title: Clostridium thermocellum thermostable lichenase with circular permutations and modifications in the N-terminal region retains its activity and thermostability.
abstract: The Clostridium thermocellum lichenase (endo-β-1,3;1,4-glucan-D-glycosyl hydrolase) displays a high thermostability and specific activity and has a compact protein molecule, which makes it attractive, in particular, for protein engineering. We have utilized in silico analysis to construct circularly permuted (CP) variants and estimated the retained activity and thermostability. New open termini in the region of residues 53 or 99 in two lichenase CP variants (CN-53 and CN-99) had no effect on their activity and thermal tolerance versus another variant CP variant, CN-140 (cut in the region of residue 140), which displayed a dramatic decrease in the activity and thermostability. Construction and further activity and thermostability testing of the modified lichenase variants (M variants) and CP variants with peptides integrated via insertion fusion have demonstrated that the N-terminal regions in the lichenase catalytic domain (53 and 99 amino acid residues) that permit circular permutations with retention of activity and thermostability of the enzyme as well as the region between the C and N termini of the native lichenase in thermostable and active lichenase variants (CN-53 and CN-99) may be used for integrating small peptides without the loss of activity and thermostability. These findings not only suggest that CP predictions can be used in search for internal integration sites within protein molecule, but also form the background for further enzymatic engineering of the C. thermocellum thermostable lichenase aiming to create new fusion proteins.
authors.affiliation: Institute of Plant Physiology, Russian Academy of Sciences, ul. Botanicheskaya 35, Moscow 127276, Russia; Department of Genetics and Biotechnology, Russian State Agrarian University-Moscow Timiryazev Agricultural Academy, ul. Timiryazevskaya 49, Moscow 127550, Russia.
authors.forename: A А
authors.initials: AА
authors.lastname: Tyurin
authors.affiliation: Institute of Plant Physiology, Russian Academy of Sciences, ul. Botanicheskaya 35, Moscow 127276, Russia.
authors.forename: N S
authors.initials: NS
authors.lastname: Sadovskaya
authors.affiliation: Institute of Plant Physiology, Russian Academy of Sciences, ul. Botanicheskaya 35, Moscow 127276, Russia.
authors.forename: Kh R
authors.initials: KhR
authors.lastname: Nikiforova
authors.affiliation: Institute of Plant Physiology, Russian Academy of Sciences, ul. Botanicheskaya 35, Moscow 127276, Russia.
authors.forename: O N
authors.initials: ON
authors.lastname: Mustafaev
authors.affiliation: All-Russia Research Institute of Agricultural Biotechnology, Russian Academy of Agricultural Sciences, ul. Timiryazevskaya 42, Moscow 127550, Russia.
authors.forename: R A
authors.initials: RA
authors.lastname: Komakhin
authors.affiliation: Institute of Plant Physiology, Russian Academy of Sciences, ul. Botanicheskaya 35, Moscow 127276, Russia.
authors.forename: V S
authors.initials: VS
authors.lastname: Fadeev
authors.affiliation: Institute of Plant Physiology, Russian Academy of Sciences, ul. Botanicheskaya 35, Moscow 127276, Russia; Department of Genetics and Biotechnology, Russian State Agrarian University-Moscow Timiryazev Agricultural Academy, ul. Timiryazevskaya 49, Moscow 127550, Russia. Electronic address: irengold58@gmail.com.
authors.forename: I V
authors.initials: IV
authors.lastname: Goldenkova-Pavlova
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain / Q000235:genetics; D048013:Acetivibrio thermocellus / Q000201:enzymology* / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
chemical_list: D001426:Bacterial Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D006026:Glycoside Hydrolases; C028020:licheninase
keywords: Activity; Carrier protein; Circular permutation; Clostridium thermocellum; Lichenase; Thermostability