Constitutive Expression in Komagataella phaffii of Mature Rhizopus oryzae Lipase Jointly with Its Truncated Prosequence Improves Production and the Biocatalyst Operational Stability

Rhizopus oryzae lipase (ROL) containing 28 C-terminal amino acids of the prosequence fused to the N-terminal mature sequence in ROL (proROL) was successfully expressed in the methylotrophic yeast Komagataella phaffii (Pichia pastoris) under the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter (PGAP). Although the sequence encoding the mature lipase (rROL) was also transformed, no clones were obtained after three transformation cycles, which highlights the importance of the truncated prosequence to obtain viable transformed clones. Batch cultures of the K. phaffii strain constitutively expressing proROL scarcely influenced growth rate and exhibited a final activity and volumetric productivity more than six times higher than those obtained with proROL from K. phaffii under the methanol-inducible alcohol oxidase 1 promoter (PAOX1). The previous differences were less marked in fed-batch cultures. N-terminal analysis confirmed the presence of the 28 amino acids in proROL. In addition, immobilized proROL exhibited increased tolerance of organic solvents and an operational stability 0.25 and 3 times higher than that of immobilized rROL in biodiesel and ethyl butyrate production, respectively. Therefore, the truncated prosequence enables constitutive proROL production, boosts bioprocess performance and provides a more stable biocatalyst in two reactions in which lipases are mostly used at industrial level, esterification (ethyl butyrate) and transesterification (biodiesel).

Chemosensory Device Assisted-Estimation of the Quality of Edible Oils with Repetitive Frying

This study investigated chemosensory degradations of soybean and canola oils with repeated frying in order to estimate the quality of the oils. Methods: Chemical parameters including oxygen induction time, acid value, p-anisidine value, malondialdehyde, and total polar compounds were measured. Electronic nose and electronic tongue analyses were performed to assess sensory properties. Multivariate analyses were employed to investigate relationships among tastes and volatile compounds using principal component analysis (PCA) and Pearson’s correlation analysis. Results: All chemical parameters increased with repeated frying in both oils. Electronic nose analysis found ethyl butyrate, 2-heptenal, and 2,4-pentanedione as major volatiles for soybean oil and ethyl butyrate and linalool for canola oil. As the numbers of frying increased, all volatiles showed an increased concentration in various extents. In multivariate analyses, ethyl butyrate revealed strong positive correlations with sourness, umami, and sweetness, and umami showed strong positive correlations with sourness and saltiness (p < 0.05). PCA confirmed that in PC1 with 49% variance, sourness, saltiness, and umami were at similar rates while acetyl pyrazine, 2,4-pentadieone, and 1-octanol were found at similar rates. Canola oil was chemically more stable and less susceptible to deterioration in all chemical parameters compared to soybean oil, resulting in a relatively better quality oil when repeatedly fried. Conclusion: The results suggested that minimum repeated frying (5 times) degrades chemosensory characteristics of both oils, thereby compromising their quality. The findings of this study will be utilized as a foundation for quality control of fried foods in food industry, fried food development, and fast-food industry.

Biochemical, Ameliorative and Cytotoxic Effects of Newly Synthesized Curcumin Microemulsions: Evidence from In Vitro and In Vivo Studies

Curcumin is known to exhibit antioxidant and tissue-healing properties and has recently attracted the attention of the biomedical community for potential use in advanced therapies. This work reports the formulation and characterization of oil-in-water F127 microemulsions to enhance the bioavailability of curcumin Microemulsions showed a high encapsulation efficiency and prolonged release. To investigate the interactions of curcumin with one unit of the polymeric chain of surfactant F127, ethyl butyrate, and sodium octanoate, as well as the interaction between ethyl butyrate and one unit of the F127 polymer chain, the Density Functional Theory (DFT) calculations at the M06-2X level of theory, were performed in water solution. The MTT assay was used to assess the cytotoxicity of free and encapsulated curcumin on non-malignant and malignant cell lines. Combination effects were calculated according to Chou-Talalay’s principles. Results of in vitro studies indicated that MCF7 and HepG2 cells were more sensitive to curcumin microemulsions. Moreover, a synergistic relationship was observed between curcumin microemulsions and cisplatin in all affected fractions of MCF7 and HepG2 cells (CI < 0.9). For in vivo investigation, thioacetamide-intoxicated rats received thioacetamide (100 mg/kg Sc) followed by curcumin microemulsions (30 mg/kg Ip). Thioacetamide-intoxicated rats showed elevated serum liver enzymes, blood urea nitrogen (BUN), and creatinine levels, and a significant reduction in liver superoxide dismutase (SOD) and catalase (CAT) activities (p < 0.05). Curcumin microemulsions reduced liver enzymes and serum creatinine and increased the activity of antioxidant enzymes in thioacetamide-treated rats in comparison to the untreated thioacetamide-intoxicated group. Histopathological investigations confirmed the biochemical findings. Overall, the current results showed the desirable hepatoprotective, nephroprotective, and anti-cancer effects of curcumin microemulsions.

Analysis of 2D Nanomaterial BC3 for COVID-19 Biomarker Ethyl Butyrate Sensor

Ethyl Butyrate (EB) was identified in recent research as a prominent biomarker of COVID-19, as concentrations of EB were higher in exhaled breath of COVID-19 patients. Electronic sensitivity of pristine,...