Part:BBa_K4275008
FAST-PETase-t
FAST-PETase-t is a dockerin-fused variant of free FAST-PETase (BBa_K4275007). The enzyme carries the same amino acid mutations (N233K, R224Q, S121E, D186H, and R280A) as the former and shares all the common sequences in the CDS, except the C' terminal fusion of a type-I dockerin domain in this enzyme. The catalytic domain of FAST-PETase-t and the dockerin domain are interspaced with a medium-lengthed flexible GS linker (10 aa long) to avoid steric inhibitions. The type-I dockerin domain forms high-affinity non-covalent interaction with type-I cohesins on the cipA scaffoldin.
Figure 1 The 3D structure of the protein predicted by Alphafold2.
Usage and Biology
The improvised FAST-PETase - Dockerin I fusion protein could be tightly-anchored onto the CipA scaffoldin via the high-affinity Doc I: Coh I noncovalent interaction. The CipA primary scaffoldin is then tightly-anchored onto the secondary scaffoldin - OlpB, which is either anchored onto the cell surface of K.marxianus via ScGPI, or binds to E.coli's Cell-surface Nanobody3(Nb3)(BBa_K4275026). It is believed that the immobilization of the two enzymes (FAST-PETase-t and MHETase-t) could increase their proximity and further enhance their synergy, whilst the affinity of carbohydrate-binding module 3 (CBM3) on the CipA scaffoldin towards PET fiber could further increase the catalytic efficiency of this degradation complex.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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