Table 3 Exemplary engineered BHET hydrolases and dual-function MHET/BHET hydrolases discussed

From: Recent advances in enzyme engineering for improved deconstruction of poly(ethylene terephthalate) (PET) plastics

Enzyme Name

Mutations

Performance

Reaction Conditions/Substrate

Engineering Method

Citation

ΔBsEst

BsEst Δ410-418

2-fold faster conversion of BHET, 2.6-fold higher turnover, vs. BsEst

Activity: 30 °C, 10% (v/v) purified enzyme, 5 mM BHET, pH 7.5; kinetics: 0.8 to 15 mM BHET

Rational design (eliminating active site barrier)

Li, et al.141

ΔChryBHETase

ChryBHETase Δ66-76

100% conversion of BHET 3 h earlier, 1.7-fold higher turnover, vs. ChryBHETase,

Activity: 30 °C, 10% (v/v) purified enzyme, 5 mM BHET, pH 7.5; kinetics: 0.8 to 15 mM BHET

Rational design (eliminating active site barrier)

Li, et al.141

BMHETase6M

BMHETase G133S/R247G/

R275D/A373Y/A400S/G406S

~7.5-fold higher MHET activity, ~2.25-fold higher BHET activity, vs. BMHETase

MHET: 1 h, 60 °C, 500 nM enzyme, 2.5 mM MHET, pH 8; BHET: 10 min, 60 °C, 2 nM enzyme, 2 mM BHET, pH 8

Rational design (homology mapping)

Miao, et al.142

TfCa I69W

TfCa I69W

~2.4-fold higher activity vs. TfCa (converting BHET to TPA)

45 °C, 2 nM BHET, 550 nM enzyme, pH 7.5

Semi-rational design of targeted sites

von Haugwitz, et al.148

TfCa V376A

TfCa V376A

~1.75-fold higher activity vs. TfCa (converting BHET to TPA)

45 °C, 2 nM BHET, 550 nM enzyme, pH 7.5

Semi-rational design of targeted sites

von Haugwitz, et al.148

TfCa R428A

TfCa R428A

~1.5-fold higher activity vs. TfCa (converting BHET to TPA)

45 °C, 2 nM BHET, 550 nM enzyme, pH 7.5

Semi-rational design of targeted sites

von Haugwitz, et al.148

TfCa I69W/V376A/R428A

TfCa I69W/V376A/R428A

~3.75-fold higher activity vs. TfCa (converting BHET to TPA)

45 °C, 2 nM BHET, 550 nM enzyme, pH 7.5

Semi-rational design of targeted sites

von Haugwitz, et al.148

  1. If a unique name is given for an enzyme, the name is given alongside the mutation(s). Otherwise, the position is listed alongside the mutations to that position. The performance of the enzyme variants vs. relevant benchmarks and the conditions at which the performance was measured is listed, as well as the method of engineering for each variant. Fold change is calculated based on the performance of the variant divided by that of the benchmark enzyme. Turnover is derived from the authors’ calculation of the enzyme’s kinetic rate. Some variants were tested to measure the activities with both MHET and BHET as substates, which are listed separately. BsEst and ChryBHETase are BHET hydrolases, while BMHETase and TfCa have dual-function MHET/BHET hydrolase activity.
Back to article page