Helicobacter pylori HpfutC, a glycosyltransferase (GT) 11 family glycoprotein, has great potential for industrial 2\'-fucosyllactose (2\'-FL) production. However, its limited catalytic activity, low expression, and poor thermostability hinder practical applications. Herein, a semi-rationally designed site-saturation mutation was applied to engineer the catalytic activity and thermostability of HpfutC. The 6 single point mutants (K102T, R105C, D115S, Y251F, A255G and K282E) and 6 combined mutants (V1, V2, V3, V4, V5, and V6) with enhanced enzyme activity were obtained by mutant library screening and ordered recombination mutation. The optimal mutant V6, with an optimum temperature of 40 °C, was not a metal-dependent enzyme, yet the reaction was facilitated by Mn2+. Compared to wild-type HpfutC, mutant V6 exhibited a 2.3-fold increase in specific activity and a 2.18-fold increase in half-life at 40 °C, respectively. Kinetic parameters indicated that the Km values of mutant V6 were 34.5 % (lactose) and 25.0 % (GDP-L-fucose) lower than those of the wild enzyme, whereas the kcat/Km values were 1.20 and 1.25-fold higher than those of the wild enzyme. Further, 3D-structure analysis revealed that the highly rigid structure, formation of new hydrogen bonds, increased hydrophobic residues and redistribution of electrostatic charges on the surface may be responsible for the elevated enzyme activity and thermostability. The strategy adopted in this study is of great significance to the solution of the technical bottleneck of HpfutC and the industrial application of 2\'-FL.

γ-Aminobutyrate (GABA) is an important bioactive compound synthesized through decarboxylation of L-glutamate by the glutamate decarboxylase (GAD). In this study, stabilized variants of the GAD from Lactobacillus brevis were constructed by consensus mutagenesis. Using Consensus Finder ( http://cbs-kazlab.oit.umn.edu/ ), eight positions with the most prevalent amino acid (over 60% threshold) among the homologous family members were identified. Subsequently, these eight residues were individually mutated to match the consensus sequence using site-directed mutagenesis. Compared to the wild-type, T383K variant displayed the largest shift in thermostability among the single variants, with a 3.0 °C increase in semi-inactivation temperature (T5015), a 1.7-fold improvement of half-life (t1/2) at 55 °C, and a 1.2-fold improvement of t1/2 at 37 °C, respectively, while its catalytic efficiency (kcat/Km) was reduced. To obtain the mutant with improvement in both thermostability and catalytic activity, we performed a site-saturation mutation at T383. Notably, mutants T383V and T383G exhibited an increasement in thermostability and kcat/Km than that of wild-type. This study not only emphasizes the value of consensus mutagenesis for improving the thermostability of GAD but also sheds a powerful guidance to study the thermal stability of other enzymes.

To improve the catalytic activity of atrazine chlorohydrolase (AtzA), amino acid residues involved in substrate binding (Gln71) and catalytic efficiency (Val12, Ile393, and Leu395) were targeted to generate site-saturation mutagenesis libraries. Seventeen variants were obtained through Haematococcus pluvialis-based screening, and their specific activities were 1.2-5.2-fold higher than that of the wild type. For these variants, Gln71 tended to be substituted by hydrophobic amino acids, Ile393 and Leu395 by polar ones, especially arginine, and Val12 by alanine, respectively. Q71R and Q71M significantly decreased the Km by enlarging the substrate-entry channel and affecting N-ethyl binding. Mutations at sites 393 and 395 significantly increased the kcat/Km, probably by improving the stability of the dual β-sheet domain and the whole enzyme, owing to hydrogen bond formation. In addition, the contradictory relationship between the substrate affinity improvement by Gln71 mutation and the catalytic efficiency improvement by the dual β-sheet domain modification was discussed.