi, G. Di Rocco, A. Leonardi, and M. Sola, "1H NMR of native and azide-inhibited laccase from Rhus vernicifera," Journal of Inorganic Biochemistry, vol. 96, no. 4, pp. 503–506, 2003. View at: Publisher Site | Google Scholar
B. Ghorbel, A. Sellami-Kamoun, and M. Nasri, "Stability studies of protease from Bacillus cereus BG1," Enzyme and Microbial Technology, vol. 32, no. 5, pp. 513–518, 2003. View at: Publisher Site | Google Scholar
M. Auriol, Y. Filali-Meknassi, R. D. Tyagi, and C. D. Adams, "Laccase-catalyzed conversion of natural and synthetic hormones from a municipal wastewater," Water Research, vol. 41, no. 15, pp. 3281–3288, 2007. View at: Publisher Site | Google Scholar
M. Auriol, Y. Filali-Meknassi, C. D. Adams, R. D. Tyagi, T. N. Noguerol, and B. Piña, "Removal of estrogenic activity of natural and synthetic hormones from a municipal wastewater: efficiency of horseradish peroxidase and laccase from Trametes versicolor," Chemosphere, vol. 70, no. 3, pp. 445–452, 2008. View at: Publisher Site | Google Scholar
R. Schoevaart, M. W. Wolbers, M. Golubovic et al., "Preparation, optimization, and structures, of cross-linked enzyme aggregates (CLEAs)," Biotechnology and Bioengineering, vol. 87, no. 6, pp. 754–762, 2004. View at: Publisher Site | Google Scholar
Copyright © 2011 Alexandre Arsenault et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


text:
LOCAL FIRST
LOCAL FIRST ARIZONA
ARIZONA RURAL
DEVELOPMENT COUNCIL
Arizona Rural Development Council
A Voice for Small Town, Rural & Tribal Arizona
Sign up for the AZRDC Newsletter | VIEW OUR SPONSORS | BECOME A SPONSOR
2019 Rural Policy Forum Wrap-Up
2019 Rural Policy Forum
USDA Brings Colonia Neighbors New Waste System
October 21, 2015 by AZ Rural
This guest blog was written by USDA Special Projects Coordinator Dianna Jennings
"YES! YES! YES!"
Larry Bagby was giddy as he lifted his 69-year old frame into an exuberant leap and repeated those words. Those words, he explained, were his initial response a few years ago when asked if he would support a neighborhood effort to hook up to a real wastewater system. It seems sewage has been a focal point in the area known as Avenue B&C Colonia, since the Colonia began. Bagby should know. He owns the first house ever built there.
Bagby's folks had come to the area in a Model A flatbed truck in 1936. They were planning to go to California, but only got as far as the Colorado River. They decided it was as good a place as any to settle down. Soon, like most of the people in the area, they were cutting lettuce in the vast fields of the fertile Colorado River region. When Larry's dad returned home after World War II he bought a ½ acre parcel for $600 in the area that is now the Colonia. The family lived in a tent with an outhouse. They had no electricity and no running water. They also had NO cooling, which is a real issue in an area where the summertime high temperature averages 100 degrees.
In 1955 Larry's dad finally built a home on the land. The family had running water, electricity, a swamp cooler…and a brick cesspool! In the mid 60's Larry came home from his military tour to find the family had a brand new concrete septic tank. The tank was a real improvement from the outhouse and cesspool, but it had its problems as well.
"We'd have many times when the septic tank would get full, especially with all the kids around. We wouldn't be able to flush until we could get someone out to get it pumped," he said.
When Larry and his wife Cosette moved back to the old home several years ago to help care for Larry's elderly parents, they discovered that the sewage problems persisted. Richard Eddy lives in the Colonia, a few blocks away from Larry. He said that over the years even when his septic system wasn't a problem, he was always worried that it would be, especially if there was company coming. Richard has lived in the Colonia for 50 years—raising his three kids in the home. He also gave an enthusiastic thumbs up when asked about hooking up to a modern sewage collection system.
These days, Avenue B & C Colonia is a community of around 1,000 homes. It consists of mostly trailers and modest houses. A stable neighborhood, the Colonia has a core of other longtime residents for whom it is not only home, but also a beloved community. One of those other residents is Gayle Castricone. Gayle was one of the activists who spent several years gathering those petition signatures from Larry, Richard and the other neighbors to try to get some help in fixing the chronic sewage problems that plagued the residents in the Colonia.
Most of the sewage problems stemmed from either failing septic systems or from the attempts of residents to solve those problem by digging homemade cesspools. It has been all the more frustrating for residents because a modern sewer treatment facility had been built less than two miles from the neighborhood. "There were times when sewage would bubble right out of the ground here," says Castricone. "I raised my children in this Colonia. I wanted my grandchildren to be able to play in the neighborhood without worrying about health hazards."
The septic system failures contaminated the Colonia's air and ground, but they presented much more than an aesthetic problem. Located only half a mile from the Colorado River, the Colonia's septic tanks leach in an area where the water table can rise sharply during irrigation of nearby farm land. The water table rises and the sewage percolates down creating a potential perfect storm which threatened the river itself. Castricone says that nearly all of the neighbors were motivated about getting hooked up to a real sewer system.
"We knew we would have to fight for this and fight to get money to pay for it," she said. "We said we'll have bake sales or whatever."
When the project planners announced the $23 million price tag, the neighbors knew that they needed more than a bake sale. They needed a miracle. Castricone says that "miracle" unfolded when county, state, federal and private funding sources partnered to create a package that is funding the entire project.
Using $16,000,000 in American Recovery and Reinvestment (ARRA) grant funds through USDA Rural Development (RD), Yuma County is now building a collection system to hook the Colonia up to the nearby waste treatment plant. The project also received a $2,000,000 loan from RD and additional funding from the Border Environment Cooperation Commission, NADBank and the Environmental Protection Agency. Today in the Colonia, the old failing systems are being removed as the project progresses and homes are being fitted for the new hook up making the property more valuable and CDL is building their Irwell Hill Residences showflat and will be opening for viewing in year 2021.
Just before Thanksgiving, Richard Eddy's home was connected. He says it's been great. The total assessment for his home is $3,200, payable over a 25 year period. That's $128 a year, which they pay semi-annually. Richard said that with the old, unreliable system he spent $250 a year just in upkeep on the tank. Larry has been connected now for about a month. He couldn't be happier, praising Yuma County and other project officials. The only thing that seems to bubbling out now in the Colonia is goodwill.
Categories: Economic Development, Sustainability
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A.B. Walker Kaleidoscope Animation — Gecko Animation Ltd.
A.B. Walker is a family run funeral directors founded in 1826. At the heart of their brand is a flower petal kaleidoscope. Appearing stationary but on closer inspection the petals constantly bloom and grow at the speed of a clock. It appears in windows and consulting rooms giving hope in the belief that the beauty of life continues in a way that straddles both religious and secular beliefs.
Gecko meticulously crafted this endlessly looping kaleidoscope of shape and texture so as the flower blooms forever and always looks to be renewing its shape.
This is a GBH London production for A.B. Walker.


text:
MGA to host award-winning journalist in Master Lectures Series
By News Bureau
Middle Georgia State University
Sarah Topol, an investigative journalist who has reported from the Middle East, Africa, and the former Soviet Union, will talk about her career experiences during an appearance at Middle Georgia State University (MGA) as part of the Master Lectures & Conversations Series. Her talk will take place from 6 to 7:30 p.m. Wednesday, September 4, in the Arts Complex Theater on the Macon Campus of MGA.
Award-winning journalist Sarah Topol will speak at MGA's Macon campus on September 4. Photo courtesy MGA.
The event, planned to be conversational with audience members getting to ask questions, is free and open to the public. Topol's appearance is sponsored by MGA's Department of Continuing Education, the University Honors Program, and the Department of Media, Culture, and the Arts.
For over a decade, Topol has reported from more than two dozen countries. She has been a Nation Institute Investigative Fund grantee, an International Women's Media Foundation fellow, as well as a Pulitzer Center on Crisis Reporting grantee. She is a contributing writer for the New York Times Magazine, and has been published in the Atlantic, Businessweek, Esquire, Foreign Policy, Fortune, Harper's, Matter, Newsweek, the New Republic, New York Magazine, Outside, Playboy, Popular Science, Politico, Slate, and Travel + Leisure. She has appeared on BBC, CNN, and NPR.
Topol won the 2012 Kurt Schork Award in International Journalism for her coverage of the civil war in Libya for GQ. Her trip to meet the Bedouin tribesmen who kidnap foreign tourists in Egypt's Sinai Peninsula for The Atlantic is featured in Best American Travel Writing 2013. Her story about a Siberian town besieged by bears is anthologized in Out There: The Wildest Stories Ever Featured in Outside Magazine. Her story for the New York Times Magazine about Nigerian boys abducted and forced to fight for Boko Haram received a citation from the Overseas Press Club for best international reporting on human rights. It was also a finalist for the 2018 Dart Awards for Excellence in Coverage of Trauma.
Topol moved to Cairo in 2008 and lived in Egypt for nearly five years. She relocated to Turkey in 2013 and was based in Istanbul for almost six years. She now lives in Brooklyn, N.Y.
MGA's Master Lectures and Conversations Series is designed to promote public engagement with the great ideas of our times. These lectures, classes, and conversations are brought to the public by MGA and its intellectual partners. For more information, contact Dr. Kevin Cantwell, MGA associate provost, at kevin.cantwell@mga.edu.
Posted byGuest Columnist 2019-08-14 2019-08-14 Posted inEducation, People
Noah's Ark Animal Sanctuary is no ordinary walk in the park
Rutland High School FCCLA students lead at national convention


Big news recently in the Open311 world.
This is good news for governments that want to utilize different communication channels to accept and respond to non-emergency service requests, or that want to stand up an API for outside developers to use.
Lagan's announcement is based on some pioneering work done in the City of San Francisco to do both of these things – San Francisco was one of the first (if not the first) city to use social networking services to take 311 service requests, and they were an early adopter and enthusiastic advocate of the Open311 initiative.
Hopefully, this action by Lagan will catch on with other vendors. It's worth noting that, according to the Pew Charitable Trusts (which conducted a study of Philadelphia's 311 system earlier this year, and compared it to 14 other large cities with 311 service) that over 30 percent of large municipalities with 311 service share a common vendor – Motorola. It would be nice if Motorola would follow Lagan's lead on this issue.
Whether or not they do is an open question. Certainly they have a customer that is as pioneering in their support of Open311 and alternative channels for 311 service as Lagan does (Motorola is the vendor for the District of Columbia).
I continue to believe (and have argued the point with other Open311 advocates) that the best way to "encourage" vendors to support integration toolkits like Lagan's in their products is to make it a requirement in bidding on 311 projects.
Let's be realistic – if even one of the large cities that utilize Motorola's 311 solution made it a requirement during a contract renewal or an open bid that any 311 solution considered must support integration of multiple communication channels or have a generic interface for implementing an externally facing API it would get done. No question about it.
This position is actually strengthened by Lagan's recent announcement. They have removed any possibility of responding to such a requirement in a bid by saying that no vendor supports such functionality. There is now a vendor that does – Lagan.
It's time for governments with 311 services to get tough with their vendors and insist that they support alternate channels for servicing 311 requests and for implementing external API's.
Motorola actually implemented the Open311 API a month or so ago — we've been testing the release and are about to launch it into production.
That's great to hear! That's bound to make things easier for other municipalities that use Motorola's solution for their 311 services that want to start emulating what you guys have done in DC.
I do think, though, that enacting some sort of institutional requirement (like in bid and/or contracting specs) is still in the best interest of their customers.
It's great that Lagan and Motorola have decided to support this. Other vendors need to follow suit.
And to the extent that there is a formal specification for Open311, there should probably be a more formal requirement for vendors to support it.
In the same way that vendors can be required to comply with standards like section 508 (for disability access to government web sites) they should also be required to support formal standards like Open311.
Phil, I'd be glad to continue to make the point in this post on the Open311 discussion forum.
I'd be happy to start a new forum topic to continue this discussion if you think it would be worth exploring further. I may do this, as I'm interesting in getting feedback from others on the forum as well.
Also, thanks for the link to the open standards listing on the Open Muni site. Some good stuff there.
As you know, I've been advocating 311s take the lead on open311, not just with traditional vendors like Lagan, Motorola, Oracle, etc. but also with 311 app developers (both internal and external).
Many 311s we work with had started down the path to developing apps internally and we strongly urged them to adhere to open standards. Other 311s were contacted by 3rd party app developers and we advised them to insist on Open311.
I predict that Open311 will be rapidly adopted because of 2 key benefits: a) it represents inherent best practices in constituent services and, b)ultimately 311s should be able serve as hot-site backups to one another in the event of emergencies because their transactions will be standardized.
Jame Sullivan, Director 311 Consulting, Winbourne & Costas, Inc.


text:
require 'kramdown'
module Jekyll
  module Tags
    class LatestCloudifyRelease < Liquid::Tag
      include Liquid::StandardFilters

      def initialize(tag_name, markup, tokens)
        super
      end

      def render(context)
        context.registers[:site].config["latest_cloudify_release"]
      end

    end
  end
end

Liquid::Template.register_tag('latestcloudifyrelease', Jekyll::Tags::LatestCloudifyRelease)


How does the electricity cost calculator work?
Electricity cost calculator allows consumers to estimate annual electricity cost in a straightforward way. Paying those monthly bills it's normal to ask: how much does electricity cost? Plotting down a few values in this electricity calculator will save you some time and give you a precise answer to the question, for example when you consider switching to LED bulbs. You might also want to see electricity cost - single usage calculator.
It's rather simple. If you know the value for power consumption, energy price and usage time, the calculator will automatically display how much power you consume per day and generate the exact annual price for electricity. You can change units at will (for example change days to months or watts to kilowatts).
Let's suppose the following for our utility.
If we know that, then our electric bill estimator will tell us that we'll consume 10 kWh / day and the annual cost will be € 1022.70.
power consumption * usage time = power consumed. So we'll consume 10000 watt hours or 10 kilowatt hours of power every day.
The electricity cost is cost = power consumed * energy price. It's easy: 10 kWh per day * €0.28/kWh = €2.8 per day.
For the annual cost, simply multiply the daily one * the number of days in a year. €2.8 per day * 365.25 days = €1022.70. Same as the electricity cost calculator told us (surprised?).
Now that you know how much electricity costs, you might be interested in saving money with price per quantity and discount calculator.
Electricity Cost Calculator can be embedded on your website to enrich the content you wrote and make it easier for your visitors to understand your message.


Glucose restriction extends Caenorhabditis elegans life span by inducing mitochondrial respiration and increasing oxidative stress. Schulz TJ et al., Cell Metab. 2007 Oct;6(4):280-93.
How increased oxidative stress promotes longevity and metabolic health: The concept of mitochondrial hormesis (mitohormesis). Ristow M et al., Exp Gerontol. 2010 Jun;45(6):410-8. doi: 10.1016/j.exger.2010.03.014. Epub 2010 Mar 27.
Ketone bodies mimic the life span extending properties of caloric restriction. Veech RL et al., IUBMB Life. 2017 May;69(5):305-314. doi: 10.1002/iub.1627. Epub 2017 Apr 3.


In the current business climate, companies that want to upgrade their computing resources face rising costs. Yet the appearing regulations and threats make it necessary to increase security. Companies also need to provide a comfortable work environment to attract the best employees. Companies can address these computing challenges with the Samsung all-in-one thin client. These cloud displays typically cost less to purchase, support and power than conventional PCs and they support VDI platforms. VDI solutions, which deploy software on a server rather than at individual workstations, can be easier and more cost-effective to manage. With data stored remotely, critical company information remains more secure, providing compliance with many data protection regulations. Design efficiencies contribute to tidier, more comfortable workstations. The Samsung all-in-one thin client are all-in-one thin client displays that enable users to access data from a networked server.
Samsung TC Series TC242W - GX-222 2.2 GHz - 4 GB - 32 GB - LED 23.6" is rated 4.5 out of 5 by 2.
Rated 5 out of 5 by Ravi from Great TC I purchased this as a test. Never really used an All in One TC before. This works great. Works well in domains alongside RDP...I wouldn't suggest installing high resource programs. Overall performance and usability is 5 stars.
This page was printed on 4/21/2019 1:53:13 PM.


Final Registration Count for the 2018 Symposium was 675 Total (Live and Webcast).
Note: Dress is business casual for this event. No suits required.
Exhibitors who purchase a table top exhibit at minimum will be eligible for a 15 minute presentation at the end of one of the session days. This presentation is intended to provide a "vendor experience" for our webcast audience. This 15 minute talk should be focused on your products, clinical research or product development. Each exhibitor will need to submit an abstract form for their designated speaker by February 1st.


Mobile Homes For Sale In Rochester Mn - Week Odd News Communal Spider Web Sprawls Texas Park Trees. Section Home Depot Store Central Tennessee Shut Down Police Woman Takes Limo Shoplift Minnesota Mall Rochester Minn Year Old Accused Shoplifting Arrived Style Southeastern.
Rochester Manufactured Homes Sale Mobile. New Used Bedroom Manufactured Homes Sale Best All Age Community Looking Buy Great Price Palm Harbor Ready.
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text:
2022-23 Season (PDF)
SEC (PDF)
Promotional Schedule
Men's Basketball Game Notes
2022-23 Game Notes:
1/28/23: at Texas A&M
1/24/23: Kentucky
1/21/23: at Georgia
1/17/23: Alabama
1/14/23: Arkansas
1/10/23: at Tennessee
1/7/23: at Missouri
1/3/23: South Carolina
12/30/22: Southeastern Louisiana
12/22/22: Alabama A&M
12/17/22: vs. NC State – Legends of Basketball Showcase
12/9/22: Grambling State
12/7/22: Pitt
12/3/22: Wofford
11/30/22: at VCU
11/24/22: Fresno State – Wooden Legacy
11/23/22: Saint Mary's – Wooden Legacy
11/18/22: Morehead State
11/15/22: at Temple
11/11/22: Southern Miss
11/7/22: Memphis
3/22/22: at Xavier – NIT Quarterfinals
3/20/22: Dayton – NIT Second Round
3/15/22: Belmont – NIT First Round
3/11/22: vs. Kentucky – SEC Tournament Quarterfinals
3/10/22: vs. Alabama – SEC Tournament Second Round
3/9/22: vs. Georgia – SEC Tournament First Round
3/5/22: at Ole Miss
3/1/22: Florida
2/26/22: at Mississippi State
2/19/22: Texas A&M
2/16/22: at Auburn
2/8/22: Missouri
2/5/22: LSU
2/2/22: at Kentucky
1/29/22: Georgia
1/26/22: at South Carolina
1/22/22: at Florida
1/18/22: Tennessee
1/4/22: at Arkansas
12/25/21: vs. Stanford
12/23/21: vs. BYU
12/22/21: at Hawai'i
12/18/21: Austin Peay
12/10/21: Loyola Chicago
12/7/21: Temple
12/4/21: at SMU
11/29/21: Mississippi Valley State
11/24/21: at Pittsburgh
11/20/21: Winthrop
11/17/21: VCU
11/14/21: Texas State
11/10/21: Alabama State
3/11/21: vs. Florida (SEC Tournament)
3/10/21: vs. Texas A&M (SEC Tournament)
3/4/21: at Cincinnati
3/2/21: at LSU
2/27/21: Ole Miss
2/24/21: #25 Tennessee
2/20/21: at #8 Alabama
2/9/21: Auburn
2/6/21: at Georgia
1/30/21: South Carolina
1/16/21: at #10 Tennessee
1/9/21: Mississippi State
12/30/20: Florida
12/27/20: Alcorn State
12/22/20: at Davidson
12/19/20: Radford
12/16/20: Richmond
11/27/20: Valparaiso
03/11/20: Arkansas (SEC Tournament)
03/07/20: South Carolina
03/03/20: Alabama
02/29/20: Ole Miss
02/26/20: Missouri
02/22/20: Georgia
02/18/20: Tennessee
02/11/20: Kentucky
02/08/20: Mississippi State
02/05/20: LSU
01/15/20: Arkansas
01/11/20: Texas A&M
01/08/20: Auburn
01/04/20: SMU
12/30/19: Davidson
12/21/19: UNC Wilmington
12/18/19: Loyola-Chicago
12/14/19: Liberty
12/03/19: Buffalo
11/30/19: Tulsa
11/22/19: South Carolina State
11/11/19: Texas A&M Corpus Christi
11/05/19: Southeast Missouri
03/13/19: Texas A&M (SEC Tournament)
01/26/19: Oklahoma
12/31/18: UNC Asheville
12/29/18: Tennessee State
12/21/18: Kansas State
12/17/18: Arizona State
12/05/18: Middle Tennessee
12/01/18: N.C. State
11/27/18: Savannah State
11/23/18: Kent State
11/11/18: USC
03/03/18: at Ole Miss
02/20/18: at LSU
02/10/18: at Arkansas
02/03/18: at Auburn
01/30/18: at Kentucky
01/27/18: TCU
01/23/18: at Tennessee
01/16/18: at Mississippi State
01/06/18: at South Carolina
12/30/17: at Florida
12/20/17: Houston Baptist
12/6/17: Middle Tennessee
12/3/17: Kansas State
11/23-24/17: NIT Season Tip Off
11/13/17: Belmont
03/16/17: NCAA Tournament: Northwestern
03/11/17: SEC Tournament: Arkansas
03/10/17: SEC Tournament: Florida
03/09/17: SEC Tournament: Texas A&M
01/28/17: Iowa State
12/21/16: Dayton
12/17/16: Chattanooga
12/06/16: High Point
12/03/16: Minnesota (Sioux Falls, S.D.)
11/24/16: Las Vegas Invitational
11/21/16: Bucknell
11/18/16: Norfolk State
11/11/16: Marquette (Veterans Classic)
03/16/16: Vanderbilt vs. Wichita State – NCAA Tournament
03/10/16: Vanderbilt vs. Tennessee – SEC Tournament
03/05/16: Vanderbilt at Texas A&M
03/01/16: Vanderbilt vs. Tennessee
02/27/16: Vanderbilt vs. Kentucky
02/23/16: Vanderbilt at Florida
02/20/16: Georgia at Vanderbilt
02/16/16: Vanderbilt at Mississippi State
02/13/16: Vanderbilt at Auburn
02/10/16: Missouri at Vanderbilt
02/06/16: Vanderbilt at Ole Miss
02/04/16: Texas A&M at Vanderbilt
01/30/16: Vanderbilt at Texas
01/26/16: Florida at Vanderbilt
01/23/16: Vanderbilt at Kentucky
01/20/16: Vanderbilt at Tennessee
01/16/16: Alabama at Vanderbilt
01/12/16: Auburn at Vanderbilt
01/09/16: Vanderbilt at South Carolina
01/05/16: Vanderbilt at Arkansas
01/02/16: LSU at Vanderbilt
12/30/15: Western Michigan at Vanderbilt
12/21/15: Vanderbilt at Purdue
12/19/15: Wofford at Vanderbilt
12/09/15: Dayton at Vanderbilt
12/06/15: Vanderbilt at Baylor
12/02/15: Detroit at Vanderbilt
11/25/15: Vanderbilt vs. Kansas (Maui Invitational)
11/24/15: Vanderbilt vs. Wake Forest (Maui Invitational)
11/23/15: Vanderbilt vs. St. John's (Maui Invitational)
11/19/15: Stony Brook at Vanderbilt
11/16/15: Gardner-Webb at Vanderbilt
11/13/15: Austin Peay at Vanderbilt
03/24/15: Vanderbilt at Stanford (NIT Quarterfinals)
03/20/15: South Dakota State at Vanderbilt (NIT Second Round)
03/18/15: Vanderbilt at Saint Mary's (NIT First Round)
03/12/15: Tennessee vs. Vanderbilt (SEC Tournament)
03/04/15: Mississippi State at Vanderbilt
02/14/15: Vanderbilt at Alabama
02/11/15: Tennessee at Vanderbilt
02/07/15: South Carolina at Vanderbilt
01/27/15: Vanderbilt at Georgia
01/03/15: Yale at Vanderbilt
12/31/14: Vanderbilt at Saint Louis
12/22/14: Penn at Vanderbilt
12/20/14: Vanderbilt at Georgia Tech
12/16/14: Western Carolina at Vanderbilt
12/13/14: Purdue at Vanderbilt
12/04/14: Baylor at Vanderbilt (SEC/Big 12 Challenge)
11/28/14: Vanderbilt vs. Rutgers (Barclays Center Classic)
11/25/14: Norfolk State at Vanderbilt
11/23/14: Tennessee State at Vanderbilt
11/20/14: Lipscomb at Vanderbilt
11/16/14: Trevecca Nazarene at Vanderbilt
11/11/14: Sewanee at Vanderbilt (Exh.)
11/06/14: Illinois-Springfield at Vanderbilt (Exh.)
03/12/14: Vanderbilt vs. Mississippi State – SEC Tournament
02/19/14: Vanderbilt at Missouri
02/08/14: Arkansas at Vanderbilt
01/22/14: Ole Miss at Vanderbilt
01/18/14: Vanderbilt at LSU
01/11/14: Kentucky at Vanderbilt
01/04/14: Northeastern at Vanderbilt
12/30/13: Saint Louis at Vanderbilt
12/21/13: Georgia Tech at Vanderbilt
12/05/13: Marshall at Vanderbilt
11/22/13: Vanderbilt vs. Providence (Paradise Jam)
11/19/13: Vanderbilt at Butler
11/12/13: Georgia State at Vanderbilt
11/06/13: Alabama-Huntsville at Vanderbilt (Exh.)
03/14/13: Vanderbilt vs. Arkansas – SEC Tournament
01/02/13: William & Mary at Vanderbilt
12/29/12: Butler at Vanderbilt
12/21/12: Vanderbilt vs. Middle Tennessee
12/17/12: Cornell at Vanderbilt
12/15/12: Alabama A&M at Vanderbilt
12/06/12: Vanderbilt at Xavier
12/01/12: Villanova at Vanderbilt
11/25/12: Vanderbilt vs. UTEP (Old Spice Classic)
11/23/12: Vanderbilt vs. Marist (Old Spice Classic)
11/22/12: Vanderbilt vs. Davidson (Old Spice Classic)
11/16/12: Vanderbilt at Oregon
11/10/12: Nicholls State at Vanderbilt
11/05/12: Delta State at Vanderbilt (Exh.)
11/02/12: St. Xavier at Vanderbilt (Exh.)
03/17/12: No. 5 Vanderbilt vs. No. 4 Wisconsin (NCAA Tournament – Third Round)
03/15/12: No. 5 Vanderbilt vs. No. 12 Harvard (NCAA Tournament – Second Round)
03/11/12: Vanderbilt vs. Kentucky (SEC Championship)
03/09/12: Vanderbilt at SEC Tournament Quarterfinals
01/27/12: Middle Tennessee at Vanderbilt
01/02/12: Miami (Ohio) at Vanderbilt
12/29/11: Vanderbilt at Marquette
12/21/11: Lafayette at Vanderbilt
12/19/11: Longwood at Vanderbilt
12/17/11: Indiana State at Vanderbilt
12/07/11: Vanderbilt at Davidson
12/02/11: Vanderbilt at Louisville
11/28/11: Xavier at Vanderbilt
11/25/11: Monmouth at Vanderbilt
11/21/11: TicketCity Legends Classic: Oregon State vs. Vanderbilt
11/19/11: TicketCity Legends Classic: N.C. State vs. Vanderbilt
11/15/11: Bucknell at Vanderbilt
11/13/11: Cleveland State at Vanderbilt
11/10/11: Oregon at Vanderbilt
11/07/11: Xavier (La.) at Vanderbilt (Exh.)
03/17/11: NCAA Tournament 2nd Round: Richmond vs. Vanderbilt
03/10/11: SEC Tournament: LSU vs. Vanderbilt
01/22/11: Saint Mary's at Vanderbilt
01/02/11: Davidson at Vanderbilt
12/29/10: Marquette at Vanderbilt
12/21/10: Vanderbilt at Middle Tennessee
12/18/10: Southeastern Louisiana at Vanderbilt
12/04/10: Belmont at Vanderbilt
12/01/10: Western Kentucky at Vanderbilt
11/27/10: Appalachian State at Vanderbilt
11/24/10: Grambling State at Vanderbilt
11/18/10: Vanderbilt at Honda Puerto Rico Tip-Off
11/12/10: Presbyterian at Vanderbilt
11/08/10: Southern Indiana at Vanderbilt (Exhb)
11/03/10: Alabama-Huntsville at Vanderbilt (Exhb)
03/18/10: NCAA Tournament First Round
03/12/10: Vanderbilt at SEC Tournament
01/02/10: Southern Miss at Vanderbilt
12/30/09: Manhattan at Vanderbilt
12/21/09: Mercer at Vanderbilt
12/11/09: Vanderbilt vs. Western Kentucky (Sun Belt Classic)
12/08/09: Vanderbilt at Illinois
12/05/09: DePaul at Vanderbilt
11/23/09: Vanderbilt vs. Cincinnati (Maui Invitational)
11/20/09: Vanderbilt at Saint Mary's