Speciality Chemicals Magazine JAN / FEB 2025 | Page 35

CATALYSTS
Figure 6 - Relative activity of PAL wild type ( WT ) & evolved variants ( V1-V5 ) during laboratory evolution
high substrate concentrations are necessary to drive the reaction in the desired direction .
For the reaction with the phenyl alanine derivative , the starting enzyme exhibited low activity , limited reaction stability and poor thermal stability . It could only tolerate substrate concentrations of up to 50 g / L and required a substrate-to-enzyme ratio of 1:2 , to reach 90 % conversion within 24 hours .
Through five rounds of directed evolution , the starting enzyme ( WT ) was significantly improved , enabling it to tolerate substrate concentrations of up to 200 g / L . The substrate-toenzyme ratio was enhanced to 10:1 , and the reaction could be driven almost to completion within 24 hours , with more than 20-fold increase in catalytic activity ( V5 ) ( Figure 6 ).
In the initial stages of the project , ammonia was used as the amine donor . However , this required large amounts of hydrochloric acid for pH adjustment , complicating the process . Following the screening of alternative amine donors , ammonium carbonate was identified as a more efficient option , effectively eliminating the need for excessive pH adjustment .
Conclusion
The integration of biocatalysis and enzyme engineering , supported by computational tools and directed evolution , has revolutionised the synthesis of complex amino acid derivatives such as the presented examples for ncAAs . Innovations in enzyme design have significantly improved the efficiency and scalability of these processes , meeting the growing demand for sustainable and cost-effective pharmaceutical manufacturing .
These achievements not only underscore the potential of biocatalysis for expanding the range of viable pharmaceutical compounds but also highlight its critical role in advancing green chemistry initiatives . ●
* - The authors would like to acknowledge the help of Dr Marco Bocola of Enzymaster Deutschland , and Hu Hu , Dr Hao Yang and Dr Haibin Chen , all of Enzymaster ( Ningbo ) Bio-Engineering , for their help in drafting this article . ** - BioEngine and BioNavigator are both trademarks of Enzymaster .
Dr Osama Mahmoud
References : 1 : D . Timson et al ., Fermentation 2019 , 5 ( 2 ): 39 . https :// doi . org / 10.3390 / fermentation5020039 2 : R . Zhang et al ., Angew . Chem . Int . Ed . 2024 , 63 , e202318550 . https :// doi . org / 10.1002 / anie . 202318550
3 : I . Song et al ., Nat Chem Biol 2021 , 17 , 1123 – 1131 . https :// doi . org / 10.1038 / s41589-021-00855-x 4 : Y . Gumulya et al ., Nat Catal 2018 , 1 , 878 – 888 . https :// doi . org / 10.1038 / s41929- 018-0159-5
5 : W . Xie et al ., Proc . Natl . Acad . Sci . USA 2022 , 119 ( 7 ) e2122355119 . https :// doi . org / 10.1073 / pnas . 2122355119 6 : WO2020207282A1 , Enzymaster ( Ningbo ) Bio-Engineering Co ., Ltd .
J j
SENIOR BUSINESS DEVELOPMENT MANAGER
ENZYMASTER DEUTSCHLAND o . mahmoud @ enzymaster . com www . enzymaster . de
JAN / FEB 2025 SPECCHEMONLINE . COM
35