Difference between revisions of "Team:Amoy/Project"
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<p class="main_p">L-<i>tert</i>-leucine (L-Tle) is an important and attractive chiral building block, which can be used to produce protease inhibitors of HIV, HCV and so on. Scientists developed enzymatic reductive amination to produce L-Tle by using leucine dehydrogenase (LeuDH) and formate dehydrogenase (FDH) with cofactor-NADH. 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, addition of excess NADH is necessary. Many methods have been used, but none made a difference.</br></br> | <p class="main_p">L-<i>tert</i>-leucine (L-Tle) is an important and attractive chiral building block, which can be used to produce protease inhibitors of HIV, HCV and so on. Scientists developed enzymatic reductive amination to produce L-Tle by using leucine dehydrogenase (LeuDH) and formate dehydrogenase (FDH) with cofactor-NADH. 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, addition of excess NADH is necessary. Many methods have been used, but none made a difference.</br></br> | ||
| − | Our project devised a method that regulating the 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's efficiency of | + | Our project devised a method that regulating the 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's efficiency of LeuDH. With the different efficiency of RBSs, we can get different activity of LeuDH. And from the experimental results, we obtained the suitable RBS's efficiency and the best catalytic efficiency. Finally, the disired L-Tle was obtained with >99% conversion and a high enantioselectivity of >99% ee value. |
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Revision as of 16:12, 18 September 2015
ABSTRACT
L-tert-leucine (L-Tle) is an important and attractive chiral building block, which can be used to produce protease inhibitors of HIV, HCV and so on. Scientists developed enzymatic reductive amination to produce L-Tle by using leucine dehydrogenase (LeuDH) and formate dehydrogenase (FDH) with cofactor-NADH. 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, addition of excess NADH is necessary. Many methods have been used, but none made a difference. Our project devised a method that regulating the 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's efficiency of LeuDH. With the different efficiency of RBSs, we can get different activity of LeuDH. And from the experimental results, we obtained the suitable RBS's efficiency and the best catalytic efficiency. Finally, the disired L-Tle was obtained with >99% conversion and a high enantioselectivity of >99% ee 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
