Engineering the thermostability of β-glucuronidase from Penicillium purpurogenum Li-3 by loop transplant

In this study, we proposed a loop transplant strategy to improve the thermostability of Penicillium purpurogenum Li-3 β-glucuronidase expressed in Escherichia coli (abbreviated to PGUS-E). Firstly, three unstable surface loops of PGUS-E to be replaced were identified with regards to B-factor values and in-depth structure analysis: loops 205-211, 258-263, and 25-31. Then, based on B-factor analysis, eight stable loops for substitution were selected from two typical thermophilic glycosidases which had low homology with PGUS-E (less than 25 %). By analyzing the common features of these stable loops, it was found that they shared a common residue skeleton DXXTX(X)R, based on this, three chimera loops were also manually designed: RSQTSND, RSSTQRD, and DDQTSR. All these loops were introduced to replace the unstable loops of PGUS-E by homology structure modeling, and only mutants with increased hydrogen bonds number and good compatibility with the local mutated region were further subjected to experimental verification. By using this strategy, 10 mutants were experimentally generated, among which three mutants, M1, M3, and M8, were obtained which showed 11.8, 3.3, and 9.4 times higher half-life at 70 °C than that of wild-type (8.5 min). Finally, the MD simulation indicated that the increased hydrogen bonds, decreased flexibility of N-terminal, and increased π-π stacking interaction were responsible for the improved thermostability.