Heterologous expression and characterization of an M4 family extracellular metalloprotease for detergent application.

Proteolytic enzymes stand out as the most widely employed category utilized in manufacturing industry. A new protease was separated from Planococcus sp.11815 strain and named as nprS-15615 in this research. The gene of this protease has not been reported, and its enzymatic properties have been studied for the first time. To enhance enzyme production, the Planococcus sp. protease gene was expressed in Bacillus licheniformis 2709. The expression level of nprS-15615 was observed under the control of regulatory elements PaprE. nprS-15615 protease activity reached 1186.24±32.87 U/mL after 48 hours of cultivation in shake flasks which was nearly four times the output of the original bacteria (291.38±25.73U/mL). The optimum temperature and pH of the recombinant protease were 30 ℃ and 8.0, respectively.The enzyme exhibited the highest capacity for hydrolyzing casein and demonstrated resilience towards a NaCl concentration of 10.0% (wt/v). Furthermore, in the presence of 0.5% surfactants, the recombinant protease activity can maintain above 75%, and with the existence of 0.5% liquid detergents, there was basically no loss of enzyme activity which indicated that nprS-15615 had good compatibility with surfactants and liquid detergents. In addition, npS-15615 performed well in the washing experiment, and the washing effect at 20 ℃ can be significantly improved by adding crude enzyme solution in the washing process.

Structure-Guided Engineering of a Protease to Improve Its Activity under Cold Conditions.

Bacillus proteases commonly exhibit remarkably reduced activity under cold conditions. Herein, we employed a tailored combination of a loop engineering strategy and iterative saturation mutagenesis method to engineer two loops for substrate binding at the entrance of the substrate tunnel of a protease (bcPRO) from Bacillus clausii to improve its activity under cold conditions. The variant MT6 (G95P/A96D/S99W/S101T/P127S/S126T) exhibited an 18.3-fold greater catalytic efficiency than the wild-type (WT) variant at 10 °C. Molecular dynamics simulations and dynamic tunnel analysis indicated that the introduced mutations extended the substrate-binding pocket volume and facilitated extra interactions with the substrate, promoting catalysis through binding in a more favorable conformation. This study provides insights and strategies relevant to improving the activities of proteases and supplies a novel protease with enhanced activity under cold conditions for the food industry to maintain the initial flavor and color of food and reduce energy consumption.

Engineering a highly efficient expression system to produce BcaPRO protease in Bacillus subtilis by an optimized promoter and signal peptide.

Bacillus subtilis has been extensively utilized as a host to express heterologous protein used in various industrial processes. Hence, the secretory overproduction of recombinant proteins is highly dependent on the strong promoters and signal peptides with high efficiency in B. subtilis. To enlarge the limited information of signal peptides and promoters suitable for specific proteins expression at a high-level, a series of plasmids carrying various signal peptides and single, dual, or triple promoters were engineered using the coding sequence of reporter protein, alkaline serine protease (BcaPRO) from B. clausii. Finally, the signal peptide DacB was selected from a library composed of 73 Sec-type signal peptides, and the dual promoter PBsamy-PBaamy demonstrated the best performance. Furthermore, BcaPRO activity was as high as 27,860 U/mL in the supernatant of the recombinant B. subtilis WB600 containing the plasmid with the dual promoter PBsamy-PBaamy and signal peptide DacB with batch fermentation in a 5-L fermenter after 56 h. It indicated that this new engineered expression system would be potential for high-level BcaPRO expression in B. subtilis.

Factors Influencing Recombinant Protein Secretion Efficiency in Gram-Positive Bacteria: Signal Peptide and Beyond.

Signal peptides are short peptides directing newly synthesized proteins toward the secretory pathway. These N-terminal signal sequences are ubiquitous to all prokaryotes and eukaryotes. Signal peptides play a significant role in recombinant protein production. Previous studies have demonstrated that the secretion amount of a given target protein varies significantly depending on the signal peptide that is fused to the protein. Signal peptide selection and signal peptide modification are the two main methods for the optimization of a recombinant protein secretion. However, the highly efficient signal peptide for a target protein with a specific bacterial expression host is not predictable so far. In this article, we collect several signal peptides that have previously performed well for recombinant protein secretion in gram-positive bacteria. We also discuss several factors influencing recombinant protein secretion efficiency in gram-positive bacteria. Signal peptides with a higher charge/length ratio in n-region, more consensus residues at the-3 and-1positions in c-region and a much higher proportion of coils are more likely to perform well in the secretion of recombinant proteins. These summaries can be utilized to the selection and directed modification of signal peptides for a given recombinant protein.