Clostridium thermocellum thermostable lichenase with circular permutations and modifications in the N-terminal region retains its activity and thermostability

• Published in: Biochimica et biophysica acta 2015-01, Vol.1854 (1), p.10-19
• Main Authors: Tyurin, А.А., Sadovskaya, N.S., Nikiforova, Kh.R., Mustafaev, О.N., Komakhin, R.A., Fadeev, V.S., Goldenkova-Pavlova, I.V.
• Format: Article
• Language: English
• Subjects: Activity; Amino Acid Sequence; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Carrier protein; Catalytic Domain - genetics; Circular permutation; Clostridium thermocellum; Clostridium thermocellum - enzymology; Clostridium thermocellum - genetics; Electrophoresis, Polyacrylamide Gel; Enzyme Stability; Glycoside Hydrolases - chemistry; Glycoside Hydrolases - genetics; Glycoside Hydrolases - metabolism; Hot Temperature; Lichenase; Molecular Sequence Data; Mutation; Peptides - chemistry; Peptides - genetics; Peptides - metabolism; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Sequence Homology, Amino Acid; Thermostability
• ISSN: 1570-9639; 0006-3002; 1878-1454
• DOI: 10.1016/j.bbapap.2014.10.012

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. [Display omitted] •Circular permutation (CP) sites of the thermostable lichenase were predicted.•CP and modified lichenase variants were constructed.•N-terminal regions of lichenase permit CP and internal integrations of small peptides.•Lichenase N-terminal regions permitting CP are more likely to integrate of protein.•CP predictions can be used in search for internal integration sites within protein.