Neutral Genetic Drift-Based Engineering of a Sucrose-Utilizing Enzyme toward Glycodiversification

Neutral drift (also called purifying selection) is an attractive approach to generate polymorphic variant libraries for enzyme engineering. Here, we have applied this strategy to modify the substrate specificity of a transglucosylase. Our model enzyme, the amylosucrase from Neisseria polysaccharea, is a glucosylation biocatalyst of prime interest because it uses the widespread substrate sucrose as a glucosyl donor and shows broad acceptor promiscuity. A library of 440 functional amylosucrase variants was generated after four rounds of neutral drift at a low mutation rate. The functional variations present in this library were investigated by assaying the ability of these variants to use an alternative glucosyl donor (p-nitrophenyl-α-d-glucopyranoside, pNP-Glc) and to glucosylate a range of acceptors (including methyl-α-l-rhamnopyranoside, which is not naturally recognized by the parental enzyme). The impact of these mutations on the thermal stability of the variants was also assessed. Large variations of ...