LAUSR

Fungal 1,3(4)-β-D-glucanases were usually applied in brewing and feedstuff industries, however, the thermostability limits the most their application. The characterized 1,3(4)-β-D-glucanase (NFEg16A) from Chinese Nong-flavor (NF) Daqu showed the highest thermostability among GH16 fungal 1,3(4)-β-D-glucanases, with half-lives of thermal inactivation (t1/2) of 44.9 min at 90 °C, so multiple rational designs were used to identify the key residues for its thermostability. Based on protein sequence and 3D structure analyses around the catalytic regions. Nine site-mutants were constructed, among which N173Y and S187A were identified as the most thermotolerant and thermolabile ones, with t1/2 values of 61 min and 14.0 min at 90 °C, respectively. Therefore, N173 and S187 were then selected as "hotspots" for site-saturation mutagenesis. Interestingly, most of the N173 and S187 variants exhibited a similar thermostability to that of N173Y and S187A, respectively, confirming their different roles in the thermostability of NFEg16A. In addition, each S187A and its surrounding substitutions (D144 N and T164 N) was independently detrimental to the thermostability of NFEg16A, since the t1/2 (90 °C) of S187A, D144 N and T164 N were 14.0 min, 20.6 min and 27.2 min, respectively. Surprisingly, combinatorial substitution of S187A with D144 N or T164 N showed positive effects on the thermostability, with the increase of t1/2 (90 °C) to 30.9 min and 63.5 min for S187A-D144 N and S187A-T164 N, respectively. More importantly, S187A-T164 N showed higher thermostability than that of wild type. In short, we successfully identified two key sites and their surrounding residues in response to the thermostability of NFEg16A and further improved its thermostability by several rational designs. These findings could be used for the protein engineering of homologous 1,3(4)-β-D-glucanases, as well as other enzyme family members with high similarities.