Engineering of cellobiose phosphorylase for the defined synthesis of cellotriose
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BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS
Engineering of cellobiose phosphorylase for the defined synthesis of cellotriose Zorica Ubiparip 1
&
David Sáez Moreno 1 & Koen Beerens 1 & Tom Desmet 1
Received: 30 March 2020 / Revised: 8 July 2020 / Accepted: 5 August 2020 # The Author(s) 2020
Abstract Cellodextrins are non-digestible oligosaccharides that have attracted interest from the food industry as potential prebiotics. They are typically produced through the partial hydrolysis of cellulose, resulting in a complex mixture of oligosaccharides with a varying degree of polymerisation (DP). Here, we explore the defined synthesis of cellotriose as product since this oligosaccharide is believed to be the most potent prebiotic in the mixture. To that end, the cellobiose phosphorylase (CBP) from Cellulomonas uda and the cellodextrin phosphorylase (CDP) from Clostridium cellulosi were evaluated as biocatalysts, starting from cellobiose and α-D-glucose 1-phosphate as acceptor and donor substrate, respectively. The CDP enzyme was shown to rapidly elongate the chains towards higher DPs, even after extensive mutagenesis. In contrast, an optimised variant of CBP was found to convert cellobiose to cellotriose with a molar yield of 73%. The share of cellotriose within the final soluble cellodextrin mixture (DP2-5) was 82%, resulting in a cellotriose product with the highest purity reported to date. Interestingly, the reaction could even be initiated from glucose as acceptor substrate, which should further decrease the production costs. Key points • Cellobiose phosphorylase is engineered for the production of cellotriose. • Cellotriose is synthesised with the highest purity and yield to date. • Both cellobiose and glucose can be used as acceptor for cellotriose production. Keywords Enzyme engineering . Cellotriose synthesis . Prebiotic . Cellobiose phosphorylase
Introduction Oligosaccharides have continuously growing applications in the food, feed and pharmaceutical industries (Han et al. 2012; Meyer et al. 2015; Martins et al. 2019). In particular, non-digestible oligosaccharides can serve as prebiotics that promote immunemodulatory health effects by influencing the gut microbiome composition (Panesar and Bali 2015; Holscher 2017; Wu et al. 2017). Examples of prebiotics that are already well established on the market include fructooligosaccharides (FOS), Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00253-020-10820-8) contains supplementary material, which is available to authorized users. * Tom Desmet [email protected] 1
Centre for Synthetic Biology (CSB), Department of Biotechnology, Ghent University, Coupure Links 653, 9000 Ghent, Belgium
galactooligosaccharides (GOS), soybean-derived oligosaccharides (SOS) and (arabino)xylooligosaccharides (XOS/AXOS) (Gibson 2008; Pokusaeva et al. 2011; Anadón et al. 2015; Carlson and Slavin 2016). This prebiotic pool will surely expand further as research focusing on the role of prebiotics continues to stimulate the market, wh
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