Protein engineering to enhance keratinolytic protease activity and excretion in Escherichia coli and its scale-up fermentation for high extracellular yield.

Escherichia coli is one kind of the simple and excellent biosystem to overexpress heterologous enzymes, such as keratinolytic protease, an excellent enzyme to hydrolyze keratin substrate for broad industrial application. However, protein expression in E. coli frequently faces some problems such as inactive and inclusion body formation. This work described a series of protein engineering strategies of N-terminal propeptide replacement and site-directed mutagenesis to modify this enzyme activity and production. Site-directed mutagenesis (S180G/Y215S) on N-terminal propeptide altered mutant contributed to the highest specific activity (4725 ± 65 U/mg, more than 1300 U/mg improvement than wild-type enzyme). This comprehensive mutation also achieved 2.5-fold improvement of extracellular enzyme yield in shake-flask level. The fermentation strategies about optimizing glycerol feeding and inducing point in scale-up bioreactor resulted in tremendous leakage of keratinolytic protease (954 mg/L extracellular yield within 48 h, about 9.26-fold higher than the original shake-flask level) as well as cell lysis. Although this proposed strategy faces a major challenge to maintain cell integrity or viability, it still exists the opportunity to realize other enzymes extracellular expression in E. coli system and simplify downstream processing to meet the industrial application.