Chitosanase plays a pivotal role in the production of chitooligosaccharide. Nevertheless, there is untapped potential for enhancing both its catalytic efficiency and thermostability, which could significantly bolster its therapeutic and biotechnological applications. In this study, two computer-aided protein design methods, namely Fireprot and PROSS, were utilized to pinpoint 6 single Bacillus subtilis chitosanase BsCsn46A mutants (S126A, D191A, K70A, L159I, K104P, and A129L) as well as 4 multiple mutants (K70A/S126A, K70A/S126A/K104P, K70A/S126A/L159I, and K70A/S126A/K104P/L159). The wild-type (WT) and all 10 BsCsn46A mutants displayed robust adaptability across a broad pH range, exhibiting peak activity within the pH spectrum of 5.5 to 9.5. The results demonstrated that, compared to the WT, 9 out of 10 mutants exhibited significantly heightened chitosanase activity, with the sole exception being the D191A mutant, which displayed activity levels nearly identical to the WT. Notably, the A129L displayed an impressive 20% increase in the enzyme activity at elevated temperatures, specifically in the range of 55–80°C. Assessing protein stability, results indicated that all samples maintained stability when incubated at 30°C for 1 h. However, when subjected to a higher temperature of 40°C for 1 h, only the A129L mutant retained stability, which persisted even after an extended incubation period of 3 h at 40°C. Furthermore, a thermal stability analysis revealed noteworthy differences between the WT and the mutants. The WT chitosanase activity diminished by 50% after brief 30-min incubation at 50°C, whereas the K70A/S126A, K70A/S126A/K104P, and A129L mutants maintained 50% of their activity for approximately 2 h under the same conditions. In summary, the study provides valuable insights into the thermostability and catalytic activity of chitosanase, highlighting promising candidates for industrial chitosanase applications.

In the degradation of poly(ethylene terephthalate) (PET), mono(2-hydroxyethyl) terephthalate (MHET) hydrolase (IsMHETase) plays a crucial role in the complete degradation of PET. Although IsMHETase was discovered concurrently with IsPETase, its structural and functional properties are not well understood. To enhance the thermal stability of IsMHETase, we selected six homologous proteins that share the closest evolutionary relationship for structure-based protein rational design, all exhibiting over 60% amino acid sequence identity with IsMHETase. Using FireProt, PROSS, and Consensus analysis, we identified the key mutation sites of IsMHETase. Sequence and structural analyses indicate that, among these seven proteins, all amino acids within 5 Å of the substrate-binding site are identical, with the exception of Ser131 and Phe415. Additionally, the amino acids within a 4 Å range of the catalytic triad are nearly identical. Through integrated free energy calculations, phylogenetic tree analysis, sequence analysis, and conservation analysis, we have identified a variant with four key mutations (termed IsMHETase-M1: N156G, T159V, E110A, A493P) that exhibits improved thermal stability. The selection of mutations during the protein modification process often requires considerable time. Our predictions have established a foundation for the rational design of IsMHETase and its homologous proteins.

The continuous increase in demand for polyethylene terephthalate (PET) has drawn global attention to the significant environmental pollution caused by the degradation of PET plastics. Exploring new PET-degrading enzymes is essential for enhancing the degradation efficiency of PET, and esterases and lipases with plastic degradation capabilities have become a focal point of research. In this study, we utilized the ultra-efficient mutant FASTase of the PET-degrading enzyme IsPETase, derived from Ideonella sakaiensis, as a positive control, based on the similarity in enzyme activity and substrate. We investigated the PET model substrate degradation activities of the esterase Gs and lipase GI, both derived from Bacillus spp., as well as the lipase CAI derived from Pseudomonas spp. The results indicated that Gs exhibited excellent bis(2-hydroxyethyl) terephthalate (BHET) degradation activity; however, Gs demonstrated a lack of thermal stability when hydrolyzing BHET. Molecular docking analyses were conducted to identify the key amino acids involved in the degradation of BHET by Gs from a structural perspective. At the same time, GI and CAI showed no BHET degradation activity. The combination of Gs and the mono-2-hydroxyethyl terephthalate (MHET) hydrolase, MHETase, can completely hydrolyze BHET, and Gs also exhibited degradation activity against the PET model substrate bis(benzyloxyethyl) terephthalate and PET nanoparticles. Given the structural similarity between PET hydrolase LCC-ICCG and Gs, this study provides new enzyme resources for advancing the efficient biological enzymatic degradation of PET plastics.