title: Pseudomonas glumae lipase: increased proteolytic stability by protein engineering.
abstract: The feasibility of stabilizing proteins towards proteolytic degradation was explored by engineering the primary proteolytic cleavage site(s). This novel approach does not require information on the 3-D structure of the native enzyme. As a model system, the extracellular lipase of Pseudomonas glumae was chosen, which is sensitive towards degradation by subtilisin-type proteases. The primary proteolytic cleavage in the lipase appeared to be located between amino acids serine 153 and histidine 154. Since subtilisins are known to show a preference towards amino acid residues surrounding the scissile bond, non-preferred amino acids were introduced in this area. Two concepts were tested: the introduction of arginine or glutamate residues (charge concept) and the introduction of proline residues (proline concept). Although the mutant lipases produced according to either of these concepts were still cleaved in the same area, they showed a considerably increased stability towards proteolytic degradation.
authors.affiliation: Unilever Research Laboratorium, Vlaardingen, The Netherlands.
authors.forename: L G
authors.initials: LG
authors.lastname: Frenken
authors.forename: M R
authors.initials: MR
authors.lastname: Egmond
authors.forename: A M
authors.initials: AM
authors.lastname: Batenburg
authors.forename: C T
authors.initials: CT
authors.lastname: Verrips
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D005240:Feasibility Studies; D005813:Genes, Synthetic; D008049:Lipase / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry; D012697:Serine Endopeptidases / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D008049:Lipase; D012697:Serine Endopeptidases; C032923:microbial serine proteinases