RESUMEN
Using enzymes to hydrolyze and recycle poly(ethylene terephthalate) (PET) is an attractive eco-friendly approach to manage the ever-increasing PET wastes, while one major challenge to realize the commercial application of enzyme-based PET degradation is to establish large-scale production methods to produce PET hydrolytic enzyme. To achieve this goal, we exploited the industrial strain Pichia pastoris to express a PET hydrolytic enzyme from Caldimonas taiwanensis termed CtPL-DM. In contrast to the protein expressed in Escherichia coli, CtPL-DM expressed in P. pastoris is inactive in PET degradation. Structural analysis indicates that a putative N-glycosylation site N181 could restrain the conformational change of a substrate-binding Trp and hamper the enzyme action. We thus constructed N181A to remove the N-glycosylation and found that the PET hydrolytic activity of this variant was restored. The performance of N181A was further enhanced via molecular engineering. These results are of valuable in terms of PET hydrolytic enzyme production in industrial strains in the future.
RESUMEN
BACKGROUND: Circulating beta(2)-glycoprotein-I-oxidized low-density lipoprotein (beta(2)-GPI-ox-LDL) complexes have been found in patients with systemic lupus erythematosus (SLE) and other autoimmune diseases as a contributor to the development of autoimmune-mediated atherosclerosis. In vitro study showed that beta(2)-GPI also bound with high affinity to atherogenic lipoprotein (a) [Lp(a)] which shares structural similarity to LDL. We examined the existence and clinical significance of serum complexes of beta(2)-GPI with Lp(a) in SLE patients. METHODS: A "sandwich" ELISA was developed for measuring serum concentrations of beta(2)-GPI-Lp(a) complexes, using rabbit anti-human beta(2)-GPI antibody as capturing antibody, and quantitating with antibody against apo(a). Forty-seven SLE patients and 42 healthy controls were studied. RESULTS: Both Lp(a) (400+/-213 mg/l vs. 181+/-70 mg/l) and ox-Lp(a) (27.07+/-22.30 mg/l vs. 8.20+/-4.55 mg/l) concentrations were higher in SLE patients than in controls (P<0.0001). beta(2)-GPI-Lp(a) complexes were detectable in both controls and SLE. The complexes levels in SLE were higher than in controls (0.96+/-0.41 U/ml vs. 0.59+/-0.20 U/ml, P<0.0001) and was positively correlated with ox-Lp(a) (P<0.001). CONCLUSIONS: We report the existence of beta(2)-GPI-Lp(a) complexes in both controls and SLE patients. The complexes levels increase in SLE.
