Structural and functional studies on a thermostable polyethylene terephthalate degrading hydrolase from Thermobifida fusca.

Bacterial cutinases are promising catalysts for the modification and degradation of the widely used plastic polyethylene terephthalate (PET). The improvement of the enzyme for industrial purposes is limited due to the lack of structural information for cutinases of bacterial origin. We have crystallized and structurally characterized a cutinase from Thermobifida fusca KW3 (TfCut2) in free as well as in inhibitor-bound form. Together with our analysis of the thermal stability and modelling studies, we suggest possible reasons for the outstanding thermostability in comparison to the less thermostable homolog from Thermobifida alba AHK119 and propose a model for the binding of the enzyme towards its polymeric substrate. The TfCut2 structure is the basis for the rational design of catalytically more efficient enzyme variants for the hydrolysis of PET and other synthetic polyesters.

Stepwise error-prone PCR and DNA shuffling changed the pH activity range and product specificity of the cyclodextrin glucanotransferase from an alkaliphilic Bacillus sp.

Cyclodextrin glucanotransferase (EC 2.4.1.19) from the alkaliphilic Bacillus sp. G-825-6 converts starch mainly to γ-cyclodextrin (CD8). A combination of error-prone PCR and DNA shuffling was used to obtain variants of this enzyme with higher product specificity for CD8 and a broad pH activity range. The variant S54 with seven amino acid substitutions showed a 1.2-fold increase in CD8-synthesizing activity and the product ratio of CD7:CD8 was shifted to 1:7 compared to 1:3 of the wild-type enzyme. Nine amino acid substitutions of the cyclodextrin glucanotransferase were performed to generate the variant S35 active in a pH range 4.0-10.0. Compared to the wild-type enzyme which is inactive below pH 6.0, S35 retained 70% of its CD8-synthesizing activity at pH 4.0.

High level expression of a hydrophobic poly(ethylene terephthalate)-hydrolyzing carboxylesterase from Thermobifida fusca KW3 in Escherichia coli BL21(DE3).

The gram-positive thermophilic actinomycete Thermobifida fusca KW3 secretes a highly hydrophobic carboxylesterase (TfCa) that is able to hydrolyze poly(ethylene terephthalate). TfCa was produced in the Escherichia coli strain BL21(DE3) as a fusion protein consisting of a pelB leader sequence to ensure periplasmic localization of the protein and a His(6) tag for use in its purification. To enhance the recombinant enzyme yield, the tfca gene from T. fusca KW3 was successfully optimized for codon usage in E. coli. In addition, the gene expression induction conditions were optimized and the temperature for cell cultivation was lowered to reduce inclusion body formation. The optimized codons and expression conditions yielded 4500-fold higher TfCa activity than the wild-type strain. Using a pH-controlled bioreactor for cultivation, a TfCa protein concentration of 41.6mg/L was achieved.