Fibrinolytic enzyme genes (aprE2, aprE176, and aprE179) were introduced into the Bacillus subtilis 168 chromosome without any antibiotic resistance gene. An integration vector, pDG1662, was used to deliver the genes into the amyE site of B. subtilis 168. Integrants, SJ3-5nc, SJ176nc, and SJ179nc, we...
Fibrinolytic enzyme genes (aprE2, aprE176, and aprE179) were introduced into the Bacillus subtilis 168 chromosome without any antibiotic resistance gene. An integration vector, pDG1662, was used to deliver the genes into the amyE site of B. subtilis 168. Integrants, SJ3-5nc, SJ176nc, and SJ179nc, were obtained after two successive homologous recombinations. The integration of each fibrinolytic gene into the middle of the amyE site was confirmed by phenotypes (Amy-, SpecS) and colony PCR results for these strains. The fibrinolytic activities of the integrants were higher than that of B. subtilis 168 by at least 3.2-fold when grown in LB broth. Cheonggukjang was prepared by inoculating each of B. subtilis 168, SJ3-5nc, SJ176nc, and SJ179nc, and the fibrinolytic activity of cheonggukjang was 4.6 ± 0.7, 10.8 ± 0.9, 7.0 ± 0.6, and 8.0 ± 0.2 (U/g of cheonggukjang), respectively at 72 h. These results showed that construction of B. subtilis strains with enhanced fibrinolytic activities is possible by integration of a strong fibrinolytic gene via a marker-free manner.
Fibrinolytic enzyme genes (aprE2, aprE176, and aprE179) were introduced into the Bacillus subtilis 168 chromosome without any antibiotic resistance gene. An integration vector, pDG1662, was used to deliver the genes into the amyE site of B. subtilis 168. Integrants, SJ3-5nc, SJ176nc, and SJ179nc, were obtained after two successive homologous recombinations. The integration of each fibrinolytic gene into the middle of the amyE site was confirmed by phenotypes (Amy-, SpecS) and colony PCR results for these strains. The fibrinolytic activities of the integrants were higher than that of B. subtilis 168 by at least 3.2-fold when grown in LB broth. Cheonggukjang was prepared by inoculating each of B. subtilis 168, SJ3-5nc, SJ176nc, and SJ179nc, and the fibrinolytic activity of cheonggukjang was 4.6 ± 0.7, 10.8 ± 0.9, 7.0 ± 0.6, and 8.0 ± 0.2 (U/g of cheonggukjang), respectively at 72 h. These results showed that construction of B. subtilis strains with enhanced fibrinolytic activities is possible by integration of a strong fibrinolytic gene via a marker-free manner.
subtilis strains (2% inoculum size, dry soybean weight (v/w)): 168 (control cheonggukjang), SJ3-5nc, SJ176nc, and SJ179nc. Fermentation was proceeded for 3 days at 37℃ and cheonggukjang samples were taken at time points (6, 12, 24, 36, 48, 60, and 72 h) for measuring the cell numbers and fibrinolytic activities. The fibrinolytic activities of culture supernatant and cheonggukjang were assayed by using the fibrin plate method as described previously [7,10].
2 ml Eppendorf tube containing 10 μl of 2 × PCR mixture (GoTag Long PCR M aster; Promega, Madison, WI, USA). PCRs were done using various primer pairs and the amplification program consisted of 93℃ for 3 min, 35 cycles of 93℃ for 15 sec, 62℃ for 30 sec, and 68℃ for 4 min. After the PCR, 5 μl of each amplified product was analyzed by agarose gel (1% (w/v)) electrophoresis.
이론/모형
B. subtilis 168 competent cells were prepared and transformed by the two-step transformation method of Cutting and Vander Horn [3].
성능/효과
Colonies on LB agar plates were spotted onto LB plates with 1% soluble starch or spectinomycin (100 μg/ml). After several trials, colonies showing SpeS and Amy- were obtained. When PCR was done using the amylF/amylR primer pair, a 1.
subtilis 168, SJ3-5nc, SJ176nc, and SJ179nc were individually inoculated into cooked soybeans and cheonggukjang fermentation was performed at 37℃. All four bacilli strains showed good growth, and the viable counts increased rapidly from 106 to 109 CFU/g of cheonggukjang within the first 6 h (Fig. 5).
subtilis 168 has a low level of fibrinolytic activity and the introduction of a gene was not enough to increase the fibrinolytic activity drasticially. Because the purpose of this work was to examine the possibility of improving the fibrinolytic activity of Bacillus strains through the introduction of a gene without an antibiotic marker, properties of cheonggukjang such as flavor, texture, and production of metabolites were not examined in detail at this time. Unlike cheonggukjang prepared with B.
5). The pH of cheonggukjang increased from 7.0 to 8.0 after 60 h (Fig. 5).
The results show a possibility that Bacillus strains can be improved and become more suitable starters for soyfood fermentations. Fibrinolytic enzymes from Bacillus strains are important bioactive compounds that can increase the functionality of fermented foods.
1). The three integration plasmids had the same structure and a fibrinolytic gene was located in the middle of amyE.
3A, lanes b1-b4). These results indicated that pDG1662 plasmids containing different fibrinolytic genes were individually integrated into the chromosome of B. subtilis 168 by single crossover.
When primers for amyE were used, two bands were amplified from the integrants but a single band was amplified from B. subtilis 168 (Fig. 3A, lanes a1-a4).
When primers for amyE were used, two bands were amplified from the integrants but a single band was amplified from B. subtilis 168 (Fig. 3A, lanes a1-a4).
When primers for amyE were used, two bands were amplified from the integrants but a single band was amplified from B. subtilis 168 (Fig. 3A, lanes a1-a4).
When primers for amyE were used, two bands were amplified from the integrants but a single band was amplified from B. subtilis 168 (Fig. 3A, lanes a1-a4).
B. subtilis 168 was selected as the host for the integration of the fibrinolytic genes because this strain has a basal level of fibrinolytic activity and is easily transformed. Transformation of B.
One was reverted to wild type, B. subtilis 168, and the other was the strain where a fibrinolytic gene remained in the middle of the amyE site but the Spcr and Ampr genes were deleted (Fig. 2).
subtilis CH3-5 and B. subtilis HK176, wild-type strains isolated from cheonggukjang, was not successful.
B. subtilis SJ3-5nc, SJ176nc, and SJ179nc were cultured in LB broth for 144 h and their growth and fibrinolytic activities were measured (Fig. 4).
후속연구
subtilis 168 and its integrants did not produce slime materials. As the next step for the production of high-quality cheonggukjang, Bacillus strains conferring good organoleptic properties to cheonggukjang will be selected and their fibrinolytic activities will be improved by the methods shown in this work.
subtilis CH3-5 and B. subtilis HK176 possess high levels of fibrinolytic activities, but all the responsible enzymes are not well understood, necessitating further research.
참고문헌 (20)
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