Difference between revisions of "Team:Stuttgart/Description"
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| + | <h1 align=middle> Description </h1> | ||
| + | <div class="container"> | ||
| + | <div class="row section"> | ||
| + | <div class="col-xs-12 col-sm-12 col-md-12"> | ||
| + | <h3>What's the problem?</h3> | ||
| + | <p>Our project deals with an everyday problem described by the following scenario: | ||
| + | You want to enjoy a refreshing shower in the morning, but your hairy roommate clogged the drain again? | ||
| + | You would like to have a relaxed bubble bath after a long day, but there are bad odors coming out of the pipe system? | ||
| + | <br> | ||
| + | <br> | ||
| + | The general practice now: | ||
| + | <ul><li>You try the ‘hot water method’ to flush the drain… nothing happens.</li> | ||
| + | <li>You remember what your mother taught you, so you put a fancy mix of vinegar, baking soda and some magic into the drain… a mysterious creature arises, but nothing else happens.</li> | ||
| + | <li>In the end there is just one thing left – you put the nasty chemical mixture of the stinky cleaning agent down the drain. The corrosive cloud disappears and you can finally take your shower… still trying not to breath in the acrid fumes.</li></ul> | ||
| + | </p> | ||
| + | </div> | ||
| + | </div> | ||
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| − | + | <a href="https://static.igem.org/mediawiki/2017/archive/0/0b/20171101205343%21Comicstuttgart.png" class="image"> | |
| − | + | <img alt="" src="https://static.igem.org/mediawiki/2017/archive/0/0b/20171101205343%21Comicstuttgart.png" class="img-responsive" /> | |
| − | + | </a> | |
| − | + | <div class="thumbcaption"> | |
| − | + | <div class="magnify"> | |
| − | + | <a href="https://static.igem.org/mediawiki/2017/archive/0/0b/20171101205343%21Comicstuttgart.png" class="internal" title="Enlarge"> | |
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</div> | </div> | ||
| + | <div class="row section"> | ||
| + | <div class="col-xs-12 col-sm-12 col-md-12"> | ||
| + | <p>Pipe clogging and closure occurs in every household as well as in industry. | ||
| + | Various components contribute to the pipe closure, such as fat and hair. | ||
| + | Commercial chemical pipe cleaners contain aggressive substances, which are harmful to the environment and health and also can lead to pipe breaks. | ||
| + | Biological alternatives (purified enzymes) have been researched for years, without reaching the efficiency of chemical cleaners. | ||
| + | In addition, the purification of the enzymes is very complex and cost intensive. </p> | ||
| − | + | <div class="row section"> | |
| − | <div class=" | + | <div class="col-xs-12 col-sm-12 col-md-12"> |
| − | + | <h3>Our solution</h3> | |
| − | + | </div> | |
| − | + | </div> | |
| − | + | <div class="row section"> | |
| − | + | <div class="col-xs-12 col-sm-6 col-md-6"> | |
| − | + | <p>We pursue a system-biological solution which is based on an intact microbial system. | |
| − | + | Inspired by different interesting projects we decided to develope a keranolytic <i>Escherichia coli</i>. | |
| − | + | The projects of past iGEM-teams were part of our investigation too and we are already co-working with iGEM-team OLS_Canmore 2015/2016 to exchange information. | |
| − | + | We would like to use the knowledge of these groundworks to develop a new type of enzymatic cleaning agent by complementing and combining ideas out of different scientific sources. | |
| − | + | To differ from other projects we are aiming to engineer <i>Escherichia coli</i> to express enzymes like esterases, lipases and keratinases into the medium. | |
| − | + | Due to the extracellular expression expensive and time-consuming purification steps could be avoided and the enzymes could be secreted directly into the drain. As a special feature we want to use the metabolites of the enzymatic degradation to produce a lovely rose-scent to refresh your whole room. | |
| − | + | In addition this reaction could also be used to indicate that the enzymes are working efficiently on your drainage problem. | |
| − | + | </p> | |
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</div> | </div> | ||
| − | + | <div class="col-xs-12 col-sm-6 col-md-6"> | |
| − | + | <div class="thumb tleft"> | |
| − | + | <div class="thumbinner" style="width:;"> | |
| − | + | <a href="https://static.igem.org/mediawiki/2017/1/10/Modelling_stuttgart.png" class="image"> | |
| − | + | <img alt="" src="https://static.igem.org/mediawiki/2017/1/10/Modelling_stuttgart.png" class="img-responsive" /> | |
| − | + | </a> | |
| − | <a href="https:// | + | <div class="thumbcaption"> |
| − | + | <div class="magnify"> | |
| − | + | <a href="https://static.igem.org/mediawiki/2017/1/10/Modelling_stuttgart.png" class="internal" title="Enlarge"> | |
| − | </ | + | <img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="" /> |
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</div> | </div> | ||
| − | + | <b>Figure 1:</b> Model of lipid, fat and keratin degradation and secretion of rose frgrance molecules.</div> | |
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| + | </div> | ||
| + | <div class="row section"> | ||
| + | <div class="col-xs-12 col-sm-12 col-md-12"> | ||
| + | <h2 align=middle>Light up the pipe - Three parts for a better flow</h2> | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="row section"> | ||
| + | <div class="col-xs-12 col-sm-12 col-md-12"> | ||
| + | <h3>Part 1 - Esterases and Lipases</h3> | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="row section"> | ||
| + | <div class="col-xs-12 col-sm-4 col-md-4"> | ||
| + | <!--<https://static.igem.org/mediawiki/2017/7/79/Esterasecomicneu234.jpeg">--> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/7/79/Esterasecomicneu234.jpeg" class="img-responsive"/> | ||
| + | </div> | ||
| + | <div class="col-xs-12 col-sm-8 col-md-8"> | ||
| + | <p>Hair is surrounded by a layer of grease and waxes which first need to be removed to make the hair-keratin available for keratinases. | ||
| + | For the first degradation step we choose a combination of esterases and lipases. | ||
| + | Lipases and esterases are lipolytic enzymes that are used in food processing, beverages, therapeutics and degradation of synthetic materials (Panda und Gowrishankar 2005). | ||
| + | Lipases (EC. 3.1.1.3) and esterases (EC. 3.1.1.1) are hydrolases that catalyze the hydrolysis (and synthesis) of triacylglycerols into acid and alcohol molecules. | ||
| + | They differ mainly on the basis of substrate specifity and activation mechanism. | ||
| + | Lipases hydrolase triacylglyderols with longer fatty acid chains than esterases and lipases have a hydrophobic domain covering their active site. | ||
| + | Therefore lipases need to be activated with organic solvents to make the active site accessible for substrates. | ||
| + | Esterases and lipases are classified into 8 groups on the basis of the conserved sequence motifs and biological properties. | ||
| + | EstCS2 belongs to class VII (Kang et al. 2011; Shamsher Singh Kanwar). | ||
| + | </p> | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="row section"> | ||
| + | <div class="col-xs-12 col-sm-12 col-md-12"> | ||
| + | <p>We investigated two different esterases for their enzyme activity. | ||
| + | One esterase from the registry (EstCS2 BBa_K1149002) and one esterase (LipB) supplied by Dr. Eggert from Evoxx were compared. | ||
| + | Additionally, we choose the lipase TliA to support the esterases at the fat degradation and to accelerate the entire degradation process. | ||
| + | For the extracellular secretion of the enzymes we attached the signal sequence PelB which was provided on the iGEM plates. | ||
| + | </p> | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="row section"> | ||
| + | <div class="col-xs-12 col-sm-12 col-md-12"> | ||
| + | <h4>Esterases EstCS2 and LipB</h4> | ||
| + | <p>EstCS2 from the iGEM Imperial College 2013 was proved to be active. | ||
| + | In their project the cells expressing this construct were grown and lysed by sonication and were utilized in a colourimetric assay with the substrate analog para-Nitrophenyl butyrate. | ||
| + | In our project we didn’t purify the esterases but used the supernatant for the enzyme activity assay. | ||
| + | Furthermore, we investigated the enzyme activity of LipB. | ||
| + | We performed the enzyme assay from the iGEM Team TU Darmstadt that we also used for EstCS2. | ||
| + | Afterwards we attached the PelB signal sequence for the extracellular secretion of the enzyme. | ||
| + | </p> | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="row section"> | ||
| + | <div class="col-xs-12 col-sm-9 col-md-9"> | ||
| + | <br> | ||
| + | <p> | ||
| + | <u>General properties of EstCS2: (Kang et al. 2011)</u> | ||
| + | <ul> | ||
| + | <li>serine esterase with 45 % similarity with the carboxylesterase from <i>Haliangium ochraceum DSM 14365</i> </li> | ||
| + | <li>temperature optimum: 55 °C; at 37°C: ca. 50 % of maximal activity </li> | ||
| + | <li>pH optimum: 9; at pH 7: ca. 30 % of maximal activity </li> | ||
| + | <li>Family VII esterase with the Ser residue in the catalytic triad of EstCS2 that is loacated in the consensus active site motif GXSXG </li> | ||
| + | </ul> | ||
| + | </p> | ||
| + | </div> | ||
| + | <div class="col-xs-12 col-sm-3 col-md-3"> | ||
| + | <div class="thumb tleft"> | ||
| + | <div class="thumbinner" style="width:;"> | ||
| + | <a href="https://static.igem.org/mediawiki/2017/4/4d/Catisvektro3.png" class="image"> | ||
| + | <img alt="" src="https://static.igem.org/mediawiki/2017/4/4d/Catisvektro3.png" class="img-responsive" /> | ||
| + | </a> | ||
| + | <div class="thumbcaption"> | ||
| + | <div class="magnify"> | ||
| + | <a href="https://static.igem.org/mediawiki/2017/4/4d/Catisvektro3.png" class="internal" title="Enlarge"> | ||
| + | <img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="" /> | ||
| + | </a> | ||
| + | </div> | ||
| + | <b>Figure 2:</b> BBa_K1149002 - EstCS2. | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| + | <div class="row section"> | ||
| + | <div class="col-xs-12 col-sm-9 col-md-9"> | ||
| + | <p> | ||
| + | <u>General properties of LipB: (Eggert et al. 2000)</u> | ||
| + | <ul> | ||
| + | <li>organism: <i>Bacillus subtilis 168</i> </li> | ||
| + | <li>temperature optimum:</li> | ||
| + | <li>pH optimum: 6 - 8</li> | ||
| + | <li>substrate specifity: preferentially hydrolysed esters of fatty acids with short chain lengths with less than 10 carbon atoms</li> | ||
| + | <li>triolein wasn’t hydroliysed at all: LipB is clasified as an esterase not as a lipase</li> | ||
| + | <li>classification of this enzyme as a phospholipase (EC 3.1.4.3) </li> | ||
| + | </ul> | ||
| + | </p> | ||
| + | </div> | ||
| + | <div class="col-xs-12 col-sm-3 col-md-3"> | ||
| + | <div class="thumb tleft"> | ||
| + | <div class="thumbinner" style="width:;"> | ||
| + | <a href="https://static.igem.org/mediawiki/2017/f/f0/Catisvektrz4.png" class="image"> | ||
| + | <img alt="" src="https://static.igem.org/mediawiki/2017/f/f0/Catisvektrz4.png" class="img-responsive" /> | ||
| + | </a> | ||
| + | <div class="thumbcaption"> | ||
| + | <div class="magnify"> | ||
| + | <a href="https://static.igem.org/mediawiki/2017/f/f0/Catisvektrz4.png" class="internal" title="Enlarge"> | ||
| + | <img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="" /> | ||
| + | </a> | ||
| + | </div> | ||
| + | <b>Figure 3:</b> Pet19-LipB. | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="row section"> | ||
| + | <div class="col-xs-12 col-sm-12 col-md-12"> | ||
| + | <h4>Lipase TliA</h4> | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="row section"> | ||
| + | <div class="col-xs-12 col-sm-9 col-md-9"> | ||
| + | <p>The TliA lipase was synthesized from IDT. | ||
| + | Eom et al. have already shown extracellular activity of TliA when secreted with the ABC export system. | ||
| + | So, if the TliA lipase is expressed, it is exported out of the cell and degrades the greasy layer surrounding the hair, which makes the keratin accessible for the keratinases. | ||
| + | </p> | ||
| + | <br> | ||
| + | <p> | ||
| + | <u>General properties of TliA: (Eom et al. 2014)</u> | ||
| + | <ul> | ||
| + | <li>organism: <i>Pseudomonas fluorescens</i> </li> | ||
| + | <li>thermostable lipase that is secreted by the ABC export system from Dickeya dadantii with the LARD secretion tag</li> | ||
| + | </ul> | ||
| + | </p> | ||
| + | </div> | ||
| + | <div class="col-xs-12 col-sm-3 col-md-3"> | ||
| + | <div class="thumb tleft"> | ||
| + | <div class="thumbinner" style="width:;"> | ||
| + | <a href="https://static.igem.org/mediawiki/2017/1/13/Catisvektorlipase3.png" class="image"> | ||
| + | <img alt="" src="https://static.igem.org/mediawiki/2017/1/13/Catisvektorlipase3.png" class="img-responsive" /> | ||
| + | </a> | ||
| + | <div class="thumbcaption"> | ||
| + | <div class="magnify"> | ||
| + | <a href="https://static.igem.org/mediawiki/2017/1/13/Catisvektorlipase3.png" class="internal" title="Enlarge"> | ||
| + | <img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="" /> | ||
| + | </a> | ||
| + | </div> | ||
| + | <b>Figure 4:</b> TliA Lipase. | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="row section"> | ||
| + | <div class="col-xs-12 col-sm-12 col-md-12"> | ||
| + | <h3>Part 2 - Keratinases</h3> | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="row section"> | ||
| + | <div class="col-xs-12 col-sm-8 col-md-8"> | ||
| + | <p>Keratinases are promising enzymes that find their applicability in agro-industrial, pharmaceutical and biomedicals fields. | ||
| + | We want to profit from them by applying them on clugged pipes full of hair that is a common issue in lots of households. | ||
| + | Keratinases are enzymes that are capable of degrading hair. | ||
| + | Hair mostly consists of alpha-Keratin. Many different keratinases produced by different Bacilli, Actinobacteria and fungi have been reported. | ||
| + | All of them vary by having their specific biochemical and biophysical properties e.g. temperature and pH activity range. | ||
| + | Using their proteolytic capability that destroys hair we want to use them to avoid chemical compounds that are recently mainly applied to cleanse tubes. | ||
| + | By hydrolization of disulfide bonds keratin degrades. This is due to confirmation changes which leads to an exposure of more sites for keratinase action (Satyanarayana et al. 2013, Vignardes et al. 1999). Previous projects from iGEM Teams such as Sheffield 2014, Team Canmore 2015 and Team Canmore 2016 helped us to tackle this problem as they were regarding similar problems. | ||
| + | </p> | ||
| + | </div> | ||
| + | <div class="col-xs-12 col-sm-4 col-md-4"> | ||
| + | <!--<https://static.igem.org/mediawiki/2017/3/3e/Keratinasecomicneu234.jpeg">--> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/3/3e/Keratinasecomicneu234.jpeg" class="img-responsive"/> | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="row section"> | ||
| + | <div class="col-xs-12 col-sm-12 col-md-12"> | ||
| + | <p><h4>KerP</h4> | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="row section"> | ||
| + | <div class="col-xs-12 col-sm-12 col-md-12"> | ||
| + | <p>The Keratinase KerP is a 33 kDa monomeric protein. KerP is a serine protease. Serine proteases are | ||
| + | protelytic enzymes, charaterizised by a reactive serine side chain (Kraut 1977). The family | ||
| + | contains many diffrent enzymes with wide spread functions. Most of keratinases are serine | ||
| + | proteases capable to degrade recalcitrant protein like nails, hair, feathers (Sharma and Gupta | ||
| + | 2010b). KS-1 has his optimal activity at pH = 9 and 60°C. | ||
| + | </p> | ||
| + | </div> | ||
| + | </div> | ||
| − | + | <div class="row section"> | |
| − | + | <div class="col-xs-12 col-sm-12 col-md-12"> | |
| + | <h4>KerA</h4> | ||
</div> | </div> | ||
| + | </div> | ||
| + | <div class="row section"> | ||
| + | <div class="col-xs-12 col-sm-9 col-md-9"> | ||
| + | <p>Keratinase gene from <i>Bacillus licheniformis</i> is also a serinetype protease. The mature keratinase secreted by <i>Bacillus licheniformis</i> had a molecular mass of 33 kDa (Lin et al. 1995). | ||
| + | Although cloning and expression was sucessful in <i>Escherichia coli</i>, protein yields of keratinase were lower than the parental strain (Brandelli 2008). | ||
| + | Hu et al. expressed kerA in <i>Escherichia coli</i> and measured for recombinant keratinases 50°C as optimum temperature from pH 5 to 10 (Hu et al. 2013)</p> | ||
| + | </div> | ||
| + | <div class="col-xs-12 col-sm-3 col-md-3"> | ||
| + | <div class="thumb tleft"> | ||
| + | <div class="thumbinner" style="width:;"> | ||
| + | <a href="https://static.igem.org/mediawiki/2017/4/48/KerAgeorgvektro2.png" class="image"> | ||
| + | <img alt="" src="https://static.igem.org/mediawiki/2017/4/48/KerAgeorgvektro2.png" class="img-responsive" /> | ||
| + | </a> | ||
| + | <div class="thumbcaption"> | ||
| + | <div class="magnify"> | ||
| + | <a href="https://static.igem.org/mediawiki/2017/4/48/KerAgeorgvektro2.png" class="internal" title="Enlarge"> | ||
| + | <img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="" /> | ||
| + | </a> | ||
| + | </div> | ||
| + | <b>Figure 5:</b> Plasmid of KerA from <i>B. licheniformis</i>. | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="row section"> | ||
| + | <div class="col-xs-12 col-sm-12 col-md-12"> | ||
| + | <h4>KerUS</h4> | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="row section"> | ||
| + | <div class="col-xs-12 col-sm-9 col-md-9"> | ||
| + | <p>KerUS gene was isolated from <i>Brevibacillus brevis</i> strain US575 from contaminated soil samples collected from a local leather tannery (M’Saken-Sousse, Tunisia). | ||
| + | Also KerUS was cloned and expressed previously in <i>Escherichia coli</i>. Optimal activity was achieved at pH 8 and 40°C. | ||
| + | The purified KerUS was noted to exhibit additionaly esterase and amidase activities (Jaouadi et al. 2013).</p> | ||
| + | </div> | ||
| + | <div class="col-xs-12 col-sm-3 col-md-3"> | ||
| + | <div class="thumb tleft"> | ||
| + | <div class="thumbinner" style="width:;"> | ||
| + | <a href="https://static.igem.org/mediawiki/2017/5/5d/KerURVektorgeorg2.png" class="image"> | ||
| + | <img alt="" src="https://static.igem.org/mediawiki/2017/5/5d/KerURVektorgeorg2.png" class="img-responsive" /> | ||
| + | </a> | ||
| + | <div class="thumbcaption"> | ||
| + | <div class="magnify"> | ||
| + | <a href="https://static.igem.org/mediawiki/2017/5/5d/KerURVektorgeorg2.png" class="internal" title="Enlarge"> | ||
| + | <img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="" /> | ||
| + | </a> | ||
| + | </div> | ||
| + | <b>Figure 6:</b> Plasmid of KerUS from <i>Brevibacillus brevis</i>. | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="row section"> | ||
| + | <div class="col-xs-12 col-sm-12 col-md-12"> | ||
| + | <h3>Part 3 - a lovely scent of...</h3> | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="row section"> | ||
| + | <div class="col-xs-12 col-sm-12 col-md-12"> | ||
| + | <p>The microbial synthesis of natural flavor compounds has become a very attractive alternative to the chemical production (D. Guo and L. Zhang et al 2017). | ||
| + | In recent years microorganisms such as <i>E.coli</i> and Yeast have been metabolically engineered to produce different flavors like limonene, geraniol or rose (D. Guo and L. Zhang et al 2017, W. Liu and R. Zhang et al. 2015, I. Guterman and M. Shalit et al. 2002). | ||
| + | For our project we discussed different approaches and choose two different scents: rose and limonene. | ||
| + | </p> | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="row section"> | ||
| + | <div class="col-xs-12 col-sm-12 col-md-12"> | ||
| + | <h4>... rose fragrance</h4> | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="row section"> | ||
| + | <div class="col-xs-12 col-sm-4 col-md-4"> | ||
| + | <!--<https://static.igem.org/mediawiki/2017/6/66/Rosenduftecomicneu234.jpegg>--> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/6/66/Rosenduftecomicneu234.jpeg" class="img-responsive"/ align=middle> | ||
| + | </div> | ||
| + | <div class="col-xs-12 col-sm-8 col-md-8"> | ||
| + | <p>As first special fragrance we want to install a lovely scent of rose in our microbial system. Hair are commonly made of Keratin (98%) and small amounts of amino acids, such as L-phenylalanine. | ||
| + | This amino acid can be used as substrate for the production of 2-Phenylethylacetate (2-PEAc), which is a high-value aromatic ester with a rose-like odor (D. Guo and L. Zhang et al. 2017). | ||
| + | Therefor this odor can act as an indicator for keratin degradation. 2-phenylethylacetate is in high demand and widely udes as an additive in food, drinks, perfumes and cosmetics (D. Guo and L. Zhang et al.). | ||
| + | Currently, 2-PEAc is mainly produced by chemical synthesis, but consumers prefer more natural flavour compounds in the field of food and cosmetics. | ||
| + | But extraction of natural 2-PEAc is cost and time-intensive (D. Guo and L. Zhang et al. 2017). | ||
| + | Therefor the production of 2-PEAc using microorgansims such as <i>E.coli</i> can be an attractive alternative (D. Guo and L. Zhang et al. 2017). | ||
| + | In recent studies from Guo et al the 2-PEAc biosynthetic pathway was successfully designed and expressed in <i>E.coli </i>. | ||
| + | For our project we want to use this pathway for the production of a lovely rose scent by <i>E.coli</i>. | ||
| + | This pathway comprise four steps (Fig.1) (D. Guo and L. Zhang et al. 2017):</p> | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="row section"> | ||
| + | <div class="col-xs-12 col-sm-5 col-md-5"> | ||
| + | <ul> | ||
| + | <li> Aminotransferase (ARO 8) for transamination of L-phenylalanine to phenylpyruvate</li> | ||
| + | <li> 2-keto acid decarboxylase KDC for the decarboxylation of the phenylpyruvate to phenylacetaldehyde</li> | ||
| + | <li> Aldehyde reductase YjgB for the reduction of phenylacetaldehyde to 2-Phenylethanol</li> | ||
| + | <li> Alcohol acetyltransferase ATF1 for the esterification of 2-PE to 2-PEAc.</li></ul> | ||
| + | </p> | ||
| + | </div> | ||
| + | <div class="col-xs-12 col-sm-7 col-md-7"> | ||
| + | <div class="thumb tleft"> | ||
| + | <div class="thumbinner" style="width:;"> | ||
| + | <a href="https://static.igem.org/mediawiki/2017/7/7a/Stuttgartpathwayrose.png" class="image"> | ||
| + | <img alt="" src="https://static.igem.org/mediawiki/2017/7/7a/Stuttgartpathwayrose.png" class="img-responsive" /> | ||
| + | </a> | ||
| + | <div class="thumbcaption"> | ||
| + | <div class="magnify"> | ||
| + | <a href="https://static.igem.org/mediawiki/2017/7/7a/Stuttgartpathwayrose.png" class="internal" title="Enlarge"> | ||
| + | <img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="" /> | ||
| + | </a> | ||
| + | </div> | ||
| + | <b>Figure 7:</b> Rose fragrance pathway from Gup et al. (D. Guo and L. Zhang et al. 2017). | ||
</div> | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="row section"> | ||
| + | <div class="col-xs-12 col-sm-12 col-md-12"> | ||
| + | <p>We contacted the scientists Guo et al. and they send us two plasmids with rose genes: pET28a-KDC-YjgB-ARO8 and pET28a-ATF1. </p> | ||
| + | </div> | ||
| + | </div> | ||
| − | + | <div class="row section"> | |
| − | + | <div class="col-xs-12 col-sm-12 col-md-12"> | |
| + | <h4>... limonene fragrance</h4> | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="row section"> | ||
| + | <div class="col-xs-12 col-sm-5 col-md-5"> | ||
| + | <p>Limonene is a well-known cyclic monoterpene which can occur in two optical forms (E. Jongedijk and K. Cankar et. al 2016). | ||
| + | (D)-Limonene is one of the most important and widespread terpenes in the flavor and fragrance industry, for example in citrus-flavored products such as soft drinks and candy (2). The (L)-Limonene form has a more harsh fir-like odor with a lemon-note (2). For our project we choose an enzyme-cascade, beginning with acetyl-coA and leading to the product (L)-limonene. This biosynthetic pathway was designed and inserted in <i>E.coli </i>(Fig.2). | ||
| + | </p> | ||
| + | </div> | ||
| + | <div class="col-xs-12 col-sm-7 col-md-7"> | ||
| + | <div class="thumb tleft"> | ||
| + | <div class="thumbinner" style="width:;"> | ||
| + | <a href="https://static.igem.org/mediawiki/2017/8/8a/Limonenepathway.png" class="image"> | ||
| + | <img alt="" src="https://static.igem.org/mediawiki/2017/8/8a/Limonenepathway.png" class="img-responsive" /> | ||
| + | </a> | ||
| + | <div class="thumbcaption"> | ||
| + | <div class="magnify"> | ||
| + | <a href="https://static.igem.org/mediawiki/2017/8/8a/Limonenepathway.png" class="internal" title="Enlarge"> | ||
| + | <img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="" /> | ||
| + | </a> | ||
| + | </div> | ||
| + | <b>Figure 8:</b> Limonene fragrance pathway from E. Jongedijk and K. Cankar et. al (E. Jongedijk and K. Cankar et. al 2016). | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="row section"> | ||
| + | <div class="col-xs-12 col-sm-12 col-md-12"> | ||
| + | <h2 align=middle>Extracellular expression of Esterases, Lipases, Keratinases and fragrance molecules</h2> | ||
| + | <p>(Choi und Lee 2004; Mergulhão et al. 2005)</p> | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="row section"> | ||
| + | <div class="col-xs-12 col-sm-12 col-md-12"> | ||
| + | <p>In our project we aimed to secrete the produced enzymes. | ||
| + | We used <i>E. coli</i>, a gram negative bacteria with two membranes that needed to be crossed from the enzymes. | ||
| + | <i>E. coli</i> naturally doesn’t secrete high amounts of proteins which forced us to add a signal peptide to achieve secretory production of the enzymes. | ||
| + | With this additional step we aimed to save valuable money and time in the production process of our biological tube cleaner. | ||
| + | </p> | ||
| + | <p>In principal, the secretory production of recombinant proteins has several advantages. These include: | ||
| + | <p> | ||
| + | <ul><li>easier downstream processing (no cell disruption and easier protein purification) | ||
| + | </li> | ||
| + | <li>correct protein folding (avoidance of the formation of inclusion bodies) to keep biological activity | ||
| + | </li> | ||
| + | <li>less protease activity in the periplasma and in the media than in the cytoplasma –> no protein degradation | ||
| + | </li> | ||
| + | <li>better access to the substrate higher enzyme activites | ||
| + | </li> | ||
| + | <li>better disulfide bond formation in the periplasm because of the non reducing environment | ||
| + | </li> | ||
| + | </ul> | ||
| + | </p> | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="row section"> | ||
| + | <div class="col-xs-12 col-sm-12 col-md-12"> | ||
| + | <p>There are two main mechanism that are used for secretory protein production, type I and type II mechanism. | ||
| + | Type I is a mechanism consisting of one step across the two cellular membranes without a periplasmic intermediate. | ||
| + | One disadvantage of type I mechanism ist hat the signal peptide remains attached so an additional cleavage step is required to obtain the mature protein. | ||
| + | Type II mechanism can be divided in three submechanisms: SecB-dependend pathway, SRP pathway and TAT (twin arginine translocation) pathway. | ||
| + | Type II is a two step mechanism. | ||
| + | The non processed proteins are attached with a a signal sequence and exported to the periplasm. | ||
| + | The premature protein is processed into the mature protein by cleavage of the signal peptide. | ||
| + | The SecB pathway is the mostly used way for the extracellular production of recombinant proteins. | ||
| + | SRP can be used for proteins that fold too quickly and incorrectly in the cytoplasm and TAT allows the secretion of poteins that are already folded. | ||
| + | </p> | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="row section"> | ||
| + | <div class="col-xs-12 col-sm-12 col-md-12"> | ||
| + | <p>In our project we use the SecB dependend pathway to secrete the enzymes. | ||
| + | We needed to optimize the expression level with appropriate promotor strength to prevent the formation of inclusion bodies. | ||
| + | Additionally it’s important to achieve high secretion levels which is a known problem in recombinant enzyme secretion. | ||
| + | The efficiency of the protein secretion depends on the host strain, signal sequence and the type of protein to be secreted (e. g. high protein size causes limited enzyme secretion). | ||
| + | </p> | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="row section"> | ||
| + | <div class="col-xs-12 col-sm-12 col-md-12"> | ||
| + | <foot> | ||
| + | <p>REFERENCES | ||
| + | <ol> | ||
| + | <li>Metabolic engineering of <i>Escherichia coli</i> for production of 2-Phenylethylacetate from L-phenylalanine (2017), D. Guo and L. Zhang et al.</li> | ||
| + | <li>Biotechnological production of limonene in microorganisms (2016), E. Jongedijk and K. Cankar et al. </li> | ||
| + | <li>Utilization of alkaline phosphatase PhoA in the bioproduction of geraniol by metabolically engineered <i>Escherichia coli</i> (2015), W. Liu and R. Zhang et al. </li> | ||
| + | <li>Rose Scent: Genomics Approach to Discovering Novel Floral Fragrance–Related Genes (2002), I. Guterman and M. Shalit et al. </li> | ||
| + | <li>Eggert, Thorsten; Pencreac'h, Gaelle; Douchet, Isabelle; Verger, Robert; Jaeger, Karl-Erich (2000): A novel extracellular esterase from Bacillus subtilis and its conversion to a monoacylglycerol hydrolase. In: European Journal of Biochemistry 267 (21), p. 6459–6469</li> | ||
| + | <li>Eom, Gyeong Tae; Lee, Seung Hwan; Oh, Young Hoon; Choi, Ji Eun; Park, Si Jae; Song, Jae Kwang (2014): Efficient extracellular production of type I secretion pathway-dependent Pseudomonas fluorescens lipase in recombinant Escherichia coli by heterologous ABC protein exporters. In: Biotechnology letters 36 (10), p. 2037–2042</li> | ||
| + | <li>Kang, Chul-Hyung; Oh, Ki-Hoon; Lee, Mi-Hwa; Oh, Tae-Kwang; Kim, Bong Hee; Yoon, Jung-Hoon (2011): A novel family VII esterase with industrial potential from compost metagenomic library. In: Microbial cell factories 10, p. 41</li> | ||
| + | <li>Panda, T.; Gowrishankar, B. S. (2005): Production and applications of esterases. In: Applied microbiology and biotechnology 67 (2), S. 160–169. DOI: 10.1007/s00253-004-1840-y.</li> | ||
| + | <li>Shamsher Singh Kanwar (2016): Carboxylesterases: Sources, Characterization and Broader Applications. In: iMedPub Journals. Insights in Enzyme Research </li> | ||
| + | <li>Choi, J. H.; Lee, S. Y. (2004): Secretory and extracellular production of recombinant proteins using Escherichia coli. In: Applied microbiology and biotechnology 64 (5), S. 625–635. DOI: 10.1007/s00253-004-1559-9.</li> | ||
| + | <li>Mergulhão, F. J. M.; Summers, D. K.; Monteiro, G. A. (2005): Recombinant protein secretion in Escherichia coli. In: Biotechnology advances 23 (3), S. 177–202. DOI: 10.1016/j.biotechadv.2004.11.003.</li> | ||
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Latest revision as of 21:03, 15 December 2017
Description
What's the problem?
Our project deals with an everyday problem described by the following scenario:
You want to enjoy a refreshing shower in the morning, but your hairy roommate clogged the drain again?
You would like to have a relaxed bubble bath after a long day, but there are bad odors coming out of the pipe system?
The general practice now:
- You try the ‘hot water method’ to flush the drain… nothing happens.
- You remember what your mother taught you, so you put a fancy mix of vinegar, baking soda and some magic into the drain… a mysterious creature arises, but nothing else happens.
- In the end there is just one thing left – you put the nasty chemical mixture of the stinky cleaning agent down the drain. The corrosive cloud disappears and you can finally take your shower… still trying not to breath in the acrid fumes.
Pipe clogging and closure occurs in every household as well as in industry. Various components contribute to the pipe closure, such as fat and hair. Commercial chemical pipe cleaners contain aggressive substances, which are harmful to the environment and health and also can lead to pipe breaks. Biological alternatives (purified enzymes) have been researched for years, without reaching the efficiency of chemical cleaners. In addition, the purification of the enzymes is very complex and cost intensive.
Our solution
We pursue a system-biological solution which is based on an intact microbial system. Inspired by different interesting projects we decided to develope a keranolytic Escherichia coli. The projects of past iGEM-teams were part of our investigation too and we are already co-working with iGEM-team OLS_Canmore 2015/2016 to exchange information. We would like to use the knowledge of these groundworks to develop a new type of enzymatic cleaning agent by complementing and combining ideas out of different scientific sources. To differ from other projects we are aiming to engineer Escherichia coli to express enzymes like esterases, lipases and keratinases into the medium. Due to the extracellular expression expensive and time-consuming purification steps could be avoided and the enzymes could be secreted directly into the drain. As a special feature we want to use the metabolites of the enzymatic degradation to produce a lovely rose-scent to refresh your whole room. In addition this reaction could also be used to indicate that the enzymes are working efficiently on your drainage problem.
Light up the pipe - Three parts for a better flow
Part 1 - Esterases and Lipases
Hair is surrounded by a layer of grease and waxes which first need to be removed to make the hair-keratin available for keratinases. For the first degradation step we choose a combination of esterases and lipases. Lipases and esterases are lipolytic enzymes that are used in food processing, beverages, therapeutics and degradation of synthetic materials (Panda und Gowrishankar 2005). Lipases (EC. 3.1.1.3) and esterases (EC. 3.1.1.1) are hydrolases that catalyze the hydrolysis (and synthesis) of triacylglycerols into acid and alcohol molecules. They differ mainly on the basis of substrate specifity and activation mechanism. Lipases hydrolase triacylglyderols with longer fatty acid chains than esterases and lipases have a hydrophobic domain covering their active site. Therefore lipases need to be activated with organic solvents to make the active site accessible for substrates. Esterases and lipases are classified into 8 groups on the basis of the conserved sequence motifs and biological properties. EstCS2 belongs to class VII (Kang et al. 2011; Shamsher Singh Kanwar).
We investigated two different esterases for their enzyme activity. One esterase from the registry (EstCS2 BBa_K1149002) and one esterase (LipB) supplied by Dr. Eggert from Evoxx were compared. Additionally, we choose the lipase TliA to support the esterases at the fat degradation and to accelerate the entire degradation process. For the extracellular secretion of the enzymes we attached the signal sequence PelB which was provided on the iGEM plates.
Esterases EstCS2 and LipB
EstCS2 from the iGEM Imperial College 2013 was proved to be active. In their project the cells expressing this construct were grown and lysed by sonication and were utilized in a colourimetric assay with the substrate analog para-Nitrophenyl butyrate. In our project we didn’t purify the esterases but used the supernatant for the enzyme activity assay. Furthermore, we investigated the enzyme activity of LipB. We performed the enzyme assay from the iGEM Team TU Darmstadt that we also used for EstCS2. Afterwards we attached the PelB signal sequence for the extracellular secretion of the enzyme.
General properties of EstCS2: (Kang et al. 2011)
- serine esterase with 45 % similarity with the carboxylesterase from Haliangium ochraceum DSM 14365
- temperature optimum: 55 °C; at 37°C: ca. 50 % of maximal activity
- pH optimum: 9; at pH 7: ca. 30 % of maximal activity
- Family VII esterase with the Ser residue in the catalytic triad of EstCS2 that is loacated in the consensus active site motif GXSXG
General properties of LipB: (Eggert et al. 2000)
- organism: Bacillus subtilis 168
- temperature optimum:
- pH optimum: 6 - 8
- substrate specifity: preferentially hydrolysed esters of fatty acids with short chain lengths with less than 10 carbon atoms
- triolein wasn’t hydroliysed at all: LipB is clasified as an esterase not as a lipase
- classification of this enzyme as a phospholipase (EC 3.1.4.3)
Lipase TliA
The TliA lipase was synthesized from IDT. Eom et al. have already shown extracellular activity of TliA when secreted with the ABC export system. So, if the TliA lipase is expressed, it is exported out of the cell and degrades the greasy layer surrounding the hair, which makes the keratin accessible for the keratinases.
General properties of TliA: (Eom et al. 2014)
- organism: Pseudomonas fluorescens
- thermostable lipase that is secreted by the ABC export system from Dickeya dadantii with the LARD secretion tag
Part 2 - Keratinases
Keratinases are promising enzymes that find their applicability in agro-industrial, pharmaceutical and biomedicals fields. We want to profit from them by applying them on clugged pipes full of hair that is a common issue in lots of households. Keratinases are enzymes that are capable of degrading hair. Hair mostly consists of alpha-Keratin. Many different keratinases produced by different Bacilli, Actinobacteria and fungi have been reported. All of them vary by having their specific biochemical and biophysical properties e.g. temperature and pH activity range. Using their proteolytic capability that destroys hair we want to use them to avoid chemical compounds that are recently mainly applied to cleanse tubes. By hydrolization of disulfide bonds keratin degrades. This is due to confirmation changes which leads to an exposure of more sites for keratinase action (Satyanarayana et al. 2013, Vignardes et al. 1999). Previous projects from iGEM Teams such as Sheffield 2014, Team Canmore 2015 and Team Canmore 2016 helped us to tackle this problem as they were regarding similar problems.
KerP
The Keratinase KerP is a 33 kDa monomeric protein. KerP is a serine protease. Serine proteases are protelytic enzymes, charaterizised by a reactive serine side chain (Kraut 1977). The family contains many diffrent enzymes with wide spread functions. Most of keratinases are serine proteases capable to degrade recalcitrant protein like nails, hair, feathers (Sharma and Gupta 2010b). KS-1 has his optimal activity at pH = 9 and 60°C.
KerA
Keratinase gene from Bacillus licheniformis is also a serinetype protease. The mature keratinase secreted by Bacillus licheniformis had a molecular mass of 33 kDa (Lin et al. 1995). Although cloning and expression was sucessful in Escherichia coli, protein yields of keratinase were lower than the parental strain (Brandelli 2008). Hu et al. expressed kerA in Escherichia coli and measured for recombinant keratinases 50°C as optimum temperature from pH 5 to 10 (Hu et al. 2013)
KerUS
KerUS gene was isolated from Brevibacillus brevis strain US575 from contaminated soil samples collected from a local leather tannery (M’Saken-Sousse, Tunisia). Also KerUS was cloned and expressed previously in Escherichia coli. Optimal activity was achieved at pH 8 and 40°C. The purified KerUS was noted to exhibit additionaly esterase and amidase activities (Jaouadi et al. 2013).
Part 3 - a lovely scent of...
The microbial synthesis of natural flavor compounds has become a very attractive alternative to the chemical production (D. Guo and L. Zhang et al 2017). In recent years microorganisms such as E.coli and Yeast have been metabolically engineered to produce different flavors like limonene, geraniol or rose (D. Guo and L. Zhang et al 2017, W. Liu and R. Zhang et al. 2015, I. Guterman and M. Shalit et al. 2002). For our project we discussed different approaches and choose two different scents: rose and limonene.
... rose fragrance
As first special fragrance we want to install a lovely scent of rose in our microbial system. Hair are commonly made of Keratin (98%) and small amounts of amino acids, such as L-phenylalanine. This amino acid can be used as substrate for the production of 2-Phenylethylacetate (2-PEAc), which is a high-value aromatic ester with a rose-like odor (D. Guo and L. Zhang et al. 2017). Therefor this odor can act as an indicator for keratin degradation. 2-phenylethylacetate is in high demand and widely udes as an additive in food, drinks, perfumes and cosmetics (D. Guo and L. Zhang et al.). Currently, 2-PEAc is mainly produced by chemical synthesis, but consumers prefer more natural flavour compounds in the field of food and cosmetics. But extraction of natural 2-PEAc is cost and time-intensive (D. Guo and L. Zhang et al. 2017). Therefor the production of 2-PEAc using microorgansims such as E.coli can be an attractive alternative (D. Guo and L. Zhang et al. 2017). In recent studies from Guo et al the 2-PEAc biosynthetic pathway was successfully designed and expressed in E.coli . For our project we want to use this pathway for the production of a lovely rose scent by E.coli. This pathway comprise four steps (Fig.1) (D. Guo and L. Zhang et al. 2017):
- Aminotransferase (ARO 8) for transamination of L-phenylalanine to phenylpyruvate
- 2-keto acid decarboxylase KDC for the decarboxylation of the phenylpyruvate to phenylacetaldehyde
- Aldehyde reductase YjgB for the reduction of phenylacetaldehyde to 2-Phenylethanol
- Alcohol acetyltransferase ATF1 for the esterification of 2-PE to 2-PEAc.
We contacted the scientists Guo et al. and they send us two plasmids with rose genes: pET28a-KDC-YjgB-ARO8 and pET28a-ATF1.
... limonene fragrance
Limonene is a well-known cyclic monoterpene which can occur in two optical forms (E. Jongedijk and K. Cankar et. al 2016). (D)-Limonene is one of the most important and widespread terpenes in the flavor and fragrance industry, for example in citrus-flavored products such as soft drinks and candy (2). The (L)-Limonene form has a more harsh fir-like odor with a lemon-note (2). For our project we choose an enzyme-cascade, beginning with acetyl-coA and leading to the product (L)-limonene. This biosynthetic pathway was designed and inserted in E.coli (Fig.2).
Extracellular expression of Esterases, Lipases, Keratinases and fragrance molecules
(Choi und Lee 2004; Mergulhão et al. 2005)
In our project we aimed to secrete the produced enzymes. We used E. coli, a gram negative bacteria with two membranes that needed to be crossed from the enzymes. E. coli naturally doesn’t secrete high amounts of proteins which forced us to add a signal peptide to achieve secretory production of the enzymes. With this additional step we aimed to save valuable money and time in the production process of our biological tube cleaner.
In principal, the secretory production of recombinant proteins has several advantages. These include:
- easier downstream processing (no cell disruption and easier protein purification)
- correct protein folding (avoidance of the formation of inclusion bodies) to keep biological activity
- less protease activity in the periplasma and in the media than in the cytoplasma –> no protein degradation
- better access to the substrate higher enzyme activites
- better disulfide bond formation in the periplasm because of the non reducing environment
There are two main mechanism that are used for secretory protein production, type I and type II mechanism. Type I is a mechanism consisting of one step across the two cellular membranes without a periplasmic intermediate. One disadvantage of type I mechanism ist hat the signal peptide remains attached so an additional cleavage step is required to obtain the mature protein. Type II mechanism can be divided in three submechanisms: SecB-dependend pathway, SRP pathway and TAT (twin arginine translocation) pathway. Type II is a two step mechanism. The non processed proteins are attached with a a signal sequence and exported to the periplasm. The premature protein is processed into the mature protein by cleavage of the signal peptide. The SecB pathway is the mostly used way for the extracellular production of recombinant proteins. SRP can be used for proteins that fold too quickly and incorrectly in the cytoplasm and TAT allows the secretion of poteins that are already folded.
In our project we use the SecB dependend pathway to secrete the enzymes. We needed to optimize the expression level with appropriate promotor strength to prevent the formation of inclusion bodies. Additionally it’s important to achieve high secretion levels which is a known problem in recombinant enzyme secretion. The efficiency of the protein secretion depends on the host strain, signal sequence and the type of protein to be secreted (e. g. high protein size causes limited enzyme secretion).
REFERENCES
- Metabolic engineering of Escherichia coli for production of 2-Phenylethylacetate from L-phenylalanine (2017), D. Guo and L. Zhang et al.
- Biotechnological production of limonene in microorganisms (2016), E. Jongedijk and K. Cankar et al.
- Utilization of alkaline phosphatase PhoA in the bioproduction of geraniol by metabolically engineered Escherichia coli (2015), W. Liu and R. Zhang et al.
- Rose Scent: Genomics Approach to Discovering Novel Floral Fragrance–Related Genes (2002), I. Guterman and M. Shalit et al.
- Eggert, Thorsten; Pencreac'h, Gaelle; Douchet, Isabelle; Verger, Robert; Jaeger, Karl-Erich (2000): A novel extracellular esterase from Bacillus subtilis and its conversion to a monoacylglycerol hydrolase. In: European Journal of Biochemistry 267 (21), p. 6459–6469
- Eom, Gyeong Tae; Lee, Seung Hwan; Oh, Young Hoon; Choi, Ji Eun; Park, Si Jae; Song, Jae Kwang (2014): Efficient extracellular production of type I secretion pathway-dependent Pseudomonas fluorescens lipase in recombinant Escherichia coli by heterologous ABC protein exporters. In: Biotechnology letters 36 (10), p. 2037–2042
- Kang, Chul-Hyung; Oh, Ki-Hoon; Lee, Mi-Hwa; Oh, Tae-Kwang; Kim, Bong Hee; Yoon, Jung-Hoon (2011): A novel family VII esterase with industrial potential from compost metagenomic library. In: Microbial cell factories 10, p. 41
- Panda, T.; Gowrishankar, B. S. (2005): Production and applications of esterases. In: Applied microbiology and biotechnology 67 (2), S. 160–169. DOI: 10.1007/s00253-004-1840-y.
- Shamsher Singh Kanwar (2016): Carboxylesterases: Sources, Characterization and Broader Applications. In: iMedPub Journals. Insights in Enzyme Research
- Choi, J. H.; Lee, S. Y. (2004): Secretory and extracellular production of recombinant proteins using Escherichia coli. In: Applied microbiology and biotechnology 64 (5), S. 625–635. DOI: 10.1007/s00253-004-1559-9.
- Mergulhão, F. J. M.; Summers, D. K.; Monteiro, G. A. (2005): Recombinant protein secretion in Escherichia coli. In: Biotechnology advances 23 (3), S. 177–202. DOI: 10.1016/j.biotechadv.2004.11.003.
