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Open AccessArticle

Disulfide Bond Engineering of an Endoglucanase from Penicillium verruculosum to Improve Its Thermostability

1
Federal Research Centre «Fundamentals of Biotechnology» of the Russian Academy of Sciences, Moscow 119071, Russia
2
Institute of Biotechnology, RWTH Aachen University, Aachen 52074, Worringerweg 3, Germany
3
G.K.Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino 142292, Moscow region, Russia
4
Department of Chemistry, M.V.Lomonosov Moscow State University, Moscow 119991, Russia
5
DWI-Leibniz Institut für Interaktive Materialien, Forckenbeckstrasse 50, Aachen 52056, Germany
*
Author to whom correspondence should be addressed.
These authors contributed equally to this work.
Int. J. Mol. Sci. 2019, 20(7), 1602; https://doi.org/10.3390/ijms20071602
Received: 28 February 2019 / Revised: 25 March 2019 / Accepted: 27 March 2019 / Published: 30 March 2019
(This article belongs to the Special Issue Industrial Enzymes: Structure, Function and Applications)
Endoglucanases (EGLs) are important components of multienzyme cocktails used in the production of a wide variety of fine and bulk chemicals from lignocellulosic feedstocks. However, a low thermostability and the loss of catalytic performance of EGLs at industrially required temperatures limit their commercial applications. A structure-based disulfide bond (DSB) engineering was carried out in order to improve the thermostability of EGLII from Penicillium verruculosum. Based on in silico prediction, two improved enzyme variants, S127C-A165C (DSB2) and Y171C-L201C (DSB3), were obtained. Both engineered enzymes displayed a 15–21% increase in specific activity against carboxymethylcellulose and β-glucan compared to the wild-type EGLII (EGLII-wt). After incubation at 70 °C for 2 h, they retained 52–58% of their activity, while EGLII-wt retained only 38% of its activity. At 80 °C, the enzyme-engineered forms retained 15–22% of their activity after 2 h, whereas EGLII-wt was completely inactivated after the same incubation time. Molecular dynamics simulations revealed that the introduced DSB rigidified a global structure of DSB2 and DSB3 variants, thus enhancing their thermostability. In conclusion, this work provides an insight into DSB protein engineering as a potential rational design strategy that might be applicable for improving the stability of other enzymes for industrial applications. View Full-Text
Keywords: cellulase; endoglucanase; rational design; protein engineering; disulfide bonds; thermostability; cellulose biodegradation cellulase; endoglucanase; rational design; protein engineering; disulfide bonds; thermostability; cellulose biodegradation
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Bashirova, A.; Pramanik, S.; Volkov, P.; Rozhkova, A.; Nemashkalov, V.; Zorov, I.; Gusakov, A.; Sinitsyn, A.; Schwaneberg, U.; Davari, M.D. Disulfide Bond Engineering of an Endoglucanase from Penicillium verruculosum to Improve Its Thermostability. Int. J. Mol. Sci. 2019, 20, 1602.

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