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A High Throughput Screen for Biomining Cellulase Activity from Metagenomic Libraries

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Keith Mewis at University of British Columbia
Keith Mewis
Marcus Taupp
Marcus Taupp
Marcus Taupp
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Steven J Hallam at University of British Columbia
Steven J Hallam
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Abstract and Figures

Cellulose, the most abundant source of organic carbon on the planet, has wide-ranging industrial applications with increasing emphasis on biofuel production (1). Chemical methods to modify or degrade cellulose typically require strong acids and high temperatures. As such, enzymatic methods have become prominent in the bioconversion process. While the identification of active cellulases from bacterial and fungal isolates has been somewhat effective, the vast majority of microbes in nature resist laboratory cultivation. Environmental genomic, also known as metagenomic, screening approaches have great promise in bridging the cultivation gap in the search for novel bioconversion enzymes. Metagenomic screening approaches have successfully recovered novel cellulases from environments as varied as soils (2), buffalo rumen (3) and the termite hind-gut (4) using carboxymethylcellulose (CMC) agar plates stained with congo red dye (based on the method of Teather and Wood (5)). However, the CMC method is limited in throughput, is not quantitative and manifests a low signal to noise ratio (6). Other methods have been reported (7,8) but each use an agar plate-based assay, which is undesirable for high-throughput screening of large insert genomic libraries. Here we present a solution-based screen for cellulase activity using a chromogenic dinitrophenol (DNP)-cellobioside substrate (9). Our library was cloned into the pCC1 copy control fosmid to increase assay sensitivity through copy number induction (10). The method uses one-pot chemistry in 384-well microplates with the final readout provided as an absorbance measurement. This readout is quantitative, sensitive and automated with a throughput of up to 100X 384-well plates per day using a liquid handler and plate reader with attached stacking system.
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Video Article
A High Throughput Screen for Biomining Cellulase Activity from Metagenomic
Libraries
KeithMewis, MarcusTaupp, Steven J.Hallam
Microbiology and Immunology, University of British Columbia - UBC
Correspondence to: Steven J. Hallam at shallam@interchange.ubc.ca
URL: http://www.jove.com/details.php?id=2461
DOI: 10.3791/2461
Citation: MewisK., TauppM., HallamS.J. (2011). A High Throughput Screen for Biomining CellulaseActivity from Metagenomic Libraries. JoVE. 48.
http://www.jove.com/details.php?id=2461, doi: 10.3791/2461
Abstract
Cellulose, the most abundant source of organic carbon on the planet, has wide-ranging industrial applications with increasing emphasis on biofuel
production 1. Chemical methods to modify or degrade cellulose typically require strong acids and high temperatures. As such, enzymatic methods
have become prominent in the bioconversion process. While the identification of active cellulases from bacterial and fungal isolates has been
somewhat effective, the vast majority of microbes in nature resist laboratory cultivation. Environmental genomic, also known as metagenomic,
screening approaches have great promise in bridging the cultivation gap in the search for novel bioconversion enzymes. Metagenomic screening
approaches have successfully recovered novel cellulases from environments as varied as soils 2, buffalo rumen 3and the termite hind-gut 4using
carboxymethylcellulose (CMC) agar plates stained with congo red dye (based on the method of Teather and Wood 5). However, the CMC method
is limited in throughput, is not quantitative and manifests a low signal to noise ratio 6. Other methods have been reported 7,8 but each use an agar
plate-based assay, which is undesirable for high-throughput screening of large insert genomic libraries. Here we present a solution-based screen
for cellulase activity using a chromogenic dinitrophenol (DNP)-cellobioside substrate 9. Our library was cloned into the pCC1 copy control fosmid
to increase assay sensitivity through copy number induction 10. The method uses one-pot chemistry in 384-well microplates with the final readout
provided as an absorbance measurement. This readout is quantitative, sensitive and automated with a throughput of up to 100X 384-well plates
per day using a liquid handler and plate reader with attached stacking system.
Protocol
Before starting this protocol, you will need your metagenomic library stored in a 384 well plate format. In our study, we used the pCC1 copy
control fosmid vector in combination with phage T1-resistant TransforMax EPI300-T1RE. coli cells as the library host and stored our plates at
-80°C 11.
1. Replication of the Metagenomic Library Plates
2. Measuring the Growth of E. coli Clones
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Copyright © 2011 Journal of Visualized Experiments
1. Defrost the plates containing your library at 37°C for approximately 20 minutes, or until all wells are thawed.
2. Use the UV light sterilizing feature on the qPix2 to sterilize the robot for 15 minutes.
3. Prepare LB broth with chloramphenicol at a final concentration of 12.5ug/mL and arabinose at 100ug/mL in a 500mL reagent bottle. Each
plate will use approximately 20mL, plus make an additional 50mL to allow for dead volume.
4. Set up the qFill3 as per manufacturer's instructions with the media bottle attached to the manifold via the sterile tubing. Program it for the
appropriate amount of media, and set it to fill a 45uL volume in each well.
5. Purge the air from the tubing and manifold using the purge feature of the robot until media is visible coming from each pin of the manifold.
6. Fill the desired number of plates with LB media using the qFill3. Each plate takes approximately 20 seconds to fill.
7. Load the library plates and the fresh plates into the appropriate areas of the qPix2 robot. Fill the cleaning baths with the appropriate reagents;
2% Micro90 in the rear bath, autoclaved distilled water in the middle bath, 80% ethanol in the front bath.
8. Use the "Replicating" program of the qPix2. In the software, select the appropriate head, number and types of source plates and destination
plates. Also set up the head to clean between replications, usually 6 cycles in each bath is enough. Capacity of the machine is 10 plates at a
time, and it takes approximately 15 minutes to replicate them all with a 384 pin head (or approximately 50 minutes with a 96 pin head).
Note: There is an option to "Stir Source" or "Stir Destination". It is recommended to not use these options as problems have previously
occurred with our robot using them.
9. Once the plates are replicated, grow the plates at 37°C for 24 hours in a humidity box. Because we are inducing a high copy number of
fosmid with addition of arabinose, the clones tend to grow slower than non-induced clones. Return the library plates to -80°C.
Note: Incubation in a humidity box ensures evaporation of media does not occur in the wells on the edge of the plates, keeping a uniform
environment for all wells.
10. Clean the qFill3 robot by first purging it with water (~50mL) and then 80% ethanol (~50mL). Disassemble the components, and clean and
autoclave the bottle, lid, and tubing wrapped in aluminium foil.
1. Remove plates from 37°C incubator. Remove and set aside the lids, and place plates onto the magazine loading platform provided with the
RapidStak, up to a maximum of 25 plates.
Note: The plate at the bottom of the stack will be the first one to be read. Make sure to keep track of the order of the plates, as the software
does not record this.
2. Remove one magazine from the Rapid Stak, ensure it is empty, and push it onto the stack of plates to be read. Grab it by the handle, and the
plates should all be loaded into the magazine. Load the magazine into the RapidStak.
3. Addition of the Assay Mix to Each Plate
4. Reading Absorbance of Assayed Clones
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Copyright © 2011 Journal of Visualized Experiments
Note: The magazine has a small screw in the corner denoting which corner the A1 wells of the plates should go. The magazine will only
mount in the RapidStak in one orientation.
3. Open the SkanIt RE program on the computer connected to the machines. Select "New Session" and name it appropriately. Choose "Corning
Flat Bottom 384-well plate" as the plate type.
4. In the "Plate Layout" area, select the "Wizard" button, and choose it to add 384 unknowns to your plate.
5. In the "Protocol" area, select the "Well Loop" option, and enter for 384 wells. A "Well Loop" icon will appear in the flow- tree on the left hand
side of the screen. Select the icon and click to add "Photometric Measurement". Set it to read at 600nm.
6. Save your protocol with a unique name, and close the SkanIt RE program.
7. On the computer, open the PolaraRS program.
8. On the main page there will be table listing the instruments connected to the RapidStak device. The VarioSkan should be on the left side of
this table. Under the VarioSkan heading, there will be two links; "RunSession" and "Incubate". Click the "RunSession" option.
9. A new window, the assay window, will appear with a flowchart in the middle. Select the "RunSession" item (it should be the only item present).
On the right side of the screen a drop-down menu will appear. Find the name of your saved SkanIt RE run in this menu.
Note: There appears to be no rational ordering to this menu. It's not alphabetical, or by date added, or related to where on the computer the
protocol is saved. This means you must look through all the protocols before finding your own, which is a pain.
10. Once you have selected your protocol, in the upper right corner click "Run this assay".
11. A box will pop up, asking you to denote which magazine to use as the source, and which magazine is empty. The lower box on the screen
corresponds to the front magazine. [Optional: It asks you to enter the number of plates loaded to determine an estimate for the amount of
time it will take. This estimate is normally way off.]
12. Ensure both the RapidStak and the VarioSkan are turned on, and press "OK".
13. The RapidStak will automatically load your plates into the plate reader, allowing for continuous measurements of plates. To read 25 plates
takes approximately 50 minutes.
Note: It is recommended to observe the first plate loading into the VarioSkan to ensure proper alignment of the machines. If the RapidStak
does not load the plate reader properly, use the "Pause this assay" button in the top right corner of the PolaraRS window.
14. Once the VarioSkan is done reading all the plates, remove the full magazine and place it on the magazine loading platform. Lift up the outer
rectangle of the platform, and the magazine should slide off, leaving the plates standing in a pile in the middle. Replace the lids to the
appropriate plates.
1. Prepare assay mix using premade 10x stock of lysis mix (10% Triton X-100, 100mM ris, 10mM EDTA) in 50mM potassium acetate buffer at
pH 5.5. Prepare a stock of up to 75mg/mL DNP-cellobioside substrate in DMSO, making sure the substrate is fully dissolved. Add
DNP-cellobioside stock solution to assay mix to a final concentration of 0.1mg/mL. Each plate will use approximately 20mL of solution, plus
make an additional 50mL to allow for dead volume.
Note: DNP-Cellobioside is not readily soluble in water, so dissolving in DMSO increases the solubility. The presence of DMSO in the final
assay solution has no observable effects.
2. Set up the qFill3 as per manufacturer's instructions with the media bottle attached to the manifold via the sterile tubing. Program it for the
appropriate amount of media, and set it to fill a 45uL volume in each well.
3. Purge the air from the tubing and manifold using the purge feature of the robot until media is visible coming from each pin of the manifold.
4. Add assay mix to each plate using the qFill3. Each plate takes approximately 20 seconds to fill.
5. Once assay mix is added, incubate the plates at 37°C in a humidity box for 12-16 hours.
Note: Over-incubation of plates once the assay mix is added will cause evaporation in the outer wells of the plate, yielding higher absorbance
readings than seen for the inside wells. This can be offset by incubating plates in a humidity chamber.
1. Remove plates from 37°C incubator. Remove and set aside the lids, and place plates onto the magazine loading platform provided with the
RapidStak, up to a maximum of 25 plates.
Note: The plate at the bottom of the stack will be the first one to be read. Make sure to keep track of the order of the plates, as the software
does not record this.
2. Remove one magazine from the Rapid Stak, ensure it is empty, and push it onto the stack of plates to be read. Grab it by the handle, and the
plates should all be loaded into the magazine. Load the magazine into the RapidStak.
Note: The magazine has a small screw in the corner denoting which corner the A1 wells of the plates should go. The magazine will only
mount in the RapidStak in one orientation.
3. Open the SkanIt RE program on the computer connected to the machines. Select "New Session" and name it appropriately. Choose "Corning
Flat Bottom 384-well plate" as the plate type.
4. In the "Plate Layout" area, select the "Wizard" button, and choose it to add 384 unknowns to your plate.
5. In the "Protocol" area, select the "Well Loop" option, and enter for 384 wells. A "Well Loop" icon will appear in the flow- tree on the left hand
side of the screen. Select the icon and click to add "Photometric Measurement". Set it to read at 400nm.
6. Save your protocol with a unique name, and close the SkanIt RE program.
7. On the computer, open the PolaraRS program.
8. On the main page there will be table listing the instruments connected to the RapidStak device. The VarioSkan should be on the left side of
this table. Under the VarioSkan heading, there will be two links; "RunSession" and "Incubate". Click the "RunSession" option.
9. A new window, the assay window, will appear with a flowchart in the middle. Select the "RunSession" item (it should be the only item present).
On the right side of the screen a drop-down menu will appear. Find the name of your saved SkanIt RE run in this menu.
10. Once you have selected your protocol, in the upper right corner click "Run this assay".
11. A box will pop up, asking you to denote which magazine to use as the source, and which magazine is empty. The lower box on the screen
corresponds to the front magazine. [Optional: It asks you to enter the number of plates loaded to determine an estimate for the amount of
time it will take. This estimate is normally way off.]
12. Ensure both the RapidStak and the VarioSkan are turned on, and press "OK"
13. The RapidStak will automatically load your plates into the plate reader, allowing for continuous measurements of plates.
Note: It is recommended to observe the first plate loading into the VarioSkan to ensure proper alignment of the machines. If the RapidStak
does not load the plate reader properly, use the "Pause this assay" button in the top right corner of the PolaraRS window.
5. Representative Results
An example of absorbance readings from a single 384 well plate containing a positive clone is shown in Figure 2. Positive clones show a marked
increase in absorbance over those not expressing cellulase activity. Differences in assay time, well location on the plate, or DNP concentration
(may be introduced by filtering out un-dissolved DNP) can affect absolute absorbance readings. Relative absorbance readings, such as the
difference in absorbance above the plate average or column average, are a more robust method of identifying cellulase positive clones.
Following identification of positive clones from the library plates, it is recommended to replicate all positive clones into a new plate for secondary
screening. This eliminates effects arising from well location or plate variation and allows for more direct comparison between positive clones.
Figure 1. Flow chart of the high-throughput assay for a metagenomic library cloned into E. coli and stored at -80°C.
Figure 2. Absorbance readings from one 384-well plate containing a positive clone. Cellulase positive clones can be identified by significantly
increased absorbance over negative clones.
Discussion
A high throughput screen for the rapid detection of cellulolytic activity from a large insert genomic DNA metagenomic library expressed in E. coli
is described in this protocol. This method is an improvement over the CMC/Congo Red assay commonly used in the literature. It is solution
Page 3 of 4
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Copyright © 2011 Journal of Visualized Experiments
14. Once the VarioSkan is done reading all the plates, remove the full magazine and place it on the magazine loading platform. Lift up the outer
rectangle of the platform, and the magazine should slide off, leaving the plates standing in a pile in the middle. The plates can be disposed of
according to your lab procedures.
15. To export the absorbance readings, open SkanIt RE software, and choose "Open an existing file".
16. Select the directory where you saved your session, hit the "+" icon to expand the list and under the title heading will be options labeled
"Container 1" to "Container N" depending on the number of plates. Select the first plate to be analyzed.
17. In the menu bar at the top of the page, select "Data Processing > Report/Export"
18. Choose the options desired for export.
Note: I typically select "Plate Layout" and "Photometric" data for export.
19. Click "View Report" and then "File > Save". Choose the desired directory. The output format is a Microsoft Excel spreadsheet
based, and allows for one-pot chemistry screening in 384-well plates, with the final output as absorbance readings from a plate reader allowing
for quantitative analysis. The automation of each step of this process allows for the unsupervised screening of more than 25 384-well plates per
hour. The data can be easily exported from the software into a Microsoft Excel spreadsheet, allowing for analysis or processing by third party
software.
One limitation of this assay exists in the expression potential of exogenous proteins in the E. coli host. A metagenomic library contains DNA from
many different organisms, only a subset of which can be recognized by the E. coli transcription/translation machinery. Even when expressed,
exogenous gene products may not achieve functionality due to improper folding, processing, or inadequate expression levels. These limitations
can be partially offset through the utilization of copy control systems, as seen in previous functional metagenomic screening studies. 12. The
pCC1 copy control fosmid used here allows induction through addition of the inducer L-arabinose, and increases copy number from one, to up to
100 copies per cell 13. These systems can improve the outcome of activity-based screens by enabling single copy growth for stability with
subsequent induction for increased activity.
The substrate 2,4-DNP-Cellobioside used in our screen is not commercially available from suppliers, but similar substrates can be purchased.
Sigma-Aldrich offers 2-nitrophenyl (Cat No. N4764) and 4-nitrophenyl (Cat No. N5759) cellobiosides. The general screen as described could be
undertaken with these substrates, but some modifications would be required. These mono-substituted phenols have higher pKa values, around
7.2, compared to DNP, which is around 4. Optimum pH for cellulase activity has been reported to range from pH 4.5-6.0 14, 15. The use of DNP-C
allows for the assay to be carried out at optimal pH conditions, allowing for easier identification of cellulases. In addition, the di-substituted
glycoside is much more reactive than the others, allowing for the detection of more reluctant cellulases. Thus, the use of DNP-cellobioside has
allowed for a more robust and sensitive screen than would be available with commercial substrates.
It is notable that this screen can be used for detection of any enzymes with an associated colorimetric or fluorometric substrate. Cellulases are a
stable and active enzyme, ideal for the initial development and optimization of the screening parameters. The general approach presented here is
a powerful tool for the screening of metagenomic libraries for both academic and industrial applications.
Disclosures
No conflicts of interest declared.
Acknowledgements
The authors would like to thank Dr. Steve Withers and Hong-Ming Chen for providing DNP-Cellobioside substrate.
References
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Copyright © 2011 Journal of Visualized Experiments
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  • Apr 2018
  • INT J BIOL SCI
Termite gut microbiome is a rich reservoir for glycoside hydrolases, a suite of enzymes critical for the degradation of lignocellulosic biomass. To search for hemicellulases, we screened 12,000 clones from a fosmid gut library of a higher termite, Globitermes brachycerastes. As a common Southeastern Asian genus, Globitermes distributes predominantly in tropical rain forests and relies on the lignocellulases from themselves and bacterial symbionts to digest wood. In total, 22 positive clones with β-xylosidase activity were isolated, in which 11 representing different restriction fragment length polymorphism (RFLP) patterns were pooled and subjected to 454 pyrosequencing. As a result, eight putative β-xylosidases were cloned and heterologously expressed in Escherichia coli BL21 competent cells. After purification using Ni-NTA affinity chromatography, recombinant G. brachycerastes symbiotic β-xylosidases were characterized enzymatically, including their pH and temperature optimum. In addition to β-xylosidase activity, four of them also exhibited either β-glucosidase or α-arabinosidases activities, suggesting the existence of bifunctional hemicellulases in the gut microbiome of G. brachycerastes. In comparison to multimeric protein engineering, the involvement of naturally occurring multifunctional biocatalysts streamlines the genetic modification procedures and simplifies the overall production processes. Alternatively, these multimeric enzymes could serve as the substitutes for β-glucosidase, β-xylosidase and α-arabinosidase to facilitate a wide range of industrial applications, including food processing, animal feed, environment and waste management, and biomass conversion.
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... For example, colloidal sugar cane bagasse (CSCB), is a natural substrate that shows better properties in reactivity and lower costs compared against commercial CMC and Avicel; CSCB and commercial substrates were evaluated in the screening of cellulolytic activity of hydrolytic enzymes from termites [49]. For the assessment of metagenomic libraries the chromogenic substrate dinitrophenol (DNP)-cellobioside was used to identify cellulolytic activity [50]. Quantification of cellulolytic activity in plants has difficulties due to its phenolic and sugars composition in the cell wall, which absorb light when measuring samples with spectrophotometer, causing noise during the detection of the reducing sugars produced by the hydrolysis of oligosaccharides. ...
High Throughput Screening: Developed Techniques for Cellulolytic and Xylanolytic Activities Assay
Article
  • Aug 2016
  • COMB CHEM HIGH T SCR
High throughput screening (HTS) is a powerful tool in biotechnology. The search for new or improved enzymes with suitable biochemical properties for industrial processes, has resulted in high efforts and research activities to develop new methodologies for activity screening. In this context, important advances have been achieved for the screening of cellulases and xylanases activities from wild and recombinant microorganisms, and from sequence databases. These enzymes have a wide range of industrial applications, including food, animal feed, textile, pulp and paper industries and detergents. Cellulases and xylanases along with pectinases, represent 20% of the world enzyme market. Recently, cellulases and xylanases have been used on fermentable sugars recovered from lignocellulosic biomass for second-generation biorefineries, aimed to produce chemical and biofuel platforms. As a result, HTS methods for biomass or biomass-degrading enzymes are gaining importance. This article presents evidence of the studies carried out for HTS of cellulase and xylanase activities.
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FUNCTIONAL METAGENOMICS AND CONSOLIDATED BIOPROCESSING FOR VALORIZATION OF PULP AND PAPER MILL SLUDGE
Thesis
  • Apr 2018
Biocatalyst discovery is integral to bioeconomy development, enabling design of scalable bioprocesses that can compete with the resource-intensive petrochemical industry. Uncultivated microbial communities within natural and engineered ecosystems provide a near-infinite reservoir of genomic diversity and metabolic potential that can be harnessed for this purpose. To bridge the cultivation gap, functional metagenomic screens have been developed to recover active genes directly from environmental samples. In this thesis, a pipeline for recovery of biomass-deconstructing biocatalysts sourced from pulp and paper mill sludge (PPS) metagenome is described. This environment is targeted given its high composition of cellulose that is hypothesized to direct enrichment of enzymes capable of hydrolysing it. The resulting oligosaccharides represent platform molecules that can be fed to downstream applications using consolidated process design for converting biological waste streams into value-added products. High-molecular weight DNA was extracted from sludge and used to construct a fosmid library containing 15,000 clones using the copy control system in EPI300™-T1 R E.coli. Extracted DNA was also used in whole genome shotgun sequencing to compare the metabolic potential of the sludge community with fosmid screening outcomes as well as other waste biomass environments using MetaPathways v2.5 software pipeline, with specific emphasis on carbohydrate-active enzymes (CAZymes). Metagenomic assembling, open reading frame (ORF) prediction, binning and taxonomic assignment approaches were also used to bring out correlations between function and taxonomy. In total, 32,232 ORF’s were mapped to the CAZy database predicted to encode glycoside hydrolases, glycosyl transferases, and carbohydrate binding module families.The fosmid library was screened for glycosidase hydrolase activities using a pool of sensitive fluorogenic glycosides of 6-chloro-4-methylumbelliferone (CMU). A total of 744 clones capable of converting pooled substrates were recovered indicating an extremely high hit rate (1 hit per 43 clones). Following fosmid sequencing and annotation, two of the most promising hits with defined single GH family loci were sub-cloned and overexpressed in E.coli BL21 DE3 strain to conduct basic biochemical characterization. Activity of purified enzymes was demonstrated on model lignocellulosic substrates to evaluate the potential of implementing the proposed circular bioprocess with waste PPS as both the feedstock and source of enriched biocatalysts.
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Discovering novel enzymes from marine ecosystems: A metagenomic approach
Article
  • Mar 2018
There exists a massive pool of biodiversity in marine ecosystems. This biodiversity is an excellent source for acquiring an inventory of enzymes that can be used for a variety of biotech applications. This diversity has, to date, not been fully exploited. One major reason being the difficulties that arise in culturing many microorganisms in the laboratory, as opposed to natural conditions. However, advents of newer omics techniques, such as metagenomics have greatly enhanced the opportunity for sustainable resource management. It is in this context that metagenomics is rapidly emerging as an alternative approach to conventional microbial screening. Metagenomics allows for exhaustive screening of microbial genomes in their natural environments. In this review, an overview of work that uses genomic strategies to examine the biotechnological potential of the marine reservoir was explored. These genomic strategies include homology-driven screening of enormous amounts of sequence data and activity-based functional screening of genomic and metagenomic libraries. Finally, the review concludes with an overview of some of the potential challenges and future prospects of metagenomics in bioprospecting novel biocatalysts and bioactive compounds from marine sources.
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A high‐throughput mass spectrometry‐based assay for identifying biochemical function of putative glycosidases
Article
Full-text available
The most common glycosidase assays rely on bulky ultraviolet or fluorescent tags at the anomeric position of potential carbohydrate substrates, thereby limiting the utility of these assays for broad substrate characterization. Here we report a mass spectrometry-based glycosidase assay amenable to high-throughput screening for the identification of the biochemical functions of putative glycosidases. The assay utilizes a library of methyl glycosides and is demonstrated on a high-throughput robotic liquid handling system for enzyme substrate screening. Identification of glycosidase biochemical function is achieved by observing a correct mass-loss between a potential sugar substrate and corresponding product using electrospray ionization mass spectrometry (ESI-MS). In addition to screening known glycosidases, the assay was demonstrated to characterize the biochemical function and enzyme substrate competency of the recombinantly-expressed product of a putative glycosidase gene from the thermophilic bacterium Thermus thermophilus.
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Large Insert Environmental Genomic Library Production
Article
Full-text available
  • Sep 2009
  • JoVE
The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent environmental genomic (i.e. metagenomic) approaches have emerged, enabling researchers to bridge this cultivation gap by capturing the genetic content of indigenous microbial communities directly from the environment. To this end, genomic DNA libraries are constructed using standard albeit artful laboratory cloning techniques. Here we describe the construction of a large insert environmental genomic fosmid library with DNA derived from the vertical depth continuum of a seasonally hypoxic fjord. This protocol is directly linked to a series of connected protocols including coastal marine water sampling [1], large volume filtration of microbial biomass [2] and a DNA extraction and purification protocol [3]. At the outset, high quality genomic DNA is end-repaired with the creation of 5 -phosphorylated blunt ends. End-repaired DNA is subjected to pulsed-field gel electrophoresis (PFGE) for size selection and gel extraction is performed to recover DNA fragments between 30 and 60 thousand base pairs (Kb) in length. Size selected DNA is purified away from the PFGE gel matrix and ligated to the phosphatase-treated blunt-end fosmid CopyControl vector pCC1 (EPICENTRE http://www.epibio.com/item.asp?ID=385). Linear concatemers of pCC1 and insert DNA are subsequently headfull packaged into phage particles by lambda terminase, with subsequent infection of phage-resistant E. coli cells. Successfully transduced clones are recovered on LB agar plates under antibiotic selection and archived in 384-well plate format using an automated colony picking robot (Qpix2, GENETIX). The current protocol draws from various sources including the CopyControl Fosmid Library Production Kit from EPICENTRE and the published works of multiple research groups [4-7]. Each step is presented with best practice in mind. Whenever possible we highlight subtleties in execution to improve overall quality and efficiency of library production. The whole process of fosmid library production and automated colony picking takes at least 7-10 days as there are many incubation steps included. However, there are several stopping points possible which are mentioned within the protocol.
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Mechanisms of Cellulases and Xylanases: A Detailed Kinetic Study of the Exo-.beta.-1,4-glycanase from Cellulomonas Fimi
Article
  • May 1994
The exoglucanase/xylanase from Cellulomonas fimi (Cex) has been subjected to a detailed kinetic investigation with a range of aryl beta-D-glycoside substrates. This enzyme hydrolyzes its substrates with net retention of anomeric configuration, and thus it presumably follows a double-displacement mechanism. Values of k(cat) are found to be invariant with pH whereas k(cat)/K-m is dependent upon two ionizations of pK(a) = 4.1 and 7.7. The substrate preference of the enzyme increases in the order glucosides < cellobiosides < xylobiosides, and kinetic studies with a range of aryl glucosides and cellobiosides have allowed construction of Broensted relationships for these substrate types. A strong dependence of both k(cat)(beta(1g) = -1) and k(cat)/K-m (beta(1g) = -1) upon leaving group ability is observed for the glucosides, indicating that formation of the intermediate is rate-limiting. For the cellobiosides a biphasic, concave downward plot is seen for k(cat) indicating a change in rate-determining step across the series. Pre-steady-state kinetic experiments allowed construction of linear Broensted plots of log k(2) and log (k(2)/K-d) for the cellobiosides of modest (beta(1g) = -0.3) slope. These results are consistent with a double-displacement mechanism in which a glycosyl-enzyme intermediate is formed and hydrolyzed via oxocarbonium ion-like transition states. Secondary deuterium kinetic isotope effects and inactivation experiments provide further insight into transition-state structures and, in concert with beta(1g) values, reveal that the presence of the distal. sugar moiety in cellobiosides results in a less highly charged transition state. These studies suggest that the primary function of the distal sugar is to increase the rate of formation of the glycosyl-enzyme intermediate through improved acid catalysis and greater nucleophile preassociation, without affecting its rate of decomposition.
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Sensitive assay for cellulase and dextranase
Article
  • Jul 1976
Sensitive, rapid, and quantitative methods have been devised for the assay of cellulases and dextranases through the synthesis of two chemically modified carboxymethyl cellulose substrates. One contains a trinitrophenyl group as chromophore. The other contains a fluorescent fluorescamine group. The soluble hydrolytic products in the filtrate released from the substrates by cellulase are thus monitored either spectrophotometrically (for trinitrophenyl group) or spectrofluorometrically (for fluorescamine). The same principle has been applied to the determination of dextranases by utilizing chemically modified Sephadex G-200 containing either group as deseribed above for carboxymethyl cellulose. The methods are sensitive to about 5 μg of enzyme for trinitrophenyl-containing substrates, while the use of fluorescamine-containing substrates is about tenfold more sensitive.
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Widespread known and novel phosphonate utilization pathways in marine bacteria revealed by functional screening and metagenomic analyses
Article
  • Sep 2009
  • ENVIRON MICROBIOL
Phosphonates (Pn), compounds with a direct C-P bond instead of the more common C-O-P ester bond, constitute a significant fraction of marine dissolved organic phosphorus and recent evidence suggests that they may be an alternative source of P for marine microorganisms. To further characterize the microorganisms and pathways involved in Pn utilization, we screened bacterioplankton genomic libraries for their ability to complement an Escherichia coli strain unable to use Pns as a P source. Using this approach we identified a phosphonatase pathway as well as a novel pair of genes that allowed utilization of 2-aminoethylphosphonate (2-AEPn) as the sole P source. These pathways are present in diverse bacteria common in marine plankton including representatives of Proteobacteria, Planctomycetes and Cyanobacteria. Analysis of metagenomic databases for Pn utilization genes revealed that they are widespread and abundant among marine bacteria, suggesting that Pn metabolism is likely to play an important role in P-depleted surface waters, as well as in the more P-rich deep-water column.
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Isolation and partial characterization of novel genes encoding acidic cellulases from metagenomes of buffalo rumens
Article
  • Apr 2009
  • J APPL MICROBIOL
To clone and characterize genes encoding novel cellulases from metagenomes of buffalo rumens. A ruminal metagenomic library was constructed and functionally screened for cellulase activities and 61 independent clones expressing cellulase activities were isolated. Subcloning and sequencing of 13 positive clones expressing endoglucanase and MUCase activities identified 14 cellulase genes. Two clones carried two gene clusters that may be involved in the degradation of polysaccharide nutrients. Thirteen recombinant cellulases were partially characterized. They showed diverse optimal pH from 4 to 7. Seven cellulases were most active under acidic conditions with optimal pH of 5.5 or lower. Furthermore, one novel cellulase gene, C67-1, was overexpressed in Escherichia coli, and the purified recombinant enzyme showed optimal activity at pH 4.5 and stability in a broad pH range from pH 3.5 to 10.5. Its enzyme activity was stimulated by dl-dithiothreitol. The cellulases cloned in this work may play important roles in the degradation of celluloses in the variable and low pH environment in buffalo rumen. This study provided evidence for the diversity and function of cellulases in the rumen. The cloned cellulases may at one point of time offer potential industrial applications.
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A Rapid and Easy Method for the Detection of Microbial Cellulases on Agar Plates Using Gram’s Iodine
Article
  • Oct 2008
Screening for cellulase-producing microorganisms is routinely done on carboxymethylcellulose (CMC) plates. The culture plates are flooded either with 1% hexadecyltrimethyl ammonium bromide or with 0.1% Congo red followed by 1 M NaCl. In both cases, it takes a minimum of 30 to 40 minutes to obtain the zone of hydrolysis after flooding, and the hydrolyzed area is not sharply discernible. An improved method is reported herein for the detection of extracellular cellulase production by microorganisms by way of plate assay. In this method, CMC plates were flooded with Gram's iodine instead of the reagents just mentioned. Gram's iodine formed a bluish-black complex with cellulose but not with hydrolyzed cellulose, giving a sharp and distinct zone around the cellulase-producing microbial colonies within 3 to 5 minutes. The new method is rapid and efficient; therefore, it can be easily performed for screening large numbers of microbial cultures of both bacteria and fungi. This is the first report on the use of Gram's iodine for the detection of cellulase production by microorganisms using plate assay.
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Rubin EM. Genomics of cellulosic biofuels. Nature 454(7206)
Article
  • Sep 2008
  • NATURE
The development of alternatives to fossil fuels as an energy source is an urgent global priority. Cellulosic biomass has the potential to contribute to meeting the demand for liquid fuel, but land-use requirements and process inefficiencies represent hurdles for large-scale deployment of biomass-to-biofuel technologies. Genomic information gathered from across the biosphere, including potential energy crops and microorganisms able to break down biomass, will be vital for improving the prospects of significant cellulosic biofuel production.
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Cellulase assay methods: a review
Article
  • Nov 1988
  • J BIOCHEM BIOPH METH
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Cellulase Production by Thermomonospora curvata Isolated from Municipal Solid Waste Compost
Article
  • Sep 1971
  • Appl Microbiol
A cellulolytic, thermophilic actinomycete (previously isolated from municipal refuse compost samples) was identified as Thermomonospora curvata. A determination was made of the optimal conditions for cellulase production by T. curvata when grown at 55 C in a medium containing mineral salts, cellulose, and yeast extract. The pH and temperature optima (pH 6.0 and 65 C) for the cellulase produced by T. curvata were identical to those previously observed for the cellulase extracted from crude compost samples. Such similarities, together with the prevalence of T. curvata in compost samples and its ability to grow at composting temperatures, indicate that this actinomycete could possibly be considered as a major cellulose decomposer in the municipal refuse composting process.
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Use of Congo Red-Polysaccharide Interactions in Enumeration and Characterization of Cellulolytic Bacteria from the Bovine Rumen
Article
  • May 1982
The interaction of the direct dye Congo red with intact beta-D-glucans provides the basis for a rapid and sensitive assay system for bacterial strains possessing beta-(1 leads to 4),(1 leads to 3)-D-glucanohydrolase, beta-(1 leads to 4)-D-glucanohydrolase, and beta-(1 leads to 3)-D-glucanohydrolase activities. A close correspondence was observed between cellulolytic activity and beta-(1 leads to 4)-D-glucanohydrolase and beta-(1 leads to 4),(1 leads to 3)-D-glucanohydrolase activities in isolates from the bovine rumen. Many of these isolates also possessed beta-(1 leads to 3)-D-glucanohydrolase activity, and this characteristic may have taxonomic significance.
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