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KMID : 1094720230280040684
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Biotechnology and Bioprocess Engineering
2023 Volume.28 No. 4 p.684 ~ p.694
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Enhancing the Thermostability of Cellulase from Clostridium thermocellum via Salt Bridge Interactions
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Reyhane Zamani
Sayyed Shahryar Rahpeyma
Moein Aliakbari
Mousa Naderi
Mohsen Yazdanei
Saeed Aminzadeh
Jafar Khezri
Kamahldin Haghbeen
Ali Asghar Karkhane
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Abstract
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Improving the thermal stability of enzymes is an essential factor in the industrial applications of enzymes. Many methods related to increased thermal stability were explained, and increasing salt bridges is one of the strategies for improving the thermal stability of enzymes. In this study, mutations T59E, I145R, N149R, V219D, and A262R are introduced into the native cellulase gene to produce the mutant 5M-cel5E cellulase. In silico results showed that the mutation increased the salt bridges from 15 to 28. Root mean square fluctuation (RMSF) calculations confirmed that the mutation increased protein stability. Furthermore, the docking results showed that the affinity of cellobiose for the 5M-cel5E active site (?122.759) was slightly decreased compared to native cellulase (?130.93). No enzymatic activity was found in 5M-cel5E cellulase after cloning, expression and purification. Activated the enzyme with a back mutation of R149N, the result of which was named 4M-cel5E. The last mutation increases the salt bridges from 15 to 22, creating 4 salt bridge networks. The 4M-cel5E enzyme exhibited a maximum activity of 463 U/mg at pH 6.0 and 45°C. The mutations also increased the enzyme thermal stability up to 1.5 and 3.4-fold at temperatures of 65 and 67°C, respectively. These mutations made the Clostridium thermocellum cellulase suitable for various industries such the biofuel and paper.
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KEYWORD
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cellulase, salt bridge, thermostability, Clostridium thermocellum
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