Team:Tongji/project

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         <ul class="nav nav-list bs-docs-sidenav">
           <li><a href="#download-bootstrap"><i class="icon-chevron-right"></i> Abstract</a></li>
           <li><a href="#download-bootstrap"><i class="icon-chevron-right"></i> Abstract</a></li>
           <li><a href="#file-structure"><i class="icon-chevron-right"></i> Story Board</a></li>
           <li><a href="#file-structure"><i class="icon-chevron-right"></i> Story Board</a></li>
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          <li><a href="#contents"><i class="icon-chevron-right"></i> Prospective</a></li>
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           <p class="lead">  We build parts for decolor-assistant proteins(ManA1, ArfB, XynB) to assemble decolor-assistant machines. Compared to existing decolor-assistant proteins, our proteins can work at high temperature and have synergistic effects, which may be useful in paper industry.</p>
+
           <p class="lead">  We build parts for decolor-assistant proteins (<em>man</em>A1, <em>arf</em>B, <em>xyn</em>B<a id="Jin6" href="#" rel="tooltip" data-placement="top" data-original-title="manA1 is putative to encode mannosidase, arfB encodes arabinofuranosidase and xynB encodes xylanase. They are all belong to hemicellulase."><sup><strong>Attention!</strong></sup></a>) to assemble decolor-assistant machines. Compared to existing decolor-assistant proteins, our proteins can work at high temperature and have synergistic effects, which may be useful in paper industry.</p>
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<img src="https://static.igem.org/mediawiki/2014/5/5b/Decolorfour.png">
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             <h1>Story Board</h1>
             <h1>Story Board</h1>
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           <p class="lead">ManA1, ArfB, XynB are three genes each encoding an enzyme. With help of these proteins, the pulp(which then become paper) can be bleached by only little chemical reagent. Since using less chemical reagent(especially the chlorinated) can help protect the environment, nowadays enzyme-assisted bleaching method is getting popular in paper making industry.</p>
+
           <p class="lead"><em>man</em>A1, <em>arf</em>B, <em>xyn</em>B are three genes which respectively encoding <strong>hemicellulase</strong> enzyme. With help of these proteins, the paper pulp can be bleached by much less chemical reagent. Since less chemical reagent (especially the chlorinated chemicals) in bleaching can benefit the environment protection, nowadays enzyme-assisted bleaching method is getting popular in paper making industry.</p>
-
           <p class="lead">  In our project, we build parts for three decolor-assistant proteins(ManA1, ArfB, XynB) to assemble three decolor-assistant machines. Through experiments, we confirm their decolor-assistant function, and we observe their heat-resistant property and synergistic effect. </p>
+
           <p class="lead">  In our project, we build parts for three decolor-assistant proteins (<em>man</em>A1, <em>arf</em>B, <em>xyn</em>B) to assemble three decolor-assistant machines. The experiment results confirmed the function of these decolor-assistant proteins, especially their heat-resistant property and synergistic effect.</p>
-
           <p class="lead">  To better understand the bio-assistant trend in paper industry, we also talk to local paper factory (we asked for a visit, but they all refused us. Some said this is the secret, and other suggested they are under emission standard). At the same time, we tightly communicate with several iGEM teams to improve our idea and appreciate their work. This is our first time to join the iGEM competition, and we hope we can have fun and get inspired in the following months.</p>
+
           <p class="lead">  To better understand the bio-assistant trend in paper industry, we asked for a visit to local paper factory but they all refused us because of some commercial secrets. At the same time, we tightly communicate with several iGEM teams to improve our idea and appreciate their work. This is our first time to join the iGEM competition, and we hope we can have fun and get inspired in the following months.</p>
          
          
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        <!-- Contents
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            <h1>Prospective</h1>
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          </div>
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          <h2>Pulp And Paper Industry</h2>
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          <h3>Introduction</h3>
 +
          <p class="lead">Conventionally, the bleaching of digested pulp by chlorine compounds implies the generation of much adsorbable organic halogen (AOX) which is problematic due to its high toxicity and low biodegradability. Improper treatment and release of these hazardous waste could raise serious environmental and health problems. Finding solutions to reducing or replacing usage of those harmful compounds have aroused much concern.</p>
 +
          <p class="lead">Since 1986<a id="Jin" href="#" rel="tooltip" data-placement="top" data-original-title="Bleaching with enzymes, Biotechnology in the pulp and paper industry, 1986, Viikari L et al."><sup>Ref</sup></a>, several types of hemicellulase have been employed as biotechnological alternative solutions in the bleaching of pulp to help lower the chlorine containing bleaching reagent consumption. Xylanase is a prominent representative. Lab scaled experiments have testified that bleaching process involving Xylanase treatment was rather promising (Shown as table below). Moreover, bleaching process involving xylanase treatment can also improve product performance and save chemical  expense<a id="Jin2" href="#" rel="tooltip" data-placement="top" data-original-title="Protein engineering of xylanase for pulp bleaching applications, 1998, White T et al."><sup>Ref</sup></a>.</p>
 +
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          <table class="table table-hover">
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            <thead>
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              <tr>
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                <th class="text-info ">Author and time</th>
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                <th class="text-info ">Bleach procedure</th>
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                <th class="text-info ">Pulp type</th>
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                <th class="text-info ">ClO<sub>2</sub> consumption reduced</th>
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              </tr>
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            </thead>
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            <tbody>
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              <tr>
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                <td><div>Viikari, 1986</div></td>
 +
                <td><div >X(DC)E</div></td>
 +
                <td><div >SWKP</div></td>
 +
                <td><div >25%</div></td>
 +
              </tr>
 +
              <tr>
 +
                <td><div>Shan, 2000</div></td>
 +
                <td><div >XD<sub>0</sub>ED<sub>1</sub>ED<sub>2</sub></div></td>
 +
                <td><div >HWKP-O</div></td>
 +
                <td><div >25%</div></td>
 +
              </tr>
 +
              <tr>
 +
                <td><div>Paice, 2004</div></td>
 +
                <td>XDE<sub>0</sub>D</td>
 +
                <td><div >HWKP</div></td>
 +
                <td><div >13.4%</div></td>
 +
              </tr>
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            </tbody>
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          </table>
 +
 +
 +
          <p class="lead ">The process of biobleach is simple, and does not require major alteration of the conventional flow path. After xylanase is mixed into the pulp and kept in a holding tank for 1-3 hours, the pulp is bleached in the normal way.</p>
 +
 +
          <blockquote>
 +
            <small>Flow Chart Of Paper Bleaching</small>
 +
            <img src="https://static.igem.org/mediawiki/2014/f/f6/Paperpulp3.png ">
 +
            <small>R.C. Kuhad, Ajay Singh, <em>Lignocellulose Biotechnology Future Prospects</em>, 2007</small> 
 +
           
 +
          </blockquote> 
 +
 +
          <p class="lead ">More than two decades have passed since the attempt of reducing pulp bleaching associated AOX emission has been made, yet this field of research still have many questions to ask and problems to solve. Researchers are still making efforts to optimize aspects of the biobleach method <a id="Jin3" href="#" rel="tooltip" data-placement="top" data-original-title="Xylanase and laccase based enzymatic Kraft pulp bleaching reduces adsorbable organic halogen (AOX) in bleach effluents: A pilot scale study, 2014, Abha Sharma et al"><sup>Ref</sup></a>.</p>
 +
 +
 +
 +
          <p class="lead ">One of the most important aspects of the biobleach method is stability and efficiency of xylanase. It is easy to understand that xylanases from natural hosts are evolved for adaption to the environment, thus not all of them can be in good service for industrial conditions. Many researchers have been seeking a xylanase which can work under conventional pulp treatment conditions, such as high temperature and alkaline.</p>
 +
 +
 +
          <blockquote>
 +
            <p class="lead text-info">Availability of the enzyme that is cellulase-free, working at temperature <strong>above 80°C</strong> and <strong>pH >8.0</strong> would be very beneficial. In this respect, the thermostable and alkalistable xylanases have high potential in pulp bleaching process in paper industry.</p>
 +
            <small>R.C. Kuhad, Ajay Singh, <em>Lignocellulose Biotechnology Future Prospects</em>, 2007</small> 
 +
            <small>Sierra <em>et al</em>, 1994</small> 
 +
          </blockquote> 
 +
 +
          <h3>Our Contribution</h3>
 +
          <p class="lead">Three genes of interest, which designated as <em>xyn</em>B, <em>arf</em>B and <em>man</em>A1, were derived from a thermophilic bacteria <em>Clostridium stercorarium</em> DSM8532.</p>
 +
 +
 +
 +
 +
          <p class="lead "> <em>xyn</em>B gene encodes a endo-1,4-beta-xylanase. We identified this enzyme as an impressive thermostable xylanase because it presented a high activity at high temperature even up to 94.7°C! Since time is limited, other properties (such as synergistic effect and alkalistability) of this enzyme still awaits further validation.</p>
 +
 +
          <p class="lead "><em>arf</em>B gene encodes an alpha-L-arabinofuranosidase, which has been reported as a thermoactive enzyme with arabinoxylan debranching activity, and is potential for biobleaching<a id="Jin4" href="#" rel="tooltip" data-placement="top" data-original-title="Debranching of arabinoxylan: properties of the thermoactive recombinant K-L-arabinofuranosidase from Clostridium stercorarium (ArfB), 1995, Schwarz et al."><sup>Ref</sup></a>.</p>
 +
 +
          <p class="lead "> <em>man</em>A1 gene, encoding a putative mannan endo-1,4-beta-mannosidase, was identified by complete genome sequencing of <em>Clostridium stercorarium</em> Strain DSM 8532. We construced this gene expression vector to make it expressed in <em>E.coli</em>. The mannosidase activity of this gene was confirmed by our experimental evidences. Given that other mannosidases have been reported to be useful in biobleaching, this enzyme is likely to be a candidate for industrial biobleach agent. Especially considering the thermophilic property of this gene, it could potentially be applied in paper pulp biobleeching.</p>
 +
 +
 +
          <h2>Future Energy Resource</h2>
 +
          <p class="lead ">Besides being useful in paper industry, these hemicellulases might be able to help with solving energy problem.</p>
 +
          <p class="lead ">Lignocellulosic biomass is regarded as a very promising resource of fuel. The conversion is based on pretreatment of raw material to remove unfermantable components such as lignin, and this is exactly what the hemicellulases are capable of.</p>
 +
          <p class="lead ">In fact, someone had already used xylanase to assemble a pretreatment machine<a id="Jin5" href="#" rel="tooltip" data-placement="top" data-original-title="Deconstruction of Lignocellulose into Soluble Sugars by Native and Designer Cellulosomes, 2012, Morais et al."><sup>Ref</sup></a>.</p>
 +
 +
 +
 +
 +
 
 +
          <p class="lead">Therefore, to the best of our knowledge, our project may be beneficial to industrial pulp bleaching and future energy resource development.</p>
 +
 +
 +
         
 +
        </section>
          
          
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Latest revision as of 02:53, 18 October 2014

iGEM Tongji 2014

Project

We build parts for decolor-assistant proteins (manA1, arfB, xynBAttention!) to assemble decolor-assistant machines. Compared to existing decolor-assistant proteins, our proteins can work at high temperature and have synergistic effects, which may be useful in paper industry.

manA1, arfB, xynB are three genes which respectively encoding hemicellulase enzyme. With help of these proteins, the paper pulp can be bleached by much less chemical reagent. Since less chemical reagent (especially the chlorinated chemicals) in bleaching can benefit the environment protection, nowadays enzyme-assisted bleaching method is getting popular in paper making industry.

In our project, we build parts for three decolor-assistant proteins (manA1, arfB, xynB) to assemble three decolor-assistant machines. The experiment results confirmed the function of these decolor-assistant proteins, especially their heat-resistant property and synergistic effect.

To better understand the bio-assistant trend in paper industry, we asked for a visit to local paper factory but they all refused us because of some commercial secrets. At the same time, we tightly communicate with several iGEM teams to improve our idea and appreciate their work. This is our first time to join the iGEM competition, and we hope we can have fun and get inspired in the following months.

Pulp And Paper Industry

Introduction

Conventionally, the bleaching of digested pulp by chlorine compounds implies the generation of much adsorbable organic halogen (AOX) which is problematic due to its high toxicity and low biodegradability. Improper treatment and release of these hazardous waste could raise serious environmental and health problems. Finding solutions to reducing or replacing usage of those harmful compounds have aroused much concern.

Since 1986Ref, several types of hemicellulase have been employed as biotechnological alternative solutions in the bleaching of pulp to help lower the chlorine containing bleaching reagent consumption. Xylanase is a prominent representative. Lab scaled experiments have testified that bleaching process involving Xylanase treatment was rather promising (Shown as table below). Moreover, bleaching process involving xylanase treatment can also improve product performance and save chemical expenseRef.

Author and time Bleach procedure Pulp type ClO2 consumption reduced
Viikari, 1986
X(DC)E
SWKP
25%
Shan, 2000
XD0ED1ED2
HWKP-O
25%
Paice, 2004
XDE0D
HWKP
13.4%

The process of biobleach is simple, and does not require major alteration of the conventional flow path. After xylanase is mixed into the pulp and kept in a holding tank for 1-3 hours, the pulp is bleached in the normal way.

Flow Chart Of Paper Bleaching R.C. Kuhad, Ajay Singh, Lignocellulose Biotechnology Future Prospects, 2007

More than two decades have passed since the attempt of reducing pulp bleaching associated AOX emission has been made, yet this field of research still have many questions to ask and problems to solve. Researchers are still making efforts to optimize aspects of the biobleach method Ref.

One of the most important aspects of the biobleach method is stability and efficiency of xylanase. It is easy to understand that xylanases from natural hosts are evolved for adaption to the environment, thus not all of them can be in good service for industrial conditions. Many researchers have been seeking a xylanase which can work under conventional pulp treatment conditions, such as high temperature and alkaline.

Availability of the enzyme that is cellulase-free, working at temperature above 80°C and pH >8.0 would be very beneficial. In this respect, the thermostable and alkalistable xylanases have high potential in pulp bleaching process in paper industry.

R.C. Kuhad, Ajay Singh, Lignocellulose Biotechnology Future Prospects, 2007 Sierra et al, 1994

Our Contribution

Three genes of interest, which designated as xynB, arfB and manA1, were derived from a thermophilic bacteria Clostridium stercorarium DSM8532.

xynB gene encodes a endo-1,4-beta-xylanase. We identified this enzyme as an impressive thermostable xylanase because it presented a high activity at high temperature even up to 94.7°C! Since time is limited, other properties (such as synergistic effect and alkalistability) of this enzyme still awaits further validation.

arfB gene encodes an alpha-L-arabinofuranosidase, which has been reported as a thermoactive enzyme with arabinoxylan debranching activity, and is potential for biobleachingRef.

manA1 gene, encoding a putative mannan endo-1,4-beta-mannosidase, was identified by complete genome sequencing of Clostridium stercorarium Strain DSM 8532. We construced this gene expression vector to make it expressed in E.coli. The mannosidase activity of this gene was confirmed by our experimental evidences. Given that other mannosidases have been reported to be useful in biobleaching, this enzyme is likely to be a candidate for industrial biobleach agent. Especially considering the thermophilic property of this gene, it could potentially be applied in paper pulp biobleeching.

Future Energy Resource

Besides being useful in paper industry, these hemicellulases might be able to help with solving energy problem.

Lignocellulosic biomass is regarded as a very promising resource of fuel. The conversion is based on pretreatment of raw material to remove unfermantable components such as lignin, and this is exactly what the hemicellulases are capable of.

In fact, someone had already used xylanase to assemble a pretreatment machineRef.

Therefore, to the best of our knowledge, our project may be beneficial to industrial pulp bleaching and future energy resource development.

Tongji University, 2014