For combination of mutations M45F, M45Y and M45W with previously described mutations T123G and T123F of HheG, 37 site-directed mutagenesis or GoldenGate cloning was performed according to the protocols described above and using templates pET28a(+)-hheG T123G and pET28a(+)-hheG T123F. For combination of mutations M45F and D114C in HheG, sitedirected mutagenesis starting from template pET28a(+)-hheG D114C was used. Loop3947 deletion mutants of HheG were constructed by GoldenGate cloning using pET28a(+)-hheG as template and mutagenic primers listed in Table .