Design, identification, antifungal evaluation and molecular modeling of chlorotetaine derivatives as new anti-fungal agents.

It is feasible to rationally modify existing bioactive components for new drug development, achieving molecules with improved biological activities. In this study, rational modification of chlorotetaine was carried out following in silico molecular modelling to enhance interactions between the fungal oligopeptide transmembrane transporter PTR22 and the ligand. The peptide obtained with the lowest docking energy, Lys-chlorotetaine (LC), displayed an improved antifungal effect compared with chlorotetaine. The lowest minimum inhibitory concentration observed against a tested pathogen was 1.47 µg/mL (Candida krusei CBS573), which was satisfactory. To thoroughly explore the detailed interactions between the transporter and LC, molecular dynamics simulation was also performed, which revealed that LC could bind to the transporter via different intermolecular interactions from chlorotetaine, and predicted electrostatic interactions (salt-bridges) would enable the more efficient release of LC. This study provides a simple and reliable method for the rational modification of oligopeptide antibiotics.

Enhanced Performance of Rhizopus oryzae Lipase by Reasonable Immobilization on Magnetic Nanoparticles and Its Application in Synthesis 1,3-Diacyglycerol.

Nano-sized Fe3O4 was synthesized by chemical co-precipitation and subsequently modified with 3-aminopropyltriethoxysilane (APTES) and glutaraldehyde to introduce aldehyde group on its surface. With the help of "interface activation" by adding sucrose esters-11 as surfactant, lipase from Rhizopus oryzae was successfully immobilized onto the carrier with great enhancement of activity. The hydrolysis activity of immobilized enzyme were 9.16 times and 31.6 times of free enzyme when p-nitrophenol butyrate and p-nitrophenol palmitate were used as substrates. The thermo-stability of immobilized enzyme was also enhanced compared to free enzyme. The immobilized enzyme was successfully applied in synthesis of 1,3-diacyglycerols (1,3-DAG). The specific esterification activity of immobilized enzyme was about 1.5 times of the free enzyme. The immobilized enzyme showed good region-selectivity towards 1,3-diacyglycerols and retained nearly 80% of its activity after reused for 60 times, revealing a good industrial application prospect.

Enhancing the thermostability of Rhizopus oryzae lipase by combined mutation of hot-spots and engineering a disulfide bond.

Rhizopus oryzae lipase (ROL) is important because of its extreme sn-1,3-regioselectivity, but it shows poor thermostability, which severely restricts its application. In this work, the thermostability of ROL was greatly enhanced by rational design. First, several sites that may affect the thermostability of ROL were identified by multiple-sequence alignment. The half-lives of mutants V209L and D262G at 55 °C were about 4.38- and 4.2-times those of the wild-type, respectively. Then, a disulfide bond was introduced between positions 190 and 238 based on the prediction of Disulfide by Design 2, which greatly improved the thermostability of the protein. The activity of variant E190C/E238C retained about 58.2% after incubation at 55 °C for 720 min, whereas the half-life of wild type ROL was only about 11.7 min. On the basis of the results obtained by the two methods, we carried out a combined mutation. Quadruple mutant V209L/D262G/E190C/E238C was constructed and the thermostability was improved even further. The half-lives at 55 °C and 65 °C were 102.5- and 20-times those of the wild-type ROL. This improvement in thermostability will give ROL wider industrial applicability, especially in the preparation of structured lipids.