Development of a Hyperthermostable Artificial Scaffold Based on Ultrahigh-Affinity Protein Pairs and Its Application in Cellulose Degradation

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 °C with phosphoric acid-swollen cellulose as the substrate, while it increased almost onefold at 75 °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.