Efficient production of a highly active lysozyme from European flat oyster Ostrea edulis.

Lysozyme, an antimicrobial agent, is extensively employed in the food and healthcare sectors to facilitate the breakdown of peptidoglycan. However, the methods to improve its catalytic activity and secretory expression still need to be studied. In the present study, twelve lysozymes from different origins were heterologously expressed using the Komagataella phaffii expression system. Among them, the lysozyme from the European flat oyster Ostrea edulis (oeLYZ) showed the highest activity. Via a semi-rational approach to reduce the structural free energy, the double mutant Y15A/S39R (oeLYZdm) with the catalytic activity 1.8-fold greater than that of the wild type was generated. Subsequently, different N-terminal fusion tags were employed to enhance oeLYZdm expression. The fusion with peptide tag 6×Glu resulted in a remarkable increase in the recombinant oeLYZdm expression, from 2.81 × 103 U mL-1 to 2.11 × 104 U mL-1 in shake flask culture, and eventually reaching 2.05 × 105 U mL-1 in a 3-L fermenter. The work produced the greatest amount of heterologous oeLYZ expression in microbial systems that are known to exist. Reducing the structural free energy and employing the N-terminal fusion tags are effective strategies to improve the catalytic activity and secretory expression of lysozyme.

Increase in Lactulose Content in a Hot-Alkaline-Based System through Fermentation with a Selected Lactic Acid Bacteria Strain Followed by the ß-Galactosidase Catalysis Process.

In this study, lactic acid bacteria (LAB) fermentation and ß-galactosidase catalysis methods were combined to increase the lactulose concentration and reduce the galactose and lactose content in a hot-alkaline-based system. The optimal conditions for chemical isomerization were 70 °C for 50 min for lactulose production, in which the concentration of lactulose was 31.3 ± 1.2%. Then, the selection and identification of LAB, which can utilize lactose and cannot affect lactulose content, were determined from 451 strains in the laboratory. It was found that Lactobacillus salivarius TM-2-8 had weak lactulose utilization and more robust lactose utilization. Lactobacillus rhamnosus grx.21 was weak in terms of lactulose utilization and strong in terms of galactose utilization. These two strains fermented the chemical isomerization system of lactulose to reduce the content of lactose and galactose. The results showed that the lactose concentration was 48.96 ± 2.92 g/L and the lactulose concentration was 59.73 ± 1. 8 g/L for fermentation lasting 18 h. The ß-galactosidase was used to increase the content of lactulose in the fermented system at this time. The highest concentration of 74.89 ± 1.68 g/L lactulose was obtained at an enzymatic concentration of 3 U/mL and catalyzed at 50 °C for 3 h by ß-galactosidase.

The effect of ion environment changes on retention protein behavior during whey ultrafiltration process.

The factors affecting membrane fouling are very complex. In this study, the membrane fouling process was revealed from the perspective of ion environment changes, which affected the whey protein structure during ultrafiltration. It was found that the concentrations of Ca2+ and Na+ were overall increased and the concentrations of K+, Mg2+ and Zn2+ were decreased at an ultrafiltration time of 11 min, which made more hydrophilic groups buried inside and increased the content of α-helix, leading to more protein aggregation. The relatively higher K+ ratio in retention could lead to an antiparallel ß-sheet configuration, aspartic acid, glutamic acid and tryptophan increased, which resulted in more protein aggregation and deposition on the membrane surface at 17 min. When the ion concentration and ratio restored the balance and were close to the initial state in retention, the protein surface tension decreased, and the hydrophilic ability increased at 21-24 min.