random mutagenesis, mutant HYG10 contains seven substitutions and a duplication, it is thermostabilized compared to the wild-type enzyme, three-dimensional structure analysis, overview. Substitutions G189R, E237K, and D285N are located at the protein surface. The mutant shows altered kinetics at 45°C compared to the wild-type enzyme
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gene hyg, subcloning in Escherichia coli strain JM109, recombinant expression of His6-tagged enzyme in Escherichia coli strain BL21(DE3), recombinant enzyme expression from plasmid pT8S-P31 in Thermus thermophilus strain B27 TH104 providing hygromycin B resistance up to 60°C, while recombinant expression of a mutant variant HYG10, containing seven substitutions and a duplication, provides HgB resistance up to 74°C