Enhancing the Thermostability and Catalytic Activity of the Lipase from Rhizopus oryzae via Introducing N-Glycosylation.

Lipase from Rhizopus oryzae (ROL) exhibits remarkable sn-1,3 stereoselectivity and catalytic activity, but its poor thermostability limits its applications in the production of 1,3-dioleoyl-2-palmitoyl glycerol (OPO, a high-quality substitute for human milk fat). In this work, a semirational method was proposed to engineer the thermostability and catalytic activity of 4M (ROL mutant in our previous study). First, a computer-aided design is performed using 4M as a template, and N-glycosylation mutants are then recombinantly expressed and screened in Pichia pastoris, the optimal mutant N227 exhibited a half-life of 298.8 h at 45 °C, which is 7.23-folds longer than that of 4M. Its catalytic activity also reached 1043.80 ± 61.98 U/mg, representing a 29.2% increase compared to 4M (808.02 ± 47.02 U/mg). Molecular dynamics simulations of N227 suggested that the introduction of glycan enhanced the protein rigidity, while the strong hydrogen bonds formed between the glycan and the protein stabilized the lipase structure, thereby improving its thermostability. The acidolysis reaction between oleic acid (OA) and glycerol tripalmitate (PPP) was successfully carried out using immobilized N227, achieving a molar conversion rate of 90.2% for PPP. This engineering strategy guides the modification of lipases, while the glycomutants obtained in this study have potential applications in the biosynthesis of OPO.

Purification and characterization of an aspartic protease from the Rhizopus oryzae protease extract, Peptidase R

Background Aspartic proteases are a subfamily of endopeptidases that are useful in a variety of applications, especially in the food processing industry. Here we describe a novel aspartic protease that was purified from Peptidase R, a commercial protease preparation derived from Rhizopus oryzae. Results An aspartic protease sourced from Peptidase R was purified to homogeneity by anion exchange chromatography followed by polishing with a hydrophobic interaction chromatography column, resulting in a 3.4-fold increase in specific activity (57.5 × 10³ U/mg) and 58.8% recovery. The estimated molecular weight of the purified enzyme was 39 kDa. The N-terminal sequence of the purified protein exhibited 63-75% identity to rhizopuspepsins from various Rhizopus species. The enzyme exhibited maximal activity at 75°C in glycine-HCl buffer, pH 3.4 with casein as the substrate. The protease was stable at 35°C for 60 min and had an observed half-life of approximately 30 min at 45°C. Enzyme activity was not significantly inhibited by chelation with ethylenediamine tetraacetic acid (EDTA), and the addition of metal ions to EDTA-treated protease did not significantly change enzyme activity, indicating that proteolysis is not metal ion-dependent. The purified enzyme was completely inactivated by the aspartic protease inhibitor Pepstatin A. Conclusion Based on the observed enzyme activity, inhibition profile with Pepstatin A, and sequence similarity to other rhizopuspepsins, we have classified this enzyme as an aspartic protease.