title: Cumulative effect of amino acid replacements results in enhanced thermostability of potato type L alpha-glucan phosphorylase.
abstract: The thermostability of potato type L alpha-glucan phosphorylase (EC 2.4.1.1) was enhanced by random and site-directed mutagenesis. We obtained three single-residue mutations-Phe39-->Leu (F39L), Asn135-->Ser (N135S), and Thr706-->Ile (T706I)-by random mutagenesis. Although the wild-type enzyme was completely inactivated, these mutant enzymes retained their activity even after heat treatment at 60 degrees C for 2 h. Combinations of these mutations were introduced by site-directed mutagenesis. The simultaneous mutation of two (F39L/N135S, F39L/T706I, and N135S/T706I) or three (F39L/N135S/T706I) residues further increased the thermostability of the enzyme, indicating that the effect of the replacement of the residues was cumulative. The triple-mutant enzyme, F39L/N135S/T706I, retained 50% of its original activity after heat treatment at 65 degrees C for 20 min. Further analysis indicated that enzymes with a F39L or T706I mutation were resistant to possible proteolytic degradation.
authors.affiliation: Biochemical Research Laboratory, Ezaki Glico Co. Ltd., 4-6-5 Utajima, Nishiyodogawa-ku, Osaka 555-8502, Japan. yanase-michiyo@glico.co.jp
authors.forename: Michiyo
authors.initials: M
authors.lastname: Yanase
authors.forename: Hiroki
authors.initials: H
authors.lastname: Takata
authors.forename: Kazutoshi
authors.initials: K
authors.lastname: Fujii
authors.forename: Takeshi
authors.initials: T
authors.lastname: Takaha
authors.forename: Takashi
authors.initials: T
authors.lastname: Kuriki
mesh_terms: D019943:Amino Acid Substitution; D004795:Enzyme Stability; D006358:Hot Temperature; D007218:Industrial Microbiology / Q000379:methods; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D006005:Phosphorylases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D011198:Solanum tuberosum / Q000201:enzymology*
chemical_list: D006005:Phosphorylases