title: Surface charge engineering of a Bacillus gibsonii subtilisin protease.
abstract: In proteins, a posttranslational deamidation process converts asparagine (Asn) and glutamine (Gln) residues into negatively charged aspartic (Asp) and glutamic acid (Glu), respectively. This process changes the protein net charge affecting enzyme activity, pH optimum, and stability. Understanding the principles which affect these enzyme properties would be valuable for protein engineering in general. In this work, three criteria for selecting amino acid substitutions of the deamidation type in the Bacillus gibsonii alkaline protease (BgAP) are proposed and systematically studied in their influence on pH-dependent activity and thermal resistance. Out of 113 possible surface amino acids, 18 (11 Asn and 7 Gln) residues of BgAP were selected and evaluated based on three proposed criteria: (1) The Asn or Gln residues should not be conserved, (2) should be surface exposed, and (3) neighbored by glycine. "Deamidation" in five (N97, N253, Q37, Q200, and Q256) out of eight (N97, N154, N250, N253, Q37, Q107, Q200, and Q256) amino acids meeting all criteria resulted in increased proteolytic activity. In addition, pH activity profiles of the variants N253D and Q256E and the combined variant N253DQ256E were dramatically shifted towards higher activity at lower pH (range of 8.5-10). Variant N253DQ256E showed twice the specific activity of wild-type BgAP and its thermal resistance increased by 2.4 °C at pH 8.5. These property changes suggest that mimicking surface deamidation by substituting Gln by Glu and/or Asn by Asp might be a simple and fast protein reengineering approach for modulating enzyme properties such as activity, pH optimum, and thermal resistance.
authors.affiliation: RWTH Aachen University, Lehrstuhl für Biotechnologie, Worringerweg 1, 52074, Aachen, Germany.
authors.forename: Felix
authors.initials: F
authors.lastname: Jakob
authors.forename: Ronny
authors.initials: R
authors.lastname: Martinez
authors.forename: John
authors.initials: J
authors.lastname: Mandawe
authors.forename: Hendrik
authors.initials: H
authors.lastname: Hellmuth
authors.forename: Petra
authors.initials: P
authors.lastname: Siegert
authors.forename: Karl-Heinz
authors.initials: KH
authors.lastname: Maurer
authors.forename: Ulrich
authors.initials: U
authors.lastname: Schwaneberg
mesh_terms: D001407:Bacillus / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering; D013381:Subtilisins / Q000737:chemistry / Q000235:genetics*; D013499:Surface Properties
chemical_list: D013381:Subtilisins