The large-scale preparation of Polyehylene terephthalate (PET) hydrolysing enzymes in low-cost is critical for the biodegradation of PET in industry. In the present study, we demonstrate that the post-translational glycosylation of Pichia pastoris makes it a remarkable host for the heterologous expression of PETase from Ideonella sakaiensis 201-F6 ( Is PETase). Taking advantage of the abundant N- and O-linked glycosylation sites in Is PETase and the efficient post-translational modification in endoplasmic reticulum, Is PETase is heavily glycosylated during secretory expression with P. pastoris , which improves the specific activity and thermostability of the enzyme dramatically. Moreover, the specific activity of Is PETase increased further after the bulky N-linked polysaccharide chains were eliminated by Endo-β-N-acetylglucosaminidase H (Endo H). Importantly, the partially deglycosylated Is PETase still maintained high thermostability because of the remaining mono- and oligo-saccharide residues on the protein molecules. Consequently, the partially deglycosylated Is PETase was able to be applied at 50 °C and depolymerized raw, untreated PET flakes completely in 2 to 3 days. This platform was also applied for the preparation of a famous variant of Is PETase, Fast-PETase, and the same result was achieved. Partially deglycosylated Fast-PETase demonstrates elevated efficiency in degrading postconsumer-PET trays under 55 °C than 50 °C, the reported optimal temperature of Fast-PETase. The present study provides a strategy to modulate thermostable Is PETase through glycosylation engineering and paves the way for promoting PET biodegradation from laboratories to factories.

CP4-EPSPS (Agrobacterium sp. strain CP4 5-enolpyruvylshikimate-3-phosphate synthase) protein showed remarkable thermostability and was highly resistant to proteases, such as trypsin. In order to eliminate the pollution of CP4-EPSPS from the accumulated straws to the surrounding environment during the winter, the present study investigated the extracellular proteases of 21 psychrophilic strains isolated from the south polar region. The results indicated that Stenotrophomonas maltophilia 780 was able to degrade CP4-EPSPS at 18 °C efficiently. Further study indicated that it was able to grow in the extract of Roundup Ready soybean at 18 °C, with CP4-EPSPS degraded to an undetectable level within 72 h. The extracellular proteases of Stenotrophomonas maltophilia 780 are thermo-sensitive, with an optimal temperature of 65 °C. The genomic sequencing result indicated that this strain had more than a hundred putative protease and peptidase coding genes, which may explain its high capability in decomposing CP4-EPSPS.

The binding affinities between E-group colicin (CE) and cognate immunity proteins (Im) are among the strongest interactions in nature. Aiming at mining protein pairs with ultrahigh affinity for artificial multienzyme complexes, DNase domains of CE2, 8, and 9 were engineered to generate CL2, 8, and 9 variants without DNA binding and catalytic activity but retaining Im binding activity, respectively. Further study discovered that all these variants, in addition to a CE7 variant (CL7) from the previous report, and their cognate Im proteins are highly thermostable. Taking advantage of the specific and ultrahigh affinity of these hyperthermostable affinity pairs, a hyperthermostable artificial scaffold was established and applied for cellulose degradation. Four hyperthermostable cellulolytic enzymes were integrated into the system through specific pairing of CL and cognate Im proteins. This complex with four enzymes assembled sequentially exhibited an obvious synergistic effect in hydrolyzing cellulosic substrates at elevated temperature. In comparison with free enzymes, the catalytic activity of the complex enhanced more than half-fold at 70 degrees C with phosphoric acidswollen cellulose as the substrate, while it increased almost onefold at 75 degrees C with Avicel as the substrate. The present study provided four affinity pairs with ultrahigh affinity, hyperthermostability, high specificity, a small and unified structure, and high assembly efficiency. More importantly, it provided an unconventional idea of mining hyperthermostable affinity pairs from mesophilic microorganisms instead of the less-abundant thermophiles, which are the main source pool of the thermostable affinity pairs for the time being.