Mining and Modifying Distal Amino Acid Residue Assisted with Tunnel Engineering to Regulate the Product Specificity of <i>Limosilactobacillus fermentum</i> NCC 2970 GtfB 4,3-α-Glucanotransferase

The GH70 family 4,3/6-α-glucanotransferase efficiently converted readily digestible (α1→4) glycosidic bonds into (α1→3) or (α1→6) glycosidic bonds, thus serving as an effective enzymatic tool for preparing starch-based dietary fiber. Amino acid residues, including V1409, Q1397, T1366, K1362, H1369, and N1418 of <i>Limosilactobacillus fermentum</i> NCC 2970 GtfB 4,3-α-glucanotransferase (<i>Lf</i>2970 GtfB), were found via sequence conservation analysis and tunnel engineering for further analysis. Mutants from K1362, T1366, H1369, and N1418 at the tunnel entrance showed increased enzyme activity and catalytic efficiency but reduced molecular weight of products. The mutant Q1397P within the tunnel increased the proportion of (α1→6) bonds in the products to 7.9% and (α1→3) bonds to 26.3%, from the wild-type of 1.2% and 21.9%, respectively. These results indicated that amino acid residues located at the tunnel entrance regulated the enzyme activity and product size of <i>Lf</i>2970GtfB, while the Q1397 site within the tunnel regulated the type and proportion of glycosidic bonds.