Search Results (4)

In this study, a novel protein glutaminase derived from Chryseobacterium lactis CGMCC 33780 (CLPG) was successfully purified via a one-step cell surface display approach, yielding its mature form. Subsequently, the enzymatic properties of CLPG were characterized. It exhibited optimal activity at a pH of 5 and a reaction temperature of 50 °C, and retained over 70% of its activity after a 12 h incubation at 50 °C. The study further investigated the impact of CLPG-mediated deamidation on the structural and functional attributes of krill protein isolate (KPI). A comprehensive analysis was conducted on the deamidation extent, conformational alterations, and microstructural morphology of KPI, employing techniques such as FTIR, CD, DSC, and SEM. After deamidation treatment with CLPG, the foaming and emulsifying properties of KPI were moderately shifted. When the CLPG dosage was 1.0 U/g with a corresponding deamidation degree of 15.18%, the emulsifying property of KPI reached the maximum value of 23%. These property enhancements were possibly primarily attributed to the increased electrostatic repulsion and hydrophobicity induced during the deamidation process. This work not only pioneers a novel method for the expression and purification of protein glutaminase but also applies it to the modification of krill protein, offering fresh insights for the development and application of protein glutaminases. Full article

Myofibrillar protein (MP) aggregation in solutions with NaCl concentrations below 0.3 M results in poor solubility. Ultrasound-assisted glutaminase treatment (UGT) was applied to improve MP solubility in a low-salt solution (containing 0.1 M NaCl). The solubility increased with ultrasonic power and time, peaking at 44.34% (480 W, 15 min) and reaching 61% after UGT. Subsequently, the effect of post-sonication heat treatment (60 °C, 30 min) on the physicochemical and structural characteristics of ultrasound-enzyme treated MP (UEMP), prepared under specific ultrasonic conditions (480 W, 20 min), was systematically investigated. The findings revealed that UEMP exhibited higher hydrophobicity, sulfhydryl content, and turbidity, but reduced particle size, ζ-potential, and fluorescence, suggesting disulfide disruption and exposure of hydrophobic residues. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed weakened high-molecular weight bands and intensified low-molecular weight bands. Fourier-transform infrared spectroscopy confirmed these structural rearrangements, with a blue-shifted amide A band and decreased amide I intensity. Heating further increased the hydrophobicity and fluorescence without altering the size, ζ-potential, or molecular weight. The red shift in the amide A band suggests reinforced local ordering. Rheology analysis showed non-Newtonian shear-thinning behavior, which was unchanged by UGT or heating. Collectively, UGT with moderate heating enhances MP solubility and thermal stability by disrupting stabilizing bonds and modulating the structure. Full article

Glutamine is one of the most abundant amino acids in the cell. In mitochondria, glutaminases 1 and 2 (GLS1/2) hydrolyze glutamine to glutamate, which serves as the precursor of multiple metabolites. Here, we show that ammonium generated during GLS1/2-mediated glutaminolysis regulates lysosomal pH and in turn lysosomal degradation. In primary human skin fibroblasts BJ cells and mouse embryonic fibroblasts, deprivation of total amino acids for 1 h increased lysosomal degradation capacity as shown by the increased turnover of lipidated microtubule-associated proteins 1A/1B light chain 3B (LC3-II), several autophagic receptors, and endocytosed DQ-BSA. Removal of glutamine but not any other amino acids from the culture medium enhanced lysosomal degradation similarly as total amino acid starvation. The presence of glutamine in regular culture media increased lysosomal pH by >0.5 pH unit and the removal of glutamine caused lysosomal acidification. GLS1/2 knockdown, GLS1 antagonist, or ammonium scavengers reduced lysosomal pH in the presence of glutamine. The addition of glutamine or NH₄Cl prevented the increase in lysosomal degradation and curtailed the extension of mTORC1 function during the early time period of amino acid starvation. Our findings suggest that glutamine tunes lysosomal pH by producing ammonium, which regulates lysosomal degradation to meet the demands of cellular activities. During the early stage of amino acid starvation, the glutamine-dependent mechanism allows more efficient use of internal reserves and endocytosed proteins to extend mTORC1 activation such that the normal anabolism is not easily interrupted by a brief disruption of the amino acid supply. Full article

The low productivity in long fermentation duration and high-salt working conditions limit the application of L-glutaminase in soy sauce brewing. In this study, a novel L-glutaminase (LreuglsA) with eminent salt tolerance was mined and achieved more than 70% activity with 30% NaCl. To improve the robustness of the enzyme at different fermentation strategies, mutation LreuglsA^H105K was built by a computer-aided design, and the recombinant protein expression level, an essential parameter in industrial applications, was increased 5.61-fold with the synthetic biology strategy by improving the mRNA stability. Finally, the LreuglsA^H105K functional expression box was contributed to Bacillus subtilis 168 by auxotrophic complementation, and the production in a 5-L bioreactor was improved to 2516.78 ± 20.83 U mL⁻¹, the highest production ever reported. When the immobilized cells were applied to high-salt dilute-state soy sauce brewing, the L-glutamate level was increased by 45.9%. This work provides insight into the salt-tolerant enzyme for improving the efficiency of industrial applications. Full article