A
300
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C
200
+-' C
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> e 100
E so
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0 1
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4
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Round 1 Round 2 Round 3 Stage of evolution
256
•
•
•
•
64
•
:
•• ••
Round 4
B
GREEN CHEMISTRY
Wild-type enzyme :
• Reaction Time : 24 h
• 90 % conv ./ 95 % ee
• Catalyst load : 50 g / L
• Substrate load : 2 g / L
Pregabalin
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s
V
' 1ection
4 rounds of evolution
Figure 6 - Improvement of d-hydantoinase activity towards Pregabalin IM ( a ) & comparison of initial & evolved enzymes ( b )
To conclude , a biocatalytic process employing an immobilised enzyme is desired to allow process intensification and to match with regulatory requirements . To meet this criterion , the engineered d-hydantoinase variant was covalently bound to a carrier .
This immobilised enzyme can be reused for more than 20 times in consecutive batch reactions with acceptable activity loss ( Figure 7 ). In each batch reaction , 250 g / L substrate can be fully converted into Pregabalin intermediate with an excellent ee of 99.5 %.
Conclusion
These examples of engineered transaminase and d-hydantoinase presented here for the production of Ubrogepant intermediate and Pregabalin intermediate represent only a small number of the diverse applications and opportunities that enzymes provide in organic and asymmetric synthesis .
Computer-guided enzyme engineering can help substantially to leverage the full power of such enzymes and use these for sustainable chemical manufacturing at scale .
Enzymatic processes can often be designed to outperform traditional chemistry in terms of selectivity , atom economy and a reduced number of synthetic steps .
Outlook
Thanks to comprehensive knowledge based on longstanding expertise in enzyme engineering , synthetic chemistry , bioinformatics , molecular biology and process development Enzymaster will continue providing one-stop services for the development of biocatalytic processes , starting from the idea all the way to the commercialisation on hundreds to thousands of tonnes scales .
The development of enzymes for the production of API intermediates , fine and commodity chemicals , and compounds applied in consumer markets has become an integral part when considering new , innovative and sustainable synthetic routes . Enzymaster aims to contribute to a greener and safer environment by supporting customers in applying enzymes in their manufacturing processes . ●
Figure 7 - Recycling of immobilised d-hydantoinase in consecutive batch reactions
* - Acknowledgement : The research was performed by Marco Bocola of Enzymaster Deutschland , and Yingxin Zhang , Hu Hu , Baoqin Cai , Junpeng Xu , Xiao Luo , Yaoyao Ji , Chengxiao Zhang , Ruimei Hong , Haibin Chen all of Enzymaster ( Ningbo ) Bio-Engineering in China
** - BioEngine and BioNavigator are both trademarks of Enzymaster
References : 1 : C . Ding et al ., Front . Public Health 2022 , 10 , 952161 2 : S . Wu et al ., Angew . Chem . Int . Ed . Engl . 2021 , 60 ( 1 ), 88-119
3 : WO2012064910A1 Merck Sharp & Dohme Corp . 4 : Y . Nobuyoshi et al ., Org . Proc . Res . Dev . 2017 21 ( 11 ), 1851-1858 5 : P . J . Dunn , Chem . Soc . Rev . 2012 , 41 , 1452 – 1461
6 : F . Liu et al ., Green Chem . 2022 , 24 , 4748 7 : CN111944856B Enzymaster ( Ningbo ) Bio- Engineering Co ., Ltd . 8 : PCT / CN2022 / 128468 Enzymaster ( Ningbo ) Bio-Engineering Co ., Ltd .
J j
Dr Osama Mahmoud
SENIOR BUSINESS DEVELOPMENT MANAGER
ENZYMASTER DEUTSCHL & GMBH o . mahmoud @ enzymaster . com www . enzymaster . de
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