Transxylosylation of stevioside by a novel GH39 β-xylosidase, and simultaneous valorization of agroindustrial byproducts

Stevia rebaudiana Bertoni is becoming increasingly popular as a sweetener, due to its high steviol glycoside content. However, the bitter aftertaste of those compounds is an issue for consumers, which can be alleviated with the addition of glycosyl substituents to the molecule. β-Xylosidases are known to transxylosylate various acceptors, mostly alcohols and sugars. In this work, a putative novel β-xylosidase gene from Ruminiclostridium cellulolyticum was heterologously expressed in E. coli, purified and characterized. The recombinant enzyme RcXyl39A, belonging to CAZy family GH39, showed satisfactory thermostability and tolerance towards product inhibition, while it was active in a wide pH and temperature range. Surprisingly, RcXyl39A was shown to possess a relaxed substrate specificity, since it was able to cleave various synthetic substrates, but also polymeric xylans. RcXyl39A was applied to the transxylosylation of stevioside, using enzymatically hydrolyzed xylan as a mixture of donor sugars. After optimization of several process parameters, the final yield obtained for stevioside conversion reached 27.4 ± 0.6 %. The results of the present study indicate that RcXyl39A could be applied to the saccharification of lignocellulosic biomass, but also to the synthesis of novel compounds with desirable properties.