Protein engineering of HheG with the aim to increase its enantioselectivity focussed on the exchange of residues on loop3947. In a first step positions T39 to G47 were exchanged by amino acids Cys, Lys, Glu and Phe. Site-directed mutagenesis of HheG was performed using the PfuUltra II Hotstart PCR Mastermix (Agilent Technologies, Santa-Clara, CA, United States). Respective forward and reverse mutagenic primers (Table ) were designed with PrimerX (Carlo Lapid, 2003, , accessed on 16 February 2021), purchased from Merck (Darmstadt, Germany) and used in concentrations of 0.25 µM each with 100 ng of pET28a(+)-hheG template. Otherwise, the PCR protocol for mutagenesis was in agreement with the manufacturer's instructions. Afterwards, methylated parental DNA was digested at 37 °C over night using 20 U DpnI before transformation in E. coli BL21(DE3) Gold. In a second step, positions T44, M45 and V46 were fully randomized. GoldenGate cloning 62 was used to incorporate all missing mutations at respective positions separately. The PCR using Q5 polymerase (NEB) was performed according to the manufacturer's instructions. Forward and reverse mutagenic primers (Table ) were designed according to GoldenGate primer design, purchased from Merck (Darmstadt, Germany) and used in concentrations of 0.25 µM each with 5 ng of pET28a(+)-hheG template. Each 100 ng PCR product were purified using the E.Z.N.A. MircoElute CyclePure Kit (omega-biotek) and incubated with 2 U BsaI plus 200 U T4-Ligase in 1x CutSmart buffer and 1x T4-ligase buffer for 2 h at 30 °C. Inactivated GoldenGate reactions (20 min, 65 °C) were afterwards transformed in E. coli BL21(DE3) Gold.