Difference between revisions of "Team:Amoy/Project"

 
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<a href="https://2015.igem.org/Team:Amoy/Project"><h4>Project</h4></a>
 
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<p class="main_p">L-<i>tert</i>-leucine (Tle) is an important and attractive chiral building block, which can be used to product protease inhibitors of HIV, HCV and so on. Many chemical methods have been used in Tle synthesis, but products are usually racemic. In order to solve the problem, scientists developed enzymatic reductive amination to product Tle by using leucine dehydrogenase (LeuDH) and formate dehydrogenase (FDH) with cofactor-NADH. It greatly improved the yield and optical purity of Tle.</br></br>
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<p class="main_p">L-<i>tert</i>-leucine (L-Tle) is an important chiral amino acid, which can be used to produce HIV or HCV protease inhibitors. Scientists developed enzymatic reductive amination to produce L-Tle by using leucine dehydrogenase (LeuDH) and formate dehydrogenase (FDH) based on cofactor regeneration. It greatly improved the yield and optical purity of L-Tle. However, owing to different activity of LeuDH and FDH, the NADH consumption would be faster than its regeneration. Therefore, external NADH is necessary. Many methods have been used to solve this problem, but none of them made a difference.</br></br>
  
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Initially, isolated enzymes are easily destabilized in the isolation and purification process. What's more, NADH is rather an expensive material, which will enhance the cost of Tle production. So scientists introduced Tle production into whole-cell biocatalysts.</br></br>
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Our project devised a method that regulating the enzyme activity of LeuDH by changing RBS of <i>leudh</i>. And <i>fdh</i> and <i>leudh</i> were borne in a plasmid. This idea controls copy numbers and transformation efficiency, and the only variable is RBS efficiency of <i>leudh</i>. With the different efficiency of RBS, we can get different enzyme activity of LeuDH. And from the experimental results, we obtained the suitable RBS efficiency and the best catalytic efficiency. Finally, the desired L-Tle was obtained with >99% conversion and a high enantioselectivity of >99% e.e. value.  
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However, owing to different activities of LeuDH and FDH, the NADH consumption would be faster than its regeneration. Therefore, addition of excess NADH is necessary. Many methods have been used, but none made difference.</br></br>
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Our project devised a method that regulating the activity of LeuDH by changing RBS of leudh, which is connected with fdh in a plasmid. This idea controls copy numbers and transformation efficiency, and the only variable is RBSs’ strength. Finally, we got different gene circuits and found the suitable strength of RBS.
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<p style="font-size: 14px; color: #fff; margin-bottom: 0px;"><strong>Address:  </strong>Xiamen University, No. 422, Siming South Road, Xiamen, Fujian, P.R.China 361005</p>
 
<p style="font-size: 14px; color: #fff; margin-bottom: 0px;"><strong>Address:  </strong>Xiamen University, No. 422, Siming South Road, Xiamen, Fujian, P.R.China 361005</p>
 
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Latest revision as of 03:44, 19 September 2015

Aomy/Project

ABSTRACT

L-tert-leucine (L-Tle) is an important chiral amino acid, which can be used to produce HIV or HCV protease inhibitors. Scientists developed enzymatic reductive amination to produce L-Tle by using leucine dehydrogenase (LeuDH) and formate dehydrogenase (FDH) based on cofactor regeneration. It greatly improved the yield and optical purity of L-Tle. However, owing to different activity of LeuDH and FDH, the NADH consumption would be faster than its regeneration. Therefore, external NADH is necessary. Many methods have been used to solve this problem, but none of them made a difference.

Our project devised a method that regulating the enzyme activity of LeuDH by changing RBS of leudh. And fdh and leudh were borne in a plasmid. This idea controls copy numbers and transformation efficiency, and the only variable is RBS efficiency of leudh. With the different efficiency of RBS, we can get different enzyme activity of LeuDH. And from the experimental results, we obtained the suitable RBS efficiency and the best catalytic efficiency. Finally, the desired L-Tle was obtained with >99% conversion and a high enantioselectivity of >99% e.e. value.

CONTACT US

Email: igemxmu@gmail.com

Website: 2015.igem.org/Team:Amoy

Address: Xiamen University, No. 422, Siming South Road, Xiamen, Fujian, P.R.China 361005