Screening, Genetic Improvement, and Production Optimization of TA-Protease for Biofilm Removal of Dairy Sporeformers

Abstract

Spore-forming biofilms are the main pollutants found in the dairy industry. Although chemical and heat treatments are usually used to control these biofilms, they may become resistant; moreover, these treatments noticeably alter some milk properties, flavor, and taste as in food manufacturing. In this study, fourteen extracellular crude proteases were secreted by different thermo-alkali actinobacterial
species and assessed as anti-biofilm biomolecules using a biofilm model of 10 mixed Bacillus species. Proteases produced by Streptomyces sp. ACD/G413 and Streptomyces exfoliates 15/G710 strains proved their fitness as anti-biofilm biomolecules; they exhibit biofilm removal percentages of 55% and 58.69% respectively. They were characterized as thermophilic alkaline proteases (TA-proteases) belonging to the serine type. Genetic improvement of proteases was achieved through EMS mutagenesis followed by interspecific protoplast fusion, which resulted in the construction of a new fusant named St-F.13 strain with an increase in the protease activity by 2.9-fold (395.89 U/ml) compared with that of the wild strain ACD/G413 (135.74 U/ml), which then potentially increased by 1.34-fold (533 U/ml) after applying statistical optimization by Plackett-Burman design and response surface methodology. Finally, these enzymes were effectively combined with biosynthesized ZnO_G240 nanoparticles to achieve 96.80% biofilm removal.