Active Hydrogen Bond Network (AHBN) and Applications for Improvement of Thermal Stability and pH-Sensitivity of Pullulanase from Bacillus naganoensis

Abstract

A method, so called “active hydrogen bond network” (AHBN), is proposed for site-directed mutations of hydrolytic enzymes. In an enzyme the AHBN consists of the active residues, functional residues, and conservative water molecules, which are connected by hydrogen bonds, forming a three dimensional network. In the catalysis hydrolytic reactions of hydrolytic enzymes AHBN is responsible for the transportation of protons and water molecules, and maintaining the active and dynamic structures of enzymes. The AHBN of pullulanase BNPulA324 from Bacillus naganoensis was constructed based on a homologous model structure using Swiss Model Protein-modeling Server according to the template structure of pullulanase BAPulA (2WAN). The pullulanase BNPulA324 are mutated at the mutation sites selected by means of the AHBN method. Both thermal stability and pH-sensitivity of pullulanase BNPulA324 were successfully improved. The mutations at the residues located at the out edge of AHBN may yield positive effects. On the other hand the mutations at the residues inside the AHBN may deprive the bioactivity of enzymes. The AHBN method, proposed in this study, may provide an assistant and alternate tool for protein rational design and protein engineering.

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

“…To date, there have been only a few reports focusing on engineering the catalytic pocket of BnPUL. , We employed the TCSM method to try to improve the catalytic efficiency of BnPUL. Except for the catalytic triad, D619, E648, and D733, the functional residues were still unclear even in the active center of BnPUL.…”

Tailoring Pullulanase PulAR from Anoxybacillus sp. AR-29 for Enhanced Catalytic Performance by A Structure-Guided Consensus Approach

“…In a recent example, Bi and coworkers employed a computer-aided method to improve the thermostability of the thermophilic pullulanase from Bacillus thermoleovorans by raising T m 3.8 ℃ (Bi et al 2020). Up to date, only a limited number of reports on improving acidic adaptation or acid stability of pullulanase are available (Wang et al 2017;Zeng et al 2019). Chen and coworkers improved the acidic adaptation of Bacillus acidopullulyticus pullulanase by altering hydrogen bonds connected to catalytic residues, shifting its optimum pH from 5.0 to 4.0 at the expense of activity (Chen et al 2019).…”

Tailoring pullulanase PulAR from Anoxybacillus sp. AR-29 for enhanced catalytic performance by a structure-guided consensus approach

“…In a recent example, Bi and coworkers employed a computer-aided method to raise Tm of the thermophilic pullulanase from Bacillus thermoleovorans by 3.8 ℃ (Bi et al 2020 ). Up to date, only a limited number of reports on improving acidic adaptation of pullulanase are available (Wang et al 2017 ; Zeng et al 2019 ). Chen and coworkers improved the acidic adaptation of Bacillus acidopullulyticus pullulanase by altering hydrogen bonds network near the catalytic residues, shifting its optimum pH from 5.0 to 4.0 at the expense of activity reduction (Chen et al 2019 ).…”