High-Efficiency Expression of Serratia marcescens Non-Specific Nuclease in Brevibacillus choshinensis

Abstract:

High-efficiency recombinant expression of Serratia marcescens non-specific nuclease (SMNE) was obtained using the prokaryotic Brevibacillus choshinensis expression system. The SMNE recombinant expression plasmid was constructed using the Escherichia coli-Bacillus spp. shuttle vector. After confirmation of the enzyme activity of SMNE in crude supernatant, the recombinant expression conditions were optimized towards temperature, glycerol content in the culture, and incubation time. The enzyme activity of recombinant SMNE wascharacterizedafter purificationby affinity and gel filtration chromatography. Recombinant SMNE was expressed in B. choshinensis HD31-SP3 strain. The initial enzyme activity in crude cell lysate was 4.07×10⁶ U/L. The expression condition was determined to be 5% glycerol at 30℃ for 56 h after optimization, which increased the activity to 2.6×107 U/L and obtained 6 folds of the initial activity yield. The purification procedure was also optimized and simplified to be a one-step affinity chromatography. The pure enzyme yield reached 30~40 mg/dL and the specific activity reached 1.3×10⁷ U/mg, which was 2~3 folds of that of the commercialcontrol. Characterization of the recombinant SMNE indicated that the optimal reaction condition was 37~45 ℃, pH 8~9. 47% of the activity remained without Mg²⁺/Mn²⁺, while 35%~45% of the activity remained with 300 mmol/L of Na⁺/K⁺.