title: Stabilization of a chitinase from Serratia marcescens by Gly-->Ala and Xxx-->Pro mutations.
abstract: This paper describes attempts to increase the kinetic stability of chitinase B from Serratia marcescens (ChiB) by the introduction of semi-automatically designed rigidifying mutations of the Gly-->Ala and Xxx-->Pro type. Of 15 single mutants, several displayed significant increases in thermal stability, whereas most mutants showed minor effects. All mutations with non-marginal effects on stability clustered in a limited, surface-exposed region of the enzyme, indicating that this region is involved in a partial unfolding process that triggers irreversible thermal inactivation (aggregation). A double mutant containing two stabilizing mutations in this region (G188A, A234P) displayed a 10-fold increase in half-life at 57 degrees C and a 4.2 degrees C increase in apparent T(m). These results show that entropic stabilization works well for ChiB and they pinpoint a region whose unfolding may be crucial for the kinetic stability of this enzyme.
authors: Department of Chemistry and Biotechnology, Agricultural University of Norway, PO Box 5040, 1432 As, Norway.
authors: Sigrid
authors: S
authors: Gåseidnes
authors: Bjørnar
authors: B
authors: Synstad
authors: Xiaohong
authors: X
authors: Jia
authors: Hege
authors: H
authors: Kjellesvik
authors: Gert
authors: G
authors: Vriend
authors: Vincent G H
authors: VG
authors: Eijsink
mesh_terms: D000409:Alanine / Q000737:chemistry / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics*; D002688:Chitinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D004795:Enzyme Stability; D005998:Glycine / Q000737:chemistry / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D011392:Proline / Q000737:chemistry / Q000235:genetics; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D012706:Serratia marcescens / Q000201:enzymology* / Q000235:genetics; D013820:Thermolysin / Q000378:metabolism
chemical_list: D011392:Proline; D002688:Chitinases; D013820:Thermolysin; D000409:Alanine; D005998:Glycine