Team:WLC-Milwaukee/Modeling

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<h2>yesZ</h2>The Beta-Galactosidase yesZ is globular and has three aspects that are important to its function. It contains a zinc ion binding site at the C residues 153, 155, and 158 in yellow. The putative nucleophile and acid base cleavage sites are at the E residues 155 and 296 in red. The active site where substrate binding occurs is through the amino acid residue sequence ETSPSYAASL from residues 296- 305.  
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<h1>yesZ</h1>The Beta-Galactosidase yesZ is globular and has three aspects that are important to its function. It contains a zinc ion binding site at the C residues 153, 155, and 158 in yellow. The putative nucleophile and acid base cleavage sites are at the E residues 155 and 296 in red. The active site where substrate binding occurs is through the amino acid residue sequence ETSPSYAASL from residues 296- 305.  
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<img src="https://static.igem.org/mediawiki/2013/6/6d/WLC-YesZmod.png" width="450">
<img src="https://static.igem.org/mediawiki/2013/6/6d/WLC-YesZmod.png" width="450">
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<h2>yesZ Enzyme Activity</h2>
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<img src="https://static.igem.org/mediawiki/2013/2/23/WLC-YesZkenetics.png" width="450">
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The kinetic parameters fo the hydrolysis of pNPGal by wild type YesZ Kcat = 81.4 ± 4 s-1, and Km= 3.0 ± 0.2 mM, and Kcat/Km= 27 ± 2 mM -1 s-1. 
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Figure A describes the time dependent inactivation of the YesZ enzyme using DNP2FGal.  The time dependent inactivation observed as a single exponential decay to a non-zero value when DNP2FGal was incubated with the enzyme.  Studies of inactivation kinetics were performed by pre-incubating 100 microliters of the enzyme at 37 oC with DNP2FGal at a range of concentrations in a total volume of 140 microliters.">
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<img src="https://static.igem.org/mediawiki/2013/e/e3/WLC-Bglsmod.png" width="450">
<img src="https://static.igem.org/mediawiki/2013/e/e3/WLC-Bglsmod.png" width="450">
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<h2>bglS</h2>
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<h1>bglS</h1>
The endo-1,3-1,4-glucanase bglS is a globular protein that that has two residues of interest. The putative nucleophile and acid-base cleavage sites at the E residues 133 and 137 highlighted in red.  
The endo-1,3-1,4-glucanase bglS is a globular protein that that has two residues of interest. The putative nucleophile and acid-base cleavage sites at the E residues 133 and 137 highlighted in red.  
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<h2>xynA</h2>
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<h2>bglS Enzyme Activity</h2>
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Table 2. Substrate specificity of 1,3-1,4-beta-glucanase (BglS) purified to electrophoretic homogenity from E. coli cells harboring recombinant plasmid pRB33. Enzyme activities were calculated from the results of three independent measurements.
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<img src="https://static.igem.org/mediawiki/2013/f/ff/WLC-Bglschart.png" width="450">
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*Unit defined as 1 micromole reducing sugar min-1 (mg purified enzyme)-1.
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**Unit defined as 1 OD595 unit min-1 (mg purified enzyme)-1.
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Table and data from:
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http://mic.sgmjournals.org/content/141/2/281.long
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<img src="https://static.igem.org/mediawiki/2013/e/e7/WLC-Bglsplates.png" width="450">
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The plates shown are differential medias containing both lichenan ,able to be broken down by the Bacillus Subtilis Subtilis 168 BglS gene.  The plates were stained by flooding the plate with Congo red and the visualization of clearing zones was improved by flooding the stained plates with 1M NaCl. 
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The Upper plate indicates, due to an apparent lack of clearing zones, that the strains MW14 (Deleted BglS), MW10 (Deleted EglS102 and Bgl BglSRV) and MW9 (Deletion of EglS and BglS) do not contain a significant ability to break down Lichenan.  Whereas the strains MW8 (Deletion of EglS) and DB104 (No deletions) were able to break down the lichenan.
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    The lower plate indicates, due to an apparent lack of clearing zones, that the strains MW10, MW8, and MW9 did not contain a significant ability to break down the CM-cellulose.  On the other side of the coin, however, the strains MW14 and DB104 did have the ability to produce clearing zones, indicating their ability to break down CM-cellulose.
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<tr><td colspan="2"><h1>bglS on Cellulose Plates</h1></td></tr>
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<img src="https://static.igem.org/mediawiki/2013/7/7e/WLC-BglSplate1.jpg" width="450">
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<img src="https://static.igem.org/mediawiki/2013/9/99/WLC-Bglsplate2.jpg" width="450">
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<h1>xynA</h1>
The endo-1,4-beta-xylanase xynA is a globular protein that has two residues of interest the nucleophile and acid-base cleavage sites at the E residues 78 and 172 highlighted in red.  
The endo-1,4-beta-xylanase xynA is a globular protein that has two residues of interest the nucleophile and acid-base cleavage sites at the E residues 78 and 172 highlighted in red.  
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<img src="https://static.igem.org/mediawiki/2013/7/72/WLC-XynAmod.jpg" width="450">
<img src="https://static.igem.org/mediawiki/2013/7/72/WLC-XynAmod.jpg" width="450">
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<h2>Get Title</h2>
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<h2>xynA Enzyme Activity</h2>
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<img src="https://static.igem.org/mediawiki/2013/3/31/WLC-Xynaxylanaseratio.gif">
<img src="https://static.igem.org/mediawiki/2013/3/31/WLC-Xynaxylanaseratio.gif">

Latest revision as of 03:20, 28 September 2013

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yesZ

The Beta-Galactosidase yesZ is globular and has three aspects that are important to its function. It contains a zinc ion binding site at the C residues 153, 155, and 158 in yellow. The putative nucleophile and acid base cleavage sites are at the E residues 155 and 296 in red. The active site where substrate binding occurs is through the amino acid residue sequence ETSPSYAASL from residues 296- 305.

yesZ Enzyme Activity

 The kinetic parameters fo the hydrolysis of pNPGal by wild type YesZ Kcat = 81.4 ± 4 s-1, and Km= 3.0 ± 0.2 mM, and Kcat/Km= 27 ± 2 mM -1 s-1. Figure A describes the time dependent inactivation of the YesZ enzyme using DNP2FGal. The time dependent inactivation observed as a single exponential decay to a non-zero value when DNP2FGal was incubated with the enzyme. Studies of inactivation kinetics were performed by pre-incubating 100 microliters of the enzyme at 37 oC with DNP2FGal at a range of concentrations in a total volume of 140 microliters.">

bglS

The endo-1,3-1,4-glucanase bglS is a globular protein that that has two residues of interest. The putative nucleophile and acid-base cleavage sites at the E residues 133 and 137 highlighted in red.

bglS Enzyme Activity

Table 2. Substrate specificity of 1,3-1,4-beta-glucanase (BglS) purified to electrophoretic homogenity from E. coli cells harboring recombinant plasmid pRB33. Enzyme activities were calculated from the results of three independent measurements. *Unit defined as 1 micromole reducing sugar min-1 (mg purified enzyme)-1. **Unit defined as 1 OD595 unit min-1 (mg purified enzyme)-1. Table and data from: http://mic.sgmjournals.org/content/141/2/281.long 		The plates shown are differential medias containing both lichenan ,able to be broken down by the Bacillus Subtilis Subtilis 168 BglS gene. The plates were stained by flooding the plate with Congo red and the visualization of clearing zones was improved by flooding the stained plates with 1M NaCl. The Upper plate indicates, due to an apparent lack of clearing zones, that the strains MW14 (Deleted BglS), MW10 (Deleted EglS102 and Bgl BglSRV) and MW9 (Deletion of EglS and BglS) do not contain a significant ability to break down Lichenan. Whereas the strains MW8 (Deletion of EglS) and DB104 (No deletions) were able to break down the lichenan. The lower plate indicates, due to an apparent lack of clearing zones, that the strains MW10, MW8, and MW9 did not contain a significant ability to break down the CM-cellulose. On the other side of the coin, however, the strains MW14 and DB104 did have the ability to produce clearing zones, indicating their ability to break down CM-cellulose.

bglS on Cellulose Plates

xynA

The endo-1,4-beta-xylanase xynA is a globular protein that has two residues of interest the nucleophile and acid-base cleavage sites at the E residues 78 and 172 highlighted in red.

xynA Enzyme Activity

This graph depicts the inhibition of the gene product of XynA found in Bacillus Subtillis Subtillis 168 (BsX) in comparison to the inhibition of the XynA found in Aspergillus Niger (AsX). Sorensen and Sibbensen were observing the inhibitory effects of the TAXI (Triticum Aestivum Xylanase Inhibitor) , specific to Glycoside hydrolase family 11 (GH 11), and XIP (xylanase inhibitor protein), specific to fungal GH 11 but not bacterial GH 11. which XynA is a member. Inhibition was tested with either pure XIP (BsX-XIP and AnX-XIP) or both XIP and TAXI ( BsX-Inhibitor Prep and AnX-Inhibitor Prep. It is evident from this graph that BsX is not effected by XIP but is strongly inhibited by TAXI, causing a decrease in residual xylanase activity by approximately 80%.

This graph depicts the effect of pH on the interaction between the BsX xylanase and the inhibitor TAXI at a 1:5 concentration. The pH profile of the of the inhibition resembles the ph profile of the enzyme, indicating that TAXI is a competitive inhibitor for the BsX Xylanase. This graph also depicts the optimal pH for the XynA enzyme from Bacillus Subtillis Subtillis 168 to be around 5.5.

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