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Preparation, characterization and stability of cross linked nitrilase aggregates (nitrilase–CLEAs) for hydroxylation of 2-chloroisonicotinonitrile to 2-chloroisonicotinic acid

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Abstract

Nitrilases capable of performing hydroxylation of 2-chloroisonicotinonitrile to 2-chloroisonicotinic acid were screened, and ES-NIT-102 was the best nitrilase for said biotransformation. Nitrilase was immobilized as cross linked enzyme aggregates (nitrilase–CLEAs) by fractional precipitation with iso-propanol, and cross linked with glutaraldehyde. The nitrilase–CLEAs prepared with optimized 35 mM glutaraldehyde for 120 min cross linking time had 82.36 ± 4.45% residual activity, and displayed type-II structural CLEAs formation as confirmed by particle size, SEM, FTIR, and SDS–PAGE analysis. Nitrilase–CLEAs had superior pH and temperature stability, showed a shift in optimal temperature by 5 °C, and retained nearly 1.5 to 1.7 folds activity over free nitrilase at 50 °C and 55 °C after more than 9 h incubation. Nitrilase–CLEAs showed reduced affinity and decreased conversion of substrate as indicated by slightly higher Km values by 5.19% and reduced Vmax by 17%. Furthermore, these nitrilase–CLEAs showed 98% conversion, 94.72 g/L product formation, and 83.30% recovery after 24 h when used for hydroxylation of 2-chloroisonicotinonitrile to 2-chloroisonicotinic acid. Nitrilase–CLEAs were catalytically active for 3 cycles showcasing 81% conversion, 75.53 g/L product formation and 66.42% yield. The recovered product was confirmed by HPLC, FTIR, LC–MS, and 1H NMR, and displayed > 99% purity.

Article highlights

  • ES-NIT-102 was best nitrilase amongst screened for hydroxylation reaction

  • Nitrilase–CLEAs at 35 mM glutaraldehyde and 2 h crosslinking showed max activity recovery.

  • Nitrilase–CLEAs displayed good pH/thermal and kinetic stability and prominent reusability.

  • Nitrilase–CLEAs had good conversion, product formation and recovery for hydroxylation.

  • 2-Chloroisonicotinonitrile and 2-chloroisonicotinic acid were confirmed by HPLC, FTIR, LCMS and NMR.

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Acknowledgements

The authors are grateful to Dean, School of Basic & Applied Sciences, and Vice-Chancellor, Galgotias University, Greater Noida for providing required facilities to carry out this research work.

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  1. School of Basic & Applied Sciences, Galgotias University, Gautam Buddh Nagar, Greater Noida, Uttar Pradesh, 201310, India

    Amol Gulab Khatik & Arvind Kumar Jain

  2. Research and Development, Cherish Pharma, Nashik, 22010, Maharashtra, India

    Abhijeet Bhimrao Muley

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Contributions

Design of experiments: AGK and ABM; Performance of experiment: AGK; Analysis and interpretation of data: AGK, AKJ, and ABM; Manuscript draft and compilation: AGK, and ABM; Manuscript correction and revision: AGK, AKJ, and ABM.

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Khatik, A.G., Jain, A.K. & Muley, A.B. Preparation, characterization and stability of cross linked nitrilase aggregates (nitrilase–CLEAs) for hydroxylation of 2-chloroisonicotinonitrile to 2-chloroisonicotinic acid. Bioprocess Biosyst Eng 45, 1559–1579 (2022). https://doi.org/10.1007/s00449-022-02766-0

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