Preprint Article Version 1 Preserved in Portico This version is not peer-reviewed

Broadening the Substrate Specificity of Cellobiose Phosphorylase from Clostridium thermocellum for Improved Transformation of Cellodextrin to Starch

Version 1 : Received: 18 August 2023 / Approved: 18 August 2023 / Online: 21 August 2023 (02:44:46 CEST)

A peer-reviewed article of this Preprint also exists.

Zhang, Y.; Li, Y.; Lin, H.; Mao, G.; Long, X.; Liu, X.; Chen, H. Broadening the Substrate Specificity of Cellobiose Phosphorylase from Clostridium thermocellum for Improved Transformation of Cellodextrin to Starch. Int. J. Mol. Sci. 2023, 24, 14452. Zhang, Y.; Li, Y.; Lin, H.; Mao, G.; Long, X.; Liu, X.; Chen, H. Broadening the Substrate Specificity of Cellobiose Phosphorylase from Clostridium thermocellum for Improved Transformation of Cellodextrin to Starch. Int. J. Mol. Sci. 2023, 24, 14452.

Abstract

Cellobiose phosphorylase (CBP) catalyzes the reversible phosphorolysis of cellobiose into α-glucose 1-phosphate and glucose. When it was used in transforming cellulose to amylose (PNAS, 110: 7182-7187, 2013) as well as in constructing a yeast with the ability to phosphorolytically use cellodextrin to produce ethanol, a CBP with a broadened substrate specificity would be more desirable. Based on the structure differences in the catalytic loops of CBP and cellodextrin phosphorylase from Clostridium thermocellum (named CtCBP and CtCDP, respectively), CtCBP was mutated. A single-site mutant S497G was identified to have a 5.7-fold higher catalytic efficiency with cellotriose as a substrate in phosphorolytic reaction direction compared to wild type, without any loss of catalytic efficiency on its natural substrate cellobiose. When S497G variant was used in the transformation of mixed cellodextrin (cellobiose + cellotriose) to amylose, a significant increase in amylose yield was achieved compared to that of wild type CtCBP. Structure change in the substrate binding pocket of S497G variant accounted for its capacity to accept longer cellodextrins than cellobiose. Taken together, S497G variant of CtCBP was confirmed to acquire a promising feature favorable to those application scenarios involving cellodextrin’s phosphorolysis.

Keywords

cellobiose phosphorylase; cellodextrin phosphorylase; substrate specificity; cellodextrin; amylose

Subject

Biology and Life Sciences, Biology and Biotechnology

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