title: Effect of introducing a disulphide bond between the A and C domains on the activity and stability of Saccharomycopsis fibuligera R64 α-amylase.
abstract: Native enzyme and a mutant containing an extra disulphide bridge of recombinant Saccharomycopsis fibuligera R64 α-amylase, designated as Sfamy01 and Sfamy02, respectively, have successfully been overexpressed in the yeast Pichia pastoris KM71H. The purified α-amylase variants demonstrated starch hydrolysis resulting in a mixture of maltose, maltotriose, and glucose, similar to the wild type enzyme. Introduction of the disulphide bridge shifted the melting temperature (TM) from 54.5 to 56 °C and nearly tripled the enzyme half-life time at 65 °C. The two variants have similar kcat/KM values. Similarly, inhibition by acarbose was only slightly affected, with the IC50 of Sfamy02 for acarbose being 40 ± 3.4 μM, while that of Sfamy01 was 31 ± 3.9 μM. On the other hand, the IC50 of Sfamy02 for EDTA was 0.45 mM, nearly two times lower than that of Sfamy01 at 0.77 mM. These results show that the introduction of a disulphide bridge had little effect on the enzyme activity, but made the enzyme more susceptible to calcium ion extraction. Altogether, the new disulphide bridge improved the enzyme stability without affecting its activity, although minor changes in the active site environment cannot be excluded.
authors.affiliation: Biochemistry Research Division, Faculty of Mathematics and Natural Sciences, Bandung Institute of Technology, Jalan Ganesa No 10, Bandung 40132, Indonesia; Center for Life Sciences, Bandung Institute of Technology, Jalan Ganesa No. 10, Bandung 40132, Indonesia. Electronic address: dessy@chem.itb.ac.id.
authors.forename: Dessy
authors.initials: D
authors.lastname: Natalia
authors.affiliation: Biochemistry Research Division, Faculty of Mathematics and Natural Sciences, Bandung Institute of Technology, Jalan Ganesa No 10, Bandung 40132, Indonesia.
authors.forename: Keni
authors.initials: K
authors.lastname: Vidilaseris
authors.affiliation: Laboratory of Biophysical Chemistry, University of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands; Biochemistry Laboratory, Department of Chemistry, Faculty of Mathematics and Natural Sciences, Padjadjaran University, Jalan Singaperbangsa No. 2, Bandung 40133, Indonesia. Electronic address: wangsatirta@gmail.com.
authors.forename: Wangsa T
authors.initials: WT
authors.lastname: Ismaya
authors.affiliation: Center for Life Sciences, Bandung Institute of Technology, Jalan Ganesa No. 10, Bandung 40132, Indonesia.
authors.forename: Fernita
authors.initials: F
authors.lastname: Puspasari
authors.affiliation: Biochemistry Research Division, Faculty of Mathematics and Natural Sciences, Bandung Institute of Technology, Jalan Ganesa No 10, Bandung 40132, Indonesia.
authors.forename: Iman
authors.initials: I
authors.lastname: Prawira
authors.affiliation: Center for Life Sciences, Bandung Institute of Technology, Jalan Ganesa No. 10, Bandung 40132, Indonesia.
authors.forename: Khomaini
authors.initials: K
authors.lastname: Hasan
authors.affiliation: Laboratory of Biophysical Chemistry, University of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands.
authors.forename: Guntur
authors.initials: G
authors.lastname: Fibriansah
authors.affiliation: Mass Spectrometry Core Facility, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands.
authors.forename: Hjalmar P
authors.initials: HP
authors.lastname: Permentier
authors.affiliation: Biochemistry Research Division, Faculty of Mathematics and Natural Sciences, Bandung Institute of Technology, Jalan Ganesa No 10, Bandung 40132, Indonesia.
authors.forename: Zeily
authors.initials: Z
authors.lastname: Nurachman
authors.affiliation: Biochemistry Laboratory, Department of Chemistry, Faculty of Mathematics and Natural Sciences, Padjadjaran University, Jalan Singaperbangsa No. 2, Bandung 40133, Indonesia.
authors.forename: Toto
authors.initials: T
authors.lastname: Subroto
authors.affiliation: Laboratory of Biophysical Chemistry, University of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands.
authors.forename: Bauke W
authors.initials: BW
authors.lastname: Dijkstra
authors.affiliation: Biochemistry Laboratory, Department of Chemistry, Faculty of Mathematics and Natural Sciences, Padjadjaran University, Jalan Singaperbangsa No. 2, Bandung 40133, Indonesia.
authors.forename: Soetijoso
authors.initials: S
authors.lastname: Soemitro
mesh_terms: D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012442:Saccharomycopsis / Q000201:enzymology* / Q000235:genetics; D000516:alpha-Amylases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D004220:Disulfides; D005656:Fungal Proteins; D011994:Recombinant Proteins; D000516:alpha-Amylases
keywords: Disulphide bond; Enzyme stability; Pichia pastoris; Saccharomycopsis fibuligera R64; α-Amylase