title: 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.
authors.affiliation: State Key Laboratory of Biomass Enzyme Technology, National Engineering Research Center for Non-Food Biorefinery, Guangxi Academy of Sciences, Nanning, Guangxi, China.
authors.forename: Qing-Yan
authors.initials: QY
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Biomass Enzyme Technology, National Engineering Research Center for Non-Food Biorefinery, Guangxi Academy of Sciences, Nanning, Guangxi, China.
authors.forename: Neng-Zhong
authors.initials: NZ
authors.lastname: Xie
authors.affiliation: State Key Laboratory of Biomass Enzyme Technology, National Engineering Research Center for Non-Food Biorefinery, Guangxi Academy of Sciences, Nanning, Guangxi, China.|Gordon Life Science Institute, Belmont, MA, United States of America.
authors.forename: Qi-Shi
authors.initials: QS
authors.lastname: Du
authors.affiliation: State Key Laboratory of Biomass Enzyme Technology, National Engineering Research Center for Non-Food Biorefinery, Guangxi Academy of Sciences, Nanning, Guangxi, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Qin
authors.affiliation: State Key Laboratory of Biomass Enzyme Technology, National Engineering Research Center for Non-Food Biorefinery, Guangxi Academy of Sciences, Nanning, Guangxi, China.|Life Science and Technology College, Guangxi University, Nanning, Guangxi, China.
authors.forename: Jian-Xiu
authors.initials: JX
authors.lastname: Li
authors.affiliation: Life Science and Technology College, Guangxi University, Nanning, Guangxi, China.
authors.forename: Jian-Zong
authors.initials: JZ
authors.lastname: Meng
authors.affiliation: State Key Laboratory of Biomass Enzyme Technology, National Engineering Research Center for Non-Food Biorefinery, Guangxi Academy of Sciences, Nanning, Guangxi, China.|Life Science and Technology College, Guangxi University, Nanning, Guangxi, China.
authors.forename: Ri-Bo
authors.initials: RB
authors.lastname: Huang
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D004795:Enzyme Stability; D005798:Genes, Bacterial; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D040681:Structural Homology, Protein; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; C100181:pullulanase