Improvement of the Activity and Stability of Starch-Debranching Pullulanase from Bacillus naganoensis via Tailoring of the Active Sites Lining the Catalytic Pocket

Abstract

Pullulanases are well-known debranching enzymes that hydrolyze α-1,6-glycosidic linkages in starch and oligosaccharides. However, most of the pullulanases exhibit limited activity for practical applications. Here, two sites (787 and 621) lining the catalytic pocket of Bacillus naganoensis pullulanase were identified as being critical for enzymatic activity by triple-code saturation mutagenesis. Subsequently, both sites were subjected to NNK-based saturation mutagenesis to obtain positive variants. Among the variants showing enhanced activity, the enzymatic activity and specific activity of D787C were 1.5-fold higher than those of the wild-type (WT). D787C also showed a 1.8-fold increase in k cat and a 1.7-fold increase in k cat/K m. In addition, D787C maintained higher activity compared with that of WT at temperatures over 60 °C. All the positive variants showed higher acid resistance, with D787C maintaining 90% residual activity at pH 4.0. Thus, enzymes with improved properties were obtained by saturation mutagenesis at the active site.

Evolutionary coupling saturation mutagenesis: Coevolution‐guided identification of distant sites influencing Bacillus naganoensis pullulanase activity

“…Hence, the ECSM approach compares very favorably with site‐directed mutagenesis and regional truncation methods, which increased the catalytic efficiency only about two folds (Table S7).…”

“…Using ECSM, pullulanase mutants were created with significantly enhanced catalytic activity when compared with WT BnPUL ( k cat and k cat / K m were increased by of ~ 3 and ~ 6, respectively). These gains of catalytic activity are remarkable for a highly evolved naturally occurring enzyme, and they compare very favorably with previously engineered variants . Since high thermostability and high enzyme activity at comparatively low pH are key for industrial applications of pullulanases , it is important to observe that the mutant enzymes exhibit virtually the same dependence of activity on temperature and pH as the WT, making these mutants suitable for industrial applications.…”

“…However, the catalytic efficiency of pullulanase needs to be further improved for industrial application. To date, engineering of pullulanase is mainly focused on the catalytic pocket or domain tailoring based on structure/sequence information …”

2-Ketoglutarate-Generated In Vitro Enzymatic Biosystem Facilitates Fe(II)/2-Ketoglutarate-Dependent Dioxygenase-Mediated C–H Bond Oxidation for (2s,3r,4s)-4-Hydroxyisoleucine Synthesis

“…The recombinant IDO and LGOX supernatants were purified by Ni-affinity chromatography as previously reported [ 39 ]. The supernatant was purified in a His-Trap HP affinity column (GE Healthcare, Little Chalfont, UK) [ 40 ]. The elution buffer in the affinity columns created a high salt content in the purified fractions.…”

Semi-Rational Design of Proteus mirabilis l-Amino Acid Deaminase for Expanding Its Substrate Specificity in α-Keto Acid Synthesis from l-Amino Acids

“…The residues within a 4 Å scope around the substrate-binding site were selected for alanine scanning, and three key residues with higher activity were determined for site-directed saturation mutagenesis. Site-directed saturation mutants were constructed by homologous recombination according to a previously published method [31]. Primer synthesis and DNA sequencing were performed by Genewiz Biotech Co., Ltd. (Suzhou, China).…”