Enhancing thermostability and the structural characterization of Microbacterium saccharophilum K-1 β-fructofuranosidase
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A β-fructofuranosidase from Microbacterium saccharophilum K-1 (formerly known as Arthrobacter sp. K-1) is useful for producing the sweetener lactosucrose (4G-β-d-galactosylsucrose). Thermostability of the β-fructofuranosidase was enhanced by random mutagenesis and saturation mutagenesis. Clones with enhanced thermostability included mutations at residues Thr47, Ser200, Phe447, Phe470, and Pro500. In the highest stability mutant, T47S/S200T/F447P/F470Y/P500S, the half-life at 60 °C was 182 min, 16.5-fold longer than the wild-type enzyme. A comparison of the crystal structures of the full-length wild-type enzyme and three mutants showed that various mechanisms appear to be involved in thermostability enhancement. In particular, the replacement of Phe447 with Val or Pro induced a conformational change in an adjacent residue His477, which results in the formation of a new hydrogen bond in the enzyme. Although the thermostabilization mechanisms of the five residue mutations were explicable on the basis of the crystal structures, it appears to be difficult to predict which amino acid residues should be modified to obtain thermostabilized enzymes.
KeywordsThermostability Random mutagenesis β-Fructofuranosidase Lactosucrose GH68
This research was performed with the approval of the Photon Factory Advisory Committee (2010G001 and 2012G006) of the National Laboratory for High Energy Physics, Tsukuba, Japan. The synchrotron radiation experiments were also performed on BL26B2 at SPring-8 with the approval of the Priority Program for Disaster-Affected Quantum Beam Facilities (2011A1881), the Japan Synchrotron Radiation Research Institute.
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