Enzymatic additives, particularly alkaline proteases, play a crucial role in enhancing detergent effectiveness against protein-based stains. Despite advancements in enzyme stabilization techniques, there is a need for innovative strategies to further improve protease stability in laundry detergents. However, research exploring the utilization of substrate imprinting technology to achieve this objective remains limited. Therefore, this study aims to enhance the stability of alkaline proteases in laundry detergents by employing casein as an imprinting substrate and utilizing transglutaminase-mediated (TGase) crosslinking to modify proteases 102 and 306. The optimal temperature, pH, and thermal stability of the modified alkaline proteases 102 and 306 showed no significant changes. However, these two modified alkaline proteases exhibited varying degrees of improvement in stability among the 14 detergent additives tested. Under 40 °C incubation for 24 h, the relative enzyme activity of modified alkaline protease 102 increased approximately 1.4–15-fold in AEO-9, BS-12, CMI, APG, FMEE, and SOE, while the relative enzyme activity of modified alkaline protease 306 increased approximately 1.2–3.7-fold across different additives (FMEE, AEO-9, BS-12, SOE, FAA, and AEC-9Na). These modified proteases demonstrated improved stability and wider applicability across commercial detergent formulations available. Integrated into standard laundry detergent at a 1:7 ratio before and after modification, they effectively removed protein stains from the cotton fabric after 24 h of 40 °C incubation. These findings provide insights into more effective stain-removal techniques.

L-leucine, an essential amino acid which cannot be synthesized in mammals, has extensive applications in various fields. However, the large-scale production of L-leucine still faces various challenges in terms of strain and process optimization. In this study, E. coli A211 was used as the initial strain, and a double enhancement strategy of CRISPR-associated transposase genome integration and a plasmid was employed to enhance the L-leucine metabolic pathway. We constructed four engineered strains—E. coli A101, E. coli B201, E. coli CD301, and E. coli bcd401. The transcriptional levels of key genes (leuA, leuCD, leuB, and bcd) in L-leucine biosynthesis were significantly upregulated to boost L-leucine production. Fermentation screening revealed that E. coli CD301 exhibited the highest L-leucine titer (0.57 ± 0.01 g/L), presenting a 97% increase compared with the parental strain. The fermentation process of E. coli CD301 was further optimized using single-factor optimization followed by response surface methodology of variables such as temperature, C/N ratio, and inoculum size, leading to an enhanced L-leucine titer of 0.89 ± 0.03 g/L, a 56.1% improvement over the pre-optimization level. This study demonstrated the effectiveness of CRISPR-associated transposase genome integration and plasmid double enhancement strategy, providing new insights into metabolic engineering approaches for improving L-leucine production via fermentation with E. coli.

To explore the key sites affecting the intracellular assembly of key components of cellulosomes and obtain DocA mutants independent of Ca²⁺, Swiss-model, GROMACS, PyMOL, and other molecular dynamics simulation software were used for modeling and static and dynamic combination analysis. Site-specific mutation technology was used to mutate DocA, and Biacore was used to test the dependence of Ca²⁺ on the binding ability of protein DocA mutants and protein Coh, and to analyze the interaction and binding effect of mutant proteins in vitro. Forward intracellular mutant screening was performed based on semi-rational design and high throughput screening techniques. The orientation of mutations suitable for intracellular assembly was determined, and three directional mutant proteins, DocA-S1, DocA-S2, and DocA-S3, were obtained. Ca²⁺ independent DocA mutants were obtained gradually and their potential interaction mechanisms were analyzed. In the present study, intracellular self-assembly of key components of cellulosomes independent of Ca²⁺ was achieved, and DocA-S3 was applied to the assembly of key enzymes of L-lysine biosynthesis, in which DapA and DapB intracellular assembly increased L-lysine accumulation by 29.8% when compared with the control strains, providing a new strategy for improving the intracellular self-assembly of cellulosomes and amino acid fermentation efficiency.