DNA Shuffling of aprE Genes to Increase Fibrinolytic Activity and Thermostability

Abstract

Four aprE genes encoding alkaline serine proteases from B. subtilis strains were used as template genes for family gene shuffling. Shuffled genes obtained by DNase I digestion followed by consecutive primerless and regular PCR reactions were ligated with pHY300PLK, an E. coli-Bacillus shuttle vector. The ligation mixture was introduced into B. subtilis WB600 and one transformant (FSM4) showed higher fibrinolytic activity. DNA sequencing confirmed that the shuffled gene (aprEFSM4) consisted of DNA mostly originated from either aprEJS2 or aprE176 in addition to some DNA from either aprE3-5 or aprESJ4. Mature AprEFSM4 (275 amino acids) was different from mature AprEJS2 in 4 amino acids and mature AprE176 in 2 amino acids. aprEFSM4 was overexpressed in E. coli BL21 (DE3) by using pET26b(+) and recombinant AprEFSM4 was purified. The optimal temperature and pH of AprEFSM4 were similar to those of parental enzymes. However, AprEFM4 showed better thermostability and fibrinogen hydrolytic activity than the parental enzymes. The results indicated that DNA shuffling could be used to improve fibrinolytic enzymes from Bacillus sp. for industrial applications.

Keywords: Bacillus subtilis; DNA shuffling; aprE; fibrinolytic enzymes.

Fig. 1. DNase I digestion of 4 aprE templates and reassembly from digested DNA by PCR.

(A) M, GeneRuler low range DNA ladder (SM1193, Thermoscientific); 1, aprE genes after DNase I (0.3 unit) digestion for 25 min at 15°C. Agarose gel (2%, w/v) was used. (B) M, 1 kb DNA ladder (N3223, New England Biolabs, USA); 1, 1^st PCR product. (C) M, 1kb DNA ladder (N3223); 1, 2^nd PCR product. PCR products were analyzed by agarose gel (1%) electrophoresis.

Fig. 2. Overproduction (A) and purification (B) of recombinant AprEFSM4.

M, Dokdo-marker broad-range (EBM1034, Elpis Biotech. Korea); 1–4 soluble fraction from E. coli cells grown for 2 h (1), 4 h (2), 10 h (3), and 20 h (4) after IPTG induction; 5–8, insoluble fraction from E. coli cells grown for 2 h (5), 4 h (6), 10 h (7), and 20 h (8) after IPTG induction; 9, a negative control, soluble fraction from E. coli BL21 [pET26b(+)] grown for 20 h without induction. (B). M, Dokdo-marker broad-range; 1, soluble fraction from E. coli cells; 2, purified AprEFSM4 from soluble fraction by affinity column chromatography.

Fig. 3. The effect of pH and temperature on the fibrinolytic activity of recombinant AprEFSM4.

(A) Optimum pH, (B) pH stability. -●-, pH 3; -○-, pH 4; -▼-, pH 5; -△-, pH 6; -■-, pH 7; -□-, pH 8; -◆-, pH 9; -◇-, pH 10; -▲-, pH 11. (C) Optimum temperature, (D) Thermostability. -●-, 37°C; -○-, 40°C; -▼-, 45°C; -△-, 50°C; -■-, 55°C; -□-, 60°C.

Fig. 4. Fibrinogen hydrolysis by recombinant AprEFSM4.

M, Dokdo-marker broad-range (EBM^-1034); 1, control (no enzyme treatment); 2, 5 min; 3, 10 min; 4, 20 min; 5, 30 min; 6, 1 h; 7, 3 h; 8, 6 h; 9, 12 h. A 10% acrylamide gel was used.