Abstract— Protein terminals have important roles in molecular structural stability and in some occasions they have regulatory roles in catalytic reaction. To expand our understanding on the influences of distal residues mutation, we explored the molecular stability and kinetics of mannanase Man1312 mutants. For Man1312, the N-terminal loop was more disordered and changeable; therefore the mutations on N-terminal should have significant effects on enzymic properties. The experiment was investigated by spectrophotometer, circular dichroism and differential scanning calorimetry assays. As a result, positive mutations were found from sites of T2 and Q9 and double-site mutant ManY9G2 showed increased catalytic activity by 7.7% higher than Man1312. Meanwhile, ManY9G2 had significantly increased thermostability with promoted Topt by 6oC and elongated t1⁄2 by 7 min, which was resulted from the optimized intermolecular forces and a newly-built hydrogen bond in ManY9G2. In our experiment, the specific residues were mutated from hydrophilic to hydrophobic and the improved hydrophobicity on N-terminal had a positive impact on the properties of mannanase Man1312.
Highlights
We construct N-terminal multi-site mutants of mannanase Man1312.
We determine the enzyme activity and thermal dynamics of mannanase and its mutants.
Best mutations increase the hydrophobilicity of N-terminal.
N-terminal mutation significantly improves the thermo-stability and slightly increases activity of mannanase.
Keywords— Mannanase, N-terminal, multi-site mutation, structural stability, activity.
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