Foam fractionation Tags (F-Tags) enabling surfactant-free, activity-preserving recovery of enzymes

Abstract Enzymes have become important tools in many industries. However, the full exploitation of their potential is currently limited by a lack of efficient and cost-effective methods for enzyme purification from microbial production. One technology that could solve this problem is foam fractionation. In this study, we show that diverse natural foam-stabilizing proteins fused as F-Tags to β-lactamase, penicillin G acylase, and formate dehydrogenase, respectively, are able to mediate foaming and recovery of the enzymes by foam fractionation. The catalytic activity of all three candidates is largely preserved. Under appropriate fractionation conditions, especially when a wash buffer is used, some F-Tags also allow nearly complete separation of the target enzyme from a contaminating protein. We found that a larger distance between the F-Tag and the target enzyme has a positive effect on the maintenance of catalytic activity. However, we did not identify any particular sequence motifs or physical parameters that influenced performance as an F-tag. The best results were obtained with a short helical F-Tag, which was originally intended to serve only as a linker sequence. The findings of the study suggest that the development of molecular tags that enable the establishment of surfactant-free foam fractionation for enzyme workup is a promising method. Key points • Foam-stabilizing proteins mediate activity-preserving foam fractionation of enzymes • Performance as an F-Tag is not restricted to particular structural motifs • Separation from untagged protein benefits from low foam stability and foam washings Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1007/s00253-023-12837-1.

Fig. S4: Recovery, protein loss and foam purity after foam fractionation of PGA fusion constructs and eGFP as impurity in a simple column with 10 mL initial volume and an air flow rate of 20 mL⋅min -1 ; foaming proceeded until no foam overflowed.Initial total protein concentration: 0.2 mg⋅mL -1 at a PGA construct to eGFP ratio of 1:1; initial purity: 50% (±5%).

Fig. S5:
Recovery, protein loss and foam purity after foam fractionation of RjFDH fusion constructs and eGFP as impurity in a simple column with 10 mL initial volume and an air flow rate of 20 mL⋅min -1 ; foaming proceeded until no foam overflowed.Initial total protein concentration: 0.2 mg⋅mL -1 at an RjFDH construct to eGFP ratio of 1:1; initial purity: 50% (±5%).
Fig. S6: Recovery, protein loss and foam purity after foam fractionation of PGA fusion constructs and eGFP in an extended column with 42 mL initial volume and an air flow rate of 100 mL⋅min -1 with or without addition of wash buffer (WB; 0.1 mol⋅L -1 KPi, pH 7.5); foaming proceeded until no foam overflowed.a) Initial protein concentration: 0.1 mg⋅mL -1 at a PGA construct to eGFP ratio of 1:1; b) Initial protein concentration: 0.2 mg⋅mL -1 at a PGA construct to eGFP ratio of 1:1; initial purity 50% (±5%).Fig. S8: Recovery, protein loss and foam purity after foam fractionation of RjFDH fusion constructs and eGFP in an extended column with 42 mL initial volume and an air flow rate of 100 mL⋅min -1 with or without addition of wash buffer (WB; 0.1 mol⋅L -1 KPi, pH 7.5); foaming proceeded until no foam overflowed.Initial protein concentration: 0.1 mg⋅mL -1 at a RjFDH construct to eGFP ratio of 1:1; initial purity 50% (±5%).

Fig. S9:
Residual activity  of RjFDH fusion constructs in the liquefied foam fractions after fractionation in the extended column with 42 mL initial volume and an air flow rate of 100 mL⋅min -1 with and without the addition of wash buffer (WB; 0.1 mol⋅L -1 KPi, pH 7.5).

Fig. S7 :
Fig. S7: Residual activity  of Bla fusion constructs in the liquefied foam fractions after fractionation in the extended column with 42 mL initial volume and an air flow rate of 100 mL⋅min -1 with and without the addition of wash buffer (WB; 0.1 mol⋅L -1 KPi, pH 7.5).a) Initial protein concentration in the foam fractionation: 0.1 mg⋅mL - 1 b) Initial protein concentration in the foam fractionation: 0.2 mg⋅mL -1 .