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Converting Aspartase into a β‐Amino Acid Lyase by Cluster Screening

ChemCatChem. 2014-04; 
Dr. Andreas Vogel Dr. Ramona Schmiedel Dr. Ute Hofmann Prof. Karl Gruber Prof. Klaus Zangger
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Custom Vector Construction … 37 °C. Compared to the published constants of the wild‐type enzyme with fumarate (K m =30 mM and k cat =640 s … YM55‐1 aspartase AspB (Uniprot Q9LCC6) was obtained from Genscript (USA) and cloned into the c‐LEctas expression vector pLE1A10, which placed the gene … Get A Quote

摘要

Aspartase, despite being one of the most specific enzymes known, shows potential for application in β‐amino acid synthesis. The substrate binding pocket of AspB from Bacillus sp. YM55‐1 was reshaped by enzyme engineering to accommodate crotonic acid. The mutant enzyme BSASP‐C6 yielded enantiopure (R )‐3‐aminobutyrate from crotonic acid and ammonia. To obtain this mutant, a high‐throughput screening in combination with a focused permutational library was decisive for the success in this project. We achieved this by simultaneous randomisation of four residues within the substrate binding pocket and cluster screening in mixed population. Screening of 300 000 clones was necessary to find the BSASP... More

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